Role of (pro)renin receptor in cyclosporin A-induced nephropathy

Abstract

Calcineurin inhibitors such as cyclosporin A (CsA) have been widely used to improve graft survival following solid-organ transplantation. However, the clinical use of CsA is often limited by its nephrotoxicity. The present study tested the hypothesis that activation of the (pro)renin receptor (PRR) contributes to CsA-induced nephropathy by activating the renin-angiotensin system (RAS). Renal injury in male Sprague-Dawley rats was induced by a low-salt diet combined with CsA as evidenced by elevated plasma creatinine and blood urea nitrogen levels, decreased creatinine clearance and induced renal inflammation, apoptosis and interstitial fibrosis, and elevated urinary N-acetyl-β-d-glucosaminidase activity and urinary kidney injury molecule-1 content. Each index of renal injury was attenuated following 2 wk of treatment with the PRR decoy inhibitor PRO20. Although CsA-treated rats with kidney injury displayed increased renal soluble (s)PRR abundance, plasma sPRR, renin activity, angiotensin II, and heightened urinary total prorenin/renin content, RAS activation was attenuated by PRO20. Exposure of cultured human renal proximal tubular HK-2 cells to CsA induced expression of fibronectin and sPRR production, but the fibrotic response was attenuated by PRO20 and siRNA-mediated PRR knockdown. These findings support the hypothesis that activation of PRR contributes to CsA-induced nephropathy by activating the RAS in rats. Of importance, we provide strong proof of concept that targeting PRR offers a novel therapeutic strategy to limit nephrotoxic effects of immunosuppressant drugs.

NEW & NOTEWORTHY The present study reports, for the first time, that activation of the (pro)renin receptor drives the renin-angiotensin system to induce renal injury during cyclosporin A administration. More importantly, our study has identified that antagonism with PRO20 offers a novel intervention in the management of side effects of cyclosporin A.

INTRODUCTION

Cyclosporin A (CsA), a potent immunosuppressant, has improved allograft survival and quality of solid-organ transplant recipients. However, the therapeutic benefits of CsA are limited by its main side effect, that is, nephrotoxicity (1). This complication is manifested histologically by afferent arteriolopathy, striped interstitial fibrosis, inflammatory cell infiltration, and tubular atrophy (2–5). Furthermore, CsA-induced nephropathy is associated with a marked increase in apoptosis of tubular and interstitial cells (6, 7). In a reproducible chronic CsA nephrotoxicity model, CsA treatment in rats on a low-salt diet (LSD) induced a histological feature similar to that described in patients on long-term CsA therapy (8). Although the molecular mechanisms responsible for CsA-induced nephrotoxicity likely are multifactorial, the renin-angiotensin system (RAS) has been implicated (2, 8). In this regard, previous studies have shown that RAS stimulation plays an important role in the pathogenesis of CsA nephrotoxicity (9, 10), as evidenced by elevated plasma renin activity and angiotensin II (ANG II), together with heightened renal ANG II type 1 receptor expression following CsA treatment (11–13). Interestingly, intrarenal renin and angiotensinogen (AGT) protein expression is activated in CsA-treated mice, indicating the involvement of the intrarenal RAS (14). Blockade of the RAS with either angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril) or ANG II receptor type I antagonists (e.g., losartan) confers renoprotection during chronic CsA nephrotoxicity (13, 15–19). However, the precise mechanism(s) whereby RAS activation occurs remains elusive. This is a clinically relevant area of inquiry because nephrotoxicity associated with the immunosuppressant agents has not been adequately addressed.

The (pro)renin receptor (PRR), a single transmembrane protein composed of 350 amino acids, was discovered and cloned by Nguyen et al. (20) in human mesangial cells. Full-length PRR (fPRR) is a 39-kDa protein that contains a protease cleavage site. fPRR is mainly cleaved by furin or site-1 protease (S1P) to generate soluble (s)PRR, a 28-kDa NH₂-terminal region (21). Both PRR and sPRR bind renin or prorenin to enhance renin activity (three- to fivefold), with the latter involving conformational changes that lead to nonproteolytic activation (20). Within the kidney, a previous immunocytochemical study has indicated that PRR is expressed predominantly in intercalated cells of the collecting duct with a lower abundance in proximal and distal tubules (22).

