Discussion

1. A B Methods Conclusion Acknowledgments SMAD3­dependent and independentpathways in podocyte de­differentiationA. Ghayur, P.J. Margetts Department of Medicine, McMaster University, Hamilton, Ontario, Canada Introduction Results Discussion • Proteinuria is associated with the progression to end stage renal disease • Transforming growth factor beta 1 (TGFB1) is a key cytokine in mediating proteinuria • Not much is known about the downstream pathways that mediates the renal damage and proteinuria • I used an anti-glomerular basement membrane (GBM) glomerulonephritis model in SMAD3+/+ and SMAD3-/-mice to induce glomerulonephritis and proteinuria • Synaptopodin expression. (A) Control SMAD3+/+ and (B) SMAD3-/-mice. In anti-GBM antibody treated (C) SMAD3+/+ and (D) SMAD3-/-mice, there was a significant decrease in synaptopodin staining. Quantification is shown in (E) • We used both SMAD3+/+ and SMAD3-/-mice to study proteinuria and podocyte de-differentiation • In both SMAD3+/+ and SMAD3-/- treated groups, there was significant foot process effacement and decrease in synaptopodin expression • SMAD3-/-mice had transient proteinuria which recovered despite persistence of podocyte effacement and synaptopodin downregulation • Blocking SMAD3 or SMAd2 using SiRNA did not protect nephrin or synaptopodin from TGFB1 induced downregulation in cultured podocytes • Proteinuria (A), or protein: creatinine ratio (B) was significantly elevated in SMAD3+/+ mice • SMAD3-/-mice treated with anti-GBM antibody also demonstrated a very transient proteinuric response • Animal study • Eight week old C57Bl/6 (SMAD3+/+) mice and SMAD3 knockout (SMAD3-/-) transgenic mice were used • On day -4 we sensitized mouse by normal sheep serum by intraperitoneal(IP) administration in complete adjuvant • Four days later (day 0), animals received 5 mg/20 g of rabbit anti-mouse GBM via tail vein injection • Urine was collected on days 3, 7, 21, and 56. • Time points – day 7, day 21 and 56 • Analysis • Kidney taken for histology, urine collected for 24 hrs for protein concentration • Protein and gene expression of treated and control animals was compared • Immortalized podocytes were cultured and treated with SiRNA for SMAD3 and SMAD2 and studied for the effects of TGFB1 administration • Cultured podocytes were treated with SMAD2 or SMAD3 siRNA. There is significant, specific, downregulation of corresponding proteins by siRNA • In summary we hypothesized that there are both SMAD dependent and independent pathways that mediate podocyte injury and by using SMAD knockout animals we have clearly shown that SMAD3-/-mice are not protected from renal damage and proteinuria. • Other pathways that could be activated by TGFB1 are detrimental in kidney diseases and further work is required to identify these pathways. • Electron micrographs of glomeruli. A) Control Smad3 +/+ and B) SMAD3-/-mice. Anti-GBM treated C) SMAD3+/+ and D) SMAD3-/-mice show significant foot process effacement quantified by the number of foot processes per μm of GBM (E). • This work was supported by Kidney Foundation of Canada and St. Joseph’s Healthcare • Ayesha Ghayur is a PhD candidate • Recombinant TGFB1 significantly suppressed nephrin and synaptopodin protein (A) concentration in podocyte cell culture. SiRNA to SMAD2 or SMAD3 was unable to rescue TGFB mediated suppression of nephrin (B), or synaptopodin (C).