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Experimental Transplantation

Combined Effects of Losartan and Pravastatin on Interstitial Inflammation and Fibrosis in Chronic Cyclosporine-Induced Nephropathy

Li, Can1,2; Sun, Bo Kyung1; Lim, Sun Woo1; Song, Joon Chang1; Kang, Shin-Wook3; Kim, Yu Seun4; Kang, Duk Hee5; Cha, Jung Ho6; Kim, Jin6; Yang, Chul Woo1,7

Author Information
doi: 10.1097/01.TP.0000155305.49439.4C

Abstract

Most immunosuppressive drug regimens depend on the use of cyclosporine (CsA); however, CsA-induced nephropathy is the major dose-limiting adverse effect (1). Long-term administration of CsA causes progressive renal failure with striped interstitial fibrosis, tubular atrophy, inflammatory cell infiltration, and hyalinosis of the afferent arterioles (2). The pathogenesis of chronic CsA-induced nephropathy is multifactorial, and in vitro and in vivo studies have shown that the predominant factors include activation of the intrarenal renin-angiotensin system (RAS), increased release of endothelin-1, dysregulation of nitric oxide (NO) and NO synthases, an imbalance of prostaglandins and thromboxane levels, stimulation of the sympathetic nervous system, and increases in transforming growth factor (TGF)-β1 and inflammatory cytokines (3).

The RAS plays an important role in the pathogenesis of chronic CsA-induced nephropathy. We have previously demonstrated that the administration of CsA to salt-depleted rats activates the RAS, as shown by a significant increase in intrarenal angiotensin II (Ang II) immunoreactivity (4). Blocking this system with either losartan (LSRT), an antagonist of Ang II type I receptor blocker (ARB), or angiotensin-converting enzyme (ACE) inhibitors suppresses the expression of pro-inflammatory mediators and profibrogenic cytokines and thus prevents the fibrosis induced by CsA (5). These findings suggest that blockade of the RAS, using ARBs or ACE inhibitors, confers a level of renoprotection beyond their blood–pressure-lowing effects in this model of chronic CsA-induced nephropathy.

Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the key enzyme that regulates the synthesis of cholesterol from mevalonic acid by suppressing the conversion of HMG-CoA (6). However, mevalonate is the precursor not only of cholesterol but also of many nonsteroidal compounds. Thus, inhibition of HMG-CoA by statins may lead to pleiotropic effects, and statins may thereby exert anti-inflammatory and anti-arteriosclerotic actions independently of lipid reduction (7). The beneficial effect of statins on the kidney have been shown by studies of ischemia-reperfusion injury (8), subtotal renal ablation (9), streptozotocin-induced diabetic nephropathy (10), puromycin-induced nephrosis (11), unilateral ureteral obstruction (12), and chronic CsA-induced nephropathy (13).

Combined treatments using statins and ARBs are now known to elicit better protective effects than each agent individually in reducing neointimal formation and the proliferation of vascular smooth-muscle cells (VSMCs) (14) and uninephrectomized Heymann nephritis (15). We therefore hypothesized that a combined treatment of LSRT with pravastatin (PRVT) may provide superior renoprotection in a rat model of chronic CsA-induced nephropathy. To test this hypothesis, LSRT and PRVT were administered separately or in combination to CsA-treated rats. Our results clearly demonstrate that PRVT enhances the effects of LSRT in inhibiting the inflammatory and fibrotic processes of chronic CsA-induced nephropathy.

MATERIALS AND METHODS

Drugs

CsA, provided by Novartis Pharma (Basel, Switzerland), was diluted in olive oil (Sigma Co., St. Louis, MO) to a final concentration of 15 mg/mL. LSRT, provided by MERCK Research Laboratories (Pahway, NJ), was dissolved in sterile water to a final concentration of 100 mg/L. PRVT (Bristol-Myers Squibb Pharmaceutical Co., Korea) was dissolved in drinking water and administered at a dose of 5 mg/kg.

Experimental Protocol

The Animal-Care Committee of the Catholic University of Korea approved the experimental protocol. Male Sprague-Dawley rats (Charles River, Technology, Korea), weighing 225 to 250 g, were housed in individual cases in a temperature- and light-controlled environment with free access to a low salt diet (0.05% sodium, Teklad Premier, Madison, WI) and tap water. Rats were randomized into eight subgroups and treated daily for 4 weeks as follows.

  1. Vehicle (VH) group (n=8): subcutaneous injection with olive oil (1 mL/kg).
  2. VH+L (LSRT) group (n=8): simultaneous treatment with olive oil and LSRT (100 mg/L in drinking water).
  3. VH+P (PRVT) group (n=8): simultaneous treatment with olive oil and PRVT (5 mg/kg in drinking water).
  4. VH+L+P group (n=8): simultaneous treatment with olive oil, LSRT, and PRVT.
  5. CsA group (n=8): subcutaneous injection with CsA (15 mg/kg).
  6. CsA+L group (n=8): simultaneous treatment with CsA and LSRT.
  7. CsA+P group (n=8): simultaneous treatment with CsA and PRVT.
  8. CsA+L+P group (n=8): simultaneous treatment with CsA, LSRT, and PRVT.

