Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling.

The Rho family of small GTPases (RhoA, Rac1 and Cdc42) controls signal-transduction pathways that influence many aspects of cell behaviour, including cytoskeletal dynamics. At the leading edge, Rac1 and Cdc42 promote cell motility through the formation of lamellipodia and filopodia, respectively. On the contrary, RhoA promotes the formation of contractile actin-myosin-containing stress fibres in the cell body and at the rear. Here, we identify synaptopodin, an actin-associated protein, as a novel regulator of RhoA signalling and cell migration in kidney podocytes. We show that synaptopodin induces stress fibres by competitive blocking of Smurf1-mediated ubiquitination of RhoA, thereby preventing the targeting of RhoA for proteasomal degradation. Gene silencing of synaptopodin in kidney podocytes causes the loss of stress fibres and the formation of aberrant non-polarized filopodia and impairment of cell migration. Together, these data show that synaptopodin is essential for the integrity of the podocyte actin cytoskeleton and for the regulation of podocyte cell migration.

Translocation of dendrin to the podocyte nucleus in acute glomerular injury in patients with IgA nephropathy.

BACKGROUND: It has been reported that podocytopenia has been occurring with increasing disease severity in patients with IgA nephropathy (IgAN). Dendrin is localized at the slit diaphragm (SD) in podocytes. We showed that dendrin translocates to the nucleus of injured podocytes in experimental nephritis and the nuclear dendrin promotes podocyte apoptosis. It is still unknown whether dendrin translocates from the SD to podocyte nucleus in IgAN. We investigated the presence of nuclear dendrin in patients with IgAN and the association between the translocated dendrin to the podocyte nucleus and disease activity. METHODS: Fourteen adult patients with IgAN were enrolled. The pathological parameters were analyzed. Immunostaining of renal biopsy specimens and urinary sediments from IgAN or minimal change nephrotic syndrome (MCNS) as the control was performed. RESULTS: A positive correlation was observed between an acute extracapillary change and the number of dendrin-positive nuclei. The location of dendrin in the nuclei was found in urinary podocytes of IgAN. The number of dendrin-positive nuclei in urinary podocytes of IgAN was significantly higher than that of MCNS. Urinary podocytes, which expressed the apoptosis marker annexin V, were also detected in IgAN. The translocation of dendrin to the podocyte nucleus as well as strong cathepsin L staining were detected in the glomeruli of IgAN. CONCLUSION: An increasing number of dendrin-positive nuclei in the glomeruli suggest acute glomerular injury in IgAN. Apoptotic podocytes were detectable in the urine of IgAN. It appears that the translocation of dendrin to the podocyte nuclei enhances podocyte apoptosis in acute glomerular injury and leads to podocytopenia in patients with IgAN.

Doxorubicin-induced glomerulosclerosis with proteinuria in GFP-GABARAP transgenic mice.

Autophagy is a process of cellular degradation, and its dysfunction elicits many pathological symptoms. However, the contribution of autophagy to kidney glomerular function has not been fully clarified. We previously reported that LC3, a promising executor of autophagy, played an important role in recovery from podocyte damage in an experimental nephrosis model (Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y. FASEB J 17: 1165-1167, 2003). γ-Aminobutyric acid A receptor-associated protein (GABARAP), has recently been characterized as another homolog of LC3, although its precise role in autophagy remains unclear. We recently generated green fluorescent protein (GFP)-GABARAP transgenic mice, in which GFP-GABARAP is abundantly expressed in glomerular podocytes. We found that the transgenic mice showed no obvious phenotype, and podocytes isolated from these mice manifested autophagic activity almost equivalent to that of wild-type mice when measured in vitro. Surprisingly, a single injection of doxorubicin caused a greater increase in proteinuria and sclerotic glomeruli in transgenic mice compared with wild-type mice. Under these conditions, neither GFP-GABARAP nor endogenous GABARAP appeared to be recruited to autophagosomes, and both remained in the cytosol. Moreover, the cytosolic GFP-GABARAP was significantly colocalized with p62 to form aggregates. These results indicate that the GFP-GABARAP/p62 complex is responsible for impairment of glomerular function and that it retards recovery from the effects of doxorubicin.

