Recovery and maintenance of nephrin expression in cultured podocytes and identification of HGF as a repressor of nephrin

Abstract

Cultured podocytes easily lose expression of nephrin. In this report, we developed optimum media for recovery and maintenance of nephrin gene expression in murine podocytes. Using reporter podocytes, we found that activity of the nephrin gene promoter was enhanced by DMEM/F12 or alpha-MEM compared with RPMI-1640. In any of these basal media, addition of 1,25-dihydroxyvitamin D(3), all-trans-retinoic acid or dexamethasone significantly increased activity of the nephrin promoter. The effects of the supplemental components were synergistic, and the maximum activation was achieved by DMEM/F12 supplemented with three agents. This culture medium was designated as vitamin D(3), retinoic acid and dexamethasone-supplemented DMEM/F12 (VRADD). In reporter podocytes that express nephrin, VRADD induced activation of the nephrin gene promoter up to 60-fold. Even in podocytes that have lost nephrin expression during multiple passages, expression of nephrin mRNA was dramatically recovered by VRADD. However, VRADD caused damage of podocytes in prolonged cultures, which was avoided in the absence of dexamethasone (designated as VRAD). VRAD maintained expression of nephrin for extended periods, which was associated with the differentiated phenotype of podocytes. Using the VRAD-primed podocytes, we revealed that expression of nephrin mRNA as well as nephrin promoter activity was suppressed by a putative dedifferentiation factor of podocytes, hepatocyte growth factor.

Combination therapy with AT1 blocker and vitamin D analog markedly ameliorates diabetic nephropathy: Blockade of compensatory renin increase

“…Although losartan or paricalcitol alone can partially restore the expression of some of these proteins, in comparison, their combination restored more proteins (␣-actinin-4, ZO-1, nephrin, and Neph-1) to higher levels, consistent with the much better antiproteinuric effect. In previous studies, blocking the RAS was shown to normalize nephrin expression (47,48), and vitamin D can up-regulate nephrin expression in podocytes (49). Together, these data clearly indicate that the podocytes are a major target of the RAS and vitamin D in renoprotection.…”

MicroRNA-193a Regulates the Transdifferentiation of Human Parietal Epithelial Cells toward a Podocyte Phenotype

“…hPECs with miR-193 KD located at the rim of cell assemblies developed processes. Strikingly, culture of hPECs with miR-193a KD on fibronectinor laminin-coated plates at low density in VRADD medium 18 resulted in morphologic changes toward an arborized phenotype typical for podocytes. Control hPECs maintained their polygonal nonarborized morphology under the same culture conditions ( Figure 4F).…”

Regeneration of Glomerular Podocytes by Human Renal Progenitors

“…Taken together, these results suggest that CD133ϩCD24ϩ renal progenitors represent a heterogeneous population, including a subset of cells (CD133ϩCD24ϩPDXϩ) that displays initial signs of podocyte commitment. 24 ( Figure 3D). Differentiation toward tubular cells resulted in the acquisition of binding properties of Lotus tetragonolobus agglutinin (LTA), a specific property of proximal tubular epithelia ( Figure 3D).…”

“…Cells were plated in EGM-MV (Lonza Ltd., Basel, Switzerland) with 20% FBS (Hyclone, Logan, Utah) or in VRAD medium 24 containing DMEM-F12 (Sigma) supplemented with 10% FBS, vitamin D3 100 nM (Sigma), and all-trans retinoic acid (100 M; Sigma). Generation of clones was achieved by limiting dilution in 96-well plates and in four-chamber glass slides (VWR International, West Chester, Pennsylvania).…”