Multiple clinical studies have reported that circulating sPRR is elevated in early pregnancy, preeclampsia, gestational diabetes mellitus, patients with heart failure with renal dysfunction, and individuals with chronic kidney disease due to hypertension or type 2 diabetes mellitus. As a result, strong support exists that sPRR be considered as a biomarker for cardiovascular and renal diseases (23–26). New evidence is also emerging to support a biological function of sPRR in the regulation of fluid homeostasis as well as pathogenesis of kidney diseases (25, 27–30).

PRO20 has been validated as an effective and specific PRR inhibitor (31–34). PRO20 interacts with the first 20amino acid residues of the prorenin prosegment (L¹PTDTASFGRILLKKMPSVR²⁰), antagonizing PRR/sPRR binding to prorenin and renin. The main purpose of the present study was to explore the role of PRR in CsA nephropathy using PRO20. Specifically, we tested the hypothesis that PRR contributes to CsA-induced nephropathy by activating the RAS.

MATERIALS AND METHODS

Materials

DMEM/F-12 was purchased from Life Technologies (Grand Island, NY). FBS was purchased from Quacell Biotechnology (Zhongshan, Guangdong, China). CsA was purchased from Cell Signaling Technology (Danvers, MA). PRO20 was generated by BGI (Shenzhen, China) (27).

Animals

Male 7-wk-old Sprague-Dawley rats (150–170 g) were purchased from the Laboratory Animal Center of Southern Medical University. All animals were cage housed and maintained in a temperature-controlled room with a 12:12-h light-dark cycle with free access to tap water. The animal protocols were approved by the Animal Care and Use Committee of Sun Yat-sen University.

Treatments

Sprague-Dawley rats were fed a LSD containing 0.05% Na⁺ (Guangdong Medical Laboratory Animal Center) starting 1 wk before the administration of CsA and for the entire experimental period. Rats were treated with either 1) vehicle (olive oil, 2 mL via 2 ig administration, control), 2) CsA (50 mg/kg/day dissolved in olive oil, 2 mL via 2 ig administration, Zhongmei Huadong Pharmaceutical), or 3) CsA + PRO20 (700 μg/kg/day). Three days before CsA treatment, PRO20 was administered via subcutaneous injection three times per day (700 μg/kg/day, dissolved in 0.9% NaCl) and for the entire experimental period. Other groups received an equal amount of saline as the vehicle control. Following the 2-wk treatment, rats were placed in metabolic cages to collect 24-h urine and metabolic data. Urine samples were snap frozen in liquid nitrogen and stored at −80°C to measure RAS components and kidney injury-related indexes. Kidney injury molecule-1 (KIM-1; CSB-E08808r, CUSABIO, Wuhan, China) content and N-acetyl-β-d-glucosaminidase (NAG) activity (A031, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were determined using ELISA kits (35). After urine collection, the animals were anesthetized with isoflurane (2.5%, 5 min, muzzle inhaled), blood was collected, and kidneys were harvested for the analysis of kidney injury-related protein and mRNA expression as well as histological analysis. All rats were euthanized by CO₂ inhalation. Creatinine and blood urea nitrogen (BUN) were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute). Creatinine clearance (CCL) was calculated with the following formula: CCL (mL/min) = [C_U (μmol/L) × urine flow (mL/min)]/C_S (μmol/L), where C_U is the concentration of creatinine in the urine and C_S is the creatinine concentration in the serum (36).

Histopathology

One-fourth of the kidney samples were fixed in 10% buffered formalin, embedded in paraffin, and cut in 5-μm-thick slices. After deparaffinization, the sections were stained with hematoxylin and eosin and Masson trichrome stain (all reagents were from Nanjing Jiancheng Bioengineering Institute). To evaluate the histological score of CsA-induced nephropathy, a semiquantitative scoring system was used, as previously described (35). In brief, the scores for interstitial lymphocytic infiltrates, interstitial cell fibrosis, tubular atrophy, and injury ranged from 0 to 3 as follows: 0, normal; 1, lesions involving <25% of the cortical area; 2, lesions involving 25%–50% of the cortical area; and 3, lesions involving >50% of the cortical area, in a blinded manner. The score index in each rat was expressed as a mean value of all scores obtained. The random pictures per kidney section (10 fields, ×200) were quantified. Masson trichrome staining was performed to analyze renal fibrosis. The amount of cortical collagen fiber was measured by quantifying the percentage of blue positive staining areas.

