Evidence suggests that loss of podocytes into urine contributes to development of glomerular diseases; shed podocytes are frequently viable and proliferate in culture conditions. To determine the phenotypic characteristics of viable urinary cells derived from human subjects, we established long-term urinary cell culture from two patients with focal segmental glomerulosclerosis and two healthy volunteers, via transformation with the thermosensitive SV40 large T antigen (U19tsA58) together with human telomerase (hTERT). Characterization of arbitrarily selected two clonal cell lines from each human subject was carried out. mRNA expression for the podocyte markers synaptopodin, nestin, and CD2AP were detected in all eight clones. Podocin mRNA was absent from all eight clones. The expression of nephrin, Wilms' tumor 1 (WT1), and podocalyxin mRNA varied among the clones, which may be due to transformation and/or cloning. These results suggest that podocyte cell lines can be established consistently from human urine. The generation of podocyte cell lines from urine of patients and healthy volunteers is novel and will help to advance studies of podocyte cell biology. Further improvements in the approaches to cell transformation and/or cell culture techniques are needed to allow cultured podocytes to fully reproduce in vivo characteristics.

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.cyclin-dependent kinase 4; SV40 large T antigen; nephrin; synaptopodin; Wilms tumor-1; P-cadherin PODOCYTE CULTURES, ESPECIALLY podocyte cell lines transformed by thermosensitive SV40 large T antigen (SV40 T), have been widely used to study podocyte biology (19,25). However, as we recently reported in studies on a generation of mouse and human podocyte cell lines, podocytes transformed by thermosensitive SV40 T (tsT) exhibit phenotypic differences compared with podocytes in vivo, with reduced expression of certain differentiation marker genes (13, 24).Cyclin-dependent kinase 4 (CDK4), a catalytic subunit of the cyclin D-CDK4 serine/threonine kinase complex, is a critical regulator of the cell cycle. CDK4 is the first kinase activated by mitogenic signals. The kinase activity of CDK4 is specifically involved in progression through G1, via phosphorylation of retinoblastoma (Rb) family members (4). Recently, Ramirez et al. (22) immortalized human bronchial epithelial cells by ectopic expression of CDK4 and human telomerase (hTERT). Microarray analysis showed that CDK4/hTERTimmortalized cells showed significantly different gene expression profiles compared with cells immortalized with human papiloma viral oncoproteins E6/E7 or SV40T and that CDK4-transformed cells clustered with primary bronchial epithelial cells. In addition, recent studies showed that cells immortalized with CDK4 are useful tools to study physiology and pathophysiology (8,23,30).We set out to develop an alternative approach to transforming podocytes by comparing the phenotype among primary cells, CDK4-transformed podocytes (CDK4-podocytes) and thermosensitive SV40 T mutant tsA58U19 (tsT-podocytes). We found that CDK4-podocytes and tsT-podocyt...