The dose and method of CsA (16), LSRT (4), and PRVT (13, 17) administration were chosen on the basis of previous reports. At the end of the study, the animals were killed under ketamine anesthesia, and the kidney tissues were rapidly removed for morphologic and molecular examinations.

Basic Parameters

Rats were pair-fed and daily body weight (BW) was monitored. Systolic blood pressure (SBP) was recorded in conscious rats by the tail-cuff method with plethysmography, using a tail manometer-tachometer system (BP-2000, Visitech Systems, Apex, NC), and at least three readings for each rat were averaged. Before sacrifice, animals were individually housed in metabolic cages (Tecniplast, Gazzada, Italy) for 24-hour urine collection, and blood samples were obtained to evaluate serum creatinine (Scr). The creatinine clearance (Ccr) was calculated with standard formula. Whole-blood CsA concentrations were measured by monoclonal radioimmunoassay (Incstar, Stillwater, MN). Serum total cholesterol and triglycerides levels were determined with an auto-analyzer (Coulter-STKS, Coulter Electronics). High-sensitivity C-reactive protein (hs-CRP) was measured by a particle-enhanced immunoturbidimetric method using a Cobas Integra 700 (Roche Diagnostic System, Basel, Switzerland) (13).

Histopathology

Kidney tissues were fixed in periodate-lysine-paraformaldehyde solution, dehydrated, and embedded in wax. After dewaxing, 4-μm sections were processed and stained with periodic acid Schiff (PAS) or Masson’s trichrome and hematoxylin. Arteriolopathy of the afferent arterioles was manifested as the expansion of the cell cytoplasm of terminal arteriolar smooth-muscle cells by eosinophilic and granular material. The percentage of arteriolopathy was quantified by counting at least 100 juxtaglomerular afferent arterioles in each rat kidney using a computer program (TDI Scope Eye Version 3.0 for Windows, Olympus, Japan), and the results were expressed as the percentage of affected arterioles in the total number of arterioles. A finding of tubulointerstitial fibrosis (TIF) was defined as a matrix-rich expansion of the interstitium with tubular dilatation, tubular atrophy, tubular cast formation, sloughing of tubular epithelial cells, or thickening of the tubular basement membrane. A minimum of 20 fields per section was assessed using a color image analyzer (TDI Scope Eye Version 3.0 for Windows, Olympus, Japan). The image was captured, and the extent of TIF was quantified using the Polygon program by counting the percentage of areas injured per field of cortex under ×100 magnification. Histopathologic analyses were performed in randomly selected cortical fields of sections by a pathologist blinded to the identity of the treatment groups.

Immunohistochemistry

Dewaxed sections were incubated with 0.5% Triton X100/phosphate-buffered saline (PBS) solution for 30 minutes and washed with PBS three times. Nonspecific binding sites were blocked with normal horse serum diluted 1:10 in 0.3% bovine serum albumin for 30 to 60 minutes and then incubated for 2 hours at 4°C in mouse antiserum against osteopontin (OPN, MPIIIB10, obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) diluted in 1:1,000 in a humid environment. After rinsing in Tris-buffered saline (TBS), sections were incubated in peroxidase-conjugated rabbit anti-mouse immunoglobulin G (Amersham Pharmacia Biotech, Piscataway, NJ) for 30 minutes. For coloration, sections were incubated with a mixture of 0.05% 3,3′-diaminobenzidine containing 0.01% H2O2 at room temperature until a brown color was visible, washed with TBS, counterstained with hematoxylin, and examined under light microscopy. The procedure of immunostaining for ED-1 (Serotec, UK), CRP (Sigma), and Ang II (Peninsula Labs, San Carlos, CA) was similar to that for OPN. The number of ED–1- or CRP-positive cells was counted in at least 20 fields of cortex per section under ×200 magnification. Analysis of Ang II immunostaining was semiquantitatively evaluated by counting the number of Ang II-positive juxtaglomerular afferent arterioles per total number of juxtaglomerular afferent arterioles available for examination, using a ×20 objective; at least 50 glomeruli (unoverlapped) were assessed per specimen.