Apoptosis

An in situ TUNEL assay was performed according to the Situ TUNEL Apoptosis Detection Kit instruction manual (G002-2, Nanjing Jiancheng Bioengineering Institute) (30). In brief, deparaffinized kidney sections were incubated with the following: terminal deoxynucleotidyl transferase (TdT) reaction mix (45 μL balanced solution + 1 μL fluorescent liquid + 4 μL TdT per section) for 60 min at 37°C, 1× PBS three times (5 min/once) at room temperature, and a drop of antifade mounting medium with DAPI. The fragment was covered and observed under a fluorescence microscope. TUNEL-positive cells showed green fluorescence.

Renin Activity, sPRR, AGT, ANG II, and Total Prorenin/Renin Analyses

A renin activity assay was performed as previously described (37). In short, plasma samples were centrifuged at 4,000 revolutions/min at 4°C for 20 min, and the supernatant was collected. Renin activity was determined by the Δ value of ANG I generation after 1 h of incubation at 37°C versus 4°C using an ANG I ELISA kit (S-1188, Peninsula Laboratories) according to the manufacturer’s instructions (37). The sPRR concentration in plasma was measured using a commercial ELISA kit (PRPENKT-TOT, Molecular Innovations) (32). Urinary total prorenin/renin content was measured using an ELISA kit (RPRENKT-TOT-5, Molecular Innovations). The ANG II concentration in plasma was measured using an ELISA kit (CEA005Ra, Cloud-Clone) (27).We set up five points of diluted standard from 24.69 to 2,000 pg/mL, and standard diluent was the blank at 0 pg/mL. We added 50 µL of standard or samples to each well and then added 50 µL of prepared detection reagent A immediately with incubation for 1 h at 37°C. Samples were aspirated and washed three times with wash buffer, and then 100 µL of prepared detection reagent B was added with an incubation for 30 min at 37°C. Finally, we added 50 µL of stop solution and conducted measurements at 450 nm immediately using a microplate reader. We plotted the optical density value of the standard (x-axis) against the log value of the concentration of the standard (y-axis), of which the r² value was 0.99750.

Cell Culture

HK-2 cells (Procell Life Science, Wuhan, China) were derived from immortalized primary human proximal tubular cells isolated from the normal male adult kidney and were immortalized with human papillomavirus (HPV 16) E6/E7 genes. Cells were cultured in DMEM/F-12 containing 10% FBS and 1% penicillin-streptomycin (Gibco BRL, Grand Island, NY) at 37°C under 5% CO₂ in a humidified incubator. For all experiments, cells were grown on six-well plates to reach 80%−90% confluence and serum starved for 12 h in serum-free DMEM/F-12 containing no drugs or hormones before stimulation with CsA.

RNA Interference

Knockdown of endogenous PRR was performed using predesigned siRNA from Origene (SR306857, Rockville, MD). HK-2 cells were cultured to 80% confluence and transfected with PRR siRNA or nontargeting control siRNA (SR30004). The siRNA was transfected 24 h before CsA treatment, and cells were harvested for RNA or Western blot analysis 24 h after CsA treatment.

Immunoblot Analysis

Western blot analysis was completed as previously described (38). Briefly, 30 μg of protein for each sample was denatured in boiling water for 10 min, separated by SDS-PAGE, and then transferred to polyvinylidene fluoride membranes (Immobillion-P, Millipore, Bedford, MA). Next, the membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature followed by incubation with the following primary antibodies: anti-PRR (1:1,000, Sigma-Aldrich), anti-fibronectin (FN; 1:1,000, Sigma-Aldrich), anti-α-smooth muscle actin (α-SMA; 1:500, Boster), anti-TNF-α (1:1,000, Santa Cruz Biotechnology), anti-bax (1:1,000, Cell Signaling Technology), anti-bcl-2 (1:1,000, Cell Signaling Technology), anti-cleaved caspase-3 (1:1,000, Cell Signaling Technology), anti-osteopontin (OPN; 1:1,000, Proteintech), and anti-β-actin (1:10,000, Sigma-Aldrich) overnight at 4°C. After being washed three times with TBST, membranes were incubated with secondary antibodies (goat anti-rabbit/mouse horseradish peroxidase-conjugated secondary antibody) for 1 h at room temperature and visualized with enhanced chemiluminescence (Thermo Scientific). Signals on immunoblots were quantitated by Image-Pro Plus version 6.0 software. β-Actin was used as an internal control.

