Fig. 3.

Generation and validation of tubular cell specific Nox4 knock-in mice.

NOX4 protein expression in renal cortex harvested from wild-type (Wt) mice and mice subjected to 10 days of Unilateral Ureteral Obstruction (A). B–C: Gene expression and Western blot analysis of renal cortex and medulla dissected from Wt and tubular cell specific Nox4^KI kidneys. Nox4 mRNA expression (B) and NOX4 protein levels (C) are increased in both cortex and medulla of Nox4^KI kidneys. HE-higher exposure of the blot. Quantification of high (70 kDa) and low molecular weight (35 kDa) NOX4 protein bands are shown. D–F: Detection of H₂O₂ production in the primary epithelial cells and renal organoids isolated from Wt and Nox4^KI kidneys. Fold increase in Amplex red fluorescence in the primary epithelial cells is shown in D. Expression levels of Nox4 mRNA in the cultured primary epithelial cells (E). Panel showing the visualization of increased H₂O₂ generation in renal organoids isolated from Nox4^KI kidney by the production of intense florescent pink color and the fold increase in H₂O₂ production (F). Representative Sirius red images of Wt and Nox4^KI kidney sections demonstrating that the overall kidney structure was not modified (G). Transcutaneous glomerular filtration rate measurement by sinistrin-FITC excretion rate on Wt and Nox4^KI mice (H). Estimation of glomerular filtration rate by endogenous creatinine clearance (I). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)