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Abstract

Glomerulosclerosis is a hallmark of diabetes (D) and hypertension (HBP) induced kidney failure. Here, we studied the combined effect of D-HBP on the kidney using expansion microscopy for nanoscale imaging of podocyte foot processes (FP). Method: In 6 weeks old male double transgenic rats (dTGRNeph-hAT1R;Cyp1a1mRen2dF1=dTGR) and TGRCyp1a1mRen2d (WT) we induced diabetes by streptozotocin (60mg/kg i.p.) for 8 weeks, and Hypertension by indole-3-carbinol (IC3) (0.0125% in chow), alone or in combination (D-HBP). After perfusion with 2%PFA, kidneys were ~4.5 times expanded using acrylamide/sodium-acrylate gel embedding, SDS/75°C+90°C denaturation, podocin immunohistochemistry and imaging by confocal microscopy after expansion in H2O. We measured FP width as the distance between two podocin signals and normalized for the expansion factor. Results: D-HBP rats developed massive albuminuria, and shown a drop in GFR which correlated with histological alterations in glomeruli, including mesangial expansion, podocyte loss and adhesion of the glomerular tuft to the Bowman’s capsule. HBP rats showed very mild histological injury. D rats did not differ from controls. AT1R overexpression in podocyte aggravated the glomerular damage, drop in renal function in HBP and D-HBP but did not affect controls. Glomerular size increased similarly in both HBP and D-HBP and did not differ between dTGR and WT. In dTGR- and WT-controls (CTRL), podocyte FP architecture was regular with long thin processes (200±20 nm width). In HBP, FPs were shortened and widened, but regularly organized. In D-HBP, FP structure within one glomerulus varied from regular with shortened and widened FP to irregular structures to regions with the total loss of FP (average FP width 400±20 nm). Conclusions: In contrast to D and HBP, combined D-HBP induced rapid progression of glomerulosclerosis and podocyte damage. Podocyte’s AT1Rs aggravate damage index. To able to visualize those alterations, we established a method, by modifying ExM, that enables the nanoscale evaluation of podocyte FPs and slit membrane in 3-D by using traditional immunohistochemistry and confocal microscopy.