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

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

doi:10.1172/jci.insight.142740

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.

Highlights

Dysfunction of the glomerular filtration barrier often manifests as nephrotic syndrome, a kidney disorder characterized by heavy proteinuria
We observed a striking 6.947fold reduction in G associated kinase (GAK) expression that correlated with a reduction in glomerular filtration rate (GFR) in this chronic kidney disease (CKD) cohort (Figure 1A), which was further validated in other CKD and diabetic kidney disease (DKD) cohorts in different data sets (Figure 1, B and C)
To determine if GAK transcript reduction was associated with reduced GAK protein expression in patients with CKD, we performed immunofluorescence on human focal segmental glomerulosclerosis (FSGS) biopsies costained for GAK and the podocyte-specific marker nephrin

Summary

Introduction

Dysfunction of the glomerular filtration barrier often manifests as nephrotic syndrome, a kidney disorder characterized by heavy proteinuria. Glomerular diseases account for approximately 80% of chronic kidney disease (CKD) cases, the therapeutic interventions aiming to prevent the development or slow the progression are very limited. We have previously demonstrated that proteins critical for clathrin-mediated endocytosis — dynamin 1 and 2, synaptojanin, and endophilin — are critical to maintaining the glomerular filtration barrier [1]. To further investigate clathrin-mediated endocytosis in human disease, in this study we mined the Nephroseq v5 transcriptomic database. In order to understand the pathobiology of Gak in glomerular disease, we generated mice that exhibited podocyte-selective loss of Gak, using a podocin promoter-driven Cre recombinase transgenic and floxed Gak mouse lines.

Methods

Results

Conclusion