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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Hypertension. 2009 Dec 21;55(2):459–467. doi: 10.1161/HYPERTENSIONAHA.109.144568

CHBPR N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Attenuates Renal Injury and Dysfunction in Hypertensive Rats with Reduced Renal Mass

Tang-Dong Liao 1, Xiao-Ping Yang 1, Martin D’Ambrosio 1, Yanlu Zhang 1, Nour-Eddine Rhaleb 1, Oscar A Carretero 1,*
PMCID: PMC2809792  NIHMSID: NIHMS166952  PMID: 20026760

Abstract

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring peptide whose plasma concentration is increased 4- to 5-fold by angiotensin-converting enzyme inhibitors. We previously reported that in models of both hypertension and postmyocardial infarction, Ac-SDKP reduces cardiac inflammation and fibrosis. However, it is unknown whether Ac-SDKP can prevent or reverse renal injury and dysfunction in hypertension. In the present study, we tested the hypothesis that in rats with 5/6 Nephrectomy (5/6Nx) -induced hypertension, Ac-SDKP reduces renal damage, albuminuria and dysfunction by decreasing inflammatory cell infiltration and renal fibrosis and increasing nephrin protein. Ac-SDKP (800 μg/kg/day, i.p. via osmotic mini-pump) or vehicle was either a) started 7 days before 5/6Nx (prevention) and continued for 3 weeks or b) started 3 weeks after 5/6Nx (reversal) and continued for up to 6 weeks. Rats with 5/6Nx developed high blood pressure (BP), left ventricular hypertrophy (LVH), albuminuria, decreased glomerular filtration rate (GFR) and increased macrophage infiltration (inflammation) and renal collagen content (fibrosis). Ac-SDKP did not affect BP or LVH in either group; however, it significantly reduced albuminuria, renal inflammation and fibrosis and improved GFR in both prevention and reversal groups. Moreover, slit diaphragm nephrin protein expression in the glomerular filtration barrier was significantly decreased in hypertensive rats. This effect was partially prevented or reversed by Ac-SDKP. We concluded that Ac-SDKP greatly attenuates albuminuria and renal fibrosis and improves renal function in rats with 5/6Nx. These effects may be related to decreased inflammation (macrophages) and increased nephrin protein.

Keywords: Ac-SDKP, albuminuria, renal dysfunction, fibrosis, inflammation, nephrin

INTRODUCTION

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring anti-inflammatory and anti-fibrotic peptide. Ac-SDKP is hydrolyzed almost exclusively by angiotensin-converting enzyme (ACE), and its plasma concentration is increased substantially following treatment with ACE inhibitors (ACEi)1. Ac-SDKP mediates some of the anti-inflammatory and anti-fibrotic effects of ACEi2,3. Rats overexpressing cardiac ACE have decreased Ac-SDKP and increased fibrosis in the heart4. Inhibition of prolyl oligopeptidase, an enzyme responsible for Ac-SDKP release from thymosin-β4, promotes cardiac and renal perivascular fibrosis and nephrosclerosis5. We and others have shown that in vitro Ac-SDKP inhibits fibroblast and mesangial cell proliferation and collagen synthesis6-8. Treatment with Ac-SDKP has been shown to reduce inflammation and collagen deposition in the heart, aorta and kidney in animal models of hypertension, myocardial infarction and diabetes 3,9-13.

The glomerular filtration barrier is a three-layered structure consisting of a) fenestrated endothelial cells lining the renal capillaries, b) the glomerular basement membrane (GBM), and c) visceral epithelial cells (podocytes) whose interdigitating foot processes form the slit diaphragm.14 The slit diaphragm is the final barrier that prevents protein leakage into the urinary space. Nephrin is a slit pore protein expressed between foot processes of podocytes in the glomeruli and is critical in maintaining permeability and preventing proteinuria15,16. Mutation of the nephrin gene leads to congenital nephrotic syndrome of the Finnish type, which specifically affects the kidney and is characterized by massive proteinuria17. In addition to glomerular filtration barrier, mesangial cells and their matrix form the central stalk of the glomerulus and are part of a functional unit, which interacts closely with endothelial cells and podocytes. Alterations in one cell type can produce changes in the others18.