Northern Blot Analysis

A 1 kb cRNA probe was generated from 2B7 cDNA clone of rat smooth-muscle OPN. Sense and antisense cRNA probe were labeled with digoxigenin (DIG)-UTP using a T7 RNA polymerase kit (Boehringer Mannheim GmbH, Mannheim, Germany). Probes were precipitated, and incorporation of DIG was determined by dot blotting. Northern blotting was performed as previously described by our laboratory (18) and others (19). Kidney cortex was homogenized. Total RNA was extracted using the RNAzol reagent (Tel-Test, Inc. Texas), and 20 μg samples were denatured with glyoxal and dimethylsulphoxide, size fractionated on 1.2% agarose gels, and capillary blotted onto positively-charged nylon membranes (Boehringer Mannheim GmbH, Mannheim, Germany). Membranes were hybridized overnight at 68°C or 42°C with DIG-labeled cRNA (or 32P labeled cDNA probes for TGF-β1) in a DIG wash and Block Buffer Set solution (Boehringer Mannheim GmbH, Mannheim, Germany). After hybridization, membranes were washed finally in 0.1× standard saline citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) at 68°C or 0.2XSSC/0.1% SDS at 42°C. Bound probes were detected using sheet anti-DIG antibody (Fab) conjugated with alkaline phosphatase (Boehringer Mannheim GmbH, Mannheim, Germany) and development with CSPD-star enhanced chemiluminescence (Boehringer Mannheim GmbH, Mannheim, Germany). The densitometry analysis was performed using the NIH ImagePC program. Three determinations for each band were averaged and referenced to 18S.

Statistical Analyses

Data are expressed as mean±SEM. Multiple comparisons among groups were performed by one-way analysis of variance with the post hoc Bonferroni test (SPSS software version 9.0). Statistical significance was accepted when P<0.05.

RESULTS

Synergistic Effect of LSRT and PRVT on Arteriolopathy in Chronic CsA-Induced Nephropathy

CsA-treated rats showed typical afferent arteriolopathy, as shown in Figure 1. Smooth-muscle cells in the afferent glomerular arteriole were replaced by PAS-positive material, resulting in the typical circumferential appearance of the lesion. Using our quantitative analysis, the percentage of arteriolopathy was significantly higher in the CsA-treated group than in the VH-treated group (38±5% vs. 7±1%, P<0.01). Administration of LSRT or PRVT significantly decreased arteriolopathy compared with the CsA-treated group (21±2%; 23±3%, P<0.05 vs. CsA), and combined treatment using LSRT with PRVT further decreased the incidence of this lesion compared with each drug alone (11±4%, P<0.05 vs. CsA+L or CsA+P).

FIGURE 1.
FIGURE 1.:
Synergistic effects of pravastatin (PRVT) and losartan (LSRT) on arteriolopathy in chronic cyclosporine (CsA)-induced nephropathy. (A) CsA-treated rat kidney shows characteristic degenerative lesion of the afferent glomerular arteriole with the accumulation of eosinophilic material (arrow, periodic acid Schiff [PAS], ×400). (B) Quantitative analysis of arteriolopathy in the experimental groups. Note the significant decrease in arteriolopathy after treatment with either LSRT or PRVT; a further decrease was observed with the combined treatment of LSRT plus PRVT. aP<0.01 vs. vehicle (VH); bP<0.05 vs. CsA; cP<0.05 vs. CsA+losartan (L) or CsA+pravastatin (P).

Synergistic Effect of LSRT and PRVT on Macrophage Infiltration and Intrarenal CRP Expression in Chronic CsA-Induced Nephropathy

Quantitative immunohistochemistry analysis (Fig. 2A) showed that ED–1-positive cells were rare in the VH-treated group, but CsA treatment increased the numbers (56±1, P<0.01 vs. VH). Co-administration of LSRT or PRVT significantly decreased this incidence (31±3; 35±4, P<0.01 vs. CsA), and the decrease was more pronounced with the combination of LSRT plus PRVT (17±3, P<0.05). Similarly, a significant increase in CRP expression and immunoreactivity was observed in the CsA-treated rat kidneys (Fig. 2B). The numbers of CRP-positive cells were markedly increased in the CsA group compared with the VH group (65±5 vs. 25±2, P<0.01), whereas their numbers were significantly decreased in the CsA+L (42±2 vs. CsA, P<0.05) and CsA+P (35±1, P<0.05 vs. CsA) groups, and a further decrease was observed in the CsA+L+P group (27±3, P<0.05 vs. CsA+L or CsA+P).

FIGURE 2.
FIGURE 2.:
Synergistic effects of PRVT and LSRT on macrophage infiltration and intrarenal C-reactive protein (CRP) expression in chronic CsA-induced nephropathy. The figures show immunohistochemistry of ED-1 in VH (A1), CsA (A2), and LSRT+PRVT-treated rat kidneys (A3), and quantitative analyses of ED-1-positive cells in the experimental groups (A4). Note the significant decrease in ED-1-positive cells after treatment with PRVT or LSRT and a further decrease when PRVT and LSRT were administered concurrently. Immunohistochemistry of intrarenal CRP in VH (B1), CsA (B2), and LSRT+PRVT-treated rat kidneys (B3) and quantitative analyses of intrarenal CRP in the experimental groups (B4). Note the significant decrease in CRP-positive cells after treatment with PRVT or LSRT and a further decrease in CRP-positive cells after the combined treatment. aP<0.01 vs. VH; bP<0.01 vs. CsA; cP<0.01 vs. CsA+L or CsA+P (magnifications, A1–A3 ×200; B1–B3 ×400).

