Role of TGF-β1/ILK/FSP1 signaling pathway in cyclosporin A-induced epithelialmesenchymal transition in cultured renal tubular epithelial cells / 南方医科大学学报

OBJECTIVE@#To explore the role of transforming growth factor-β1/integrin-linked kinase/fibroblast-specific protein 1 (TGF- β1/ILK/FSP1) signaling pathway in cyclosporine A (CsA)-induced renal tubular epithelial cell transdifferentiation.@*METHODS@#Rat renal tubular epithelial NRK-52E cells were induced with 1 mg/L CsA, treated with TGF-β1 inhibitor (SB431542, 10 μmol/L), or transfected with the ILK-RNAi lentiviral expression vector (ILKshRNA) or a negative control vector before CsA induction. The expressions of TGF-β1, ILK and FSP-1 mRNAs and proteins in the cells were detected using real-time PCR and Western blotting. The positive cells for α-SMA expression were detected by immunohistochemistry.@*RESULTS@#Compared with the blank control cells, the cells treated with CsA showed significantly increased levels of TGF-β1, ILK and FSP-1 mRNAs and proteins ( < 0.05). The expressions of TGF-β1, ILK and FSP-1 were significantly lower in TGF-β1 inhibitor group than in CsA group ( < 0.05). The levels of ILK and FSP-1 were significantly decreased after shRNA-mediated ILK silencing ( < 0.05). The number of positive cells for -SMA was significantly lower in cells treated with SB431542 and in cells with ILK silencing than in the cells treated with CsA alone ( < 0.05).@*CONCLUSIONS@#The activation of TGF-β1/ILK/FSP-1 signaling pathway is an important mechanism for CsA-induced transdifferentiation in rat renal tubular epithelial cells. ILK participates in CsA-induced epithelialmesenchymal transition of renal tubular epithelial cells.

Effect of inhibiting TGF-β1 on Smad2/3 and ILK signaling molecules in chronic cyclosporine A nephropathy in mouse / 中国免疫学杂志

Objective:To investigate the effect of specific inhibition of transforming growth factor-β1 (TGF-β1) with SB-431542 on the Smad2/3 and integrin-linked kinase (ILK) signaling molecules in tubule interstitial fibrosis(TIF)-induced cyclosporine A(CsA) in mouse.Methods:50 BALB/c mice were randomly divided into 5 groups (10 mice per group):the CsA model group (CMG),the interventional model group (IMG),the solvent control group (SCG),the low-salt control group (LCG),and the normal control group (NCG).The model mouse was established with low-sodium diet and intragastric administration of cyclosporine A,which was dissolved in olive oil at a dose of 60 mg/(kg·d).After 4 weeks,a specific inhibitor of TGF-β1 (SB-431542)was administered intraperitoneally with 10 mg/(kg·2 d) for 10 days (every other days).Mice were sacrificed at day 38.Serum creatinine (Scr) was measured,hydroxyp roline (Hyp)level and morphological changes of renal tissue were analyzed,expression levels of TGF-β1,P-Smad 2/3 and ILK were respectively detected by immunohistochemistry or Western blot,mRNA levels of TGF-β1,Smad 2/3 and ILK were respectively detected real-time polymerase chain reaction (RT-PCR).Results:Compared with three control groups (NCG,LCG and SCG),mice weight was decreased significantly,Scr level was increased significantly in two modeling groups (CMG and IMG) (P<0.01),and these changes in CMG were more obvious than those of IMG (P<0.05).Different levels of tubulointerstitial injury,interstitial infiltration of inflammatory cells and blue collagen staining in two modeling groups were observed,and particularly evident in CMG.TGF-β1,P-Smad2/3 and ILK immunostaining were mainly expressed in tubulointerstitium.The TGF-β1,P-Smad2/3 and ILK mRNA and immunostaining levels in two modeling groups were significantly increased as compared with three control groups (P<0.01),but their levels in IMG were significantly lower than those of CMG (P<0.05).The level of Hyp in renal tissue was positively correlated with Scr,TGF-β1,Smad2/3 and ILK (r=0.860,0.711,0.776,0.676,P<0.01).Conclusion:The activation of the TGF-β1/Smads signaling pathway plays an important role in the development of chronic CsA-induced TIF.The activation of ILK is closely correlated with the development of TIF,and may be used as a downstream factor of TGF-β1/Smads signaling pathway in regulating CsA-induced TIF.

Induction of Acute Cyclosporine Nephrotoxicity in Pig / 대한이식학회지

PURPOSE: Pigs are promising donor species for xenotransplantation. This study was performed to examine acute cyclosporine (CsA) nephrotoxicity in a pig model. METHODS: Adult pigs were treated daily for 1 week with vehicle (VH), or 7.5 mg/kg (CsA7.5), 15 mg/kg (CsA15), and 30 mg/kg (CsA30) CsA. The renal function, electrolyte levels, whole- blood CsA levels, and histopathological results (vacuolization and tubulointerstitial fibrosis) were compared among the different treatment groups. RESULTS: After 1 week of treatment, it was found that CsA induced characteristic lesions that were remarkably similar to those of chronic CsA nephropathy in humans. Compared with the results obtained for the VH group, CsA reduced renal function and yielded poor histopathological results. With an increase in the CsA concentration, the renal function and histological parameters worsened in a dose-dependent manner. CONCLUSION: The study showed that CsA dose-dependently induced renal injury in a pig model. These results provide basic data to estimate or prevent the adverse renal effects of immunosuppressants during porcine xenotransplantation in the future.