Effects of 22-oxa-calcitriol on podocyte injury in adriamycin-induced nephrosis.

BACKGROUND: In various animal studies, vitamin D has been shown to have a significant effect on reduction of proteinuria and the progression of kidney disease. However, little is known on its renoprotective effect in adriamycin (ADR)-induced nephrosis mice. The present study was intended to determine the therapeutic benefit of 22-oxa-calcitriol (OCT), a vitamin D analog, in reducing proteinuria and its renoprotective effect, i.e. preventing podocyte injury on ADR-induced nephrosis mice. METHODS: Three experimental groups were used as follows: (1) nephrosis mice, established by a single intravenous injection of ADR; (2) ADR+OCT mice, nephrosis mice treated with OCT, and (3) mice treated only with OCT as the control group. Podocyte injury was assessed by podocyte apoptosis using the TUNEL assay, podocyte counting, podocyte-specific expressed protein by immunofluorescence and Western blot analysis, and foot process effacement using electron microscopy. RESULTS: Lower proteinuria was observed in ADR+OCT mice. Improvement in glomerulosclerosis and interstitial fibrosis, and prevention of glomerular hyperfiltration were observed in ADR+OCT mice. Immunofluorescence and Western blot analyses showed restoration of downregulated expression of nephrin, CD2AP and podocin. Nevertheless, dendrin expression was not restored. An insignificant reduction in podocyte numbers was found in ADR+OCT mice. Complete foot process effacement was partially prevented in ADR+OCT mice. CONCLUSIONS: The results indicate that OCT reduces podocyte injury and has renoprotective effects in ADR nephrosis mice.

Dendrin location in podocytes is associated with disease progression in animal and human glomerulopathy.

BACKGROUND: Adriamycin (ADR) nephrosis in mice has been extensively studied and has enabled a greater understanding of the processes underlying the progression of renal injury. Dendrin is a novel component of the slit diaphragm with proapoptotic signaling properties, and it accumulates in the podocyte nucleus in response to glomerular injury in mice. The present study re-evaluated chronic progressive nephropathy in ADR mice and the localization of dendrin in mice and in human glomerulopathy. METHODS: To investigate the localization of dendrin, a mouse model of nephrosis and glomerulosclerosis was used, in which ADR was injected once. WT-1-positive cells and apoptotic cells were counted in vivo and in vitro. To check the expression of dendrin in ADR mice, immunostaining and Western blot were performed. A survey of dendrin staining was performed on human kidney biopsy specimens. RESULTS: The injection of ADR induced proteinuria, podocyte loss and glomerulosclerosis. It also caused the relocation of dendrin from the slit diaphragm to the podocyte nucleus. We demonstrated the location of dendrin to podocyte nuclei in several cases of human glomerulopathy. The mean occurrence of dendrin-positive nucleus per glomerulus increased in several cases of human glomerulopathy. CONCLUSIONS: These findings suggest that the relocation of dendrin to the podocyte nuclei is useful as a novel marker of podocyte injury in human glomerulopathy.

Combination therapy with telmisartan and oxacalcitriol suppresses the progression of murine adriamycin nephropathy.

BACKGROUND: Blockade of the renin-angiotensin system plays a key role in suppressing the progression of renal diseases. It has not been well established whether this therapy provides additional effects when combined with vitamin D or its analog in a model of adriamycin (ADR)-induced nephropathy. METHODS: We evaluated the effect of an angiotensin II subtype 1 receptor blocker (telmisartan) combined with a vitamin D analog (oxacalcitriol) on mice ADR-induced nephropathy (9.5 mg/kg single intravenous injection). We also tested immortalized murine podocytes to examine the effects on podocyte apoptosis. RESULTS: Mice with ADR-induced nephropathy developed progressive albuminuria and glomerulosclerosis within 30 days accompanied by decreased expression of slit diaphragm (SD)-associated proteins (nephrin and podocin), reduced numbers of podocytes, and increased systolic blood pressure. Treatment with telmisartan or oxacalcitriol alone moderately ameliorated kidney injury. The combined treatment most effectively reduced the albuminuria and glomerulosclerosis. These effects were accompanied by the restoration of SD-associated proteins, reduction of podocyte apoptosis, and prevention of podocyte depletion in the glomeruli. Treatment with telmisartan, oxacalcitriol, and the combination therapy resulted in similar reductions in systolic blood pressure. In cultured murine podocytes, ADR stimulated the expression of Bax/Bcl-2 and apoptosis as determined by Hoechst 33342 staining. These changes were effectively inhibited by telmisartan or oxacalcitriol, but the combination treatment most effectively reduced these effects. CONCLUSIONS: These data demonstrated that application of a renin-angiotensin system blocker plus a vitamin D analog effectively prevented renal injury in ADR-induced nephropathy. The observed amelioration of renal injury may be partly attributable to antiapoptotic effects in podocytes.