RNA Isolation and Quantitative RT-PCR

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. Total RNA concentrations were determined using a NANODROP 2000 spectrophotometer (Thermo Scientific) according to the manufacturer’s instructions. Total RNA (1,000 ng) was reverse transcribed into cDNA using the cDNA synthesis kit (Takara Bio, Shiga, Japan), and quantitative PCR was carried out using real-time PCR system (Applied Biosystems, Life Technologies) and FastStart Universal SYBR Green Master (ROX, Roche) according to the manufacturer’s instructions. Relative mRNA expression levels were calculated from threshold cycle (C_T) numbers, i.e., 2^−ΔΔCT, according to the manufacturer’s suggestions. GAPDH was detected as an internal control. Primer sequences are shown in Table 1 (27, 28, 30).

Table 1.

Primer sequences for real-time PCR

Target Gene	Primer Sequence
Human GAPDH
Forward	5′-TCATTGACCTCAACTACATG-3′
Reverse	5′-CAAAGTTGTCATGGATGACC-3′
Human PRR
Forward	5′-CAGACGTGGCTGCATTGTCC-3′
Reverse	5′-CTGGGGGTAGAGCCAGTTTGTT-3′
Human FN
Forward	5′-TGGGCGAGGGAGAATAAG-3′
Reverse	5′-CCACATAGGAAGTCCCAGCA-3′
Rat GAPDH
Forward	5′-GTCTTCACTACCATGGAGAAGG-3′
Reverse	5′-TCATGGATGACCTTGGCCAG-3′
Rat PRR
Forward	5′-ATCCTTGAGACGAAACAAGA-3′
Reverse	5′-AGCCAGTCATAATCCACAGT-3′
Rat FN
Forward	5′-ACCAGTGGGATAAGCAGCAT-3′
Reverse	5′-CCTTCCAGCGACCCGTAGAG-3′
Rat α-SMA
Forward	5′-TGGCTGATGGAGTACTTC-3′
Reverse	5′-TGGCTGATGGAGTACTTC-3′
Rat TGF-β1
Forward	5′-CTCAACACCTGCACAGCTCC-3′
Reverse	5′-CTCAACACCTGCACAGCTCC-3′
Rat TNF-α
Forward	5′-CACAGTGAAGTGCTGGCAAC-3′
Reverse	5′-ACATTGGGTCCCCCAGGATA-3′
Rat IL-6
Forward	5′-AGAGACTTCCAGCCAGTTGC-3′
Reverse	5′-AGTCTCCTCTCCGGACTTGT-3′
Rat IL-1β
Forward	5′-GGCCAGTGATGGTGGGTCAG-3′
Reverse	5′-TCAACAACGCCACCTTGTGTA-3′

α-SMA, α-smooth muscle actin; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; PRR (ATP6AP2), (pro)renin receptor; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α.

Statistical Analyses

All data are reported as means ± SE. Comparison among three means was performed using one-way ANOVA. If a significant P value was obtained (i.e., <0.05), the location of the differences was identified using a Tukey’s post hoc test. Comparison of two means was completed using an unpaired Student’s t test. Analyses were performed using GraphPad Prism 6 software (GraphPad Software).

