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WO2016019226A1 - Compositions et procédés d'utilisation d'antagonistes du récepteur de la (pro)rénine - Google Patents

Compositions et procédés d'utilisation d'antagonistes du récepteur de la (pro)rénine Download PDF

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Publication number
WO2016019226A1
WO2016019226A1 PCT/US2015/043085 US2015043085W WO2016019226A1 WO 2016019226 A1 WO2016019226 A1 WO 2016019226A1 US 2015043085 W US2015043085 W US 2015043085W WO 2016019226 A1 WO2016019226 A1 WO 2016019226A1
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Prior art keywords
prr
amino acid
seq
acid sequence
polypeptide
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Yumei Feng EARLEY
Tianxin Yang
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University of Utah Research Foundation Inc
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University of Utah Research Foundation Inc
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Priority claimed from US14/449,714 external-priority patent/US9586995B2/en
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Priority to US15/501,159 priority Critical patent/US10780143B2/en
Publication of WO2016019226A1 publication Critical patent/WO2016019226A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • Hypertension is the most important risk factor for cardiovascular (CV) diseases and remains the number one cause of morbidity and mortality in the United States. Despite the variety of traditional antihypertensive agents available, the blood pressure of about 40% of patients is still difficult to manage. A major component of drug resistant hypertension is neurogenic hypertension with increased
  • RAS renin-angiotensin system
  • PRR prorenin receptor
  • PRR promotes Angiotensin II (Ang II) generation and activates both Ang II- dependent and -independent signaling pathways through binding to renin and prorenin. It is now well recognized that Ang II is produced and acts locally in the central nervous system (CNS) and serves a crucial role in CV function. So far, the beneficial effects of RAS blockade have been attributed to the inhibition of the vasoconstriction and hypertrophy- inducing properties of Ang II. Thus, PRR provides a new target to study the effect of the brain RAS in hypertension because it is a novel component of the RAS, controlling the production of the vasoconstrictor Ang II and the hypertrophic signaling pathways through both Ang Il-dependent and -independent signaling pathways.
  • RAS components include angiotensin-converting enzyme (ACE) inhibitors, Ang II type 1 receptor (AT1R) blockers, and direct renin inhibitors.
  • ACE angiotensin-converting enzyme
  • AT1R Ang II type 1 receptor
  • renin inhibitors all of these compounds cause dramatic increases of plasma renin levels due to the negative feedback loop (decrease of Ang II levels) on renin production. Renin and prorenin directly bind to PRR and can activate signaling pathways independent of Ang II. The clinical relevance of PRR is particularly significant in situations where there are increases in renin and prorenin levels.
  • PRR The activation of PRR initiates an intracellular signaling pathway involving mitogen-activated protein kinase which increases the synthesis of profibrotic molecules such as plasminogen activator inhibitor- 1, fibronectin, collagen and transforming growth factor- ⁇ .
  • mitogen-activated protein kinase which increases the synthesis of profibrotic molecules such as plasminogen activator inhibitor- 1, fibronectin, collagen and transforming growth factor- ⁇ .
  • prorenin an activating ligand of PRR, is found at levels one hundred times higher in the plasma of a diabetic patient than the amount of prorenin found in healthy individuals.
  • PRR expression was increased in diabetic retinopathy, nephropathy, and in hypertension.
  • Thioether bridges have been used in modifying small peptides in the past to increase stability.
  • compounds or thioether bridge-modified peptides for treating hypertension that act on the (pro)renin receptor.
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a (pro)renin receptor (PRR) antagonist.
  • PRR renin receptor
  • the PRR antagonist is a polypeptide.
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • PRR renin receptor
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3).
  • PRR renin receptor
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • PRR renin receptor
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a (pro)renin receptor (PRR) antagonist, wherein the composition further comprises a
  • Also disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist.
  • PRR renin receptor
  • the PRR antagonist is a polypeptide.
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX.
  • PRR renin receptor
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX.
  • PRR renin receptor
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • a composition comprising (pro)renin receptor (PRR) antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • PRR renin receptor
  • Also disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist.
  • PRR renin receptor
  • the PRR antagonist is a polypeptide.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • PRR renin receptor
  • composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence
  • composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • PRR renin receptor
  • composition comprising (pro)renin receptor (PRR) antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • PRR renin receptor
  • Also disclosed are methods of decreasing proteinuria comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist.
  • PRR renin receptor
  • the PRR antagonist is a polypeptide.
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of decreasing proteinuria comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO: 3).
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • PRR renin receptor
  • Also disclosed are methods of promoting wound healing comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist.
  • PRR renin receptor
  • the PRR antagonist is a polypeptide.
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3).
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the composition further comprises a
  • Also disclosed are methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising (pro)renin receptor (PRR) antagonist.
  • PRR renin receptor
  • the PRR antagonist is a polypeptide.
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the
  • polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID O: l).
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3).
  • PRR renin receptor
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • compositions comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • PRR renin receptor
  • Also disclosed are methods of reducing Erkl/2 activation comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist.
  • PRR renin receptor
  • the PRR antagonist is a polypeptide.
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • PRR renin receptor
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3).
  • PRR renin receptor
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • PRR renin receptor
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject an effective amount of a composition comprising (pro)renin receptor (PRR) antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • a composition comprising (pro)renin receptor (PRR) antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • Figure 1 is a schematic illustrating that hypertensive stimuli increase brain PRR expression, leading to Ang II formation and/or activation of Ang 11-independent signaling, and thus hypertension. Therefore deletion of PRR in the brain will mitigate the hypertension.
  • FIG. 2 is an image illustrating prorenin and renin protein expression in brains of C57BI/6J mice.
  • the prorenin and renin protein was extracted from whole brain lysate. Samples incubated with (pro)renin antibody showed strong bands at 46 kD for prorenin and thin bands at 38 kD for matured renin.
  • Figure 3 is a graph illustrating an increase of brain PRR expression in deoxycorticosterone acetate (DOCA)-salt hypertensive mice.
  • the PRR mRNA expression levels varied in CV regulatory regions of the brain and increased following 21 days of DOCA-salt treatment. *P ⁇ 0.05 vs. Sham Cortex; #P ⁇ 0.05 vs. Sham treatment in the same brain nucleus.
  • Figure 4 is a graph illustrating that aldosterone and high salt regulate PRR expression in Neuro-2A cells.
  • Neuro-2A cells were incubated with either normal salt (146mM) or high salt (160mM) for 5 days without or without aldosterone ( ⁇ ⁇ ), MR inhibitor (Eplerenone, 10 ⁇ ), or ENaC inhibitor (Amiloride, 10 ⁇ ) for 6 hours.
  • Figure 5A is a diagram illustrating the PRR gene deleted by Cre recombinase under the control of neurofilament-H promoter in the Nefh-PRR mouse model for brain-targeted PRR deletion.
  • Figure 5B is a series of representative immuno-fluorescent images of the subfornical organ (SFO) and paraventricular nucleus (PVN) in WT and Nefh-PRR mice.
  • SFO subfornical organ
  • PVN paraventricular nucleus
  • Figure 6 is a series of graphs illustrating the pressor response to
  • WT and Nefh-PRR mice were implanted with telemetric transmitters and ICV cannula.
  • mice Two weeks after recovery, mice were ICV infused (0.3 ⁇ 1/ ⁇ ) with carbachol (lOOng/ ⁇ ), Ang II (lOOng/ ⁇ ), Ang II (lOOng/ ⁇ ) + losartan (10 g/ ⁇ ), mouse renin (lOOng/ ⁇ ), mouse prorenin (lOOng/ ⁇ ), or mouse prorenin (lOOng/ ⁇ ) +losartan (lOug/ ⁇ ) over 10 minutes.
  • Blood pressure (BP) was recorded in conscious freely moving mice. *P ⁇ 0.05 vs. WT.
  • Figure 7 is a graph illustrating reduced BP in Nefh-PRR mice in DOCA-salt hypertension. Mice were implanted with telemetry transmitters for BP recording and treated with DOCA-salt for 21 days.*P ⁇ 0.05 VS. WT.
  • Figure 8 is a graph illustrating Ang II levels in DOCA-salt hypertensive mice. Mice were treated with DOCA-salt or Sham for 21 days. Brain hypothalamus and kidney tissues were harvested for Ang II measurement using fluorescent ELISA KIT. *P ⁇ 0.05 vs. WT.
  • FIGs 9 A and 9B are graphs illustrating that ICV administration of AAV- Cre-eGFP reduces pressor response induced by ICV mouse prorenin in PRR-Floxed mice.
  • the AAV-PRR-eGFP(lOOnl) was administered ICV to PRR-Floxed mice. After 7 days, mice were ICV infused (0.3 1/min) with mouse prorenin (lOOng/ ⁇ ) over 10 minutes. BP was recorded in conscious freely moving mice (Figure 9A). At the end of experiment, brain cortex, hypothalamus, and brainstem were harvested for PRR mRNA measurement ( Figure 9B). *P ⁇ 0.05 vs. AAV-eGFP.
