Inhibiting calpain 1 and 2 in cyclin G associated kinase–knockout mice mitigates podocyte injury
Xuefei Tian, Kazunori Inoue, Yan Zhang, Ying Wang, C. John Sperati, Christopher E. Pedigo, Tingting Zhao, Meihua Yan, Marwin Groener, Dennis G. Moledina, Karen Ebenezer, Wei Li, Zhenhai Zhang, Dan A. Liebermann, Lois Greene, Peter Greer, Chirag R. Parikh, Shuta Ishibe
Xuefei Tian, Kazunori Inoue, Yan Zhang, Ying Wang, C. John Sperati, Christopher E. Pedigo, Tingting Zhao, Meihua Yan, Marwin Groener, Dennis G. Moledina, Karen Ebenezer, Wei Li, Zhenhai Zhang, Dan A. Liebermann, Lois Greene, Peter Greer, Chirag R. Parikh, Shuta Ishibe
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Research Article Cell biology Nephrology

Inhibiting calpain 1 and 2 in cyclin G associated kinase–knockout mice mitigates podocyte injury

Abstract

Evidence for reduced expression of cyclin G associated kinase (GAK) in glomeruli of patients with chronic kidney disease was observed in the Nephroseq human database, and GAK was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak-knockout mice (Gak-KO), which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mice carrying the mutation to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to progressive podocyte damage. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak-KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak-KO mice. Increased podocyte calpain activity–mediated proteolysis of IκBα resulted in increased NF-κB p65–induced expression of growth arrest and DNA-damage-inducible 45 beta in the Gak-KO mice. Our results suggest that loss of podocyte-associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.

Authors

Xuefei Tian, Kazunori Inoue, Yan Zhang, Ying Wang, C. John Sperati, Christopher E. Pedigo, Tingting Zhao, Meihua Yan, Marwin Groener, Dennis G. Moledina, Karen Ebenezer, Wei Li, Zhenhai Zhang, Dan A. Liebermann, Lois Greene, Peter Greer, Chirag R. Parikh, Shuta Ishibe

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Figure 7

Loss of Capns1 results in increased GADD45B expression due to IκBα cleavage and NF-κB p65 signaling in Gak-KO mice.

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Loss of Capns1 results in increased GADD45B expression due to IκBα cleav...
(A) Representative Western blot images from control, Capns1-KO, Gak-KO, and Gak/CapnsS1-DKO mouse primary podocytes immunoblotted with GADD45B and Wilms tumor 1 (WT1). (B) Quantification of A by densitometry. n = 6. *P < 0.05. (C) Representative immunofluorescence image of Gak-KO mouse glomeruli demonstrates increased GADD45B expression (GADD45B in green and WT1 in red). Scale bar: 50 μm. (D) Representative immunofluorescence image of glomeruli from control and FSGS patient stained with GADD45B (green) and TLE4 (red). Scale bar: 10 μm. (E) Quantification of C. n = 5 mice; 20 glomeruli per mouse were evaluated. (F) Quantification of D. n = 3 patients; 16 glomeruli per group were evaluated. (G) Gak-KO mice podocytes show IκBα cleavage (upper panel) and talin1 cleavage (middle panel) as determined by Western blots; these cleavage events were reduced by the podocyte-specific loss of Capns1. (H) ChIP assay using phospho-NF-κB p65 and primer sets for Gadd45b promoter in the primary podocytes of control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice. n = 7. (I) Representative Western blot images for phospho-NF-κB p65 (pNF-κB p65) (Ser536) and total NF-κB p65 (NF-κB p65) from primary podocytes in control, Capns1-KO, Gak-KO, and Gak/Capns1-DKO mice. (J) Quantification of the nuclear NF-κB p65 activities of primary podocytes isolated from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice at the age of P14. n = 6. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (E, F, H, and J). Statistically analyzed via a 1-way ANOVA with Dunnett’s correction.