Effect of calcineurin inhibitors on extracellular matrix turnover in isolated human glomeruli.
ABSTRACT Although chronic cyclosporine toxicity is mainly characterized by tubular atrophy and interstitial fibrosis, glomerular injury with expansion of mesangial matrix and sclerosis is not uncommon. Tacrolimus is a newer calcineurin inhibitor that has been used in renal transplant recipients as primary or rescue therapy. Clinical trials suggest an improved long-term graft survival among patients treated with tacrolimus. Recently we have shown that tacrolimus and cyclosporine have similar effects on extracellular matrix turnover in cultured cells. The present study was performed to investigate the effects of the calcineurin inhibitors on whole glomeruli extracellular matrix turnover.
Human glomeruli isolated from kidney biopsies just before transplantation were incubated with culture media containing either cyclosporine (200 ng/mL) or tacrolimus (10 ng/mL) for 24 hours. Glomeruli incubated only with culture medium were used as control.
The expressions of (alpha2)IV collagen, metalloprotease 9 (MMP9), tissue inhibitors of metalloproteases 2 (TIMP-2), and TGFbeta were evaluated by in situ reverse transcription and polymerase chain reactions (RT-PCR). beta-actin was used as a control gene. Cyclosporine (but not tacrolimus) increased the expression of (alpha2)IV collagen and TIMP2 in isolated glomeruli. TGF-beta was markedly increased by cyclosporine. MMP9 expression was not affected by the calcineurin inhibitors. By light microscopy kidney biopsies did not show pathologic changes.
Cyclosporine treatment modulates extracellular matrix turnover in isolated human glomeruli, inducing an imbalance between synthesis and degradation. This effect, not observed in tacrolimus-treated human glomeruli, may induce the extracellular matrix deposition and sclerosis characteristic of chronic cyclosporine toxicity.
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ABSTRACT: Exploring the expression of Toll-like receptor (TLR) in cyclosporine (CsA)-induced renal injury in humans, we evaluated the expression of TLR4 in both biopsied renal tissue and cultured tubular cells. Immunohistochemical stains for TLR4, heat shock protein (HSP) 47, and HSP70 were performed in both pre- and post-treatment biopsies obtained from 18 patients of minimal change nephrotic syndrome or IgA nephropathy treated with CsA, and the percentage of positive tubules was compared in each case. For in vitro experiments, HK-2 cells were treated with CsA (2, 5, and 10 microg/ml) for 24, 48, and 72 h. TLR4 mRNA and protein were measured using real-time RT-PCR and Western blot. In addition, hypoxic effect was added by GasPak System. The tubular expressions of TLR4 (2.2 +/- 1.2% vs 4.4 +/- 2.0%, p < 0.001) and HSP70 (2.6 +/- 2.8% vs 6.1 +/- 4.2%, p = 0.002) were increased after CsA treatment. TLR4 mRNA and protein expression were also increased in a dose-dependent manner. Hypoxia enormously increased TLR4 expression. In summary, CsA increased tubular expression of TLR4 and its ligand HSP70. As hypoxia was shown to be a strong stimulus for TLR4 expression, it can be said that TLR4 is influenced by both direct toxicity and impediment of renal microcirculation in human CsA nephrotoxicity.Apmis 08/2009; 117(8):583-91. · 2.07 Impact Factor
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ABSTRACT: The aim of this study was to determine the effects of cyclosporine A (CyA) on urinary levels of matrix metalloproteinase 2 and 9 (MMP2, MMP9) and their tissue inhibitors 1 and 2 (TIMP1, TIMP2) in steroid-dependent nephrotic syndrome (SDNS). The study group (1) consisted of 18 children SDNS aged 3.5-17.0 years treated with CyA. All NS children were examined three times: (A) at proteinuria relapse, before CyA treatment, (B) after 6 months, and (C) after 12 months of CyA administration. The control group (2) consisted of 18 healthy children. Serum CyA level was assessed by immunofluorescence. Enzyme-linked immunosorbent assay kits for total human MMP2 and 9 and TIMP1 and 2 were obtained from R&D Systems. Compared with healthy controls, urinary MMP9/Cr in NS children before CyA was on the same level and increased during CyA treatment, and urinary TIMP1/Cr was twice as high and increased significantly during CyA treatment. MMP9/TIMP1 in NS children treated with CyA increased, but the difference was not statistically significant. Urinary MMP2/Cr was similar, and urinary TIMP2/Cr was significantly higher in children treated with CyA (p < 0.01). The MMP2/TIMP2 ratio in NS children treated with CyA was significantly lower in comparison with healthy controls (p < 0.01). A negative correlation was noted between urinary MMP2/TIMP2 ratio and serum CyA in NS children (r = -0.541, p < 0.01). An imbalance within the MMP2 and TIMP2 and MMP9 and TIMP1 system may play a role in the pathogenesis CyA nephropathy.Pediatric Nephrology 08/2008; 23(10):1795-802. · 2.94 Impact Factor
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ABSTRACT: The MMPs and their inhibitors [tissue inhibitor of MMPs (TIMPs) ] form the mainstay of extracellular matrix homeostasis. They are expressed in response to numerous stimuli including cytokines and GPCR activation. This review highlights the importance of adrenoceptors and phosphoprotein phosphatases (PPP) in regulating MMPs in the cardiovascular system, which may help explain some of the beneficial effects of targeting the adrenoceptor system in tissue remodelling and will establish emerging crosstalk between these three systems. Although - and β-adrenoceptor activation increases MMP but decreases TIMP expression, MMPs are implicated in the growth stimulatory effects of adrenoceptor activation through transactivation of epidermal growth factor receptor. Furthermore, they have recently been found to catalyse the proteolysis of β-adrenoceptors and modulate vascular tone. While the mechanisms underpinning these effects are not well defined, reversible protein phosphorylation by kinases and phosphatases may be key. In particular, PPP (Ser/Thr phosphatases) are not only critical in resensitization and internalization of adrenoceptors but also modulate MMP expression. The interrelationship is complex as isoprenaline (ISO) inhibits okadaic acid [phosphoprotein phosphatase type 1/phosphoprotein phosphatase type 2A (PP2A) inhibitor]-mediated MMP expression. While this may be simply due to its ability to transiently increase PP2A activity, there is evidence for MMP-9 that ISO prevents okadaic acid-mediated expression of MMP-9 through a β-arrestin, NF-κB-dependent pathway, which is abolished by knock-down of PP2A. It is essential that crosstalk between MMPs, adrenoceptors and PPP are investigated further as it will provide important insight into how adrenoceptors modulate cardiovascular remodelling, and may identify new targets for pharmacological manipulation of the MMP system.British Journal of Pharmacology 05/2012; 166(4):1225 - 1243. · 5.07 Impact Factor