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