  • Figure 10 is a diagram illustrating the 3D structure of PR10 (SEQ ID NO:4).
  • Figure 11 shows the amino acid sequence and chemical structure of modified peptide PR 103 (SEQ ID NO: 7) with a thioether bridge and one non-standard amino acid, dehydroalanine (Dha), at position 7. dehydroalanine (Dha), 2-aminobutyric acid (Abu), and dehydrobutyrine
  • Figure 12 is a diagram illustrating the 3D structure of modified peptide PR103.
  • Figure 13 shows the amino acid sequence and chemical structure of modified peptide PR 105 (SEQ ID NO: 8) with two thioether bridges and one non-standard amino acid, dehydroalanine (Dha), at position 7.
  • Figure 14 is a diagram illustrating the 3D structure of modified peptide PR105.
  • Figure 15 shows the amino acid sequence and chemical structure of modified peptide PR 107 (SEQ ID NO: 9) with two thioether bridges and one non-standard amino acid, dehydroalanine (Dha), at position 7.
  • Figure 16 shows the amino acid sequence and chemical structure of modified peptide PR201 (SEQ ID NO: 10) with three thioether bridges and four non-standard amino acids, 2-aminobutyric acid (Abu, at positions 3 and 7), dehydrobutyrine (Dhb, at position 5), and dehydroalanine (Dha, at position 18).
  • Figure 17 shows the amino acid sequence and chemical structure of modified peptide PR202 (SEQ ID NO: 11) with two thioether bridges and four non-standard amino acids, 2-aminobutyric acid (Abu, at position 3), dehydrobutyrine (Dhb, at positions 5 and 6), and dehydroalanine (Dha, at position 18).
  • Figure 18 shows the amino acid sequence and chemical structure of modified peptide PR203 (SEQ ID NO: 12) with two thioether bridges and one non-standard amino acid, dehydroalanine (Dha, at position 10).
  • Figure 19 is a series of graphs showing the exemplary dose responses for peptide PR 10 and PR20.
  • Figure 20 is a series of fluorescence microscopy images showing PRR binding in the PR20 alanine replacement assay. Each of the amino acid positions of PR20 was substituted with alanine. PR20 and the alanine substituted peptides were labeled with FITC. Fluorescence indicates binding of the peptide to PRR.
  • Figure 21 shows the core amino acid sequence for the PR30 peptide (SEQ ID NO: 5) and modified thioether bridge containing peptides PR301 (SEQ ID NO: 13), PR302 (SEQ ID NO: 14), and PR303 (SEQ ID NO: 15).
  • PR301 comprises five nonstandard amino acid residues
  • PR302 and PR303 each comprise four non-standard amino acid residues.
  • Figure 22 shows the core amino acid sequence for the PR40 peptide (SEQ ID NO: 6) and modified thioether bridge containing peptides PR401 (SEQ ID NO: 16), PR402 (SEQ ID NO: 17), and PR403 (SEQ ID NO: 18).
  • PR401 comprises four nonstandard amino acid residues, and PR402 and PR403 each comprise three nonstandard amino acids.
  • Figure 23 shows FVB mice that were sham-operated or implanted with DOCA pellet alone or in combination with PRR decoy peptide PRO20 and were given saline as drinking fluid. After 7 days, the wound in the DOCA group was opened after grabbing the neck for urine collection. In contrast, the wound in the DOCA + PRO20 group remained closed despite the same animal handling.
  • Figures 24A and B show the effect of PRO20 on lipopolysaccharide (LPS)- induced hypotension and bradycardia.
  • LPS lipopolysaccharide
  • Male 8-week-old C57 mice were treated subcutaneously administered for 7 days with PRO20 via osmotic mini-pump (600 ⁇ g/kg/d) and instrumented with radiotelemetric devices. One week later, they received a single dose of LPS (10 mg/kg, i.p.) or vehicle.
  • MAP mean arterial pressure
  • HR Hourly monitoring heart rate
  • N 3-4 per group. Data are mean + SE. *, p ⁇ 0.05 vs. vehicle at the corresponding period.
  • Figures 25A and 25B show the effect of PRO20 on LPS-induced renal dysfunction.
  • C57 mice were pretreated with PRO20 and then treated with LPS or vehicle.
  • blood was withdrawn from vena cava under general anesthesia and plasma was assayed for creatinine (A) and BUN (B).
  • N 3-4 per group. Data are mean + SE.
  • Figures 26A and 26B show the characterization of PRO20 and examination of its effect on Angll-induced hypertension.
  • A Comparison of PRO20 and the HRP in inhibiting prorenin-induced signaling. Primary rat IMCD cells were pretreated for 1 h with PRO20 (1.5 ⁇ ) or the HRP (2.0 ⁇ ) and then were exposed to 100 nM prorenin for 10 min. ERKl/2 phosphorylation was determined by immunoblotting.
  • B Effect of intramedullary delivery of PRO20 on Angll-induced hypertension.
  • Uninephrectomized SD rats were divided into the following three groups: (1) Angll, (2) Angll + IM PRO (intramedullary PRO20 infusion), and (3) Angll + IV PRO20 (intravenous PRO20 infusion).
  • Angll was subcutaneously infused at 100 ng/kg/min via an osmotic mini-pump.
  • IM PRO PRO20 at 120 ⁇ g/kg/d
  • IV PRO (PRO20 at 120 ⁇ g/kg/d) was performed via catheterization of jugular vein. Telemetry was performed to monitor mean arterial pressure (MAP) and it was turned on 4 h per day from 5:00PM to 9:00PM for 7 days.
  • MAP mean arterial pressure
  • N 3-6 per group. Data are mean + SE.
  • Figures 27A-C show the effect of IM PRO on Angll- induced kidney injury.
  • A Measurement of urinary protein excretion. Following 1-wk Angll infusion, rats were placed in metabolic cages for urine collection. Urine protein was measured by using Commassie blue.
  • B Representative photographs of PAS staining of kidney sections.
  • C Renal injury scores from semi-quantitative analysis of
  • Figures 28A-E show the effect of IMPRO on renal inner medullary ENaC expression during Angll-induced hypertension.
  • Figure 29 shows immunostaining of AQP2 in the renal inner medulla of control, Angll, and Angll + IMPRO rats. Shown are representatives from 6 animals per group.
  • Figures 30A-F show the role of PRR in Angll-induced renin response in the renal medulla in vivo and in vitro.
  • In vivo studies examined the effects of IMPRO on renal medullary renin activity and prorenin/renin expression in Angll-infused rats (A- E) and in vitro studies probed the direct action of PRO20 in renin regulation in cultured renal CD cells
  • A Renin activity in the inner medulla.
  • B Active renin content in the inner medulla.
  • C Prorenin content in the inner medulla.
  • D ELISA detection of prorenin/renin in the inner medulla.
  • Plasma aldosterone concentrations (A) and urinary aldosterone excretion (B) were determined in control, Angll, Angll + IM PRO20, and Angll + IV PRO20 rats.
  • the direct action of PRO20 in aldosterone regulation was tested in primary culture of rat IMCD cells (C).
  • C primary culture of rat IMCD cells
  • the cells were exposed to vehicle, Angll, or Angll + PRO20 and the medium were assayed for aldosterone release.
  • N 3-6 per group. Data are means ⁇ SE.
  • Figures 32A-H show the role of PRR in the acute regulation of ENaC activity in cultured CD cells.
  • Confluent mpkCCD cells grown on Transwell membrane were pretreated for 30 min with PRO20 (1.5 ⁇ ), losartan (1.0 ⁇ ) , apocynin (1 mM), or eplerenone (10 mg/L) and then treated with Angll (500 nM) or prorenin (10 nM).
  • Ieq was measured by using EVOM and amiloride-sensitive Ieq was determined at the end of experiments.
  • A Time course of Ieq changes in control, Angll, and Angll + PRO20 groups.
  • Figures 33A-D show the role of PRR in chronic regulation of ENaC by prorenin in cultured CD cells.
  • A Time course of Ieq changes in control, prorenin, prorenin + PRO20, and prorenin + losartan groups over 24 h in mpkCCD cells.
  • B Time course of changes in Ieq during 24-h aldosterone treatment in mpkCCD cells.
  • C Amiloride-sensitive Ieq in mpkCCD cells after exposure for 24 h to 10 nM prorenin in presence or absence of eplerenone.
  • D Medium aldosterone concentration in primary rat IMCD cells exposed for 24 h to 10 nM prorenin alone or in combination with 1.5 ⁇ PRO20 or 1.0 ⁇ losartan.
  • Figure 34 shows the schematic illustration of the central role of prorenin/PRR in regulation of ENaC activity in the renal CD cells during Angll-induced
  • Angll treatment elevates renal medullary expression of prorenin and PRR.
