Characterisation of pathophysiological function of NEDD4-2 in kidney

Abstract

Nedd4-2 (NEDD4L, neural precursor cell expressed, developmentally down regulated 4-like) belongs to the Nedd4 family of ubiquitin ligases. These ligases aid in maintaining cellular homeostasis by binding to, and ubiquitinating a number of membrane proteins to initiate their internalization and turnover. Previous work from our laboratory has suggested that Nedd4-2 plays an essential role in regulating ion channels, especially the epithelial sodium channel and voltage gated sodium channels. The misregulation of these channels has been implicated in multiple channelopathies, including hypertension and cystic fibrosis like disease. This study characterises a previously unknown function of Nedd4-2 in the kidney. In order to understand this significance of Nedd4-2 in renal homeostasis, the previously generated Nedd4-2⁻ʹ⁻ (Nedd4-2 knockout) mice (Boase et al., 2011) were characterised. The initial histological examination of postnatal kidneys suggested renal cyst formation in Nedd4-2⁻ʹ⁻ animals. Further analysis revealed that Nedd4-2 loss results in renal dysplasia. Nedd4-2⁻ʹ⁻ mice showed variable renal cystic index, onset of cyst formation starting from postnatal day 2 and progressing until the Nedd4-2⁻ʹ⁻ animals die due to respiratory distress around day 19-21. To investigate the prevalence of the cystic phenotype in other tissues histological analysis was performed in pancreas, liver, spleen, colon, stomach and thymus with no significant pathological differences observed in the knockout mice. The Nedd4-2⁻ʹ⁻ kidneys showed increased cell proliferation, with no apoptotic differences in the cells lining the cystic epithelia suggesting an imbalance between cell proliferation and apoptosis in cyst formation. The cyst formation and kidney development disorders are associated with malformation in the kidney tissue leading to extracellular matrix modification with enhanced accumulation of collagens causing increased interstitial fibrosis. The Nedd4-2⁻ʹ⁻ kidneys showed increased interstitial fibrosis, collagen-1 accumulation and expression during progression of the disease. The renal tissue membrane is made up of polysaccharides, glycogen and mucin, the Nedd4-2⁻ʹ⁻ kidneys were found to have decreased accumulation of polysaccharides. The cysts in the Nedd4-2⁻ʹ⁻ kidneys originated from different parts within the nephron. The larger cysts originated from loop of Henle and with the smaller cysts from collecting ducts and distal convoluted tubules. The cystic progression is dependent on cAMP flux initiated by fluid secretion within the cyst. The postnatal day 19 cystic kidneys in Nedd4-2⁻ʹ⁻ animals showed increased cAMP levels suggesting cystic disease progression. As renal cystic disorders may arise from abnormal cilia, ciliary anomalies were found in the Nedd4-2⁻ʹ⁻ around the cysts suggesting importance of cilia in kidney cyst formation. Polycystins are known to be involved in renal cyst development with polycystin-1 and polycystin-2 together known to form calcium ion channel. To investigate the role of Nedd4-2 in the regulation of these polycystins, in vitro and in vivo studies were conducted. In vitro studies suggested that depletion of Nedd4-2 results in increased expression of polycystin-1 on the cell membrane with a decrease in polycystin-2 levels. Further, polycystin-1 was found to be ubiquitinated by Nedd4-2 in vitro providing the first evidence of Nedd4-2-mediated regulation of polycystins. In vivo Polycystin-1 was up-regulated in the Nedd4-2⁻ʹ⁻ kidneys suggesting an important role of Nedd4-2 in regulation of polycystins in cyst formation. To analyse the transcriptional signature of the phenotype seen in the knockout kidneys, postnatal day 19 kidneys from wild-type and Nedd4-2⁻ʹ⁻ mice were subjected to RNA sequencing highlighting 537 genes that were differentially expressed between wild-type and knockout kidneys, with 167 genes down-regulated and 370 genes significantly up-regulated in the absence of Nedd4-2. DAVID and Ingenuity pathway analyses was used to highlight the importance of genes involved in extracellular matrix modification, cell junction formation and cell-cell communication. The work presented in this thesis thus provides new information on the pathophysiological role of Nedd4-2 in kidney and identifies polycystin-1 as a Nedd4-2 target, along with transcriptional changes which may partially explain the cystic phenotype associated with renal dysplasia.