Podocytes, proteinuria, nephrotic syndrome, miscellaneous

2005 Nephrology, Dialysis and Transplantation  
Colchicine has been reported to inhibit collagen deposition by suppressing fibroblast growth factors such as TGF-Beta. We hypothesized that besides of its ability to prevent amyloid deposition, colchicine may prevent the development of interstitial fibrosis (IF) in amyloidosis patients. For this purpose we design a study to evaluate the influence of colchicine therapy on the development of IF in 25 FMF patients (Group 1). For control group 25 non-amyloidotic patients that did not received
more » ... cine therapy was included in the study (Group 2). Patients were carefully matched for risk factors predisposing to IF. The incidence of recurrence and the development of IF in the 1 st , 2 nd and 3 rd year posttransplantation were evaluated from the follow-up biopsies of all cases. Only 4 patients showed amyloid recurrence in renal allograft. The development of IF was 44% (11/25) in group 1 patients and 80% (20/25) in group 2 patients during 36 months (p<0.01). The incidence of the development of IF in the 1 st , 2 nd and 3 rd years posttransplantation was found statistically higher in group 2 recipients when compared with group 1 recipients (p<0.01). The overall 1-, 2-and 3-year graft survival rates for the group 1 recipients were 96%, 92%, and 80%, respectively. The corresponding graft survival rates were 96%, 88%, and 60% for group 2 recipients respectively. In conclusion our results support the thesis that colchicine therapy may have a beneficial effect on preventing the development of renal interstitial fibrosis. Connective tissue growth factor (CTGF) is up-regulated in fibrotic kidney diseases. It is one of the key factors contributing to excess synthesis of extracellular matrix proteins. Regulation of CTGF expression has been addressed in vitro in cells cultured on plastic dishes, which do not reflect the in vivo cellular environment. To mimic the in vivo situation more closely, regulation of CTGF was investigated in human renal fibroblasts embedded in three dimensional collagen-I gels. Fibroblasts in collagen gels in serum containing medium developed a phenotype characterized by punctuate staining pattern of focal adhesion proteins, absence of cell spanning actin stress fibres, and barely detectable levels of CTGF. For comparison, cells on collagen-coated dishes were characterized by focal adhesions, strong fibres of F-actin and high basal levels of CTGF. Changes in cell morphology and the actin cytoskeleton have been shown to modulate CTGF expression. Therefore, the cells were treated with colchicine, a microtubule disrupting agent, which leads to activation of the small GTPase RhoA and rearrangement of the actin cytoskeleton. Cells embedded in collagen rounded, whereas cells in culture dishes spread forming enlarged focal adhesions and a net of dense stress fibres. In spite of the different morphological alterations, CTGF was strongly up-regulated in a RhoA-dependent manner as shown by the inhibition by the RhoA kinase inhibitor Y27632, supporting the critical role of RhoA signalling in the regulation of CTGF. Treatment of fibroblasts embedded in collagen gels with colchicine also initiated clustering of focal adhesion proteins, e.g. paxillin or vinculin. The morphological changes were associated with the activation of signalling proteins such as FAK or Cas, as indicated by increased tyrosine phosphorylation. Inhibition of Src like kinases by the inhibitor PP2 had no effect on the morphological changes, but interfered with tyrosine phosphorylation and completely prevented up-regulation of CTGF. This signalling pathway was only detectable in cells cultured in collagen gels. The strong phosphorylation of focal adhesion proteins of cells interacting with a rigid collagen matrix or plastic obviously masked the regulatory potential of Src/FAK signalling. In addition to the RhoA signalling pathway, Src/FAK signalling may play an essential role in the up-regulation of CTGF, when cells are exposed to morphological constraints as happens in closing wounds or fibrotic tissue. Cell proliferation is governed by cell cycle. Cyclins and cyclin dependent kinases (CDKs) constitute the major positive regulatory proteins. Integrins, a number of adhesion molecule family, regulate many signaling pathways including mitogen-activated proteins (MAP) kinases that regulate cell survival, proliferation, apoptosis and migration. There is a corelation between activation of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinase and induction of the cyclin D1 promotor. Our previous studies found decreased podocyte expression of integrinα3β1 and podocytopenia in human with primary focal segmental glomerulosclerosis and chronic PAN-treated rats. Despite an increased understanding of intracellular signaling pathways that promote integrin-mediated survival and proliferation in many cell types, mechanisms whereby integrinα3β1 regulate podocyte survival and proliferation, and the role of ERK signaling in this process, remain unclear. The present study was to investigate the effect of inte-grinα3β1 on podocyte proliferation, and its regulation at the level of cell cycle. Our study included renal specimens from 15 patients with classic primary FSGS, five normal kidneys obtained from total nephropathy due to renal cell carcinoma, and primary culture of rat podocytes. The immunohistochemistry was performed to analyze the expression of inte-grinα3β1, cyclin D1, ERK, and p-ERK in normal control and classic FSGS patients. The western blot analysis was used to measure protein levels of ERK, p-ERK, and cyclin D1 in quiescent condition after Gly-Arg-Gly-Asp (GRGD) treatment to block integrin and ECM interaction on cultured rat podocytes. The α3 subunit of integrin decreased in FSGS (60.8±28.57) than control (250.2±23.73, p<0.01). The β1 subunit of integrin also decreased in FSGS (40.6±27.93) than control (197±60.64, p<0.01). The ERK was less in FSGS than control (0±0 v.s 0.52±0.30, p<0.01). The p-ERK was less in FSGS than control (0.04±0.08 v.s 0.41±0.23, p<0.01). The cyclin D1 decreased in FSGS than control (0.02±0.04 v.s 0.75±0.35, p<0.01). The activation of ERK (p-ERK/ERK)decreased gradually in 12, 24 and 48 hours after GRGD treatment. The cyclin D1 protein expression was found to decrease in 12 hours after GRGD treatment, than increase in 24 hours after GRGD treatment. According above results, we found that integrin α3 subunit, β1 subunit, ERK, p-ERK and cyclin D1 decreased in FSGS patients. The activation of ERK decreased after integrin-ECM interaction blocked by GRGD. But the protein expression of cyclin D1 decreased in initial 12 hours after GRGD treatment, then increased in 24 hours after GRGD treatment. So, initial block of integrinα3β1-ECM interaction can inhibit cyclin D1 expression, thereafter, other factors also regulate cyclin D1 expression. v210 Podocytes, proteinuria, nephrotic syndrome, miscellaneous Monday,
doi:10.1093/ndt/20.suppl_5.v209 fatcat:gy4jf2efcbe5xhumy4ba37wwbi