Characterization of novel glomerular proteins : role in physiology and disease

Abstract

Glomerular damage accounts for the majority of chronic kidney disease cases, the prevalence of which is dramatically escalating worldwide, mainly due to increase in diabetes and associated nephropathy. The costs for treatment of CKD represent an enormous burden to the health care system. Chronic kidney disease frequently progresses to end-stage renal disease with the only curative treatment options today are lifelong dialysis or kidney transplantation. In order to better understand and treat glomerular disease before it has reached this irreversible stage, it is imperative to understand the cell biology and physiology of the glomerulus including delineation of the molecular make-up of glomerular filtration barrier. This barrier constitutes the endothelial cells of the glomerular capillaries with its bound surface layer of a carbohydrate-rich meshwork (glycocalyx), the glomerular basement membrane, and the podocytes with the sub podocytes space and the attached slit diaphragm spanning between neighboring and interdigitating foot processes. Even though the concerted interplay of all layers of the glomerular filtration barrier is important for the ultra-filtration of the plasma, the podocytes and their injuries have recently been acknowledged as the major culprit of glomerular disease.

In this thesis work, I describe two novel podocyte specific proteins, TDRD5 and RHPN1, which we show in vivo to be important for the integrity of the glomerular filtration barrier.

TDRD5 has previously been considered a male germ cell line specific protein important for retro transposon silencing and spermiogenesis in mice. Here I show that it is expressed in mouse and human podocytes and is enriched in the zebrafish glomerulus. By using the in vivo morpholino knock down technique in zebrafish larvae it was demonstrated that TDRD5 expression is required for the proper formation of the zebrafish pronephros.

RHPN1 is a RHOA binding protein which functions as an attenuator of the polymerization of actin stress fibers by regulating the phosphorylation of nonmuscle myosin II regulatory light chain through RHOA downstream effectors. Deletion of the Rhpn1 gene leads to neonatal albuminuria and glomerular basement membrane abnormality. The phenotype was characterized.

As a part of efforts to elucidate expression signatures of glomerular diseases, the global glomeruli expression pattern of Adriamycin treated nephrotic mice was analyzed by RNA sequencing. The Adriamycin induced nephropathy mouse model mimics the human disease focal segmental glomerular sclerosis which constitutes one of the main causes of chronic kidney disease.

The diabetic nephropathy associated single nucleotide polymorphism 3q locus was shown in our work to be a remote cis-acting variant differentially regulating glomerular NCK1 expression, implicating an important role for glomerular NCK1 in diabetic nephropathy pathogenesis.