RESULTS

PRO20 Attenuates CsA-Induced Nephrotoxicity

We assessed the effect of PRO20 on CsA-induced renal injury in rats on a LSD. At the end of the experiment, plasma samples were first assayed for creatinine and BUN. The increases of plasma creatinine (Fig. 1A) and BUN (Fig. 1B) were significantly attenuated by PRO20. Urinary NAG activity and KIM-1 were monitored to reflect the degree of renal injury (36, 39, 40). Both the KIM-1 level (Fig. 1C) and NAG activity (Fig. 1D) were significantly elevated in CsA rats, and both were improved by PRO20. In agreement with these findings, CCL, an index of glomerular filtration rate, was lower in CsA-treated rats, which was partially restored following PRO20 treatment (Fig. 1E). Similarly, CsA treatment decreased body weight, which was reversed by PRO20 (Fig. 1F). By hematoxylin and eosin staining analysis, the CsA-treated group displayed interstitial inflammatory cell infiltration, tubular vacuolization, and atrophy (Fig. 2A). PRO20 improved the histological damage induced by CsA (Fig. 2A). The results were semiquantitatively analyzed by determining the tubulointerstitial injury score (Fig. 2B). These findings collectively substantiate that CsA-induced renal injury is mitigated by PRO20.

Figure 1.

Analysis of renal function and injury. Sprague-Dawley rats were fed a low-salt diet (0.05% Na⁺) for 3 wk and treated for 14 days with vehicle [control (CTR)], cyclosporin A (CsA), or CsA + (pro)renin receptor decoy inhibitor (PRO20). A: plasma creatinine. B: plasma blood urea nitrogen (BUN). ELISA measurement of urinary kidney injury molecule-1 (KIM-1; C) and urinary N-acetyl-β-d-glucosaminidase (NAG) activity (D). E: creatinine clearance. F: body weight gain (ΔBW). Data are means ± SE; n = 5 animals. *P < 0.05 and **P < 0.01 vs. CTR; #P < 0.05 and ##P < 0.05 vs. CsA.

Figure 2.

Histological analysis of renal injury. A: hematoxylin and eosin (HE) staining of the renal cortex (magnification: ×200). B: renal tubulointerstitial injury score from semiquantitative analysis of renal pathologies (average of 10 fields of area per rat). Data are means ± SE; n = 5. **P < 0.01 vs. control (CTR); ##P < 0.01 vs. cyclosporin A (CsA).

PRO20 Inhibits CsA-Induced RAS Activation

The RAS plays an important role in inflammation and fibrosis during chronic CsA therapy (41). However, the mechanism of how CsA activates the RAS remains largely unknown. The present study examined the potential role of PRR in mediating the activation of the RAS induced by CsA. Renal sPRR protein expression and plasma sPRR were elevated in CsA-treated versus control rats (P < 0.05; Fig. 3, A and B). Furthermore, CsA-induced elevations of plasma sPRR levels (Fig. 3B), renin activity (Fig. 3C), and ANG II concentrations (Fig. 3D) were suppressed by PRO20. It was necessary to measure urinary excretion of total prorenin/renin because the ELISA kit cannot distinguish between prorenin and renin. In this regard, CsA-induced elevations of urinary prorenin/renin were attenuated by PRO20. These findings provide solid evidence that PRO20 inhibits CsA-induced RAS activation.

Figure 3.

Analysis of renin-angiotensin system components. A: renal full-length and soluble (pro)renin receptor (fPRR and sPRR, respectively) protein expression was analyzed by immunoblot analysis and by densitometry with normalization by β-actin. B: plasma sPRR concentration. C: plasma renin activity. D: plasma angiotensin (ANG) II concentration. E: urinary total prorenin/renin excretion. Data are means ± SE; n = 5. *P < 0.05 and **P < 0.01 vs. control (CTR); #P < 0.05 and ##P < 0.01 vs. cyclosporin A (CsA).

PRO20 Blunts CsA-Induced Renal Fibrosis, Inflammation, and Apoptosis

Renal fibrosis is a common pathological change shared by multiple types of renal diseases including CsA nephropathy (1, 2). Supporting our hypothesis, PRO20 attenuated CsA-induced fibrosis in the renal cortex, protein expression of FN and α-SMA expression, and mRNA expression of FN and transforming growth factor-β (Fig. 4, A–C).

Figure 4.

Analysis of renal fibrosis. A: Masson trichrome staining of the kidney (magnification: ×200). Shown are representative images from 5 rats/group. B: collagen fiber area (%). C: fibronectin (FN) and α-smooth muscle actin (α-SMA) protein expression was analyzed by immunoblot analysis and by densitometry with normalization by β-actin. D: renal mRNA expression of FN, α-SMA, and transforming growth factor (TGF)-β1 was analyzed by quantitative RT-PCR with normalization by GAPDH. Data are means ± SE; n = 5. *P < 0.05 and **P < 0.01 vs. control (CTR); #P < 0.05 and ##P < 0.01 vs. cyclosporin A (CsA).