We have shown that in aldosterone-salt-induced hypertension, Ac-SDKP significantly decreased renal cell proliferation and renal fibrosis but only slightly reduced glomerular and tubular injury12; however, we did not examine the underlying mechanism(s) or test renal function. In the present study, we tested the hypothesis that Ac-SDKP prevents and reverses renal albuminuria and dysfunction in 5/6Nx-induced hypertension by decreasing inflammatory cell infiltration and renal fibrosis and increasing nephrin protein.

MATERIALS AND METHODS

Animals

Male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 275-300 g were housed in an air-conditioned room with a 12-hr light/dark cycle and received standard laboratory rat chow and tap water. They were allotted 7 days to adjust to their new environment. Prior to all surgical procedures, rats were given analgesia (butorphanol 2 mg/kg s.c.) and anesthesia (sodium pentobarbital 50 mg/kg i.p.). This study was approved by the Henry Ford Hospital Institutional Animal Care and Use Committee (IACUC).

Surgical procedure for 5/6 nephrectomy (5/6Nx)

Rats were anesthetized and 5/6Nx was performed by unilateral nephrectomy plus ligation of lower and upper renal arterial branches of the contralateral kidney with a 6-0 silk suture. Ligation was deemed successful when 2/3 of the kidney turned dark red19. The sham-operated group underwent a similar surgical procedure except that the suture around the renal artery was not tightened. An osmotic mini-pump filled with Ac-SDKP (800 μg/kg/day) or vehicle (0.01N acetic acid saline solution) was implanted s.c. between the shoulder blades.

Experimental protocols

Rats were randomly divided into two protocols: prevention and reversal.

Prevention Protocol

Rats received vehicle or Ac-SDKP beginning 7 days before surgery and continuing for 3 weeks.

Group 1: Prevention sham Nx rats given vehicle (sham-vehicle/3 weeks, n = 12).

Group 2: Prevention 5/6Nx rats given vehicle (5/6Nx-vehicle/3 weeks, n = 12). This group was also used as a control for the initial point of the reversal protocol.

Group 3: Prevention 5/6Nx rats given Ac-SDKP (5/6Nx-Ac-SDKP/3 weeks, n = 12).

Reversal Protocol

Rats received vehicle or Ac-SDKP beginning 3 weeks after surgery and continuing for up to 6 weeks.

Group 4: Reversal sham Nx rats given vehicle (sham-vehicle/6 weeks, n = 5).

Group 5: Reversal 5/6Nx rats given vehicle (5/6Nx-vehicle/6 weeks, n = 10).

Group 6: Reversal 5/6Nx rats given Ac-SDKP (5/6Nx-Ac-SDKP/6 weeks, n = 7).

Group 2 of the Prevention Protocol (5/6Nx-vehicle/3 weeks) was used as the treatment baseline for the reversal protocol.

Measurement of Ac-SDKP plasma concentration and urinary excretion and albuminuria

Blood was collected from the inferior vena cava using a heparinized syringe containing lisinopril at a final concentration of approximately 10−5M. Aliquots were centrifuged at 3,700 rpm at 4°C for 10 minutes and the supernatant was collected to measure plasma Ac-SDKP.

After adapting to the metabolic cage for 24 hours, the rats fasted during the 24 hr urine collection. To prevent Ac-SDKP degradation by urinary ACE, an ACEi (lisinopril,10−5M) was sprayed and dried on the funnel and also added to the collecting tubes (200 μl of 10−3M lisinopril per tube). Urinary volume was measured and aliquots were centrifuged twice at 132,000 rpm at 4°C for 10 minutes (Eppendorf centrifuge 5415R). The supernatant was passed through a 25mm syringe filter (0.2 μm HT Tuffryn membrane, Gelman Laboratories), and stored at −20°C. Urinary and plasma Ac-SDKP were measured by ELISA (SPI Biolaboratories, France). Urine albumin was measured with an ELISA kit (Cayman Chemical).

