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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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Targeted inactivation of Rac1 and Cdc42 in podocytes. (A) The diagram demonstrates the strategy for generation of podocyte-specific Rac1 and Cdc42 knockout mice. Mice expressing Cre-recombinase under control of the podocyte promoter (2.5P-Cre) were bred with mice carrying floxed Rac1 locus (exon 3) and floxed Cdc42 locus (exon 2). (B) PCR analysis of genomic DNA from tail clippings. The PCR product band of floxed (280 bp) and wild-type (200 bp) Rac1 as well as floxed (300 bp) and wild-type (200 bp) Cdc42 are shown. In addition, the 2.5P-Cre PCR product band (268 bp) is indicated. (C) Western blot analysis of Rac1 and Cdc42 in isolated glomeruli from podoRac1−/− and podoCdc42−/− mice with antibodies against Rac1, Cdc42, and β-actin reveals strong reduction of specific protein signal. (D) Survival curve for podoCdc42−/− and podoRac1−/− mice shows 100% mortality with loss of podocyte-specific Cdc42 by day 60. (E) SDS-PAGE analysis of urine samples demonstrates variable selective proteinuria by 10 days of age in podoCdc42−/− mice and heavy nonspecific proteinuria by 16 days of age compared to floxed control. Loss of podocyte-specific Rac1 has no effect on urine protein at 6 months. (F) Urine albumin-to-creatinine ratios in podoCdc42−/− mice were significantly increased versus floxed controls (n=5 per group). (G) Whole kidneys from podoCdc42−/− mouse at 3 weeks of age are pale yellow and display a granular surface compared to floxed control.
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Figure 1: Targeted inactivation of Rac1 and Cdc42 in podocytes. (A) The diagram demonstrates the strategy for generation of podocyte-specific Rac1 and Cdc42 knockout mice. Mice expressing Cre-recombinase under control of the podocyte promoter (2.5P-Cre) were bred with mice carrying floxed Rac1 locus (exon 3) and floxed Cdc42 locus (exon 2). (B) PCR analysis of genomic DNA from tail clippings. The PCR product band of floxed (280 bp) and wild-type (200 bp) Rac1 as well as floxed (300 bp) and wild-type (200 bp) Cdc42 are shown. In addition, the 2.5P-Cre PCR product band (268 bp) is indicated. (C) Western blot analysis of Rac1 and Cdc42 in isolated glomeruli from podoRac1−/− and podoCdc42−/− mice with antibodies against Rac1, Cdc42, and β-actin reveals strong reduction of specific protein signal. (D) Survival curve for podoCdc42−/− and podoRac1−/− mice shows 100% mortality with loss of podocyte-specific Cdc42 by day 60. (E) SDS-PAGE analysis of urine samples demonstrates variable selective proteinuria by 10 days of age in podoCdc42−/− mice and heavy nonspecific proteinuria by 16 days of age compared to floxed control. Loss of podocyte-specific Rac1 has no effect on urine protein at 6 months. (F) Urine albumin-to-creatinine ratios in podoCdc42−/− mice were significantly increased versus floxed controls (n=5 per group). (G) Whole kidneys from podoCdc42−/− mouse at 3 weeks of age are pale yellow and display a granular surface compared to floxed control.

Mentions: To define the function of Rac1 and Cdc42 in podocytes in vivo, we used mice that express Cre-recombinase under control of a podocyte-specific promoter and crossed them with mice with floxed exon 3 of the Rac1 gene, or mice with floxed exon 2 of the Cdc42 gene, resulting in targeted inactivation of either Rac1 (podoRac1−/−) or Cdc42 (podoCdc42−/−) (Figure 1A). Mouse genotype containing Cre-recombinase expressing construct and homozygous for either floxed Rac1 or Cdc42 was confirmed by PCR analysis of tail genomic DNA (Figure 1B). Western blot analysis of protein lysates obtained from glomeruli isolated from podoRac1−/− and podoCdc42−/− mice demonstrated profound reduction of Rac1 and Cdc42 protein expression compared to glomerular lysates from floxed controls (Rac1-fl/fl and Cdc42-fl/fl) (Figure 1C). As podocytes constitute a fraction of the glomerular cell population, endothelial and mesangial cells contribute to the remaining signals in podoRac1−/− and podoCdc42−/− glomeruli.