Inflammation contributes to CsA nephropathy (42), and we assessed whether PRO20 is protective in this regard. Notably, CsA-induced elevations in TNF-α protein and mRNA expression, together with IL-6 and IL-1β mRNA expression, were blunted by concurrent treatment with PRO20 (Fig. 5B).

Figure 5.

Analysis of inflammation and apoptosis. A: TNF-α protein expression was analyzed by immunoblot analysis and by densitometry with normalization by β-actin. B: renal mRNA expression of TNF-α, IL-6, and IL-1β was analyzed by quantitative RT-PCR with normalization by GAPDH. C: osteopontin (OPN) protein expression was analyzed by immunoblot analysis with densitometry and normalized by β-actin. D: renal mRNA expression of OPN was analyzed by quantitative RT-PCR with normalization by GAPDH. Bcl-2 and bax (E) as well as pro-caspase-3 and cleaved caspase-3 (F) protein expression was analyzed by immunoblot analysis with densitometry and normalized by β-actin. G: TUNEL assay. H: TUNEL-positive area (%). Scale bars = 500 μm. Data are means ± SE; n = 5. *P < 0.05 and **P < 0.01 vs. control (CTR); #P < 0.05 and ##P < 0.05 vs. cyclosporin A (CsA).

OPN levels were dramatically increased in CsA-treated rats, which correlates with interstitial macrophage infiltration and fibrosis (43, 44). We therefore examined the effect of PRO20 on OPN expression in CsA-treated rats. As shown in Fig. 5, C and D, levels of OPN mRNA and protein were significantly increased in CsA-treated rats, both of which were inhibited by PRO20.

Because renal cell apoptosis is important concerning CsA nephropathy and renal dysfunction, we assessed whether PRO20 exerts protection in this regard. Of interest, CsA decreased protein expression of the bcl-2-to-bax ratio, but PRO20 attenuated this response (Fig. 5E). Furthermore, heightened protein expression of pro-caspase-3 and cleaved caspase-3 evoked by CsA were markedly attenuated by PRO20 (Fig. 5F). The results concerning cleaved caspase-3 were substantiated by counting TUNEL-positive cells (Fig. 5G). Taken together, we provide robust evidence that PRO20 attenuates CsA-induced renal fibrosis, inflammation, and apoptosis.

CsA-Induced Fibrogenesis in HK-2 Cells Is Abrogated by Inhibiting PRR/sPRR Activation

Using a reductionist approach, we tested whether CsA heightens sPRR production in HK-2 cells. CsA treatment elevated sPRR abundance in HK-2 cells, which was accompanied by decreased protein expression of fPRR, indicating enhanced cleavage (Fig. 6A). In addition, CsA treatment induced sPRR release into the cellular media (Fig. 6B), and this was associated with greater protein expression of FN.

Figure 6.

Regulation and functional role of the (pro)renin receptor (PRR) in cultured renal epithelial cells exposed to cyclosporin A (CsA). HK-2 cells were grown on six-well plates until 80% confluence, pretreated with 10 μM PRO20, and then treated with 15 μM CsA for 24 h. A: full-length and soluble PRR [full-length (f)PRR and soluble (s)PRR, respectively] protein expression was analyzed by immunoblot analysis and by densitometry analysis with normalization by β-actin. B: ELISA measurement of medium sPRR. C: fibronectin (FN) protein expression was analyzed by immunoblot analysis and densitometry with normalization by β-actin. D: FN mRNA level was determined by quantitative RT-PCR with normalization by GAPDH. In a separate experiment, HK-2 cells were transfected with PRR siRNA or scrambled (scr) siRNA followed by CsA treatment at 15 μM for 24 h. E: fPRR, sPRR, and FN protein expression was analyzed by immunoblot analysis and by densitometry with normalization by β-actin. F: quantitative RT-PCR detection of PRR and FN mRNA levels with normalization by GAPDH. Data are means ± SE. *P < 0.05 and **P < 0.01 vs. control (CTR); #P < 0.05 and ##P < 0.05 vs. CsA.