The role of podocytes in proteinuria.

Glomerular visceral epithelial cells, also known as podocytes, are highly specialized epithelial cells that cover the outer layer of the glomerular basement membrane. Podocytes consist of cell bodies, major processes and foot processes (FP) of neighbouring cells, with the filtration slits bridged by the slit membrane between them. The function of podocytes is largely based on their specialized cell architecture and functions such as stabilization of glomerular capillaries and participation in the barrier function of the glomerular filter. Therefore, they form the final barrier to protein loss, which explains why podocyte injury is typically associated with marked proteinuria. Under pathological conditions, podocytes exhibit various changes. Among these changes, FP effacement represents the most characteristic change in cell shape of podocytes. FP effacement is dependent on disruption of the actin cytoskeletal network in the podocytes, The mechanisms of organization and re-organization of actin in the FP of podocytes are discussed in this review.

Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton.

Podocytes of the renal glomerulus are unique cells with a complex cellular organization consisting of a cell body, major processes and foot processes. Podocyte foot processes form a characteristic interdigitating pattern with foot processes of neighboring podocytes, leaving in between the filtration slits that are bridged by the glomerular slit diaphragm. The highly dynamic foot processes contain an actin-based contractile apparatus comparable to that of smooth muscle cells or pericytes. Mutations affecting several podocyte proteins lead to rearrangement of the actin cytoskeleton, disruption of the filtration barrier and subsequent renal disease. The fact that the dynamic regulation of the podocyte cytoskeleton is vital to kidney function has led to podocytes emerging as an excellent model system for studying actin cytoskeleton dynamics in a physiological context.

Synaptopodin protects against proteinuria by disrupting Cdc42:IRSp53:Mena signaling complexes in kidney podocytes.

The actin-based foot processes of kidney podocytes and the interposed slit diaphragm form the final barrier to proteinuria. Mutations affecting several podocyte proteins cause disruption of the filtration barrier and rearrangement of the highly dynamic podocyte actin cytoskeleton. Proteins regulating the plasticity of the podocyte actin cytoskeleton are therefore of critical importance for sustained kidney barrier function. Synaptopodin is an actin-associated protein essential for the integrity of the podocyte actin cytoskeleton because synaptopodin-deficient mice display impaired recovery from protamine sulfate-induced foot process effacement and lipopolysaccharide-induced nephrotic syndrome. Moreover, bigenic heterozygosity for synaptopodin and CD2AP is sufficient to induce spontaneous proteinuria and focal segmental glomerulosclerosis-like glomerular damage in mice. Mechanistically, synaptopodin induces stress fibers by blocking the proteasomal degradation of RhoA. Here we show that synaptopodin directly binds to IRSp53 and suppresses Cdc42:IRSp53:Mena-initiated filopodia formation by blocking the binding of Cdc42 and Mena to IRSp53. The Mena inhibitor FP(4)-Mito suppresses aberrant filopodia formation in synaptopodin knockdown podocytes, and when delivered into mice protects against lipopolysaccharide-induced proteinuria. The identification of synaptopodin as an inhibitor of Cdc42:IRSp53:Mena signaling defines a novel antiproteinuric signaling pathway and offers new targets for the development of antiproteinuric therapeutic modalities.