Synergistic Effect of LSRT and PRVT on OPN Expression in Chronic CsA-Induced Nephropathy

CsA treatment up-regulated OPN mRNA expression approximately sevenfold (Fig. 3, A and B) (720±59% vs. 102±3%, P<0.05 vs. VH), but this was reduced when LSRT or PRVT was added (388±47%; 406±50%, P<0.05 vs. CsA). Combined treatment with LSRT and PRVT further decreased OPN mRNA expression compared with each drug alone (190±25%, P<0.05). OPN immunoreactivity was consistently up-regulated in CsA-treated rat kidneys and was mainly localized to fibrotic areas (Fig. 3D). After the combined treatment of LSRT plus PRVT, immunoreactivity decreased significantly in parallel with the improvements in renal histology (Fig. 3E).

FIGURE 3.
FIGURE 3.:
Synergistic effects of LSRT and PRVT on osteopontin (OPN) expression in chronic CsA-induced nephropathy. Northern blots of OPN mRNA (A) and densitometric analyses (B). Note the significant increase in OPN mRNA expression in the CsA-treated group but a decrease with LSRT or PRVT treatment. Combined treatment using LSRT and PRVT further decreased OPN mRNA expression compared with each drug alone. Immunohistochemistry of OPN in VH (C), CsA (D), and LSRT+PRVT-treated rat kidneys (E). Note the significant decrease in OPN immunoreactivity after the combined treatment. aP<0.01 vs. VH; bP<0.01 vs. CsA; cP<0.05 vs. CsA+L or CsA+P (magnification ×200).

Synergistic Effects of LSRT and PRVT on TIF and on TGF-β1 mRNA Expression in Chronic CsA-Induced Nephropathy

Kidney tissues from rats treated with CsA had characteristic morphologic findings similar to the renal lesions observed in humans undergoing long-term CsA therapy (Fig. 4B). Focal TIF, tubular atrophy, and inflammatory cell infiltration were evident. On our quantitative analysis system (Fig. 4D), there was a significant increase in the TIF in the CsA group compared with the VH group (0±0 vs. 42±3%, P<0.01), whereas this lesion was diminished with the concomitant administration of either LSRT (25±4%, P<0.01 vs. CsA) or PRVT (30±5%, P<0.01 vs. CsA). Combined treatment of LSRT and PRVT further decreased TIF compared with each drug alone (11±3%, P<0.05 vs. CsA+L or CsA+P).

FIGURE 4.
FIGURE 4.:
Synergistic effect of LSRT and PRVT on tubulointerstitial fibrosis (TIF) and transforming growth factor (TGF)-β1 mRNA expression in chronic CsA-induced nephropathy. Trichrome staining in VH (A), CsA (B), and LSRT+PRVT-treated rat kidneys (C). Quantitative analysis of TIF in the experimental groups. Note the significant decrease in TIF after treatment with PRVT or LSRT; a further decrease was observed when PRVT and LSRT were administered concurrently (D). Northern blot of TGF-β1 mRNA and relative densitometric analysis (E and F). Note that the increased TIF seen in CsA-treated rat kidneys was significantly decreased by LSRT or PRVT treatment, and a further decrease was seen after the combined treatment. These morphologic changes paralleled TGF-β1 mRNA expression. aP<0.01 vs. VH; bP<0.01 vs. CsA; cP<0.05 vs. CsA+L or CsA+P (Trichrome, magnification ×100).

We used Northern blotting to assess TGF-β1 mRNA expression in the treatment groups. As shown in Figure 4, E and F, CsA treatment induced a 5.9-fold increase in TGF-β1 mRNA expression (636±38% vs. 108±10%, P<0.05), whereas expression decreased significantly when LSRT (233±13%, P<0.01 vs. CsA) or PRVT (242±23%, P<0.01 vs. CsA) was administered. The combined treatment of LSRT plus PRVT decreased TGF-β1 mRNA expression more than the use of either LSRT or PRVT alone (140±33%, P<0.05 vs. CsA+L or CsA+P).