  • PRR Activation of PRR by prorenin acutely increases ENaC activity via ROS generation and chronically induces ENaC expression through release of aldosterone, ultimately leading to increased sodium reabsorption in the CD and thus blood pressure.
  • PRR mediates Angll-induced renal medullary renin response, a likely mechanism for sustaining the maximal level of local Angll.
  • Figures 35A and B show the effect of PRO20 on AQP2 expression in primary rat inner medullary collecting duct cells.
  • the cells were treated for 24 h with 10-8 M vasopressin (A) or 10 nM prorenin (B) alone or in combination with 1.5 M PRO20.
  • Figure 36 shows the effect of PRO20 on AQP2 expression in rat kidneys in vivo.
  • PRO20 was administered for 1 week at 120 ⁇ g/kg/d via an intrarenal infusion catheter connected to an osmotic mini-pump.
  • the control group received vehicle infusion. All animals were water deprived during the last 3 days to enhance renal AQP2 expression.
  • FIGs 37A and 37B shows plasma creatinine (A) and BUN (B) in control, ischemia-reperfusion (I/R), and I/R + PRO20 mice.
  • Animals were anesthetized and the left and right renal arteries were isolated and occluded for 30 min to produce ischemia. After 30 min, the reperfusion was made.
  • An hour after the commencement of ischemia the VR+ PRO20 mice received PRO20 by subcutaneous injection three times a day (total daily dose: 12.0 mg kg/day for the first 24 hour and 2.0 mg kg/day for the second 24 h).
  • the control and I/R mice received the vehicle treatment.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.efinitions
  • the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps.
  • each step comprises what is listed (unless that step includes a limiting term such as “consisting of), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.
  • Effective amount means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent.
  • the effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
  • terapéuticaally effective amount means an amount of a therapeutic, prophylactic, and/or diagnostic agent (e.g., composition comprising a PRR antagonist) that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, alleviate, ameliorate, relieve, alleviate symptoms of, prevent, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of the disease, disorder, and/or condition.
  • a therapeutic, prophylactic, and/or diagnostic agent e.g., composition comprising a PRR antagonist
  • Kidney disease refers to a variety of conditions that lead to kidney damage and deterioration of kidney function. Kidney disease includes acute kidney injury (AKI) and chronic kidney disease (CKD). AKI, previously called acute renal failure (ARF) is an abrupt loss of kidney function that develops within a short period of time. Chronic kidney disease (CKD), also known as chronic renal disease (CRD), is a progressive loss in renal function over a period of months or years.
  • AKI acute kidney injury
  • CKD chronic kidney disease
  • CRD chronic renal disease
  • treating refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition.
  • treating a microbial infection may refer to inhibiting survival, growth, and/or spread of the microbe.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • (Pro)renin receptor antagonist refers to a composition able to antagonize the action of prorenin.
  • PRR antagonists can bind to the (pro)renin receptor and prevent or block (pro)renin from binding.
  • PRR antagonists can bind to (pro)renin and prevent or block (pro) renin from binding to the (pro)renin receptor.
  • subject refers to the target of administration, e.g. an animal.
  • the subject of the disclosed methods can be a vertebrate, such as a mammal.
  • the subject can be a human.
  • the term does not denote a particular age or sex.
  • Subject can be used interchangeably with “individual” or "patient.”
  • polynucleotide as referred to herein means single-stranded or double-stranded nucleic acid polymers. In some instances, the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term “polynucleotide” specifically includes single and double stranded forms of DNA. As will be also recognized by the skilled artisan,
  • polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules.
  • sequence similarity or sequence identity between sequences are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 1 1-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range -1 from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist.
  • the PRR antagonist can be a polypeptide.
  • a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2 (LPTDTTTFKRIFLKRMPSI).
  • the polypeptide comprises an amino acid sequence having at least 75, 80, 85, 90, 95, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2
  • Disclosed are methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO:2
  • Disclosed are methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTXXXXXXXXXXXSX (SEQ ID NO: l).
  • SEQ ID NO: 1 can be any amino acid.
  • SEQ ID NO: 1 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, and 18 to amino acids 3, 4, 6, 7, and 18 of SEQ ID NO:2.
  • Disclosed are methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO: 3).
  • methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTFXRIXXXXXXXSX (SEQ ID NO:3).
  • Each of the X's in SEQ ID NO:3 can be any amino acid.
  • SEQ ID NO:3 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 to amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 of SEQ ID NO:2.
  • Methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence of SEQ ID NO:2
  • Disclosed are methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is one or more of the polypeptides described herein, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • Kidney disease can be due to a variety of conditions that lead to kidney damage and deterioration of kidney function. For example, AKI due to sepsis, drug toxicity, ischemia, and CKD due to diabetes, hypertension, and glomerulonephritis are all conditions that can lead to kidney disease.
  • Kidney disease can be determined by measurement of plasma creatinine and blood urea nitrogen (BUN), urinary analysis of albumin and casts, and immunological and histological analysis of kidney biopsy samples. The rise of plasma creatinine and BUN can indicate renal failure.
  • BUN blood urea nitrogen
  • kidney disease treatments comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, further comprising administering a common kidney disease treatment.
  • Common kidney disease treatments can be, but are not limited to, angiotensin converting enzyme inhibitor, angiotensin receptor antagonist, steroids, or an immunosuppressant.
  • the PRR antagonist can be administered in conjunction with or followed by any of the common kidney disease treatments.
  • the PRR antagonist can be administered prior to the common kidney disease treatment.
  • the common kidney disease treatment can be administered prior to the PRR antagonist.
  • Administration of the PRR antagonist and common kidney disease treatment can occur within 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, or 60 minutes of each other.
  • the administration of the PRR antagonist and common kidney disease treatment can occur within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours of each other.
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist.
  • the PRR antagonist can be a polypeptide.
  • [00130] Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2. In some instances the polypeptide comprises an amino acid sequence having at least 75, 80, 85, 90, 95, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2. In some aspects the polypeptide is the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence
  • polypeptide is the amino acid sequence the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NOT).
  • SEQ ID NO: 1 can be any amino acid.
  • SEQ ID NOT is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, and 18 to amino acids 3, 4, 6, 7, and 18 of SEQ ID NO:2.
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence
  • a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3).
  • XXTDXTTFXRIXXXXXXXSX SEQ ID NO:3
  • Each of the X's in SEQ ID NO:3 can be any amino acid.
  • SEQ ID NO:3 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, 8, 10, 11, and 18 to amino acids 3, 4, 6, 7, 8, 10, 11, and 18 of SEQ ID NO:2.
  • [00135] Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence of SEQ ID NO:2.
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • methods of treating kidney disease comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is one or more of the polypeptides described herein, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • Infectious diseases are diseases caused by infection with various pathogens such as, but not limited to, bacteria, viruses, and fungi.
  • Inflammatory diseases refer to conditions that involve the inflammation pathway. Examples of inflammatory diseases are autoimmune diseases, hypertension, diabetes, aging, asthma, allergies, sepsis, and cancer.
  • Disclosed are methods of treating infectious or inflammatory diseases comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, further comprising administering a common infectious or inflammatory disease treatment.
  • Common infectious or inflammatory disease treatments can be, but are not limited to, antibiotics and anti-inflammatory agents.
  • methods of treating infectious or inflammatory disease comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, further comprising a common infectious or inflammatory disease treatment, wherein the infectious or inflammatory disease treatment comprises the administration of an antibiotic or anti-inflammatory.
  • the PRR antagonist can be administered in conjunction with or followed by any of the common infectious or inflammatory disease treatments. In some instances, the PRR antagonist can be administered prior to the common infectious or inflammatory disease treatment. In some instances, the common infectious or inflammatory disease treatment can be administered prior to the PRR antagonist. Administration of the PRR antagonist and common infectious or inflammatory disease treatment can occur within 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, or 60 minutes of each other. In some instances, the administration of the PRR antagonist and common infectious or inflammatory disease treatment can occur within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours of each other.
  • the PRR antagonist can be a polypeptide.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2. In some instances the polypeptide comprises an amino acid sequence having at least 75, 80, 85, 90, 95, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTXXXXXXXXXXXSX (SEQ ID NO: 1).
  • SEQ ID NO: 1 can be any amino acid.
  • SEQ ID NO: 1 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, and 18 to amino acids 3, 4, 6, 7, and 18 of SEQ ID NO:2.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3).
  • methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTFXRIXXXXXXXSX (SEQ ID NO:3).
  • SEQ ID NO:3 can be any amino acid.
  • SEQ ID NO:3 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 to amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 of SEQ ID NO:2.
  • Methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence of SEQ ID NO:2.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is one or more of the polypeptides described herein, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • Acute kidney injury occurs when a subject's kidneys suddenly stop functioning properly. For example, the kidneys lose their ability to remove waste from the blood. Thus, any subject having an abrupt loss of kidney function is said to have acute kidney injury.
