Over the past several decades, cyclosporine (CsA) has been used to prevent rejection of transplanted tissue. It is particularly effective in improving 1-year renal allograft survival. The 1-year renal allograft survival rate increased 10%-20% after the cyclosporine era.1,2 But these improvements have little impact on the long-term renal allograft survival rates. Chronic administration of CsA produces renal injury, which is termed chronic CsA nephropathy (CAN), and is thought to be the major side-effect. It is one of the known nonimmunological factors causing 30%-50% of chronic allograft nephropathy in the total renal transplant population.3 Since CSA is the prime agent used in immunosuppressive therapy, lack of efficacy for the prevention of long-term allograft failure may in part be due to the side effects of CsA.4
Although the pivotal role of renin angiotensin system (RAS) has been demonstrated in the rat model with chronic CsA nephropathy, it is still unclear whether RAS activation is responsible for chronic CsA nephropathy in humans. CsA, a highly insoluble cyclic polypeptide consisting of 11 amino acids, inhibits calcineurin, a Ca2+-dependent serine-threonine phosphatases, thus reducing the production of interleukin-2. Recent study has shown that beyond the inhibition of calcineurin phosphatase activation, CsA may exert its effect by different mechanisms.5 The most important histological lesion in CsA-related chronic nephrotoxicity is represented by the pathological structural changes of the arterioles with tubulointerstitial lesion. Infusion of angiotensin II (ANGII) provoked histolgical changes in rat kidneys similar to those observed in CsA-related chronic nephrotoxicity.6 Therapeutic inhibition of the RAS with the use of angiotensin converting enzyme (ACE) inhibitors and AT-1 receptor antagonists ameliorates these structural changes, as well as improves the rate of decline of chronic renal allograft dysfunction with CAN patients.7 It address the complex interactions between CsA and the RAS and the possible role of the renin axis in the pathogenesis of CsA-nephrotoxicity.
With this background, this study was designed to investigate the levels of renin-ANGII in both renal tissue and plasma from kidney transplantation patients with established CAN, and combine with the in vitro experiments to evaluate the effects of CsA on RAS in CAN patients.
We enrolled 110 renal transplant recipients who underwent cadeveric allograft transplantation from Januanry 2001 to June 2007. The enrollment criteria included age 18 to 60 years, the serum creatinine 110–368 μmol/L (the normal range is less than 120 μmol/L), or creatinine clearance (Ccr) calculated with the Cock-Gault formula 30–90 ml/min (the normal range is more than 90 μmol/L). All patients were more than 1 year after transplant and received maintenance triple-drug immunosuppression consisting of Neoral, mycophenolate mofetil and prednisone. No patients received ACE inhibitors or angiotensin type 1 receptor blockers. All patients gave written informed consent. The histological diagnosis was made according to the Banff criteria.8 Only tissue sections with more than 10 glomeruli and two arteries were considered useful.
Twenty-six renal transplant recipients with allograft biopsy were included in this study. Thirteen patients (9 men and 4 women, aged (45±13) years) experienced CsA-related chronic nephrotoxicity (CAN group). The other thirteen patients with histological diagnosis of chronic rejection were examined as controls (control group) (Table 1). Blood samples were obtained from each patient at the time when they received the allograft biopsy. Cyclosporin levels obtained 2 hours after the morning dose (C2) were determined by fluorescence polarization immunoassay technology (Abbott AXSYM™, USA). Renin and ANGII plasma levels were measured by radioimmunoassay (Radioimmunology Institute, Beijing, China). Information about the blood pressure, serum creatinine levels and the kinds of antihypertensive medication were available.
Histopathology and immunohistochemical examination
Tissue blocks from allografts were formalin-fixed and paraffin-embedded. Three μm thick serial sections were cut and stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) for light microscopic examination. Immunohistochemical staining was performed by the standard EnVision procedure, using five-μm thick sections. After microwave pretreatment for antigen retrieval, sections were incubated with 5% goat serum to block nonspecific background staining. Primary antibodies against renin (1:00; Santa Cruz, USA) and ANGII (1:00; Santa Cruz, USA) were applied for two hours at room temperature. The EnVisionTM+system (DAKO, USA) was used, diaminobenzidine (DAB) being the enzyme and chromogen employed. Positive control sections were six cases of normal renal tissue. Negative control sections were incubated with normal serum instead of the primary antibody. The presence of positive immunohistochemical staining (IHCS) was assessed by Image Analysis System (ZEISS Axioplan2 imagine, Axiovision3.1, Germany). Positive staining area (%) in tissue was also evaluated.9
Cell culture and treatment
The HUVEC cell lines were obtained from the American Type Culture Collection (ATCC No CRL-1998). Rat mesangial cell lines (MC) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in RPMI 1640 (Gibco, USA), supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin, and maintained at 37°C and 5% CO2 atmosphere. For subsequent experiments, subcultured cells from passages 3–12 at 80%-90% confluency were used after serum depletion for 3 days. CsA (50 mg/ml, Novatis, Switzerland) was diluted in olive oil to a stock solution of 10 mg/ml, stored at -70°C and diluted in RPMI 1640 at concentrations of 250 μg/L, 500 μg/L, and 1000 μg/L.