The anti-inflammatory effects of angiotensin II blockade in animal model of cyclosporine-induced nephropathy / 대한내과학회지

BACKGROUND: Long-term treatment of immunosuppresant CsA causes interstitial inflammation and fibrosis in the kidney. Renin-angiotensin system (RAS) plays the most important role in the pathogenesis CsA-induced renal injury. Accordingly we evaluated the anti-inflammatory effect of angiotensin II blockades using losartan (LSRT) in a rat model of chronic CsA nephropathy. METHODS: Male Sprague-Dawley rats, initially weighing 225 to 250 g, were used. After 1 week of a low-salt diet (0.05% sodium), the rats were randomized into four groups and treated for 4 weeks. The Vehicle (VH) group was treated with olive oil. The VH+LSRT group was treated with olive oil and LSRT. The CsA group received CsA. The CsA+LSRT group was simultaneously treated with CsA and LSRT. The anti-inflammatory effect of LSRT was evaluated with C-reactive protein (CRP) expression, osteopontin (OPN) mRNA and protein expression, and ED-1 infiltration RESULTS: The CsA treatment caused an increase in serum creatinine and a decrease in creatinine clearance compared with that of the VH group. Intrarenal CRP positive cells were significantly decreased in the CsA+LSRT group compared with the CsA group (38.0 +/- 2.1 vs. 65.0 +/- 5.1, p<0.01). In the CsA group, the degree of OPN mRNA expression was increased compared with that of the VH group. But, OPN mRNA expression was decreased in the CsA+LSRT group (387.5 +/- 56.6% vs. 719.8 +/- 58.5%, p<0.05). In the degree of ED-1 infiltration, we had a similar results such as CRP and OPN mRNA expression (CsA group 30.5 +/- 8.0 vs. CsA+LSRT 86.0 +/- 11.0, p<0.01). CONCLUSION: We concluded that the anti-inflammatory effects of angiotensin II blockade has a potential protective effect against CsA-induced renal injury.

Expression of Transforming Growth Factor-beta1 in Cyclosporine-Induced Nephropathy in Rats

Cyclosporine nephropathy was induced by intraperitoneal injection of cyclosporine 25 mg/kg in Sprague-Dawley rats daily for 1, 4, 8, and 12 weeks to clarify the relationship between cyclosporine nephropathy and the expression of TGF-beta1 with extracellular matrix deposition. On light microscopic examination, the kidneys in the 12 week cyclosporine-treated rats showed focal or striped fibrosis, vacuolization of tubular cells, and injury of endothelial cells. Immunohistochemically, TGF-beta1 protein was strongly expressed in the cyclosporine-treated rat kidneys, especially in the glomerular endothelial cells, interstitial endothelial cells, tubular epithelial cells, and parietal cells in the Bowman's capsule of the glomerulus as well as the periglomerular arterioles. The amount of TGF-beta1 expression was correlated with the morphological change in the cyclosporine-treated rats. Extracellular matrix, such as fibronectin and collagen IV, was also expressed in the endothelial cells of the glomerulus and the interstitium. It can be concluded, therefore that TGF-beta1 protein is probably involved in the early stage of fibrogenesis in cyclosporine nephropathy. It can be postulated that cyclosporine nephropathy results from the accumulation of extracellular matrix associated with the increase of TGF-beta1 transcription. Therefore, these results could be used in reducing fibrosis in cyclosporine nephropathy.

The Effect of Angiotensin Converting Enzyme Inhibitor on Chronic Cyclosporine Nephropathyin Salt Depleted Rats

PURPOSE: Cyclosporine(CsA) is a potent immunosuppressant but the use of CsA is associated with various side effects, especially nephrotoxicity. In the kidney, salt depletion activates the renin-angiotensin-aldosteron(RAS) system and accentuates chronic CsA nephropathy. We postulate that angiotensin converting enzyme inhibitors(ACEI) can prevent chronic CsA nephropathy, since ACEI may inhibit this cascades. This study was aimed to assess the effect of ACEI on chronic cyclosporin nephropathy in salt depleted rats. METHODS: 36 Fischer-344 rats were divided into 6 groups. Group I received normal salt diet(NSD). Group II received a low salt diet(LSD). Group III received CsA with a NSD. Group IV received CsA with a LSD. Group V received NSD+CsA with ACEI. Group VI received LSD+CsA with ACEI. Rats were sacrificed after six weeks, and the glomerular filtration rate(GFR), serum sodium, potassium and whole blood cyclosporine levels were measured. Renal tissues were sampled for the observation of histological changes. RESULTS: No differences in blood CsA level and serum sodium were found between groups during the course of this experiment. Serum potassium in group VI was significantly increased compared with group IV and V(P<0.05). In groups treated with CsA only and in those where CsA was combined with ACEI, GFR was found to be significantly more decreased in LSD than NSD, and GFR in group V was significantly decreased in comparison with group III (P<0.05). Renal histologic lesions associated with CsA, which consisted of cortical interstitial fibrosis, tubular atrophy and hyalinization of arterioles, were more severe in the LSD group. But, no differences were observed between the groups treated with CsA and ACEI, and the groups treated with only CsA. CONCLUSION: Salt depletion associated with the activation of the RAS system accentuated chronic CsA nephrotoxicity, but, ACEI could not reduce the functional and morphological changes of salt depleted kidneys, in which nephropathy can be exacerbated in spite of the blocking of the angiotensin II pathway. Further studies are required to elucidate whether ACEI ameliorated the effect of salt-depleted CsA nephrotoxicity upon the effective renal blood flow.