  • Acute kidney injury can be caused by, but is not limited to, ischemia- reperfusion, disease, kidney stones, antibiotics, low blood pressure, heart failure or liver cirrhosis.
  • Disclosed are methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, further comprising administering a known therapy for acute kidney injury.
  • Common acute kidney injury treatments can be, but are not limited to, medications to control blood potassium or restore blood calcium levels, dialysis, and i.v. fluids.
  • methods of treating acute kidney injury comprising administering to a subject a therapeutically effective amount of a composition comprising PRR antagonist, wherein the PRR antagonist is a polypeptide, further comprising a known therapy for acute kidney injury, wherein the therapy for acute kidney injury comprises the administration of i.v. fluids.
  • the PRR antagonist can be administered in conjunction with or followed by any of the common infectious or inflammatory disease treatments. In some instances, the PRR antagonist can be administered prior to the common infectious or inflammatory disease treatment. In some instances, the common infectious or inflammatory disease treatment can be administered prior to the PRR antagonist. Administration of the PRR antagonist and common infectious or inflammatory disease treatment can occur within 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, or 60 minutes of each other. In some instances, the administration of the PRR antagonist and common infectious or inflammatory disease treatment can occur within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours of each other.
  • Disclosed are methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist.
  • the PRR antagonist can be a polypeptide.
  • a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2. In some instances the polypeptide comprises an amino acid sequence having at least 75, 80, 85, 90, 95, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2
  • Disclosed are methods decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • methods decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTXXXXXXXXXXXSX (SEQ ID NO: l).
  • SEQ ID NO: 1 can be any amino acid.
  • SEQ ID NO: 1 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, and 18 to amino acids 3, 4, 6, 7, and 18 of SEQ ID NO:2.
  • Disclosed are methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO: 3).
  • methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTFXRIXXXXXXXSX (SEQ ID NO:3).
  • SEQ ID NO: 3 can be any amino acid.
  • SEQ ID NO: 3 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, 8, 10, 11, and 18 to amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 of SEQ ID NO:2.
  • Disclosed are methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence of SEQ ID NO:2.
  • Disclosed are methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • methods of decreasing proteinuria comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is one or more of the polypeptides described herein, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • the PRR antagonist can be a polypeptide.
  • compositions comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2. In some instances the polypeptide comprises an amino acid sequence having at least 75, 80, 85, 90, 95, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2
  • composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of promoting wound healing comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence set forth in SEQ ID NO:2.
  • SEQ ID NO: 1 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, and 18 to amino acids 3, 4, 6, 7, and 18 of SEQ ID NO:2.
  • a composition comprising a PRR antagonist wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO: 3).
  • methods of promoting wound healing comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTFXRIXXXXXXXSX (SEQ ID NO:3).
  • Each of the X's in SEQ ID NO:3 can be any amino acid.
  • SEQ ID NO:3 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 to amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 of SEQ ID O:2.
  • compositions comprising a PRR antagonist wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • methods of promoting wound healing comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence of SEQ ID NO:2.
  • composition comprising a PRR antagonist
  • composition further comprises a pharmaceutically acceptable carrier.
  • methods of promoting wound healing comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is one or more of the polypeptides described herein, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • the PRR antagonist can be a polypeptide.
  • the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2. In some instances the polypeptide comprises an amino acid sequence having at least 75, 80, 85, 90, 95, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2
  • Disclosed are methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTXXXXXXXXXXXSX (SEQ ID NO: l).
  • SEQ ID NO: 1 can be any amino acid.
  • SEQ ID NO: 1 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, and 18 to amino acids 3, 4, 6, 7, and 18 of SEQ ID NO:2.
  • Disclosed are methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3).
  • methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTFXRIXXXXXXXSX (SEQ ID NO:3).
  • Each of the X's in SEQ ID NO: 3 can be any amino acid.
  • SEQ ID NO: 3 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, 8, 10, 11, and 18 to amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 of SEQ ID NO:2.
  • [00170] Disclosed are methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence of SEQ ID NO:2.
  • compositions comprising a PRR antagonist, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • methods of treating hypertension comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is one or more of the polypeptides described herein, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • the PRR antagonist can be a polypeptide.
  • compositions comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2. In some instances the polypeptide comprises an amino acid sequence having at least 75, 80, 85, 90, 95, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2
  • composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence set forth in SEQ ID NO:2.
  • [00175] Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l).
  • methods of reducing Erkl/2 activation comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTXXXXXXXXXXXSX (SEQ ID NO: 1).
  • SEQ ID NO: 1 can be any amino acid.
  • SEQ ID NO: 1 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, and 18 to amino acids 3, 4, 6, 7, and 18 of SEQ ID NO:2.
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide comprises the amino acid sequence XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3).
  • methods of reducing Erkl/2 activation comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence XXTDXTTFXRIXXXXXXXSX (SEQ ID NO:3).
  • SEQ ID NO:3 can be any amino acid.
  • SEQ ID NO:3 is provided as an example of a polypeptide to be used in the methods described herein, wherein the sequence comprises 100% identity at amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 to amino acids 3, 4, 6, 7, 8, 10, 1 1, and 18 of SEQ ID NO:2.
  • Disclosed are methods of reducing Erkl/2 activation comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2.
  • methods of reducing Erkl/2 activation comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is a polypeptide, wherein the polypeptide is the amino acid sequence of SEQ ID NO:2.
  • composition comprising a PRR antagonist
  • composition further comprises a pharmaceutically acceptable carrier.
  • methods of reducing Erkl/2 activation comprising administering to a subject a therapeutically effective amount of a composition comprising a PRR antagonist, wherein the PRR antagonist is one or more of the polypeptides described herein, wherein the composition further comprises a pharmaceutically acceptable carrier.
  • a PRR antagonist inhibits or blocks the ligand-receptor interaction of prorenin to PRR.
  • PRR antagonists can block prorenin from binding PRR.
  • a PRR antagonist competes with prorenin for binding to PRR.
  • PRR antagonists can be polypeptides.
  • functional PRR antagonist polypeptides include but are not limited to IFDNIISQGVLKEDVF (PR10; SEQ ID NO:4), LPTDTTTFKRIFLKRMPSI (PR20; SEQ ID NO:2),
  • LPTRTATFERIPLKKMP SVRE PR40; SEQ ID NO: 6
  • PRR antagonist polypeptides can comprise an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:2.
  • PRR antagonist polypeptides can comprise the amino acid sequence set forth in SEQ ID NO:2.
  • PRR antagonist polypeptides can consist of the amino acid sequence set forth in SEQ ID NO:2. In some instances, PRR antagonist polypeptides can is the amino acid sequence set forth in SEQ ID NO:2.
  • PRR antagonist polypeptides include modified peptides, e.g., peptides comprising a thioether bridge and/or amino acids that are not standard or naturally occurring in humans, e.g., amino acids found in polypeptides of microbial origin.
  • non-standard amino acids include, but are not limited to, dehydroalanine (Dha), 2-aminobutyric acid (Abu), and dehydrobutyrine (referred to herein interchangeably as "Dht” or "Dhb").
  • Thioether-bridge modified peptides are designed based on the core amino acid sequences of PR10, PR20, PR30, and PR40 in order to avoid peptide degradation by peptidase in vivo.
  • the introduction of one or more thioether bridges makes the resulting peptides more stable and, therefore, strong PRR antagonists.
  • the NisBTC encoding plasmid (pTU-BTC) and substrate-peptide-encoding plasmids pPR103, pPR105, pPR107, pPR201, pPR202 were constructed and introduced into the lactic acid producing bacterium L.
  • PR203 SEQ ID NO: 12 as provided in Figure 18.
  • PRR antagonist polypeptides can comprise common amino acid substitutions or modifications.
  • a PRR antagonist polypeptide derived from the core amino acid sequence of PR20 can comprise amino acid residues 3, 4, 6, 7, and 18 of the amino acid sequence set forth in SEQ ID NO:2.
  • a polypeptide can comprise the amino acid sequence XXTDXTTXXXXXXXXXXSX (SEQ ID NO: l) wherein each of the X's in SEQ ID NO: l can be any amino acid.
  • SEQ ID NO: l comprises 100% identity at amino acids 3, 4, 6, 7, and 18 to amino acids 3, 4, 6, 7, and 18 of SEQ ID NO:2, but could have any amino acid at the other positions.
  • a polypeptide can comprise XXTDXTTFXRIXXXXXXSX (SEQ ID NO:3) whereineach of the X's in SEQ ID NO:3 can be any amino acid.
  • SEQ ID NO:3 comprises 100% identity at amino acids 3, 4, 6, 7, 8, 10, 1 1, and l 8 to amino acids 3, 4, 6, 7, 8, 10, 11, and 18 of SEQ ID NO:2, but could have any amino acid at the other positions.