Synergistic Effect of LSRT and PRVT on Ang II Expression in Chronic CsA-Induced Nephropathy

Figure 5 shows the expression of intrarenal Ang II in the VH and CsA groups. Immunoreactivity of Ang II was localized to the afferent arterioles, and there was no immunoreactivity in glomerular or tubular cells. Expression of intrarenal Ang II was minimal in VH-treated rat kidneys but was increased in the kidneys with CsA treatment. Quantitative analysis of Ang II-positive glomeruli in the kidneys (Fig. 5B) revealed a significant increase in the CsA group compared with the VH group (50±6 vs. 23±6, P<0.01). Administration of LSRT (32±4, P<0.05 vs. CsA) or PRVT (38±6, P<0.05 vs. CsA) decreased the number of Ang II-positive glomeruli, and the combination of LSRT plus PRVT decreased this further (23±2, P<0.05 vs. CsA+L or CsA+P).

FIGURE 5.
FIGURE 5.:
Synergistic effect of LSRT and PRVT on intrarenal angiotensin (Ang) II expression in chronic CsA-induced nephropathy. Strong immunoreactivity for Ang II is shown in the CsA-treated rat kidney (A). Quantitative analysis of Ang II-positive glomeruli in the experimental groups (B). Note that the increased number of Ang II-positive glomeruli in the CsA-treated group was significantly decreased after LSRT or PRVT treatment, and a further decrease was observed with the combined treatment. aP<0.01 vs. VH; bP<0.05 vs. CsA; cP<0.05 vs. CsA+L or CsA+P (magnification ×400).

Effects of LSRT and PRVT Treatment on Basic Parameters in Chronic CsA-Induced Nephropathy

Table 1 shows the basic parameters for each group. The whole-blood CsA level was not significantly different from controls in rats treated with CsA (CsA, CsA+L, CsA+P, and CsA+L+P). The mean BW of CsA-treated rats was significantly lower than that for the VH-treated rats (255±5 vs. 295±4, P<0.01). Neither LSRT nor PRVT reduced the loss of BW. There were no significant differences in the levels of SBP and hs-CRP between VH and CsA groups, and neither was influenced by LSRT or PRVT treatment. Four weeks of treatment with CsA impaired renal function, as shown by an increase in Scr (1.21±0.07 vs. 0.59±0.10, P<0.01) and a decrease in Ccr (0.15±0.01 vs. 0.53±0.04, P<0.01) levels. Addition of LSRT or PRVT and combined treatment of the two did not improve renal function. There was no significant difference between groups for any of the lipid parameters.

TABLE 1
TABLE 1:
Whole-blood cyclosporine levels, body weight, systolic blood pressure, renal function, lipid parameters, and serum hs-CRP in the experimental groups

DISCUSSION

We have previously demonstrated that both LSRT (4, 5, 20) and PRVT (13) are renoprotective in a rat model of chronic CsA-induced nephropathy. Here, we tested the combined effects of LSRT and PRVT using the same model; this approach proved better than monotherapy using either drug in attenuating the tubulointerstitial inflammation and fibrosis caused by CsA. Morphologic improvement was accompanied by suppression of pro-inflammatory (intrarenal CRP and OPN) and profibrotic (TGF-β1) mediators. Interestingly, this effect was unrelated to lipid- or blood–pressure-lowering actions. Our observations thus expand the clinical use of a combination of LSRT and PRVT in the prevention of CsA-induced renal injury.

Statins enhance the inhibitory effects of ARBs on neointimal formation and the proliferation of VSMCs induced by cuff placement around the femoral artery (14). In the kidney, the combination of RAS blockers and statins gives synergistic renoprotection in puromycin-induced nephrotic syndrome (15), subtotal nephrectomy (9), and diabetic nephropathy (21). Here, we found that administration of LSRT or PRVT significantly decreased both inflammatory cell infiltration and TIF, and the combined treatment of LSRT plus PRVT further decreased these indicators. At a molecular basis, increased levels of OPN and TGF-β1 in CsA-treated rat kidneys were reduced by treatment with either LSRT or PRVT and further decreased by the combination of the two. These findings are consistent with previous studies by Lee et al. (9) and Brouhard et al. (22), which suggested that PRVT enhances the anti-inflammatory and antifibrotic effects of LSRT in CsA-induced renal injury.

CRP is an inflammatory biomarker implicated in cardiovascular diseases and abnormal renal functions (23, 24), and its production is presumably restricted to the liver and kidney (25). Diaz Padilla et al. (26) reported that rat CRP, like human CRP, can activate the autologous complement system, indicating that important biological functions of CRP are conserved between mammalian species. We have recently demonstrated that intrarenal CRP immunoreactivity is increased in the CsA-treated rat kidney and that this may reflect the degree of interstitial inflammation and injury (13). In this study, increased intrarenal CRP production was decreased by PRVT or LSRT treatments, and it was further decreased when LSRT and PRVT were administered concurrently. By contrast, neither LSRT nor PRVT affected serum hs-CRP levels. We propose that the synergistic effect of PRVT and LSRT on CsA-induced tubulointerstitial injury may be associated in part with this action on intrarenal CRP.

