α-Actinin-4 and CLP36 Protein Deficiencies Contribute to Podocyte Defects in Multiple Human Glomerulopathies

Abstract

Genetic alterations of ␣-actinin-4 can cause podocyte injury through multiple mechanisms. Although a mechanism involving gain-of-␣-actinin-4 function was well described and is responsible for a dominantly inherited form of human focal segmental glomerulosclerosis (FSGS), evidence supporting mechanisms involving loss-of-␣-actinin-4 function in human glomerular diseases remains elusive. Here we show that ␣-actinin-4 deficiency occurs in multiple human primary glomerulopathies including sporadic FSGS, minimal change disease, and IgA nephropathy. Furthermore, we identify a close correlation between the levels of ␣-actinin-4 and CLP36, which form a complex in normal podocytes, in human glomerular diseases. siRNA-mediated depletion of ␣-actinin-4 in human podocytes resulted in a marked reduction of the CLP36 level. Additionally, two FSGS-associated ␣-actinin-4 mutations (R310Q and Q348R) inhibited the complex formation between ␣-actinin-4 and CLP36. Inhibition of the ␣-actinin-4-CLP36 complex, like loss of ␣-actinin-4, markedly reduced the level of CLP36 in podocytes. Finally, reduction of the CLP36 level or disruption of the ␣-actinin-4-CLP36 complex significantly inhibited RhoA activity and generation of traction force in podocytes. Our studies reveal a critical role of the ␣-actinin-4-CLP36 complex in podocytes and provide an explanation as to how ␣-actinin-4 deficiency or mutations found in human patients could contribute to podocyte defects and glomerular failure through a lossof-function mechanism.Chronic and end stage kidney diseases, which are frequently caused by defects in glomerular filtration barrier function, have become a major global health problem. Although many factors can contribute to the development and progression of human glomerulopathies, genetic defects play important roles in the disease processes (1-6). Elucidation of how genetic alterations contribute to glomerular defects is therefore critical for understanding the molecular mechanisms underlying the development and progression of human glomerulopathies.The glomerular filtration barrier is composed of an endothelium, a glomerular basement membrane, and a layer of podocytes building the slit diaphragm for filtration with their interdigitating foot processes. Although genetic alterations can cause defects in any of the three layers of the glomerular filtration barrier, podocytes appear to be a frequent target of genetic alterations in glomerulopathies (3, 4, 7-14). ␣-Actinin-4 is a member of the actinin protein family that consists of an actinbinding domain in the N terminus, four spectrin-like repeats in the central region, and two EF-hand motifs in the C terminus (15). Although both ␣-actinin-1 and -4 are expressed in mouse podocytes, ␣-actinin-4 is the sole member of the actinin family expressed in human podocytes (16). ␣-Actinin-4 is widely expressed in mammalian tissues and organs. However, despite the widespread expression, podocytes appear to be the primary site of manifestations of diseases induced by genetic alterations of ACTN4 (16,...

Basic and Translational Concepts of Immune-Mediated Glomerular Diseases

“…Mutations in podocyte genes that regulate the slit diaphragm, cell membrane, and cytoskeleton are increasingly recognized, not only in FSGS but in other forms of GN as well. 224 African Americans with nondiabetic nephropathy express variants in the gene encoding APOL1. [225][226][227][228][229] Both clinical and experimental studies document the importance of several other genes, especially ones that regulate the podocyte actin cytoskeleton, in modulating the development of proteinuria, foot process effacement, and sclerosis, including RhoA, urokinase receptor, Pdlim2, and connective tissue growth factor.…”

α Actinin 4 (ACTN4) Regulates Glucocorticoid Receptor-mediated Transactivation and Transrepression in Podocytes

“…Multiple mutations in ACTN4 have been linked to FSGS (19,20). Additionally, ACTN4 deficiency is also found in multiple human primary glomerulopathies, including sporadic FSGS, MCD, and IgA nephropathy (21)(22)(23)(24). However, the molecular mechanisms by which ACTN4 maintains podocytes homeostasis and its biochemical activities remain largely unknown.…”

Mechanisms of Glucocorticoid Resistance in Idiopathic Nephrotic Syndrome

“…Before this, animal experiments [64,65] had shown that ACTN4 -absent mice exhibited abundant proteinuria with sclerosis of glomerular capillaries and podocyte foot process effacement, but whether humans were affected in the same way was unknown at that time. In 2011, Liu et al [62] demonstrated that mutations in the non-actin-binding region (e.g., R310Q, Q348R) result in deficiencies of α-actinin-4 and its binding protein CLP36. This disruption of the α-actinin-4-CLP36 complex hinders RhoA signaling and the generation of traction force in podocytes.…”

“…Mutations in ACTN4 are related to familial FSGS in an autosomal dominant manner [57,61]. There are two main etiological mechanisms associated with the disorders caused by the mutants: “gain of function” and “loss of function” [62]. Gain-of-function mutations promote actin binding and abnormal aggregation of α-actinin-4 leading to podocyte damage.…”

“…Furthermore, the mutant may induce proteotoxicity in podocytes, impair the proteins' function by misfolding them and eventually cause apoptosis [60]. Loss-of-function mutation was not fully recognized as a pathogenic factor in human nephropathy until 2011 [62]. Before this, animal experiments [64,65] had shown that ACTN4 -absent mice exhibited abundant proteinuria with sclerosis of glomerular capillaries and podocyte foot process effacement, but whether humans were affected in the same way was unknown at that time.…”