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)

Targeted inactivation of Rac1 and Cdc42 in podocytes. (A) The diagram demonstrates the strategy for generation of podocyte-specific Rac1 and Cdc42 knockout mice. Mice expressing Cre-recombinase under control of the podocyte promoter (2.5P-Cre) were bred with mice carrying floxed Rac1 locus (exon 3) and floxed Cdc42 locus (exon 2). (B) PCR analysis of genomic DNA from tail clippings. The PCR product band of floxed (280 bp) and wild-type (200 bp) Rac1 as well as floxed (300 bp) and wild-type (200 bp) Cdc42 are shown. In addition, the 2.5P-Cre PCR product band (268 bp) is indicated. (C) Western blot analysis of Rac1 and Cdc42 in isolated glomeruli from podoRac1−/− and podoCdc42−/− mice with antibodies against Rac1, Cdc42, and β-actin reveals strong reduction of specific protein signal. (D) Survival curve for podoCdc42−/− and podoRac1−/− mice shows 100% mortality with loss of podocyte-specific Cdc42 by day 60. (E) SDS-PAGE analysis of urine samples demonstrates variable selective proteinuria by 10 days of age in podoCdc42−/− mice and heavy nonspecific proteinuria by 16 days of age compared to floxed control. Loss of podocyte-specific Rac1 has no effect on urine protein at 6 months. (F) Urine albumin-to-creatinine ratios in podoCdc42−/− mice were significantly increased versus floxed controls (n=5 per group). (G) Whole kidneys from podoCdc42−/− mouse at 3 weeks of age are pale yellow and display a granular surface compared to floxed control.
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Figure 1: Targeted inactivation of Rac1 and Cdc42 in podocytes. (A) The diagram demonstrates the strategy for generation of podocyte-specific Rac1 and Cdc42 knockout mice. Mice expressing Cre-recombinase under control of the podocyte promoter (2.5P-Cre) were bred with mice carrying floxed Rac1 locus (exon 3) and floxed Cdc42 locus (exon 2). (B) PCR analysis of genomic DNA from tail clippings. The PCR product band of floxed (280 bp) and wild-type (200 bp) Rac1 as well as floxed (300 bp) and wild-type (200 bp) Cdc42 are shown. In addition, the 2.5P-Cre PCR product band (268 bp) is indicated. (C) Western blot analysis of Rac1 and Cdc42 in isolated glomeruli from podoRac1−/− and podoCdc42−/− mice with antibodies against Rac1, Cdc42, and β-actin reveals strong reduction of specific protein signal. (D) Survival curve for podoCdc42−/− and podoRac1−/− mice shows 100% mortality with loss of podocyte-specific Cdc42 by day 60. (E) SDS-PAGE analysis of urine samples demonstrates variable selective proteinuria by 10 days of age in podoCdc42−/− mice and heavy nonspecific proteinuria by 16 days of age compared to floxed control. Loss of podocyte-specific Rac1 has no effect on urine protein at 6 months. (F) Urine albumin-to-creatinine ratios in podoCdc42−/− mice were significantly increased versus floxed controls (n=5 per group). (G) Whole kidneys from podoCdc42−/− mouse at 3 weeks of age are pale yellow and display a granular surface compared to floxed control.
Mentions: To define the function of Rac1 and Cdc42 in podocytes in vivo, we used mice that express Cre-recombinase under control of a podocyte-specific promoter and crossed them with mice with floxed exon 3 of the Rac1 gene, or mice with floxed exon 2 of the Cdc42 gene, resulting in targeted inactivation of either Rac1 (podoRac1−/−) or Cdc42 (podoCdc42−/−) (Figure 1A). Mouse genotype containing Cre-recombinase expressing construct and homozygous for either floxed Rac1 or Cdc42 was confirmed by PCR analysis of tail genomic DNA (Figure 1B). Western blot analysis of protein lysates obtained from glomeruli isolated from podoRac1−/− and podoCdc42−/− mice demonstrated profound reduction of Rac1 and Cdc42 protein expression compared to glomerular lysates from floxed controls (Rac1-fl/fl and Cdc42-fl/fl) (Figure 1C). As podocytes constitute a fraction of the glomerular cell population, endothelial and mesangial cells contribute to the remaining signals in podoRac1−/− and podoCdc42−/− glomeruli.

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