  • Examples of polypeptides comprising SEQ ID NO: l or SEQ ID NO:3 include, but are not limited to SEQ ID NOs: 13-18 (these are PR301, PR302, PR303, PR401, PR402 and PR403).
  • the PRR antagonist can be a peptide comprising an amino acid sequence having at least 50% identity to an amino acid sequence set forth in one of SEQ ID NOs: 8- 18.
  • the PRR antagonist is a peptide comprising an amino acid sequence having at least 60% identity to an amino acid sequence set forth in one of SEQ ID NOs:8-12 and 13-18.
  • the PRR antagonist is a peptide comprising an amino acid sequence having at least 70% identity to an amino acid sequence set forth in one of SEQ ID NOs:8-12 and 13- 18.
  • the PRR antagonist is a peptide comprising an amino acid sequence having at least 80% identity to an amino acid sequence set forth in one of SEQ ID NOs:8-12 and 13-18.
  • the PRR antagonist is a peptide comprising an amino acid sequence having at least 90% identity to an amino acid sequence set forth in one of SEQ ID NOs:8-12 and 13-18. In another embodiment, the PRR antagonist is a peptide comprising the amino acid sequence set forth in one of SEQ ID Os:8-12 and 13-18.
  • PRR antagonist variants or derivatives are disclosed.
  • the PRR antagonists can be modified or altered.
  • analog is used interchangeably with “variant” and “derivative.”
  • variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. Such, amino acid sequence modifications typically fall into one or more of three classes:
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily are smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final derivative or analog. Substutitional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with Tables 1 and 2 and are referred to as conservative substitutions.
  • Substantial changes in function are made by selecting substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical
  • substitutions which in general are expected to produce the greatest changes in the protein properties are those in which: (a) the hydrophilic residue, e.g.
  • seryl or threonyl is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; Tryptophan, Tyrosinyl (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or hystidyl, is substituted for (or by) an electronegative residue, e.g.
  • variants and derivatives of the disclosed proteins herein are to define them in terms of homology/identity to specific known sequences.
  • variants of PRR antagonists herein disclosed which have at least, 70% or at least 75% or at least 80% or at least 85% or at least 90% or at least 95% homology to the PRR antagonists specifically recited herein.
  • PRR antagonists specifically recited herein.
  • polypeptides can be modified by either natural processes, such as post- translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications.
  • Modifications include, without limitation, acetylation, acylation, ADP- ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, yristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation.
  • Variants can also include peptidomimetics. As used herein,
  • peptidomimetic means a mimetic of a function of a protein which includes some alteration of the normal peptide chemistry.
  • Peptidomimetics typically are short sequences of amino acids that in biological properties, mimic one or more function(s) of a particular protein. Peptide analogs enhance some property of the original peptide, such as increases stability, increased efficacy, enhanced delivery, increased half-life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position.
  • One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic.
  • suitable amino acid mimics include ⁇ -alanine, L-a-amino butyric acid, L-y-amino butyric acid, L-a-amino isobutyric acid, L-e-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, ⁇ - ⁇ -Boc-N-a- CBZ-L-lysine, ⁇ - ⁇ -Boc-N-a-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L- norvaline, N-a-Boc-N-5CBZ-L-ornithine, ⁇ - ⁇ -Boc-N-a-a-
  • Polynucleotide variants can have substantial identity to a PRR antagonist
  • a polynucleotide variant can be a polynucleotide comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a reference polynucleotide sequence.
  • a reference polynucleotide sequence can be the polynucleotide sequence capable of encoding SEQ ID NO:2.
  • compositions comprising any of the disclosed polypeptides or polynucleotides.
  • compositions which can also include a carrier such as a pharmaceutically acceptable carrier.
  • a carrier such as a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising the peptides disclosed herein, and a pharmaceutically acceptable carrier.
  • compositions described herein can comprise a
  • pharmaceutically acceptable carrier By “pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • carriers include dimyristoylphosphatidyl (DMPC), phosphate buffered saline or a multivesicular liposome.
  • DMPC dimyristoylphosphatidyl
  • PG:PC:Cholesterol:peptide or PC:peptide can be used as carriers in this invention.
  • Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles.
  • compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the polypeptide, peptide, nucleic acid, vector of the invention is not compromised.
  • Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • vectors comprising any of the disclosed polypeptides or polynucleotides.
  • Viral and non-viral vectors can be used to administer the disclosed polypeptides or polynucleotides.
  • nanoparticles can be used to delivery any of the disclosed polypeptides or polynucleotides.
  • viral vectors such as adenoviral, adeno-associated, and retroviral vectors, can be used to delivery any of the disclosed polynucleotides.
  • the vectors are expression vectors.
  • expression of polynucleotide of interest within the vector is controlled by the vector.
  • Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.
  • compositions comprising the disclosed vectors.
  • administration or delivery of the polypeptides, polynucleotides, vectors, or compositions to cells can be via a variety of mechanisms.
  • compositions can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • Preparations of parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for optical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.
  • compositions can be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glycolic
  • Neuron-specific PRR knockout mice Nefh-PRR
  • WT wildtype littermates
  • BP blood pressure
  • ICV intracerebroventricular
  • mice prorenin 100 ng/ul
  • mouse renin 100 ng/ul
  • A1R Ang II type 1 receptor blocker
  • Nefh-PRR and WT littermates were treated with 50 mg of deoxycorticosterone acetate (DOCA) subcutaneously, plus 0.9% NaCI drinking water for 21 days.
  • the baseline BP was similar between Nefh-PRR (101 ⁇ 2) and WT (101 ⁇ 3) mice.
  • BP was increased in WT mice (132 ⁇ 6) by DOCA-salt treatment, while Nefh-PRR mice remained normotensive (108 ⁇ 3).
  • prorenin via PRR mediates Angll/ATlR-dependent pressor response in the brain.
  • Neuronspecific PRR deletion attenuates the development of DOCA-salt hypertension likely due to the lack of Ang II/AT1R activation.
  • Elevated expression and activity of RAS components in the brain CV control regions support the concept that the brain RAS is involved in the pathogenesis of hypertension, including DOCA-salt hypertension.
  • PRR participates in Ang II generation, triggering both Ang 11-dependent and -independent activation of signaling pathways, and plays a significant role in regulating CV function. Accordingly, decreasing PRR expression in CV regulatory nuclei will reduce Ang II generation and/or decrease Ang 11-independent signals, resulting in vasodilation and reduction of BP.
  • a conditional PRR knockout mouse model with deletion of PRR specifically in neurons was generated and characterized.
  • the mouse PRR exon 2 gene was deleted by breeding PRR floxed mice with mice that express Cre recombinase under the control of the neuron-specific neurofilament-H (Nefh) promoter (Nefh-Cre mice from Jackson laboratory, Maine) (Figure 5A).
  • PRR knockout mice appear to be vital and exhibit global PRR ablation in the brain regions that are involved in central regulation of BP, such as the subfornical organ (SFO) and paraventricular nucleus (PVN) ( Figure 5B), as well as the rostral ventral lateral medulla (RVLM), solitary nucleus (NTS), and non-CV regulatory nuclei.
  • SFO subfornical organ
  • PVN paraventricular nucleus
  • RVLM rostral ventral lateral medulla
  • NTS solitary nucleus
  • PRR is critical for early development. To test whether neurons are functionally intact in neuron-specific PRR knockout mice, carbachol (a cholinergic agonist), Ang II, renin, and prorenin were ICV infused to both Nefh-PRR and wild type (WT) mice.
  • Nefh-PRR and WT mice are implanted with telemetric transmitters and receive DOCA-salt, high salt only, or sham treatment as described above.
  • Spontaneous baroreflex sensitivity (SBRS), cardiac, and vasomotor sympathetic tone are assessed as described previously (see, e.g., Li W, et al. Brain-targeted (pro)renin receptor knockdown attenuates angiotensin ii-dependent hypertension. Hypertension. 2012, 59: 1 188-1 194; which is hereby incorporated by reference in its entirety).
  • the SBRS is calculated at four different time points (0, 7, 14, and 21 days) without additional animals needed using the sequence method (Hemolab software).
  • baroreflex reflex sensitivity (BRS) is assessed using a pharmacological method consisting of infusion of sodium nitroprusside (5 ⁇ g/min, iv) and
  • phenylephrine 50 ng/min iv
  • BP blood pressure
  • Autonomic function is assessed using intraperitoneal injection of propranolol ( ⁇ -blocker, 4 mg/kg), methyl-atropine (muscarinic receptor blocker, lmg/kg), and chlorisondamine (ganglionic blocker, 5 mg/kg) as described previously.
  • Plasma and urine are collected for norepinephrine (NE) measurement using a CatCombi ELISA kit (IBL International, Hamburg, Germany).