Following serum starvation for 24 hours to induce quiescence HUVECs and MCs were grown on slides and stimulated with various concentrations of CsA for 24 hours. Medium was removed, and the cell layer was rinsed with Tris-buffered saline (TBS). Then cells were fixed with freshly prepared 4% paraformaldehyde for 20 minutes at room temperature, rehydrated with TBS, blocked with normal goat serum for 30 minutes and incubated with a primary antibody against renin or ANGII overnight at 4°C. After washing with TBS, cells were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies, and bound antibodies were detected by DAB. Slides without treatment with primary antibodies served as negative controls. Positive cells were counted in ten fields of vision per slide and one marker was repeat-detected four times. Positive cells were calculated using Image Analysis System.
We used a radioimmunnoassay method to detect the contents of ANGII in the culturing solution of HUVECs and MCs after treating with different concentration of CsA for 24 hours. The secretion media from 10 cm2-plates were centrifuged at 1500×g for 10 minutes. The supernatant was subsequently collected and stored at -80°C for culture medium detection. The specified treatment cells in the plates were harvested by knife and lysed in an ice-cold buffer containing 50 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% NP40, 0.5% nadeoxycholate, 0.1% SDS, 1 mmol/L PMSF, 10 mg/L aprotinin, 1 mmol/L NaVO4. Insoluble material was removed by centrifugation at 12 000×g for 15 minutes at 4°C and stored at -80°C for cytoplasm detection. ANGII immunoreactivity was measured in duplicate (n=5 per group), according to the supplier (radioimmunoassay kits, Radioimmunology Institute, Beijing, China).
Data were expressed as mean ± standard deviation (SD). Comparisons between two groups were performed by Student's t test with SPSS 11.0. P value less than 0.05 was considered statistically significant.
Pathological characteristics of the patients
All of the CAN biopsies showed arteriolopathy, striped interstitial fibrosis and tubular atrophy. Six of thirteen (46%) biopsies showed no, or negligible, glomerular lesions. Segmetal glomerularsclerosis was observed in the other seven (54%) cases. On our semiquantitative scoring system, neither histological chronic index nor interstitial fibrosis showed significant difference between the two groups. The vascular hyalinosis was significantly higher in the CAN group than in the control group (2.7±0.5 vs 1.0±0.2, P <0.05). Whereas, a significant increase in transplant glomerulopathy was observed in the control group compared with the CAN group (7.1±1.6 vs 3.6±0.8, P <0.05).
Effect of CsA on renin and ANGII expression in the allograft
In the six normal renal tissues, renin-positive cells were found in renal tubules and the juxtaglomerular apparatus (JGA, Figure 1A). The positive staining of ANGII was minimally localized in tubular epithelial cells (Figure 2A). In CAN and control group specimens, renin-positive cells were detected in all the ducts and parts of the glomeruli (Figures 1B and 1C). ANGII-positive cells were markedly observed not only in tubules but also expressed in the glomeruli (Figure 2B). In the CAN group, ANGII was also confined to the vessels (Figure 2C). Moreover, the IHCS scores and positive staining area for renin and ANGII were significantly higher in the CAN group than the control group (Table 2).
Plasma levels of C2, renin and ANGII
Neither the ANGII nor the renin plasma levels were significantly different between the two groups. Table 3 illustrates the relationship of CSA blood concentration with plasma levels of renin and ANGII. As demonstrated in this table, although the plasma levels of C2 is higher in the CAN group than control group, there were no significant changes between the two groups. The analysis revealed that the histological lesions in the CAN group failed to correlate with either the C2 level or the plasma levels of renin and ANGII. Plasma concentrations of renin and ANGII in patients with CAN were inconsistent with renal IHCS scores of renin and ANGII expression.
Effect of CsA on renin and ANGII expression in HUVEC and MC
Low level of both renin and ANGII were observed in HUVECs and MCs without stimulation. After treatment with CsA of 1000 μg/L concertration for 24 hours, the expression of renin and ANGII were strongly positive in the two cell lines (Figures 3 and 4). Quantitative analysis showed a significant increase in the number of renin and ANGII positive cells in CsA group of HUVEC compared with the control (231.50±12.10 vs 182.34±11.51, P <0.05; 237.25±21.88 vs 86.12±9.81, P <0.05; respectively). In CSA group of MC, both renin and ANGII were also markedly higher than in the control group (176.35±14.51 vs 153.±11.11, P <0.05; 396.50±26.15 vs 123.67±24.19, P <0.05; respectively).
Effect of CsA on renin and ANGII secretion in HUVEC and MC
Table 4 shows ANGII secretion from HUVEC and MC using radioimmunity examination. CsA significantly stimulated ANGII release from cultured HUVEC and MC in a dose-dependent manner.