Pharmacological and genetic approaches substantiate that CsA-induced PRR/sPRR activation precipitates fibrogenesis. First, PRR antagonism using PRO20 inhibited CsA-induced upregulation of FN protein (Fig. 6C) and mRNA expression (Fig. 6D) accompanied by decreased sPRR abundance (Fig. 6A) in HK-2 cells. Second, transfection of HK-2 cells with PRR siRNA induced a >80% reduction of mRNA and protein expression of PRR and sPRR versus cells transfected with scrambled siRNA (Fig. 6, E and F). Of note, siRNA-mediated knockdown of PRR markedly reduced CsA-induced increases in FN mRNA and protein expression (Fig. 6, E and F). Collectively, these results support the hypothesis that CsA-induced PRR/sPRR activation precipitates fibrogenesis in cultured renal epithelial cells.

DISCUSSION

The clinical usage of CsA is often limited due to its nephrotoxicity (2). Activation of the RAS, especially the intrarenal RAS, plays an essential role in the pathogenesis of CsA nephropathy (45). The extent to which PRR, a novel component and regulator of the RAS, might be involved in CsA nephropathy is unknown. Here, we explored this issue in a rat model of CsA-induced nephropathy using the specific PRR inhibitor PRO20. Because our findings revealed an important role for PRR in nephrotoxicity associated with CsA, a reductionist approach using HK-2 cells was conducted to identify the underlying mechanism.

We provide comprehensive evidence that PRO20 mitigates myriad end points indicating renal injury induced by CsA. Plasma creatinine and CCL together with BUN were determined to reflect renal function. KIM-1, a transmembrane glycoprotein in renal proximal tubular epithelial cells, was assessed to sensitively and specifically reflect renal injury and recovery (46). NAG, an intracellular lysosomal enzyme with the highest distribution in proximal tubules, was measured to estimate the degree of renal tubular injury (47). As anticipated, CsA treatment increased plasma creatinine, BUN, and urinary KIM-1 content and NAG activity and reduced CCL. These findings clearly indicate that our model of CsA-induced kidney damage and impaired glomerular filtration rate exhibited a robust phenotype. As hypothesized, when rats were treated with PRO20 concurrent with CsA, these end points, together with the loss of body mass associated with nephrotoxicity, improved significantly. Furthermore, the renal tubule atrophy, vacuolation, infiltration of inflammatory cells, and interstitial fibrosis displayed by rats treated with CsA in our study and reported by others (48) were improved by PRO20. As such, PRO20 alleviates CsA-induced renal histological injury and interstitial fibrosis. These results provide convincing evidence concerning the renoprotective action of PRO20 in a rat model of CsA nephropathy.

Although the relationship between PRR and the RAS has been debated, increasing evidence strongly supports PRR as an important regulator of renin activity at both the systemic (49) and local levels (32, 33). In this regard, PRR-dependent activation of the intrarenal RAS contributes importantly to the pathogenesis of renal injury induced by albumin overload and adriamycin in rodents (27, 50). In addition, reports have indicated that serum sPRR is inversely associated with the estimated glomerular filtration rate in patients with chronic kidney disease caused by hypertension and diabetes (40). Inappropriate activation of the RAS has been demonstrated in the pathogenesis of CsA nephropathy, but the precipitating stimulus is unclear (45). This gap in knowledge inspired us to determine whether PRR acts in a RAS-dependent manner to mediate CsA nephropathy. Importantly, we revealed that CsA treatment upregulates plasma and renal sPRR levels, clear evidence of PRR activation, which can be blocked by PRO20. Consistent with this observation, we have previously shown that PRO20 treatment suppressed urinary sPRR excretion in rats following albumin overload (27) or 5/6 nephropathy (51). Given the well-documented pathogenic roles of sPRR in various models of renal disease, it seems reasonable to speculate that inhibition of sPRR production may at least in part contribute to the renoprotective action of PRO20. However, the mechanism of the inhibitory effect of PRO20 on sPRR abundance remains elusive. As a PRR decoy inhibitor, PRO20 presumably antagonizes PRR by interrupting binding of prorenin or renin to PRR and therefore is unexpected to affect PRR or sPRR levels. This issue warrants further investigation in the future. Furthermore, CsA treatment elevated multiple RAS components, including plasma renin activity and ANG II, together with urinary prorenin/renin excretion. Of note, PRO20 was highly effective in suppressing the rise of renin activity, prorenin/renin content, sPRR levels, and ANG II levels. These results strongly substantiate PRR as an important regulator of renin activity in the context of our rat model of CsA nephropathy. Moreover, PRR can act in a RAS-dependent and -independent manner. Apart from regulating prorenin and renin activity, activation of PRR induces multiple signaling pathways, including MAPK (52), oxidative stress (53), Wnt/β-catenin signaling (54), the fibrogenic response (55), etc. Therefore, the therapeutic value of PRO20 may go beyond RAS inhibition.