  • PRR deletion will improve SBRS and reduce sympathetic activity due to reduced Ang II generation and its stimulation of ATI receptors in the brain, and thus reverse cardiac hypertrophy and fibrosis following reduction of hypertension.
  • the Nefh-PRR and WT mice receive DOCA-salt, high salt, or sham treatment as described above. At the end of 21 days of treatment, mice are sacrificed; plasma and brain tissues are harvested for Ang II, Ang 1-7 measurement utilizing an ELISA kit (Phoenix Pharmaceutical Inc.).
  • Figure 8 shows the ability to measure the Ang II levels in WT mice receiving either DOCA-salt or sham treatment.
  • ACE, ACE2, ATIR, and MasR mRNA and protein levels may be determined using real time PCR, immunofluorescent staining, and western blotting as described.
  • the radioligand receptor binding assay for ATIR is performed to determine the levels of functional receptors as described.
  • the ACE and ACE2 activity are measured to evaluate the function of the enzymes using Fluorogenic Peptide VI.
  • Data is expressed as mean ⁇ SEM. Data is analyzed by one-way or two-way, repeated measures ANOVA followed by Student's modified t-test with Bonferroni correction for multiple comparisons between means using the modified error mean square term from the ANOVA.
  • Mouse prorenin infusion increased the BP (mmHg) in WT mice ( ⁇ : 31.3 ⁇ 1.1);
  • EXAMPLE 6 EXEMPLARY METHOD FOR CONSTRUCTING PLASMIDS FOR THIOETHER BRIDGE-MODIFIED PEPTIDES
  • thioether-bridge modified peptides were designed according to the core amino acid sequence of PR 10 and PR20.
  • the NisBTC-encoding plasmid (pTU-BTC) and substrate-peptide-encoding plasmids (pPR103, pPR105, pPR107, pPR201, pPR202) are constructed and introduced into the lactic acid producing bacterium Lactococcus lactis to produce the thioether-bridged peptides PR103, PR105, PR107, PR201 and PR202 respectively.
  • polynucleotide sequences coding for peptides PR10 and PR20 were optimized to L. lactis for higher expression efficiency using Optimizer software (available at the following web address: genomes.urv.es/OPTIMIZER/).
  • the DNA sequences coding for peptides were synthesized with Seal and Xbal restriction enzyme sites for cloning into the NICE pNZ8150 expression vector to form pPR103, pPR105, pPR107, pPR201, and pPR202 constructs.
  • Expression of PRR antagonist peptides was achieved by co-transfection of pTU-BTC with pPR103, pPR105, pPR107, pPR201, or pPR202 in the NZ9000 L. Lactis.
  • the PRR antagonist peptides are expressed in the cell culture medium.
  • Peptides with a 6xHis tag are purified through Nichel nitrilotriacetic (Ni-NTA) resin.
  • Ni-NTA Nichel nitrilotriacetic
  • the purified thioether-bridge containing peptides are tested for antagonist activity to PRR in animal models of hypertension and other diseases.
  • J. EXAMPLE 10 IDENTIFICATION OF AMINO ACID RESIDUES INVOLVED IN PRR BINDING
  • amino acids at positions 3, 4, 6, 1 , 18 are essential for PR20 binding to PRR.
  • Amino acids at positions 8, 10, 1 1 were identified as being important, but not critical, for PR20 binding to PRR.
  • PR30 SEQ ID NO:5
  • PR40 SEQ ID NO:6
  • Amino acid residues 3, 4, 6, 7, and 18 Figure 21 and 22; underlined, bold text
  • Amino acid residues 8, 10, 1 1, and 14 were determined to be involved in PRR binding, but not essential ( Figures 21 and 22, bold, italicized text).
  • the polynucleotides encoding the peptides were codon optimized for expression in L. lactis using Optimizer software, and computational modeling was used to design modified PR30 and PR40 peptides comprising thioether-bridges.
  • the thioether-bridges were designed according to the three-dimensional (3D) structure of human renin. Computational modeling was used to confirm the similarity of the thioether-bridge modified peptides to the original human renin peptide. As shown in Figure 21, two modified peptides derived from PR30 comprising a single thioether bridge were designed, PR301 (SEQ ID NO: 13) and PR302 (SEQ ID NO: 14), and one modified peptide was designed with two thioether bridges, PR303 (SEQ ID NO: 15).
  • PRR prorenin receptor
  • sPRR A soluble form of PRR (sPRR) is generated by intracellular cleavage by furin and secreted in plasma.
  • sPRR binds renin and prorenin and has been reported to activate prorenin.
  • PRR has received a great deal of attention as it is viewed as a potential regulator of the local RAS and is implicated in the pathogenesis of hypertension and other cardiovascular and renal diseases.
  • Ichihara et al. introduced an epitope of the prorenin prosegment ⁇ termed the handle region peptide (HRP) ⁇ , a decoy peptide, which presumably blocks binding of prorenin to PRR. This is the only PRR blocking peptide available in the literature. The existing results with the HRP are conflicting.
  • the HRP is reported to attenuate diabetic and hypertensive organ damages and ocular disease but have no effect on the hypertension in double-transgenic rats overexpressing human renin and angiotensinogen genes. Moreover, in cultured vascular smooth muscle cells, the HRP failed to affect PRR-mediated activation of MAP kinase, strongly arguing against its role as a PRR peptide blocker . Besides renin regulation, PRR exhibits direct signaling properties such as the activation of mitogen-activated protein kinase (MAPK) and the stimulation of fibrogenesis which are independent of Angll.
  • MAPK mitogen-activated protein kinase
  • PRO20 was designed as a 20-aa peptide containing the 10-aa sequence of the HRP. In fact, deletion analysis showed that the HRP sequence was not required for the PRO20 activity in inhibiting renin-induced calcium signaling in cultured VSMCs. The HRP failed to affect prorenin- or renin-induced Erk activation in cultured vascular smooth muscle cells.
  • PRO20 promotes wound healing. Under anesthesia, a 1 -cm-long incision was made in the neck area for implantation of the DOCA pellet and an osmotic minipump delivering vehicle or PRO20. Mice receiving no treatment of DOCA-salt and PRO20 served as controls.
  • ARF is considered a critical prognostic factor in sepsis, while the management of sepsis and sepsis-induced ARF is largely supportive. Therefore, therapies to prevent or treat this devastating disease are urgently required.
  • the therapeutic potential of PRO20 in endotoxin- induced sepsis and ARF was examined in mice. C57 mice were pretreated with PRO20 via osmotic minipump for 1 wk, and then treated with a single dose of LPS injection. Mean arterial pressure and heart rate were monitored by telemetry. Eighteen hours after the LPS injection, animals were sacrificed. LPS injection induced severe hypotension and bradycardia (Fig. 24). All of these parameters were significantly improved by PRO20 (Fig. 24).
  • Nguyen et al. cloned a specific receptor for prorenin and renin, termed (pro)renin receptor (PRR). It is a 350-amino-acid protein containing a large unglycosyated and highly hydrophobic N-terminal domain, a single transmembrane protein, and a short cytoplasmic tail of approximately 20 amino acids. The carboxyterminal tail was previously purified from chromaffin granules as an 8-9-kD accessory protein (M8-9) of the vacular-type H+-ATPase (V-ATPase) and designated ATP6AP2.
  • M8-9-kD accessory protein M8-9 of the vacular-type H+-ATPase (V-ATPase) and designated ATP6AP2.
  • prorenin can be the endogenous ligand for PRR in rat vascular smooth muscle cells.
  • Prorenin or renin bound to PRR display increases in the catalytic activity.
  • PRR-bound prorenin undergoes conformational change to unfold the enzyme- inhibitory prosegment and expose the active site, leading to non-enzymatic activation of prorenin.
  • PRR is postulated to function as a regulator of tissue renin activity.
  • PRR also exhibits signaling properties such as the activation of the mitogen-activated protein kinase. PRR-mediated activation of the signaling cascade does not depend on the canonical activity of the RAS.
  • renal medullary PRR can mediate Angll- induced local renin response and elevation of blood pressure.
  • PRR decoy peptide PRO20 which is a 21 amino-acid peptide corresponding to the prosegment of prorenin.
  • the kidney was exposed from the flank region and a catheter was placed in the renal medulla, approximately 4.0 mm underneath the surface, and secured by using vetbond glue; the other end of the catheter was connected to an osmotic mini-pump delivering vehicle or PRO20 at 120 ⁇ g/kg/d.
  • Intravenous infusion of PRO20 via jugular vein was performed to control the spillover. Telemetry was turned on 4 h per day from 5:00PM to 9:00PM.