Systolic blood pressure (SBP), left ventricle weight (LVW), body weight (BW) and kidney weight (KW)

SBP was measured in conscious rats by use of a noninvasive computerized tail-cuff system (Model-1231, IITC INC.) as described previously20. Animals were trained for 3 days before SBP measurement. At the endpoints of each study, rats were euthanized and the abdomen opened. The kidney was excised, the capsule was removed and weighed and the ratio of kidney weight to body weight determined. It was then sectioned transversely into 4 slices. One slice from the middle of the kidney was fixed in 4% paraformaldehyde for paraffin-embedded section. A lower mid-renal slice was embedded in OCT compound and immersed in cold isopentane (VWR), then snap-frozen in liquid nitrogen and stored at −80°C. One slice from the apex of the renal cortex was used for hydroxyproline assay. The remaining slice was rapidly frozen in liquid nitrogen and stored at −80°C. The heart was also excised and weighed.

Glomerular filtration rate (GFR)

GFR was measured with fluorescein isothiocyanate labeled inulin (FITC-inulin, Sigma)21. Briefly, rats were anesthetized and placed on a heating pad. FITC-inulin (10 mg/ml) was injected as a bolus at 3 μl/g body weight (BW), followed by constant infusion of 0.15 μl/min/g BW. After a 30 minute stabilization period, urine was collected for 30 minutes, taking a 100μl sample before and after urine collection. Samples of FITC-inulin standards, plasma and diluted urine were transferred to a 96-well microplate in triplicate and mixed with 10 mM HEPES buffer (N-(2-hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid), pH 7.4). Plates were examined with a microplate fluorescence reader (Labsystems Fluoroskan II) at an excitation level of 485 nm and an emission level of 538 nm. GFR was calculated using the formula: GFR = (urine fluorescence × urine volume / blood fluorescence) / collection time. GFR was corrected by kidney weight (KW), with units expressed as μl/min/100 mg KW.

Renal macrophage infiltration

Paraffin-embedded sections (6 μm) were deparaffinized and endogenous peroxidase activity blocked with 0.3% hydrogen peroxide. Antigens were revealed by microwave heat-induced epitope retrieval in citrate acid buffer (pH 6.0). First nonspecific binding was blocked with 2.5% normal horse serum; then a primary monoclonal antibody, mouse anti-rat CD68 antigen, which is a marker for macrophages (clone: ED-1, 1:200, AbD Serotec) was applied and samples were incubated overnight at 4°C. The next day, sections were incubated with a secondary biotinylated antibody, horse anti-mouse IgG. Immunoreactivity was detected with an ABC peroxidase kit (Vectastain Elite,Vector Laboratories) and visualized with 3-amino-9-ethylcarbazole (AEC, Zymed Laboratories). PBS buffer alone and a nonspecific purified mouse anti-rat IgG were used as a negative control and an isotype IgG control, respectively. Reddish-brown staining was considered positive. Sections were counterstained with hematoxylin. 12 regions of the section were examined under the 40× objective of an inverted microscope (IX81), photographed with a digital camera (DP70, Olympus America, Center Valley, PA), and evaluated by a computerized image analysis system (Microsuite Biological Imaging, Olympus America, Center Valley, PA). All images captured and analyzed in this study were obtained using the same system unless otherwise specified. Positive cells in high-power fields were counted for each section and expressed as cells/ mm2.

Renal collagen content

Collagen content of the renal cortex was determined by hydroxyproline assay as described previously12. Briefly, samples were dried, homogenized and hydrolyzed with 6 N HCI for 16 hours at 110°C. A standard curve of 0-5 μg hydroxyproline was used. Data were expressed as micrograms of collagen per milligram of dry weight, assuming that collagen contains an average of 13.5% hydroxyproline22.

