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Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte injury.

Blattner SM, Hodgin JB, Nishio M, Wylie SA, Saha J, Soofi AA, Vining C, Randolph A, Herbach N, Wanke R, Atkins KB, Gyung Kang H, Henger A, Brakebusch C, Holzman LB, Kretzler M - Kidney Int. (2013)

Bottom Line: Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood.Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state.However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.

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Glomerular mRNA and protein expression of podocyte markers from one-month-old podoRac1−/− and podoCdc42−/− mice. (A) Gene expression for podocyte markers nephrin, podocin, and synaptopodin in podoRac1−/− glomeruli does not differ from floxed control. In contrast, isolated glomeruli from podoCdc42−/− mice display a significant reduction in transcript levels of nephrin and podocin compared to floxed control mice (*p<0.05). Synaptopodin gene expression is not significantly different. (B) Immunofluorescence studies from kidney sections stained with antibodies against nephrin, podocin, and synaptopodin demonstrate continuous distribution in podocytes from floxed control and podoRac1−/− mice. In contrast, immunofluorescence for nephrin and podocin in podocytes from podoCdc42−/− mice exhibit a significantly impaired, granular distribution along the basement membrane.
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Figure 4: Glomerular mRNA and protein expression of podocyte markers from one-month-old podoRac1−/− and podoCdc42−/− mice. (A) Gene expression for podocyte markers nephrin, podocin, and synaptopodin in podoRac1−/− glomeruli does not differ from floxed control. In contrast, isolated glomeruli from podoCdc42−/− mice display a significant reduction in transcript levels of nephrin and podocin compared to floxed control mice (*p<0.05). Synaptopodin gene expression is not significantly different. (B) Immunofluorescence studies from kidney sections stained with antibodies against nephrin, podocin, and synaptopodin demonstrate continuous distribution in podocytes from floxed control and podoRac1−/− mice. In contrast, immunofluorescence for nephrin and podocin in podocytes from podoCdc42−/− mice exhibit a significantly impaired, granular distribution along the basement membrane.

Mentions: To define molecular alterations of the podocyte slit diaphragm, we measured glomerular gene expression and examined the cellular distribution of nephrin, podocin, and synaptopodin by quantitative RT-PCR (Figure 4A) and confocal laser microscopy in 4-week-old mice (Figure 4B and C ). Analysis of mRNA obtained from isolated glomeruli revealed a reduction of nephrin and podocin transcript levels in podoCdc42−/− mice (P<0.05), but no significant change in the expression of synaptopodin mRNA, compared to floxed controls. In contrast, nephrin and podocin expression in podoRac1−/− mice did not change (Figure 4A). Through an analysis of nephrin, podocin, and synaptopodin distribution in podocytes at the protein level by immunofluorescence, we observed continuous distribution along the glomerular capillary wall for each marker in wild-type animals. In contrast, nephrin and podocin immunofluorescence staining appeared discontinuous and granular in glomeruli from podoCdc42−/− mice, whereas synaptopodin remained unchanged. The intensity and pattern of these markers in podoRac1−/− mice, however, was identical to floxed controls (Figure 4B and C). These observations were consistent with the morphologic findings and lack of proteinuria described above.


Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte injury.

Blattner SM, Hodgin JB, Nishio M, Wylie SA, Saha J, Soofi AA, Vining C, Randolph A, Herbach N, Wanke R, Atkins KB, Gyung Kang H, Henger A, Brakebusch C, Holzman LB, Kretzler M - Kidney Int. (2013)

Glomerular mRNA and protein expression of podocyte markers from one-month-old podoRac1−/− and podoCdc42−/− mice. (A) Gene expression for podocyte markers nephrin, podocin, and synaptopodin in podoRac1−/− glomeruli does not differ from floxed control. In contrast, isolated glomeruli from podoCdc42−/− mice display a significant reduction in transcript levels of nephrin and podocin compared to floxed control mice (*p<0.05). Synaptopodin gene expression is not significantly different. (B) Immunofluorescence studies from kidney sections stained with antibodies against nephrin, podocin, and synaptopodin demonstrate continuous distribution in podocytes from floxed control and podoRac1−/− mice. In contrast, immunofluorescence for nephrin and podocin in podocytes from podoCdc42−/− mice exhibit a significantly impaired, granular distribution along the basement membrane.
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Related In: Results  -  Collection

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Figure 4: Glomerular mRNA and protein expression of podocyte markers from one-month-old podoRac1−/− and podoCdc42−/− mice. (A) Gene expression for podocyte markers nephrin, podocin, and synaptopodin in podoRac1−/− glomeruli does not differ from floxed control. In contrast, isolated glomeruli from podoCdc42−/− mice display a significant reduction in transcript levels of nephrin and podocin compared to floxed control mice (*p<0.05). Synaptopodin gene expression is not significantly different. (B) Immunofluorescence studies from kidney sections stained with antibodies against nephrin, podocin, and synaptopodin demonstrate continuous distribution in podocytes from floxed control and podoRac1−/− mice. In contrast, immunofluorescence for nephrin and podocin in podocytes from podoCdc42−/− mice exhibit a significantly impaired, granular distribution along the basement membrane.
Mentions: To define molecular alterations of the podocyte slit diaphragm, we measured glomerular gene expression and examined the cellular distribution of nephrin, podocin, and synaptopodin by quantitative RT-PCR (Figure 4A) and confocal laser microscopy in 4-week-old mice (Figure 4B and C ). Analysis of mRNA obtained from isolated glomeruli revealed a reduction of nephrin and podocin transcript levels in podoCdc42−/− mice (P<0.05), but no significant change in the expression of synaptopodin mRNA, compared to floxed controls. In contrast, nephrin and podocin expression in podoRac1−/− mice did not change (Figure 4A). Through an analysis of nephrin, podocin, and synaptopodin distribution in podocytes at the protein level by immunofluorescence, we observed continuous distribution along the glomerular capillary wall for each marker in wild-type animals. In contrast, nephrin and podocin immunofluorescence staining appeared discontinuous and granular in glomeruli from podoCdc42−/− mice, whereas synaptopodin remained unchanged. The intensity and pattern of these markers in podoRac1−/− mice, however, was identical to floxed controls (Figure 4B and C). These observations were consistent with the morphologic findings and lack of proteinuria described above.

Bottom Line: Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood.Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state.However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.

Show MeSH
Related in: MedlinePlus