  • Renin activity in urine, tissue homogenates, and cell culture medium was determined by measurement of Angl generation using an ELISA kit and it was performed in the native condition, active renin content with excessive angiotensinogen, and total renin content with excessive angiotensinogen plus trypsinization as previously described. Aldosterone concentrations in plasma, urine, and cell culture medium were measured using a commercial ELISA kit
  • Renal tissues were lysed and subsequently sonicated in PBS that contained 1% Triton x-100, 250 ⁇ phenylmethanesulfonyl fluoride (PMSF), 2 mM EDTA, and 5 mM dithiothrietol (DTT) (pH 7.5). Protein concentrations were determined by the use of Coomassie reagent. 40 ⁇ g of protein for each sample was denatured in boiling water for 10 min, then separated by SDS-PAGE, and transferred onto nitrocellulose membranes. The blots were blocked lh with 5% nonfat dry milk in Tris-buffered saline (TBS), followed by incubation for overnight with primary antibody.
  • TBS Tris-buffered saline
  • blots were incubated with goat anti-rabbit/mouse horseradish peroxidase (HRP)-conjugated secondary antibody and visualized using Enhanced Chemiluminescence (ECL). The blots were quantitated by using Imagepro-plus.
  • HRP horseradish peroxidase
  • Primary antibodies are as follows: rabbit anti- ⁇ -ENaC antibody (Cat#: SPC-403D, Stressmarq Biosciences Inc.), rabbit anti- ⁇ -ENaC (Cat#: SPC-404D, Stressmarq Biosciences Inc.) , rabbit anti- ⁇ -ENaC (Cat#: SPC-405D, Stressmarq Biosciences Inc.), goat anti-AQP2 (Cat#:SC-9882, Santa Cruz Biotechnology), rabbit anti- pAQP2(Cat#: abl 1 1346, Abeam ) and mouse anti-ERKl/2 (Cat#:9016, Cell Signaling Technology).
  • Primers for a-ENaC 5'- gcgacaacaatccccaag-3 ' (SEQ ID NO:26) (sense) and 5'-tgaagcgacaggtgaagatg-3 ' (SEQ ID O:27) (antisense); primers for ⁇ -ENaC: 5'-aagcacctgtaatgcccaag-3 ' (SEQ ID NO:28) (sense) and 5'- atagcccatccccaccag-3 ' (SEQ ID NO:29) (antisense);
  • primers for ⁇ -ENaC were: 5'- cgaagaaactggtgggattt-3 ' (SEQ ID NO:30) (sense) and 5'- gatggtggaaaagcgtgaag-3 ' (SEQ ID NO:31) (antisense); primers for GAPDH: 5'- gtcttcactaccatggagaagg-3 ' (SEQ ID NO:32) (sense) and 5'-tcatggatgaccttggccag-3' (SEQ ID NO:33) (antisense).
  • IMCD cells Primary cultures enriched in IMCD cells were prepared from pathogen-free male Sprague-Dawley rats (40-100 g body wt) and grown in 6-well plate as previously described.
  • Electrophysiology experiments were performed on the immortalized mpkCCD cells once the cell monolayers reached confluence.
  • the transepithelial voltage (Vte) and resistance ( te) across cell monolayers were measured with an epithelial volt-ohmmeter (World Precision Instruments).
  • Angll (500 nM) or prorenin (10 nM) was added to the apical side of the inserts, and amiloride (10 ⁇ ) was added at the end of experiments to calculate amiloride-sensitive sodium current as the difference between total current and amiloride-insensitive current.
  • the handle region peptide is the only PRR blocking peptide available in the literature but efficacy of the HRP in inhibiting PRR signaling is doubtful. Effectiveness of PRO20 and the HRP in blocking prorenin-induced ERK1/2 phosphorylation in primary rat IMCD cells was compared. As expected, the HRP was ineffective.
  • PRO20 is a novel 21 -amino-acid PRR decoy peptide that similarly targets the prosegment of prorenin but covers a longer region than the HRP (10 amino acids). Unlike the HRP, PRO20 almost completely abolished ERK1/2 activation by prorenin (Fig. 26A).
  • a catheter was chronically implanted in the renal medulla of nephrectomized rats to achieve site- specific delivery of PRO20, and intravenous infusion via jugular vein served as a control for spillover. Telemetry was used to monitor daily mean arterial pressure (MAP).
  • MAP mean arterial pressure
  • One-week Angll infusion induced immediate and sustained increases in MAP increasing it from 108 + 5.8 (day 0) to 164.7 + 6.2 (day 7) mmHg.
  • IM PRO remarkably attenuated Angll-induced hypertension and lowered the MAP to 1 10.2 + 4.8 mmHg, nearly the baseline level, on day 7.
  • IV PRO was less effective than IM PRO in lowering MAP (Fig. 26C).
  • Epithelial sodium channel is the major sodium channel on the apical membrane of the CD.
  • Immunoblotting and qRT-PCR analysis of renal ENaC expression was performed. Immunoblotting demonstrated a 2.2-fold increase in renal medullary ⁇ -ENaC protein abundance following Angll infusion and this increase was abolished by IM PRO (Fig. 28A-E). In contrast, ⁇ -ENaC protein abundance in the cortex was unaffected.
  • qRT-PCR detected 1.6-fold increase of ⁇ -ENaC mRNA expression in the inner medulla, which was abolished by IM PRO (Fig. 28F). In contrast, renal medullary mRNA expression of ⁇ - and ⁇ -ENaC remained unchanged (Fig. 28G&H).
  • Angll infusion induces localized renin response in the renal medulla whereas systemic renin activity is suppressed. Therefore, the analysis was focused on the effect of PRO20 on renin activity in the renal inner medulla in vivo and in vitro. Angll infusion elevated renal medullary renin activity that was significantly blocked by IM PRO (Fig. 30A).
  • PRO20 can primarily affect renin activity but not renin expression in the renal medulla.
  • the ELISA kit was unable to differentiate between prorenin and renin whereas the renin activity-based assay specifically detected prorenin or renin content.
  • the direct action of PRO20 its effect on Angll-induced renin activity in primary rat IMCD cells were examined. Medium renin activity was increased by Angll and this increase was blunted by PRO20 (Fig. 30F). However, PRO20 did not affect the baseline renin activity.
  • Angll is known to acutely increase ENaC activity in split-opened CCD. Electrophysiology analysis was employed to examine the effect of PRO on Angll- induced ENaC activity in cultured mpkCCD cells. Following Angll treatment, the equivalent current (Ieq), calculated as the ratio of V(te) to R(te.), was transiently increased, peaking at 5 min and returning to baseline at 15 min and the increase was prevented by PRO20 (Fig. 32A). The same results were obtained by measurement of amilori de-sensitive current, an index of ENaC activity, at the maximal effect of Angll (5 min) (Fig. 32B).
  • Angll is known to increase renin at both activity and expression levels, which likely result in increased release of both renin and prorenin.
  • the relative contribution of prorenin versus renin through PRR-mediated signals transduction versus enzyme activity to Angll-induced ENaC activation is unclear. Accordingly, the effect of PRR activation by prorenin and renin on ENaC activity was examined in mpkCCD cells exposed to Angll in the presence or absence of PRO20. Prorenin acutely induced a comparable increase of Ieq at 10 nM, a dose being 50-times lower than that of Angll (Fig. 32A-C).
  • ROS reactive oxygen species
  • Fluid retention is referred to excess fluid that accumulates in the tissues. Patients with fluid retention can develop swelling of feet and lower legs, and sometimes in arms, hands, face, or other areas of the body. Fluid retention can be mild or severe. In severe cases of fluid retention, extra water builds up in lungs, causing pulmonary edema, and excessive plasma volume expansion can lead to congestive heart failures. Both of these conditions can be life threatening.
  • liver disease includes any type of liver problem, such as hepatitis, cirrhosis and liver failure
  • side effects from certain medications including blood pressure medications, antidiabetic drug thiazolidinedione, endothelin receptor antagonist, obesity and type 2 diabetes, preeclampsia, and lymph nodes disorders.
  • Fluid retention can be mild or severe. A mild case can happen after eating a meal that is high in salt. A severe response can occur if extra water builds up inside of the lungs, a condition known as pulmonary edema that can make it very difficult to breathe.
  • diuretics which increase urine volume by inhibiting one of sodium transporters along the nephron.
  • These medications can help alleviate symptoms but sometimes have limited potency and also cause electrolyte disturbances since they only target a single sodium transporter but do not address the underlying pathophysiological mechanisms.
  • Renal collecting is the terminal part of the nephron, playing an essential role in final adjustment of urinary sodium and water excretion. Hormonal regulation of sodium and water transport primarily occurs in the collecting duct. Sodium and water reabsorption in the collecting duct are mediated by the sodium channel ENaC and water channel aquaporin-2 (AQP2). Sodium transport via ENaC and water transport via AQP2 in the collecting duct are controlled by aldosterone and vasopressin, respectively.
  • Spironolactone marketed primarily under the brand name Aldactone in most countries, is a synthetic antagonist of aldosterone receptor and is a potassium- sparing diuretic. It is less commonly used in the clinics due to its limited potency.