Glomerular matrix, interstitial and perivascular fibrosis

A transmural section was taken from the upper mid-kidney slice. Sequential 4μm paraffin-embedded sections were stained with Periodic Acid Schiff, Sigma (PAS)2 to examine the glomerular matrix. Glomeruli (25-30) were photographed under the 40× objective and the glomerular matrix (GM) determined as a percentage of the glomerular area. Picro-sirius red staining was used to quantify the renal interstitial collagen fraction (ICF) and renal perivascular fibrosis23. For ICF, 12 images were taken with the 20× objective, examining the cortex and outer medulla to avoid interference by large vessels. ICF was expressed as the ratio of collagen area to total area. For perivascular fibrosis, 10 vessels were imaged using the 40× objective and perivascular fibrosis expressed as the ratio of the fibrotic area surrounding the vessel to total cross-sectional area5.

Nephrin in the glomerulus

Frozen sections (4 μm) were immunostained with a nephrin antibody (Fitzgerald Industries) and visualized using a FITC-conjugated secondary antibody. Non-specific binding was blocked by 10% species-appropriate normal serum. Negative controls were processed in a similar fashion except that sections were incubated with an isotype IgG control instead of the primary antibody. Positive staining in high-power fields was measured in each section of the glomerulus and expressed as a percentage of the glomerular area.

All imaging analysis was conducted in a double-blind fashion. The person performing microphotography and computerized imaging analysis did not know which groups he was examining.

Data analysis

All data are expressed as mean ± SEM. Analysis of variance (ANOVA) was used to compare mean values of each parameter (Ac-SDKP, BW, LVW/BW, KW/BW, SBP, albuminuria, GFR, macrophage, renal fibrosis and nephrin) between different groups. Hochberg’s method for multiple comparisons was used to adjust the alpha level of significance 24.

The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.

RESULTS

Ac-SDKP plasma concentrations and urinary excretion

Ac-SDKP plasma concentrations were 3-fold higher in rats infused with Ac-SDKP compared to vehicle, while urinary Ac-SDKP excretions were roughly 7- to 11-fold higher in the Ac-SDKP infused group as compared to the vehicle group (Table 1).

Table 1.

Urine and plasma Ac-SDKP levels, body weight, systolic blood pressure, left ventricle weight and kidney weight

Parameters Prevention(3 weeks) Reversal(6 weeks)
Sham-
vehicle		 5/6Nx-
vehicle		 5/6Nx-Ac-
SDKP		 Sham-
vehicle		 5/6Nx-
vehicle		 5/6Nx-Ac-
SDKP
Plasma Ac-SDKP
(nM)		 2.19±0.16 2.87±0.29 7.05±1.41‡ 2.27±0.19 2.39±0.10 5.10±0.64‡
Urinary Ac-SDKP
(μg/24 hrs)		 1.79±0.75 0.67±0.20 7.40±1.94∥ 1.02±0.06 0.77±0.04 5.54±0.61§
BW (g) 356 ± 9 278 ± 23† 331±10§ 417± 15 394±14 421±15
KW/BW (mg/100 g) 434±24 482±56 462±23 484±14 486±52 454±19
PVF (ratio) 0.81±0.04 1.02±0.07* 0.83±0.07 0.78±0.01 1.20±0.15† 1.08±0.05#
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*

= p < 0.05

†

= p < 0.001, 5/6Nx-vehicle vs sham- vehicle

‡

= p < 0.05

§

= p < 0.01

∥

= p < 0.001

p = 0.062, 5/6Nx-Ac-SDKP vs 5/6Nx-vehicle.

#

(p = 0.438 for PVF reversal)

BW = body weight; KW/BW = ratio of kidney weight to body weight; PVF = perivascular fibrosis, expressed as the ratio of perivascular collagen to vessel cross-sectional area.

Systolic blood pressure (SBP), BW, cardiac and renal hypertrophy

Rats with 5/6Nx in the prevention and in the reversal groups had significantly increased SBP and cardiac weight, and Ac-SDKP did not affect either SBP or cardiac weight (Fig. 1).

Fig 1.

Fig 1

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Effect of Ac-SDKP on systolic blood pressure (SBP) and cardiac hypertrophy. A) SBP increased significantly in 5/6Nx rats given vehicle in both prevention and reversal protocols. Ac-SDKP had no effect on SBP. B) Cardiac weight (LVW/BW = ratio of left ventricle weight to body weight) increased significantly in 5/6Nx rats given vehicle in both prevention and reversal protocols. Ac-SDKP had no effect on cardiac hypertrophy (n = 5-8).