The mechanism by which the combined treatment of LSRT and PRVT attenuates interstitial inflammation and fibrosis in this model may be multifactorial, and reduction in arteriolopathy may be involved. This is one of the characteristic findings of chronic CsA-induced nephropathy (27), which ultimately leads to low-grade hypoxia-induced renal inflammation and fibrosis. Previous studies have reported that both ARBs (28) and statins (29–31) modulate vascular remodeling by inhibiting smooth-muscle cell proliferation, migration, and extracellular matrix synthesis. Furthermore, statins amplify the inhibitory effects of ARBs on vascular neointimal formation and the proliferation of VSMCs (14). Here, we found that the combined administration of LSRT and PRVT diminished arteriolopathy compared with giving each drug alone. On the basis of this study and previous reports, we assume that this decreased arteriolopathy may account for the anti-inflammatory and antifibrotic effects of LSRT and PRVT in this model.

It is also possible that the combined effect of LSRT and PRVT on tubulointerstitial inflammation and fibrosis in chronic CsA-induced nephropathy is associated with reduced intrarenal RAS activity because Ang II has been shown to directly or indirectly stimulate interstitial inflammation and fibrosis, and blockade of the RAS with ACE inhibitors or ARBs confers protection on renal structure in chronic CsA-induced nephropathy (4, 5). In contrast with LSRT, the impact of statins on the RAS remains controversial. However, inhibition of ACE activity by statins has been reported in the setting of cardiac hypertrophy (32). Moreover, statins can interfere with the RAS by decreasing Ang II type 1 receptor mRNA levels in cultured VSMCs exposed to Ang II and in aortic segments of spontaneously hypertensive rats (33). In the current study, we observed that LSRT treatment decreased Ang II immunoreactivity, and combined treatment of LSRT and PRVT decreased this further. Thus, it appears that the combination of LSRT and PRVT synergistically attenuates inflammation and fibrosis through a mechanism involving the inhibited RAS in chronic CsA-induced nephropathy.

It is noteworthy that histologic improvement in the present study was not accompanied by preservation of renal function. This discrepancy may be related to the characteristics of this animal model of chronic CsA-induced nephropathy. Rosen et al. (34) and Elzinga et al. (35) developed a reproducible rat model of chronic CsA-induced nephropathy using a low salt diet. Salt depletion activates the RAS, which is implicated in the changes in renal function (hemodynamics) and histology, mimicking those described in human patients undergoing long-term CsA therapy. However, blockade of Ang II with LSRT only protected against renal structural damage (4, 5). This suggests that other pathways may also be involved in the functional impairment induced by CsA, and this is supported by a study showing that specific anti-endothelin antibody preserves renal function without improvement of renal structure (36). This finding raises the possibility that there is dissociation between renal functional impairment and architectural damage during experimental chronic CsA-induced nephropathy (35).

Choice of drug dosage is important in evaluating its effect. In this study, LSRT was administered at a dose of 10 mg/kg, which is higher than that given to humans (usually 50–100 mg/day in clinical practice). In general, a relatively high dose of LSRT is required to exert its antihypertensive effect in rats (10 or 30 mg/kg) (37–39). The dose of LSRT used in this study appears to be relevant because this dose of LSRT has been shown to prevent CsA-induced over-expression of TGF-β1 and OPN as well as renal fibrosis (4, 5, 20). With regard to PRVT, we and others have demonstrated that PRVT at a dose of 5 mg/kg does not alter blood lipid levels but is effective in decreasing CsA-induced renal injury (13).

In clinical practice, hypertension and hyperlipidemia are common complications in transplant recipients on CsA-based immunosuppressants therapies (40, 41). RAS blockers and statins have been usually used to treat these complications. However, these drugs possess hypotension- and hypolipidemia-independent properties. Administration of either LSRT or PRVT inhibits TGF-β1 and OPN expression and thus prevents fibrosis (4, 42, 43). Moreover, the functions of statins also extend to immunomodulation, as shown by their inhibition of the expression of class II major histocompatibility antigens and of natural killer cell activity (44). Indeed, experimental and clinical studies in cardiac and renal-transplant recipients demonstrate that administration of statins successfully decreases acute or chronic rejection episodes and improves graft survival (45–47). Thus, combined treatment of LSRT plus PRVT may provide additional renoprotection not only for chronic CsA-induced nephropathy but also for rejection episodes in transplant recipients receiving CsA.

In summary, this study demonstrates that the combined treatment of LSRT and PRVT elicits anti-inflammatory and antifibrotic effects in chronic CsA-induced nephropathy beyond their blood- and lipid-lowering actions. Our findings provide potential rationale for the clinical use of LSRT and PRVT in reducing chronic human CsA-induced nephropathy.

