Effect of calcineurin inhibitors on extracellular matrix turnover in isolated human glomeruli.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Hepatocyte growth factor (HGF) reduces the expression of profibrotic factors in human isolated glomeruli]. / Hepatocyte growth factor (HGF) riduce l'espressione di fattori profibrotici in glomeruli umani isolati.

BACKGROUND: The imbalance between the synthesis and degradation of the mesangial matrix causes glomerulosclerosis and ultimately leads to chronic renal failure. HGF is a pleiotropic cytokine involved in angiogenesis, morphogenesis, organogenesis, and bone remodeling. Recently, we and other investigators have shown that HGF has a central role in the recovery of acute renal failure. Furthermore, HGF treatment halts the progression of kidney disease in a murine model of chronic renal failure. The aim of the present study was to evaluate the effect of HGF on the mRNA levels of molecules involved in the extracellular matrix turnover and of the c-met receptor in isolated human glomeruli. METHODS: Human glomeruli were isolated by microdissection from donor kidney biopsies just before transplantation. Glomeruli were extensively washed and incubated with culture media containing HGF (50 ng/mL) for 24 h at 37 C. Glomeruli incubated without HGF were used as controls. After 24 h, glomeruli were washed and freezed and thawed three times. The expression of c-met, (alpha2) IV collagen, TGF-beta, metalloproteases 9 (MMP9), and of the inhibitor of metalloproteases-1, TIMP-1 was evaluated by in situ reverse transcription (RT) and polymerase chain reaction (PCR). beta-actin was used as a housekeeping gene. RESULTS: The (alpha2)IV collagen mRNA level was decreased by HGF in human glomeruli. TGF-beta and TIMP-1 gene expression was markedly reduced by HGF treatment, whereas the expression of MMP-9 and c-met did not change. Under light-microscopic examination, kidney biopsies showed neither glomerular hypercellularity nor mesangial expansion. CONCLUSIONS: HGF treatment reduces the expression of extracellular matrix components and of profibrotic factors in human glomeruli. Our results confirm a protective role of HGF in glomerulosclerosis.

Long-term exposure to high glucose up-regulates VCAM-induced endothelial cell adhesiveness to PBMC.

BACKGROUND: The changes induced on endothelial cells by a long-term exposure to high glucose, a situation that mimics the hyperglycemia of diabetics, have not yet been determined. We compared short- and long-term effects of elevated glucose on macrovascular and microvascular endothelial cells. METHODS: Endothelial cells were grown in high-glucose media for 24 hours and for 8 weeks. Cell proliferation was evaluated by cell counting, apoptosis and expression of adhesion molecules by flow cytometry; nitric oxide (NO) by measuring the concentration of nitrite/nitrate in the cell supernatant; alpha 2(IV) collagen mRNA and protein by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The adhesion of peripheral blood mononuclear cells (PBMCs) to endothelial cells was evaluated by adhesion assay. In some experiments, endothelial cells were preincubated with anti-vascular cell adhesion molecule-1 (VCAM-1) and anti-receptor for advanced glycation end product (RAGE) blocking antibodies. RESULTS: At 24 hours, but not at 8 weeks, high glucose increased endothelial cell proliferation and apoptosis. High glucose did not modify NO synthesis at 24 hours and 8 weeks. Collagen production and expression were increased only after eight weeks. VCAM-1 but not intercellular adhesion molecule-1 was up-regulated after 8 weeks, a change not observed after 24 hours. The adhesion of PBMCs was significantly increased at eight weeks and was completely abrogated by anti--VCAM-1 and by anti-RAGE antibodies. After 24 hours, there was a modest increase of PBMC adhesion that was not blunted by anti-RAGE antibodies. CONCLUSIONS: Increased adhesion of PBMCs, caused by up-regulation of VCAM-1 with a mechanism involving advanced glycation end product (AGE) adducts, and augmented collagen deposition are critical effects of long-term high glucose on endothelial cells, and may eventually promote the atherosclerotic process.

Cyclosporine induces different responses in human epithelial, endothelial and fibroblast cell cultures.

BACKGROUND: Nephrotoxicity, accelerated atherosclerosis, and graft vascular disease are common complications of cyclosporine long-term treatment characterized by a wide disruption of organ architecture with increased interstitial areas and accumulation of extracellular matrix (ECM). How cyclosporine induces these changes is not clear, but it is conceivable that they are the sum of changes induced at the cell level. METHODS: We studied the effects of cyclosporine on human endothelial (HEC), epithelial (HK-2), and fibroblast (MRC5) cells. Cell proliferation was evaluated by cell counting, apoptosis and collagen production by enzyme-linked immunosorbent assay, and nitric oxide by measuring the concentration of nitrite/nitrate in the cell supernatant. (alpha1)I and (alpha2)IV collagen, matrix metalloprotease-9 (MMP9), and tissue inhibitors of metalloprotease-1 (TIMP-1) mRNA levels were measured by reverse transcription-polymerase chain reaction. Proteolytic activity was evaluated by zymography. RESULTS: Cyclosporine showed a marked antiproliferative and proapoptotic effect on endothelial and epithelial cells. Fibroblast growth was not affected by cyclosporine. Nitric oxide was up-regulated by cyclosporine in epithelial cells and fibroblasts but not in endothelial cells. (alpha1)I and (alpha2)IV collagen synthesis was increased in cyclosporine-treated endothelial and epithelial cells, respectively. Proteolytic activity was increased in endothelial and epithelial cells. TIMP-1 mRNA was up-regulated by cyclosporine in fibroblasts. CONCLUSIONS: Our results demonstrate that cyclosporine exhibits an antiproliferative effect on endothelial and epithelial cells. This effect is associated with induction of apoptosis probably via nitric oxide up-regulation in epithelial cell cultures. Cyclosporine treatment induces ECM accumulation by increasing collagen synthesis in endothelial and epithelial cells and reducing its degradation by up-regulating TIMP-1 expression in fibroblasts. We conclude that cyclosporine affects cell types differently and that the disruption of organ architecture is the result of multiple effects at the cell level.

Henoch-Schönlein purpura in a chronic hemodialysis patient.

We describe a patient on maintenance hemodialysis who developed purpura, abdominal pain with bloody stool, and gross hematuria. A skin biopsy revealed leukocytoclastic vasculitis with IgA deposits. This is the first report of Henoch-Schönlein purpura in a hemodialysis patient.