Activation of inflammatory, apoptotic, and fibrogenic responses is known to contribute to the pathogenesis of CsA nephropathy. For example, Thomas et al. (7) reported that accelerated apoptosis characterizes CsA-associated interstitial fibrosis. Furthermore, interstitial fibrosis was associated with significant macrophage influx, which correlated with increased cortical tubular staining for the macrophage adhesion protein OPN (4, 5). CsA nephropathy also features increased expression of OPN in tubular epithelial cells, which is closely correlated with the degree of macrophage infiltration and interstitial fibrosis (17). These results suggest that CsA may increase macrophage infiltration and apoptosis by upregulating OPN expression to an extent that promotes interstitial fibrosis, eventually leading to renal dysfunction and structural damage. Here, we observed that PRO20 treatment inhibited CsA-induced overexpression of renal proinflammatory factors including TNF-α, IL-6, and IL-1β, reduced apoptotic factors such as bax and cleaved caspase-3, and suppressed the levels of FN, α-SMA, and transforming growth factor-β. Congruent with these findings, CsA-induced upregulation of OPN mRNA and protein expression was attenuated by concurrent treatment with PRO20. As such, it is not unreasonable to conclude that PRO20 inhibition of OPN expression may contribute to the anti-inflammatory and antiapoptotic actions of PRR blockade.

Finally, cell culture experiments were conducted to examine the direct role of PRR in CsA-induced cell injury. HK-2 cells are one of the best-characterized renal epithelial cells and have been commonly used to investigate mechanisms responsible for fibrotic responses (30, 56). Following exposure to CsA, upregulated FN expression was accompanied by marked increases in sPRR release. PRO20 and PRR siRNA consistently blocked CsA-induced FN expression. These pharmacological and genetic approaches substantiate the hypothesis that sPRR mediates CsA-evoked fibrotic responses, and this is in agreement with our previous report (30). Still unresolved is the precise mechanism whereby CsA treatment triggers the cleavage process to release sPRR, and an ongoing area of inquiry in our laboratory is to ascertain the role of calcineurin-associated phosphatase activity in this regard.

Here, we provide the first evidence showing that PRR antagonism with PRO20 attenuates CsA-induced renal functional and structural damage by suppressing the RAS. Additional findings using a variety of experimental approaches provide direct evidence that CsA-induced PRR activation contributes importantly to fibrotic responses displayed by cultured renal epithelial cells. Collectively, we provide solid evidence that manipulating PRR might offer a novel therapeutic strategy for intervening/managing off-target renal effects of CsA treatment.

Perspectives and Significance

Our findings support the hypothesis that activation of PRR contributes to CsA-induced nephropathy by activating the RAS in rats. Of importance, we provide strong proof of concept that targeting PRR offers a novel therapeutic strategy to limit nephotoxic effects of immunosuppressant drugs.

GRANTS

This work was supported by National Science Foundation of China Grant 81630013 and VA Merit Review I01 BX004871 from the Department of Veterans Affairs. T.Y. is a Senior Research Career Scientist in the Department of Veterans Affairs.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.H. and T.Y. designed research; J.H., Y.T., Y.C., S.M., M.P., J.S., and A.L. performed experiments; J.H. analyzed data; J.H. and T.Y. drafted manuscript; J.D.S., B.H., and Y.D. edited manuscript; all authors approved final version of manuscript.