In the prevention protocol, rats with 5/6Nx had significantly decreased BW compared to sham operation, and this decrease was prevented by Ac-SDKP (Table 1). In the reversal protocol, BW in the 5/6 NX decrease and Ac-SDKP tended to increase, however these changes did not reach statistical significance. Kidney weight to body weight ratio was similar in all groups, indicating that 5/6Nx induced hypertrophy of the remaining nephrons (Table 1).

Albuminuria

Rats with 5/6Nx given vehicle had severe albuminuria compared to sham Nx in both prevention (3 wks) and reversal protocols (6 wks). Ac-SDKP prevented or reversed the albuminuria induced by 5/6Nx (Fig. 2A).

Fig 2.

Fig 2

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Effect of Ac-SDKP on albuminuria and glomerular filtration rate (GFR). A) Rats with 5/6Nx given vehicle developed severe albuminuria in both prevention and reversal protocols. Ac-SDKP prevented or reversed the albuminuria induced by 5/6Nx. B) GFR decreased significantly in 5/6Nx rats given vehicle. Ac-SDKP treatment improved GFR in both prevention and reversal protocols (n = 5-7).

Glomerular filtration rate

Rats with 5/6Nx given vehicle had significantly decreased GFR in both prevention (3 wks) and reversal protocols (6 wks). Ac-SDKP significantly improved GFR in both protocols (Fig. 2B).

Renal macrophage infiltration

Rats with 5/6Nx given vehicle had significant macrophage infiltration in the kidney in both prevention and reversal protocols. Ac-SDKP significantly decreased macrophage infiltration in both prevention and reversal protocols (Fig. 3). However, macrophage infiltration was not normalized by Ac-SDKP treatment.

Fig 3.

Fig 3

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Effect of Ac-SDKP on macrophage infiltration. A) Representative images of renal macrophage infiltration. Reddish staining in the cytoplasm is positive for macrophages (bar = 80 μm). B) Quantitative data show that macrophage infiltration in the kidney increased significantly in 5/6Nx rats given vehicle. Ac-SDKP decreased macrophage infiltration significantly in both prevention and reversal protocols (n = 5-6).

Renal fibrosis

Rats with 5/6Nx given vehicle had significantly increased renal collagen content (hydroxyproline assay) in both prevention (3 wks) and reversal protocols (6 wks). Ac-SDKP significantly prevented and reversed these effects, respectively (Fig. 4). Renal interstitial collagen fraction (ICF) and perivascular fibrosis were also investigated by using picro-sirius red (PSR) staining and computerized imaging analysis. Red color indicated collagen deposition. Renal ICF data were consistent with the collagen content (Fig. 6). Ac-SDKP also tended to lower perivascular fibrosis, but this decrease did not reach statistical significance (Table 1).

Fig 4.

Fig 4

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Effect of Ac-SDKP on renal collagen content. Renal collagen content increased significantly in 5/6Nx rats given vehicle. Ac-SDKP decreased renal collagen content in both prevention and reversal protocols (n = 5-8).

Fig 6.

Fig 6

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Effect of Ac-SDKP on renal interstitial fibrosis. A) Representative images of renal interstitial fibrosis. Red indicates collagen deposition revealed by picro-sirius staining (bar = 200 μm). B) Quantitative data show that renal interstitial collagen fraction increased significantly in 5/6Nx rats given vehicle. Ac-SDKP decreased glomerular matrix significantly in both prevention and reversal protocols (n = 5-6).

Glomerulosclerosis

Effect of Ac-SDKP on glomerulosclerosis was assessed on kidney sections by using periodic acid Schiff (PAS) staining and computerized image analysis. Dark purple regions indicate extracellular matrix stained by PAS. Ac-SDKP significantly prevented and reversed glomerular matrix expansion, which is an indicator of glomerulosclerosis.

