The glomerular podocyte is believed to play a role in the development and progression of albuminuria and glomerulosclerosis associated with diabetes, among other [27
]. Podocytes, and more particularly dysregulation of their differentiation, amongst other injurious stimuli, are at the centre of the pathogenesis of nephropathy. In this study, we describe the gradual modulation of pivotal characteristics of immortalized human podocytes in response to chronic exposure to high glucose. This conversion could be considered as a dedifferentiation process, since it was accompanied by increased expression of mesenchymal vimentin and reduced expression of specialized epithelial components which are podocytic markers. Our data indicated that glucose-mediated PC downregulation which occurred progressively, preceded downregulation of nephrin, the expression of which was substantially suppressed as early as 4 weeks of culture in high glucose.
Changes of podocyte structure and function have been previously described as epithelial-to-mesenchymal transition (EMT) [29
] since pro-fibrotic components appeared, concomitant with loss of markers characteristic of epithelial differentiation. However, phenotypic changes of podocytes observed in vitro
or in vivo
may not necessarily represent EMT-like changes [18
]. Podocytes are cells embryonically derived from the metanephric mesenchyme and express epithelial markers (e.g. ZO-1, cytokeratin). Following exposure to TGF-β1, epithelial markers of podocytes were reported to be increased, concomitant with increased tight junction formation [32
]. In contrast, in EMT tight junctions are reduced. The phenotypic changes observed in our in vitro
model more closely resemble a process of partial dedifferentiation. Vimentin, a component of intermediate filaments is expressed in differentiated podocytes but its expression becomes upregulated in podocytes lacking their specific markers, for example in nephrotic glomeruli, in the puromycin aminonucleoside model in rat [22
]. Hence enhanced vimentin expression in podocytes following chronic exposure to high glucose could represent a marker of dedifferentiation. Partial podocyte dedifferentiation induced by high glucose could be further supported by the observed loss of PC, nephrin and CALLA, concomitant with upregulation of mesenchymal vimentin.
Our findings are consistent with the reported effects of TGF-β1 and Ang-II resulting in podocyte dedifferentiation and apoptosis under normal and high glucose conditions [7
]. Additionally, our study demonstrates for the first time that dysregulation of the normal podocytic characteristics is an event differentially affecting the expression of function-specific podocytic markers: downregulation of the epithelial marker CD10/CALLA and PC first occurred progressively, and were followed by stably downregulated nephrin at later time intervals.
Nephrin and CD2AP are pivotal for slit diaphragm permselective properties [29
], and their loss has been linked to podocytic dysregulation and loss of the differentiated podocytic phenotype [37
]. The punctate pattern of expression of nephrin and CD2AP which was observed in our in vitro
system of podocytes (Figures , ) could represent the in vitro
equivalent of foot-like process formation [11
]. Accordingly, glucose-induced, decreased punctate staining in these cells [11
] possibly indicated the existence of fewer foot-like processes, resembling foot effacement in vivo
. Downregulation of the podocyte marker nephrin concomitant with upregulation of the mesenchymal marker vimentin occurred following 4 weeks of exposure to high glucose. Glucose-mediated downregulation of PC expression started as early as two weeks following exposure to high glucose, and gradually reached maximal suppression within 18 weeks. Hence, glucose-induced loss of the differentiated characteristics was complete by 18 weeks. In other studies, in human lung adenocarcinoma, PC downregulation appeared to be intimately associated with upregulation of vimentin and E-cadherin, both involved in mesenchymal transition [38
]. We herein report for the first time that in in vitro
cultured podocytes, PC downregulation was reversible only when the cells still expressed this component in substantial, albeit decreased amounts. At later time intervals, the observed maximal loss of PC became permanent. In agreement with our observations, in an in vivo
mouse model of podocyte injury, the decrease in nephrin and synaptopodin reflected early downregulation of these proteins in injured but still functioning podocytes, but PC expression was substantially downregulated only in severely injured or sclerotic podocytes [39
A novel finding of the present study was the observation that restoration of PC expression was not WT1-mediated, since this process was not accompanied by restoration of WT1 binding to the PC gene promoter region. Earlier in vivo
and in vitro
studies reported that increased levels of expression and activity of WT1 were associated with increased levels of PC expression in podocytes [25
]. However, it has been proposed that WT1 alone does not suffice to upregulate PC expression [12
]. Our observations suggest that WT1 is implicated in establishing basal PC levels and maintaining PC expression in differentiating and differentiated podocytes [25
]; nevertheless according to our data WT1 was not directly involved with upregulation of previously reduced PC expression. Moreover, at the late time interval, when PC downregulation had become permanent, a minor (but non-significant) increase of WT1 binding at the relative response element was observed which was not adequate for transcriptional activation. Additional transcription factors could be apparently involved in re-starting the partially or permanently suppressed expression of PC in the presence of high glucose. In this instance, WT1 may function in a manner similar to that used for regulation of the expression of the nephrin gene, since downregulation and re-expression of cell surface-associated nephrin were not accompamied by altered binding of WT1 to the nephrin gene promoter. Interestingly, it has been suggested that the Sp1 zinc finger protein can support transcriptional regulation of either nephrin or PC independently of WT1 [41
]. Another transcription factor implicated in upregulation of nephrin [43
] and PC expression (unpublished data) is the vitamin D receptor (VDR), strongly indicating that enhancement of nephrin and PC expression may be WT1 independent.