Netrin-1 increases epithelial cell proliferation and promotes renal cyst formation by activating Rac2 in collecting duct cells

Abstract

Despite recent advances in our understanding of the pathogenesis of polycystic kidney disease (PKD), the underlying molecular mechanisms involved in cystogenesis are not fully understood. Common features of PKD are excessive epithelial cell proliferation and fluid-filled cyst formation in the kidney, leading to loss of kidney function. We recently demonstrated that netrin-1 is upregulated in primary human PKD cystic cells and in the kidneys of rodent models of PKD. Moreover, overexpression of netrin-1 in tubular epithelium increases cell proliferation (p<0.01) and induces renal cyst development (p<0.0001), while decreasing netrin-1 reduces cyst growth (p<0.01) and improves kidney function (p<0.05). However, the underlying mechanism by which netrin-1 induces cystic cell proliferation is unknown. We hypothesize that netrin-1 increases cystic epithelial cell proliferation by activating Ras-Related C3 Botulinum Toxin Substrate 2 (Rac2) in collecting duct (CD). 4-week-old male tubular overexpressed netrin-1 transgenic mice (NTN-1 Tg6) and 8-week old Pkd1RC/RC mice were randomized to receive vehicle or the specific Rac2 inhibitor EHT 1864 (20 mg/kg/48hr i. p; n=4-5/group) for 4 weeks. Blood and kidneys were collected for biochemical, histological, and Western blot analysis. Renal co-localization of Rac2 with CD specific marker Auaphorin-2 in kidney of human and mouse PKD was assessed by immunofluorescence staining and protein expression was measured by Western blot analysis. Renal Rac2 activity was measured using Rac2 activation kit. Cystic index were measured using H&E staining. Cell proliferation was measured using MTT assay and renal function was measured using BUN assay kit. Rac2 is upregulated in the CD of both human PKD and Pkd1RC/RC mice (p<0.01) compared to controls. Moreover, overexpression of netrin-1 activates Rac2 in the cystic kidney (p<0.001) and in human cystic cells (p<0.05). Overexpression of Rac2 increases cell proliferation and phosphorylation of ERK in CD cells (p<0.01). Inhibition of Rac2 reduces netrin-1-induced CD cell proliferation (p<0.01) and cyst growth (p<0.01) in both netrin-1 transgenic (NTN-1 Tg6) and Pkd1RC/RC mice. Our results demonstrate that overexpression of netrin-1 increases cystic epithelial cell proliferation via activating Rac2 in CD cells and contributes to cyst growth. Future studies will be carried out to dissect the downstream signaling mechanism of netrin-1/Rac2 mediated cell proliferation cyst formation in PKD. This work is supported by Intramural grant award (IGPP00043) from Augusta University to R. Mohamed; National Institute of Health (1P01HL134604-01) to J.C. Sullivan. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.