Nephrin expression

Rats with 5/6Nx given vehicle had significantly decreased glomerular nephrin expression in both prevention and reversal groups. Ac-SDKP partially restored nephrin expression in both protocols (Fig. 7).

Fig 7.

Fig 7

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Effect of Ac-SDKP on nephrin. A) Representative images of glomerular nephrin expression. Green indicates positive nephrin staining (bar = 25 μm). B) Quantitative data show that nephrin expression decreased significantly in 5/6Nx rats given vehicle. Ac-SDKP partially restored nephrin expression in both prevention and reversal protocols (n = 5-8).

DISCUSSION

Using a model of severe hypertension, renal injury and dysfunction, we found that Ac-SDKP not only prevents but (more importantly) reverses renal injury. The effects of Ac-SDKP were independent of changes in blood pressure. This is in agreement with our previous studies showing that in various models of hypertension the anti-inflammatory and anti-fibrotic effects of Ac-SDKP in the heart, aorta and kidney were independent of any antihypertensive effect 3,11,12.

As reported previously25-27, the 5/6Nx model is characterized by severe hypertension, nephrosclerosis, renal fibrosis and inflammation (as indicated by macrophage infiltration), proteinuria and decreased GFR. In addition, we have shown that nephrin is significantly decreased in this model. All of these effects were either prevented or partially reversed by Ac-SDKP. Also, in this model of hypertension and renal disease, an increase in renin-angiotensin system (RAS) activity has previously been described26. Angiotensin II may contribute to renal damage not only by causing hypertension but also by inducing inflammatory cell infiltration and fibroblast and mesangial cell proliferation, resulting in renal fibrosis and nephrosclerosis28. It has been reported that Ac-SDKP at very high doses inhibits ACE in vitro, thus this tetrapeptide could have renal protection via inhibition of Ang II production29. However, it is unlikely that Ac-SDKP acts through inhibition of ACE, since Ac-SDKP in vivo, at similar doses used in the present study, did not affect the conversion of Ang I to Ang II30. Also in the present study, in a model of high renin dependent hypertension, Ac-SDKP did not have anti-hypertensive effect, suggesting that it did not act by blocking of Ang II formation.

The mechanism by which Ac-SDKP decreases renal injury caused by reduction of renal mass could be partially mediated by reducing inflammation, as suggested by the decrease in macrophage infiltration. It is well known that immunological and inflammatory processes play an important role in end stage renal disease31-33. Macrophage infiltration is one of the hallmarks of inflammatory renal injury33. In the present study, we demonstrated that renal macrophage infiltration was significantly increased in 5/6Nx rats and this effect was partially prevented or reversed by Ac-SDKP treatment. Ac-SDKP inhibits macrophage infiltration by directly inhibiting macrophage differentiation, activation and migration in vitro and in vivo34,35. Thus the decrease in renal inflammation caused by Ac-SDKP could involve a direct effect on macrophages either via the release of superoxide or the release of pro-inflammatory cytokines that may trigger further inflammation and oxidative stress and exacerbate renal lesions36,37. Indeed, we previously reported that in Ang II-induced hypertension, Ac-SDKP was able to reduce oxidative stress as evidenced by reduction in 4-hydroxynonenol (a by-product of lipid oxidation and a marker for oxidative stress) and nitrotyrosine (a marker for superoxide production)11. Here, we demonstrated that Ac-SDKP decreased macrophage infiltration in both prevention and reversal protocols but did not restore its concentration to baseline. We speculated that a certain level of macrophages could be beneficial for healing injury, since the inflammatory response is one of the body’s defense mechanisms37,38. Completely eliminating macrophage infiltration may lead to infection, tumor growth and other diseases38,39. The question of how we might balance the inflammatory response needs to be investigated further.