REFERENCES

1. Myers BD, Sibley R, Newton L, et al. The long-term course of cyclosporine-associated chronic nephropathy. Kidney Int 1988; 33: 590.
2. de Mattos AM, Olyaei AJ, Bennett WM. Nephrotoxicity of immunosuppressive drugs: long-term consequences and challenges for the future. Am J Kidney Dis 2000; 35: 333.
3. Olyaei AJ, de Mattos AM, Bennett WM. Nephrotoxicity of immunosuppressive drugs: new insight and preventive strategies. Curr Opin Crit Care 2001; 7: 384.
4. Yang CW, Ahn HJ, Kim WY, et al. Synergistic effects of mycophenolate mofetil and losartan in a model of chronic cyclosporine nephropathy. Transplantation 2003; 75: 309.
5. Burdmann EA, Andoh TF, Nast CC, et al. Prevention of experimental cyclosporin-induced interstitial fibrosis by losartan and enalapril. Am J Physiol 1995; 269: F491.
6. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990; 343: 425.
7. Ni W, Egashira K, Kataoka C, et al. Antiinflammatory and antiarteriosclerotic actions of HMG-CoA reductase inhibitors in a rat model of chronic inhibition of nitric oxide synthesis. Circ Res 2001; 89: 415.
8. Joyce M, Kelly C, Winter D, et al. Pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, attenuates renal injury in an experimental model of ischemia-reperfusion. J Surg Res 2001; 101: 79.
9. Lee SK, Jin SY, Han DC, et al. Effects of delayed treatment with enalapril and/or lovastatin on the progression of glomerulosclerosis in 5/6 nephrectomized rats. Nephrol Dial Transplant 1993; 8: 1338.
10. Kim SI, Han DC, Lee HB. Lovastatin inhibits transforming growth factor-beta1 expression in diabetic rat glomeruli and cultured rat mesangial cells. J Am Soc Nephrol 2000; 11: 80.
11. Harris KP, Purkerson ML, Yates J, et al. Lovastatin ameliorates the development of glomerulosclerosis and uremia in experimental nephrotic syndrome. Am J Kidney Dis 1990; 15: 16.
12. Moriyama T, Kawada N, Nagatoya K, et al. Fluvastatin suppresses oxidative stress and fibrosis in the interstitium of mouse kidneys with unilateral ureteral obstruction. Kidney Int 2001; 59: 2095.
13. Li C, Yang CW, Park JH, et al. Pravastatin treatment attenuates interstitial inflammation and fibrosis in a rat model of chronic cyclosporine-induced nephropathy. Am J Physiol Renal Physiol 2004; 286: F46.
14. Horiuchi M, Cui TX, Li Z, et al. Fluvastatin enhances the inhibitory effects of a selective angiotensin II type 1 receptor blocker, valsartan, on vascular neointimal formation. Circulation 2003; 107: 106.
15. Zoja C, Corna D, Camozzi D, et al. How to fully protect the kidney in a severe model of progressive nephropathy: a multidrug approach. J Am Soc Nephrol 2002; 13: 3024.
16. Li C, Yang CW, Ahn HJ, et al. Colchicine suppresses osteopontin expression and inflammatory cell infiltration in chronic cyclosporine nephrotoxicity. Nephron 2002; 92: 422.
17. Coelho-Filho OR, De Luca IM, Tanus-Santos JE, et al. Pravastatin reduces myocardial lesions induced by acute inhibition of nitric oxide biosynthesis in normocholesterolemic rats. Int J Cardiol 2001; 79: 215.
18. Li C, Yang CW, Kim WY, et al. Reversibility of chronic cyclosporine nephropathy in rats after withdrawal of cyclosporine. Am J Physiol Renal Physiol 2003; 284: F389.
19. Yu XQ, Wu LL, Huang XR, et al. Osteopontin expression in progressive renal injury in remnant kidney: role of angiotensin II. Kidney Int 2000; 58: 1469.
20. Yang CW, Ahn HJ, Kim WY, et al. Influence of the renin-angiotensin system on epidermal growth factor expression in normal and cyclosporine-treated rat kidney. Kidney Int 2001; 60: 847.
21. Qin J, Zhang Z, Liu J, et al. Effects of the combination of an angiotensin II antagonist with an HMG-CoA reductase inhibitor in experimental diabetes. Kidney Int 2003; 64: 565.
22. Brouhard BH, Takamori H, Satoh S, et al. The combination of lovastatin and enalapril in a model of progressive renal disease. Pediatr Nephrol 1994; 8: 436.
23. Blake GJ, Ridker PM. Inflammatory bio-markers and cardiovascular risk prediction. J Intern Med 2002; 252: 283.
24. Stuveling EM, Hillege HL, Bakker SJ, et al. C-reactive protein is associated with renal function abnormalities in a non-diabetic population. Kidney Int 2003; 63: 654.
