Rats with experimental membranous nephropathy shed podocytes into the urine that attach to tissue culture plates ex-vivo, and proliferate. These results suggest that detached podocytes are viable. These results add new perspectives into our understanding of podocyte loss in the development of glomerulosclerosis.

Background: A decrease in podocyte number contributes to the development of glomerulosclerosis in diabetic nephropathy. Although podocytes have been detected in the urine in certain glomerular diseases, their viability is poorly understood. Methods: Diabetes was induced in rats with streptozotocin. Urine was collected from control rats (given citrate), and rats with diabetic nephropathy, and cells obtained by centrifugation were resuspended in tissue culture media, and seeded onto collagen-coated tissue culture plates. Cells were grown under standard cell culture conditions ex vivo. Cell number was measured, the cell type in the urine was identified by immunostaining with specific antibodies, and morphology was assessed by light and electron microscopy. Results: Within 24 h, cells obtained from the urine of diabetic rats attached to tissue culture plates ex vivo. Cells were not detected in the urine from control rats. All cells from diabetic rats stained positive for the podocyte-specific proteins synaptopodin, nephrin, podocin and Glepp-1 and negative for mesangial (OX-7), tubular (Tamm-Horsfall protein) and endothelial (RECA) cell antigens. The cell number increased daily, which is consistent with cell growth ex vivo. Conclusions: Rats with diabetic nephropathy shed podocytes into the urine that attach and grow ex vivo. These results are consistent with the detachment of viable podocytes in diabetes and add new perspectives into our understanding of development of glomerulosclerosis in diabetes mellitus.

ATRA induces podocyte differentiation in vitro and in vivo and alters the expression of certain podocyte-specific proteins. Further studies are ongoing to delineate the mechanism of this effect.

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in renal physiology, including the prevention of proteinuria. Cyclin-dependent kinase 5 (CDK5) has been shown to influence several cellular processes in other terminally differentiated cells, in particular neurons. In this study, we examined the role of CDK5 in podocyte differentiation, proliferation, and morphology. In conditionally immortalized mouse podocytes in culture, CDK5 increased in association with podocyte differentiation. During mouse glomerulogenesis in vivo, CDK5 expression was predominantly detected in podocytes from the capillary loop stage to maturation and persisted in the podocytes of adult glomeruli. In contrast, CDK5 was markedly decreased in the proliferating and dedifferentiated podocytes of mice with anti-glomerular basement membrane nephritis and in human immunodeficiency virus transgenic mice. p35, the activator of CDK5, was also detected in podocytes and the p35/CDK5 complex was active. Cell fractionation studies showed that active p35/ CDK5 was mainly localized to the plasma membrane. Specific inhibition of CDK5 in differentiated cultured podocytes, either pharmacologically or with siRNA, induced shape changes, with cellular elongation and loss of process formation compared to the characteristic arborized phenotype. These data suggest a role for CDK5 as a regulator of podocyte differentiation, proliferation, and morphology. 1 Injury to podocytes is associated with proteinuria and progressive glomerulosclerosis. Podocytes derive from epithelial cells originating in the metanephric mesenchyme, which develop into postmitotic terminally differentiated cells, 3 and therefore have similarities to neurons. 4 During glomerulogenesis, podocytes proliferate until the S-shape body stage, and exit the cell cycle at the capillary loop stage. 5,6 Podocytes then acquire their fully differentiated phenotype, a process that in the mouse is not complete until 1 week after birth. Mature podocytes tightly regulate and maintain their quiescent and differentiated phenotype, and therefore the majority of diseases involving podocytes are not associated with proliferation and an increase in cellularity. Indeed, studies have shown that the inability to proliferate contributes to glomerular scarring. 7 In contrast, podocytes may dedifferentiate, and proliferate, in human immunodeficiency virus (HIV)-associated nephropathy, which is characterized by a rapid decline in renal function, emphasizing the importance of podocyte quiescence for glomerular function. 8 These studies show that the state of podocyte differentiation is closely linked with its proliferative potential. Studies have also shown that podocyte morphology is critical for normal function. 9,10 After injury, flattening and effacement of podocytes leads to loss of normal function.

In response to injury, the highly specialized and terminally differentiated glomerular visceral epithelial cell, or podocyte, may undergo several cell fates, including dedifferentiation and proliferation, persistent cell cycle arrest, hypertrophy, apoptosis, or necrosis. Common to these potential outcomes of injury is their ultimate regulation at the level of the cell cycle. There is now a large body of literature confirming the importance of cell cycle regulatory proteins in the cellular response to injury. Although CDK inhibitor p21 levels increase in podocytes following injury, the role of p21 is unclear in focal segmental glomerulosclerosis (FSGS), in part because its function depends heavily on the cytotoxic stimulus and the cellular context. Adriamycin (ADR) is a podocyte toxin used to induce experimental FSGS. The purpose of this study was to define the role of p21 in ADR-induced podocyte injury. BALB/c mice, a strain carrying the recessive ADR susceptibility gene, were backcrossed against c57B6 p21-/- mice to yield a 12th generation BALB/c p21-/- strain. Experimental FSGS was induced by injection of ADR 12 mg/kg × 2 doses (n = 8/group), with mice killed at 1, 2, 8, and 11 wk. Diseased p21-/- mice demonstrated worse albuminuria, more widespread glomerulosclerosis, and higher blood urea nitrogen compared with diseased p21+/+ mice. In diseased p21-/- mice vs. p21+/+ mice, apoptosis [measured by TdT-mediated dUTP nick end labeling (TUNEL) assay] was increased, and podocyte number (measured by WT-1 immunostaining) was decreased. To validate these findings in vitro, we utilized differentiated mouse podocytes, p21-/- and p21+/+, exposed to 0.125 μg/ml ADR. Apoptosis, measured by Hoechst 33342 staining and TUNEL assay, was greater in cultured p21-/- podocytes compared with p21+/+ podocytes. Reconstitution of p21 via retroviral transfection rescued the p21-/- podocytes from apoptosis. We conclude that p21 is prosurvival in the podocyte's response to ADR-induced injury. Ongoing studies are defining the mechanisms of this protective effect as it relates to DNA damage and apoptosis.