The renal protective effect of Ac-SDKP may also be partly due to a reduction in renal fibrosis and nephrosclerosis. We and others have shown that Ac-SDKP inhibited fibroblast and mesangial cell proliferation, and collagen synthesis5,6,8,13. We also have shown that Ac-SDKP decreases expression of the profibrotic cytokine TGF-β and that it blocks Smad activation2,9. The present study showed that Ac-SDKP decreased or restored renal collagen content, renal interstitial collagen fraction and glomerulosclerosis, which may be due to reduction of fibroblast and mesangial cell proliferation and inhibition of TGF-β/Smad pathways. We also found that Ac-SDKP tended to decrease perivascular fibrosis but this decrease did not reach statistical significance (Table 1). The partial effect of Ac-SDKP on perivascular fibrosis could be due to either dosage of the peptide or short period of treatment, especially in the reversal protocol. Additionally, we cannot discard the possibility that Ac-SDKP does not inhibit some of the mechanisms leading to perivascular fibrosis such as mesenchymal transformation of endothelial cells40, pericyte migration and differentiation into myofibroblasts41, or irreversible transformation of renal adventitial fibroblasts to myofibroblasts42, which could require more than 3 weeks (present experimental protocol) to resolve.

A major finding of the present study was the marked difference in the effect of Ac-SDKP treatment on albuminuria and GFR compared to vehicle in 5/6Nx rats. Ac-SDKP prevented or reversed renal injury and dysfunction. The pathogenesis of proteinuria in hypertension has not been fully delineated. Recent studies implicate the slit pore protein nephrin, which plays an important role in trafficking of albumin across the glomerular barrier43,44. Nephrin is synthesized by podocytes, glomerular epithelial cells that are reportedly reduced in hypertensive patients45 and type II diabetics with nephropathy46. Bonnet et al demonstrated that reduced nephrin expression in diabetic spontaneously hypertensive rats (SHR) was accompanied by increased albuminuria47. Our results show that 5/6Nx causes a significant decrease in nephrin expression and that Ac-SDKP partially prevents or reverses these decreases. The effects of Ac-SDKP on nephrin could be secondary to a decrease in inflammation or to inhibition of mesangial cell proliferation which could help maintain balance with the number of podocytes, however this issue needs to be studied48.

There is evidence that shortly after the onset of proteinuria, interstitial inflammation develops and fibrosis ensues, indicating that proteinuria itself may elicit pro-inflammatory and pro-fibrotic effects that directly contribute to renal damage49. Thus decreasing proteinuria may also contribute to the anti-inflammatory and anti-fibrotic effect of Ac-SDKP on subtotal renal ablation.

As discussed above, Ac-SDKP decreased glomerulosclerosis induced by 5/6Nx. Thus the renal protective effect of Ac-SDKP on GFR could be a consequence of preventing glomerulosclerosis.

In the present study, we found that Ac-SDKP attenuates albuminuria and renal dysfunction in hypertensive rats with subtotal renal ablation. These effects may be related to decreased inflammation, fibrosis, and glomerulosclerosis coupled with an increase in nephrin expression.

PERSPECTIVES

The present study demonstrates that Ac-SDKP provided strong renal protective effects in animal models of renal injury and dysfunction despite absence of any significant effects on blood pressure. Knowing that ACE inhibitors are widely used to treat hypertension and associated renal diseases, and able to increase circulating, tissue or excreted Ac-SDKP, the current observation provides significant understanding of one of the multiple mechanisms by which ACE inhibitors exert their protective effects such as anti-inflammatory, anti-fibrotic and correction of renal function (glomerular filtration and protein leakage). Therefore, development of peptide or non-peptide Ac-SDKP analogues that are resistant to peptidases could become an important tool in the treatment of renal diseases.

Fig 5.

Fig 5

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Effect of Ac-SDKP on glomerulosclerosis. A) Representative images of the glomerular matrix (GM). Dark purple regions indicate extracellular matrix stained by periodic acid Schiff (PAS) (bar = 25 μm). B) Quantitative data show that GM increased significantly in 5/6Nx rats given vehicle. Ac-SDKP decreased GM significantly in both prevention and reversal protocols (n = 5-6).

Acknowledgments

Sources of Funding

This work was supported by National Institutes of Health grants HL28982- (O.A.C.) and HL71806- (N.E.R.)

Footnotes

Disclosure: None

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