  • PRO20 is unique in that it has dual actions in inhibiting both ENaC and AQP2. Moreover, unlike existing diuretics, PRO20 reduced gene expression of the transporters and thus can be more effective. PRO20 was demonstrated to reduce ENaC expression and activity in renal medullary cells. PRO20 also effectively reduced vasopressin- and prorenin-induced AQP2 expression in cultured primary rat collecting duct cells in vivo (Fig. 35).
  • PRO20 treatment also remarkably reduced AQP2 expression in the rat kidney in vivo (Fig. 36).
  • prorenin via PRR activated both ENaC and AQP2 in the collecting duct.
  • PRO20 can be effective in management of fluid retention associated with the activation of the prorenin/PRR pathway.
  • the HRP exhibited no effect on Angll-dependent hypertension in Goldblatt rats whereas PRO20 produced a significant blood pressure-lowering effect in Angll-infused rats.
  • PRO20 a 21-amino-acid peptide, contained the entire 10- amino-acid sequence of the HRP and targeted the prosegment of prorenin. Deletion analysis can be used to determine whether the non-HRP sequence of PRO20 confers the activity of this new peptide.
  • Renin is expressed in the CD, highlighting existence of local RAS in the distal nephron. Abundant evidence demonstrated that the local RAS in the renal medulla plays a key role in mediating Angll-induced hypertension. Angll infusion induces distinct changes in systematic and renal medullary renin response with suppressed renin levels in plasma and renal cortex but increased renin levels in the inner medulla and urine, indicating distinct regulatory mechanisms of systemic and local RAS. The increases in renal medullary and urinary renin activity in response to Angll infusion were completely abolished by IM PRO, accompanied by nearly complete blockade of the hypertensive response.
  • the renal medullary renin response induced by PRR represents a dominant mechanism of the chronic pressor effect of Angll.
  • Angll-induced renin activity was similarly blocked by PRO20.
  • Prorenin exhibited a high potency in rapidly increasing ENaC activity and achieved the maximal activation at a dose 50-times lower than that of Angll. Prorenin was far more effective than renin in the rapid activation of ENaC, supporting the concept that prorenin rather than renin is a true endogenous ligand of PRR. Prorenin can act via its enzyme activity or signal transduction. Prorenin-induced ENaC activation was completely abolished by PRO20 but not losartan, indicating exclusive reliance on receptor-mediated signaling transduction but not the RAS activity.
  • prorenin was more rapid (1 min) than that of Angll (5 min) and this time lag in ENaC activation is in agreement of prorenin as a mediator of Angll action.
  • the acute effect of prorenin was dependent on ROS since it was abolished by a NADPH oxidase inhibitor apocynin. Apocynin similarly blocks the stimulatory effect of Angll on ENaC activity.
  • IM PRO was more effective that IV PRO in attenuating the increased urinary aldosterone excretion in response to Angll infusion but their effects on plasma aldosterone was similar.
  • Aldosterone is thought to be primarily produced by adrenal glands and renal production or its regulation of aldosterone has not been established.
  • the new PRR decoy peptide PRO20 effectively inhibited prorenin-induced ERK1/2 activation and remarkably attenuated hypertension and kidney injury in the absence of any noticeable toxicity.
  • PRO20 appears to be a more effective PRR inhibitor as compared with the HRP.
  • urinary sPRR correlated nicely with urinary renin activity, proteinuria, and blood pressure.
  • the present study is the first to report utility of urinary sPRR as a biomarker of intrarenal RAS activity and hypertensive kidney injury.
  • plasma sPRR is indicated to function as a biomarker in chronic heart failure, pregnancy, and chronic kidney disease, it is independent of plasma renin, prorenin, or aldosterone, nor is increased in patients with hypertension or diabetes.
  • the present study employed a newly developed PRR-decoy peptide PRO20 coupled with intramedullary infusion technique to investigate functional role of renal medullary PRR during Angll- induced hypertension. Not only was a remarkable blood pressure-lowering effect of intramedullary PRR antagonism demonstrated, but also a mechanism of this phenomenon involving prorenin/PRR- dependent, enzyme activity-independent, biphasic activation of ENaC activity was demonstrated. The rapid phase of ENaC activation occurs within minutes through ROS generation, followed by the chronic phase of ENaC activation within hours through the release of aldosterone. It has been demonstrated that PRR antagonist has the therapeutic potential for management of hypertension, fluid metabolism disorders, and kidney injury
  • PRO20 The effect of PRO20 on ischemia-reperfusion renal injury in C57/BL6 mice was examined. A 30-min ischemia followed by reperfusion caused severe renal failure as evidenced by increased plasma creatinine and BUN (Fig. 36). PRO20 administered post VR significantly attenuated the rise in both parameters (Fig. 36). This is the first evidence for protective action of PRO20 against ischemia-reperfusion-induced injury to the kidney. In light of the common mechanisms that can underlie ischemia- reperfusion-induced injury in multiple organs, PRO20 can exert similar protective action against ischemic injury in other organs such as heart and brain. REFERENCES
  • Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor. J Hypertens 25:2441-2453.
  • Prorenin has high affinity multiple binding sites for (pro)renin receptor.
  • Angiotensin II stimulates renin in inner medullary collecting duct cells via protein kinase C and independent of epithelial sodium channel and mineralocorticoid receptor activity. Hypertension 57:594-599.
  • Prostaglandin E-Prostanoid4 Receptor Mediates Angiotensin Il-Induced (Pro)Renin Receptor Expression in the Rat Renal Medulla. Hypertension.
  • Angiotensin II increases activity of the epithelial Na+ channel (ENaC) in distal nephron additively to aldosterone. J Biol Chem 287:660-671.
  • RILLKKMPSV influences the vasculature, neurons and glia, and (pro)renin receptor expression in the retina. Hypertension 55: 1454-1460.
  • Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney. Am J Physiol Renal Physiol 302:F679-687.
  • Watanabe N., Morimoto, S., Fujiwara, T., Suzuki, T., Taniguchi, K., Mori, F., Ando, T., Watanabe, D., Kimura, T., Sago, H., et al. 2013. Prediction of gestational diabetes mellitus by soluble (pro)renin receptor during the first trimester. J Clin Endocrinol Metab 98:2528-2535.
  • Plasma soluble (pro)renin receptor is independent of plasma renin, prorenin, and aldosterone concentrations but is affected by ethnicity. Hypertension 63 :297-302.

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Abstract

L'invention concerne des procédés de traitement d'affections des reins, de maladies infectieuses inflammatoires, d'insuffisance rénale aiguë et d'hypertension par l'administration d'un antagoniste du récepteur de la (pro)rénine (PRR). Dans certains exemples, l'antagoniste du PRR est un polypeptide. L'antagoniste du PRR peut être un polypeptide ayant au moins 70 % d'identité avec la séquence d'acides aminés présentée dans SEQ ID n° 2. L'invention concerne également des procédés permettant de diminuer la protéinurie et des procédés favorisant la cicatrisation d'une plaie par l'administration d'un antagoniste du récepteur PRR.
PCT/US2015/043085 2012-09-19 2015-07-31 Compositions et procédés d'utilisation d'antagonistes du récepteur de la (pro)rénine Ceased WO2016019226A1 (fr)

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Publication number Priority date Publication date Assignee Title
US10780143B2 (en) 2012-09-19 2020-09-22 University Of Utah Research Foundation Compositions and methods of use for (pro)renin receptor antagonists

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050037007A1 (en) * 1999-01-05 2005-02-17 The University Of Utah Research Methods for treating conditions associated with the accumulation of excess extracellular matrix
US20080161321A1 (en) * 2004-03-17 2008-07-03 David Louis Feldman Use of Renin Inhibitors in Therapy
US20110091427A1 (en) * 2009-10-02 2011-04-21 Baxter International Inc. Methods for treating a kidney injury
US20140094409A1 (en) * 2012-09-19 2014-04-03 Yumei FENG Antagonist for (pro)renin receptor for the treatment of hypertension and diabetes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050037007A1 (en) * 1999-01-05 2005-02-17 The University Of Utah Research Methods for treating conditions associated with the accumulation of excess extracellular matrix
US20080161321A1 (en) * 2004-03-17 2008-07-03 David Louis Feldman Use of Renin Inhibitors in Therapy
US20110091427A1 (en) * 2009-10-02 2011-04-21 Baxter International Inc. Methods for treating a kidney injury
US20140094409A1 (en) * 2012-09-19 2014-04-03 Yumei FENG Antagonist for (pro)renin receptor for the treatment of hypertension and diabetes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GARG ET AL.: "Review article: the pathophysiological roles of the renin-angiotensin system in the gastrointestinal tract.", ALIMENTARY PHARMACOLOGY & THERAPEUTICS, vol. 35, no. 4, February 2012 (2012-02-01), pages 414 - 428 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10780143B2 (en) 2012-09-19 2020-09-22 University Of Utah Research Foundation Compositions and methods of use for (pro)renin receptor antagonists

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