It is well known that RAS plays a major role in the development of chronic kidney disease. Some interesting data on the interaction of CsA and RAS has been presented by us and others during the recent years.10–12 In rats, activation of RAS by CsA is a consistent finding. Increased plasma renin-ANGII activity by CsA has been related to increased renin-ANGII expression in the renal tissue. Data on the effects of CsA on RAS in man are, however, to some extent contradictory.13 In our study an increased intra-graft expression of renin and ANGII in kidney transplant patients with histological diagnosis of CsA-related chronic nephrotoxicity was observed. The immunohistologic renin-ANGII staining was significantly higher in specimens with CAN than in those without CAN. The strongly intra-renal renin and ANGII deposits in CAN patients indicated that intra-renal RAS was activated. Contrary to this, the plasma renin-ANGII level in CAN patients was not significantly increased. It may be concluded that tissue RAS (tRAS) rather than circulating RAS (cRAS) has an important role in the development of CsA-induced adverse effects on kidney in humans.
Our findings indicate that chronic CsA treatment did not activate systemic RAS in humans. The RAS comprises two components: the classical cRAS and the recently recongnized tRAS. cRAS has a role in maintaining blood volume and pressure homeostasis. The kidney, although a major participant in the cRAS, has in addition to its own complete system of tRAS.14,15 It plays an important role, not only in the regulation of hemodynamics, but also in the pathogenesis of arteriolopathy and tubulointerstitial fibrosis. Our results support the existence of a local tRAS whose activity is not reflected by circulating levels. The pathogenic role of enhanced renin and ANGII expression intra-renal can be explained in the following ways. The observed changes might reflect a “switch” from an initial activition of the cRAS to a more sustained activation of the tRAS. Because the circulating active renin is derived exclusively from the cRAS component of the kidney, a fall in ANGII would be anticipated when the acute activation of the cRAS is replaced by the chronic activation of the tRAS.13 CsA stimulates both the cRAS and the tRAS. CsA-related acute nephrotoxicity exerts its effects by stimulating the cRAS, while CAN was in the chronic tRAS-stimulatory phase. This leads to the status of activation of intra-renal RAS without changes in systemic RAS. The other explanation of the differences in the effect of CsA on RAS observed between rats and humans may be as following. CsA stimulates renin synthesis and inhibits renin activation and excretion. In man the inhibitory effect on renin activation and excretion may override the stimulatory effect on renin synthesis.
The mechanism of RAS activation by CsA is somewhat obscure and probably multifactorial.16 It is concluded that CAN is related with vascular endothelial dysfunction.17 ANGII is the major active species of RAS. It was initially identified by its physiologic ability to regulate salt and water metabolism, blood pressure and vascular tone. More recently it was also demonstrated to function as a growth factor. Evidence has demonstrated that ANGII is one of the biologic determinants involved in the processes of neointimal proliferation.18 This effect was most likely due to ANGII-mediated proliferation and differentiation of endothelial cells. CsA stimulates renin and ANGII secretion and expression directly. CsA-induced endothelial dysfunction due to generation of reactive oxygen species could be associated with RAS activation. ANGII is the major activator of NADPH oxidase. ANGII infusion as well as genetic over-expreesion of RAS has been shown to result in generation of reactive oxidative species. Thus, CsA is able to cause oxidative stress by activation of RAS. Besides these, increased release of endothelin-1, transforming growth factor-betal and enhanced immunogenicity or inappropriate apoptosis may all be implicated in the pathogenesis of chronic CSA nephrotoxicity.19,20
CsA also resulted in histological changes in the study, which seems to be independent of serum CsA concentration C2. Long-term effects of reduction or withdrawal of CsA (n=118) improved renal function significantly in humans. But the tubulointerstitial fibrosis as well as arteriolosclerosis in the rat model of CAN is dissociated from GFR improvement.21 The tissue damage of CsA-related chronic nephrotoxicity is a non-reversible process, indicating the importance of the allograft biopsy and requiring prevention in the earliest stages of transplantation.
Taken together, we have summarized evidence supporting tRAS as one important participant in the pathogenesis of the complications of CsA administration. CsA may cause a vicious circle: RAS activation by CsA direct effect on renin production reduces renal blood flow and induces local ischemia in kidneys which further increases renin release and potentiates CsA toxicity. Thus, RAS activition may be both a cause and a consequence of CsA-induced renal damage. ANGII inhibitors and ANGII receptor antagonists provide a feasible clinical option for the prevention of CsA-related chronic nephrotoxicity.
In summary, Long-term CsA administration increased local expression of renin and ANGII. And histological lesions are related to tRAS activation rather than cRAS or C2. Both renin and ANGII secretion and expression were directly increased by CsA stimulation. Although our data did not help explain the relationship between tRAS activation and graft dysfunction, the study identified the important role of RAS in the development of CsA-related chronic nephrotoxicity in humans with kidney transplantation.
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