25. Jabs WJ, Logering BA, Gerke P, et al. The kidney as a second site of human C-reactive protein formation in vivo. Eur J Immunol 2003; 33: 152.
26. Diaz Padilla N, Bleeker WK, Lubbers Y, et al. Rat C-reactive protein activates the autologous complement system. Immunology 2003; 109: 564.
27. Franceschini N, Alpers CE, Bennett WM, et al. Cyclosporine arteriolopathy: effects of drug withdrawal. Am J Kidney Dis 1998; 32: 247.
28. Wu L, Iwai M, Nakagami H, et al. Roles of angiotensin II type 2 receptor stimulation associated with selective angiotensin II type 1 receptor blockade with valsartan in the improvement of inflammation-induced vascular injury. Circulation 2001; 104: 2716.
29. Guijarro C, Blanco-Colio LM, Ortego M, et al. 3-Hydroxy-3-methylglutaryl coenzyme a reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle cells in culture. Circ Res 1998; 83: 490.
30. Komukai M, Wajima YS, Tashiro J, et al. Carvastatin suppresses intimal thickening of rabbit carotid artery after balloon catheter injury probably through the inhibition of vascular smooth muscle cell proliferation and migration. Scand J Clin Lab Invest 1999; 59: 159.
31. Riessen R, Axel DI, Fenchel M, et al. Effect of HMG-CoA reductase inhibitors on extracellular matrix expression in human vascular smooth muscle cells. Basic Res Cardiol 1999; 94: 322.
32. Luo JD, Zhang WW, Zhang GP, et al. Simvastatin inhibits cardiac hypertrophy and angiotensin-converting enzyme activity in rats with aortic stenosis. Clin Exp Pharmacol Physiol 1999; 26: 903.
33. Wassmann S, Laufs U, Baumer AT, et al. Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells: involvement of angiotensin AT1 receptor expression and Rac1 GTPase. Mol Pharmacol 2001; 59: 646.
34. Rosen S, Greenfeld Z, Brezis M. Chronic cyclosporine-induced nephropathy in the rat. A medullary ray and inner stripe injury. Transplantation 1990; 49: 445.
35. Elzinga LW, Rosen S, Bennett WM. Dissociation of glomerular filtration rate from tubulointerstitial fibrosis in experimental chronic cyclosporine nephropathy: role of sodium intake. J Am Soc Nephrol 1993; 4: 214.
36. Perico N, Dadan J, Remuzzi G. Endothelin mediates the renal vasoconstriction induced by cyclosporine in the rat. J Am Soc Nephrol 1990; 1: 76.
37. Vaziri ND, Wang XQ, Ni ZN, et al. Effects of aging and AT-1 receptor blockade on NO synthase expression and renal function in SHR. Biochim Biophys Acta 2002; 1592: 153.
38. Zhou XJ, Vaziri ND, Zhang J, et al. Association of renal injury with nitric oxide deficiency in aged SHR: prevention by hypertension control with AT1 blockade. Kidney Int 2002; 62: 914.
39. Diep QN, El Mabrouk M, Yue P, et al. Effect of AT(1) receptor blockade on cardiac apoptosis in angiotensin II-induced hypertension. Am J Physiol Heart Circ Physiol 2002; 282: H1635.
40. Weidle PJ, Vlasses PH. Systemic hypertension associated with cyclosporine: a review. Drug Intell Clin Pharm 1988; 22: 443.
41. Mathis AS, Dave N, Knipp GT, et al. Drug-related dyslipidemia after renal transplantation. Am J Health Syst Pharm 2004; 61: 565.
42. Shihab FS, Bennett WM, Tanner AM, et al. Angiotensin II blockade decreases TGF-beta1 and matrix proteins in cyclosporine nephropathy. Kidney Int 1997; 52: 660.
43. Pichler RH, Franceschini N, Young BA, et al. Pathogenesis of cyclosporine nephropathy: roles of angiotensin II and osteopontin. J Am Soc Nephrol 1995; 6: 1186.
44. Weitz-Schmidt G, Welzenbach K, Brinkmann V, et al. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 2001; 7: 687.
45. Ji P, Si MS, Podnos Y, Chow H, et al. Prevention of chronic rejection by pravastatin in a rat kidney transplant model. Transplantation 2002; 74: 821.
46. Katznelson S, Wilkinson AH, Kobashigawa JA, et al. The effect of pravastatin on acute rejection after kidney transplantation: a pilot study. Transplantation 1996; 61: 1469.
47. Kobashigawa JA, Katznelson S, Laks H, et al. Effect of pravastatin on outcomes after cardiac transplantation. N Engl J Med 1995; 333: 621.
Keywords:

Cyclosporine; Angiotensin II; Statin; Nephropathy

© 2005 Lippincott Williams & Wilkins, Inc.