The role of RHO GTPases and of coenzyme Q10 in nephrotic syndrome. The objective of this K99/R00 application is to investigate the role of RHO GTPase signaling and of the coenzyme Q10 (CoQ10) biosynthesis pathway in the pathogenesis of nephrotic syndrome (NS). Steroid-resistant NS (SRNS) accounts for 10~20% of NS in children and 40% of NS in adults and inevitably progresses to chronic kidney disease (CKD). SRNS constitutes the second most frequent cause of CKD in children (15%) and there is no curative therapy for this disease. CKD incurs a medical cost of more than $20 billion per year in the US The discovery of genes involved in monogenic forms of NS allowed for major progress in our understanding of glomerular filtration barrier physiology and of pathomechanisms of NS. Shared mechanism of SRNS converges in dysfunction of the podocyte. Gene identification implicated specific cellular signaling pathways in the pathogenesis of SRNS. I was involved in the implication of two specific signaling pathways, RHO GTPase signaling and CoQ10 biosynthesis, which I will further characterize within the proposed work. The applicant has previously identified mutations in ARHGDIA as a monogenic cause of SRNS and implicated RHO GTPase signaling in the pathogenesis of SRNS (Gee, J Clin Invest 123:3243, 2013). RHO GTPases control a wide range of cellular processes, including cell adhesion, migration, and cell division. However, it is not well understood how RHO GTPase signaling is regulated and contributes to the actin cytoskeletal changes in podocytes. Recently, the applicant identified a homozygous truncating mutation in NEK3 (NIMA-related kinase 3) in siblings with NS by whole exome sequencing. NEK3 encodes a member of the NIMA (never in mitosis A) family of serine/threonine protein kinases. NEK3 phosphorylates RHO GTPase guanine nucleotide exchange factors (GEFs) VAV1 and VAV2, leading to activation of RAC1. Our preliminary data show that NEK3 is expressed in developing and mature podocytes. However, the role of NEK3 in podocytes and its contribution to RHO GTPase signaling has not been studied. Therefore, I will examine NEK3 function and its role in RHO GTPase signaling using cultured podocytes and zebrafish model. This will further elucidate the role of RHO GTPase signaling in podocytes and in the pathogenesis of SRNS. CoQ10 is a lipid-soluble component of virtually all cell membranes and plays an essential role in the transport of electron in the mitochondrial respiratory chain. Mutations in PDSS2, COQ2 and COQ6, of which gene products are involved in CoQ10 biosynthesis, have been identified in individuals with SRNS. The applicant has recently demonstrated that mutations in ADCK4 cause SRNS in humans via defective CoQ10 biosynthesis (Ashraf & Gee, J Clin Invest 123:5179, 2013). Interestingly, SRNS caused by mutations in COQ6, COQ2 or ADCK4 can be treated with CoQ10 supplementation. However, the pathogenesis of SRNS resulting from CoQ10 deficiency is not well understood. Even though the kidney is one of the organs with the highest energy requirements, considering the essential role of CoQ10 in energy metabolism, it is not clear why mutations in ADCK4 cause isolated renal disease. Therefore, I have generated an Adck4 mouse model that I will investigate regarding the role of ADCK4 in regulating the CoQ10 biosynthesis pathway. Accomplishment of the proposed research will provide (1) new insights into the etiology and pathomechanisms of SRNS; (2) new animal models for studying NS; and (3) definitions of new molecular targets to treat NS. Based on my previous training and research experience, I am highly motivated and confident in attaining my overall career goal, which is to establish myself as an independent investigator in the field of kidney research at an academic institution. To achieve this goal, I wil investigate the disease mechanisms of NS using both in vitro (cell culture) and in vivo (zebrafish and mice) approaches. This K99/R00 application will facilitate the transition of my research career towards an independent investigator position. The training (K99) phase of this award will be mentored by Dr. Friedhelm Hildebrandt, who is an investigator of Howard Hughes Medical Institute and internationally recognized leader in the fields of human genetics of pediatric kidney diseases, including NS. The BCH/HMS has several collaborative research groups working on kidney development and diseases, with whom I have already interacted and published, providing an ideal environment for my training and career development.
Nephrotic syndrome constitutes the second most frequent cause of chronic kidney disease and there is no curative therapy for this disease. Chronic kidney disease incurs $20 billion of medicare cost per year in the US. The recent discovery of genes involved in nephrotic syndrome offered fundamental insights into our understanding of glomerular glomerular filtration barrier physiology and of pathogenic mechanisms of nephrotic syndrome. The functions of these genes have started to coalesce to distinct pathways such as RHO GTPase signaling (ARHGDIA and ARHGAP24) and coenzyme Q10 biosynthesis (COQ2, COQ6, PDSS2 and ADCK4). Elucidation of the roles of these pathways in podocytes will allow us to understand molecular mechanisms of nephrotic syndrome pathogenesis and to search for potential therapy for this currently untreatable disease.
