Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution

Abstract

Background: NADPH oxidase 4 (NOX4) catalyzes the formation of hydrogen peroxide (H₂O₂). NOX4 is highly expressed in the kidney, but its role in renal injury is unclear and may depend on its specific tissue localization.

Methods: We performed immunostaining with a specific anti-NOX4 antibody and measured NOX4 mRNA expression in human renal biopsies encompassing diverse renal diseases. We generated transgenic mice specifically overexpressing mouse Nox4 in renal tubular cells and subjected the animals to the unilateral ureteral obstruction (UUO) model of fibrosis.

Results: In normal human kidney, NOX4 protein expression was at its highest on the basolateral side of proximal tubular cells. NOX4 expression increased in mesangial cells and podocytes in proliferative diabetic nephropathy. In tubular cells, NOX4 protein expression decreased in all types of chronic renal disease studied. This finding was substantiated by decreased NOX4 mRNA expression in the tubulo-interstitial compartment in a repository of 175 human renal biopsies. Overexpression of tubular NOX4 in mice resulted in enhanced renal production of H₂O₂, increased NRF2 protein expression and decreased glomerular filtration, likely via stimulation of the tubulo-glomerular feedback. Tubular NOX4 overexpression had no obvious impact on kidney morphology, apoptosis, or fibrosis at baseline. Under acute and chronic tubular injury induced by UUO, overexpression of NOX4 in tubular cells did not modify the course of the disease.

Conclusions: NOX4 expression was decreased in tubular cells in all types of CKD tested. Tubular NOX4 overexpression did not induce injury in the kidney, and neither modified microvascularization, nor kidney structural lesions in fibrosis.

Keywords: Chronic kidney disease; Diabetes; IgA nephropathy; Kidney fibrosis; NOX4; Renal biopsy; Tubular cells.

Graphical abstract

Fig. 1

NOX4 Antibody validation, NOX4 expression pattern in the normal human glomerular compartment, and in renal nephropathies. Representative Western blot analysis of T-Rex cells overexpressing (right lane) or not (left lane) the human NOX4 protein (A). Representative images of NOX4 immunohistochemical analysis of T-REX cells overexpressing (tetracycline inducible) (C) or not (B), the human NOX4 protein. D–F: Representative images of NOX4 immunohistochemical analysis of normal human kidney, diabetic and IgA nephropathies. In normal glomeruli, NOX4 expression is restricted to parietal glomerular epithelial cells (D, upper and lower panel). NOX4 expression is observed in the glomerular mesangium of diabetic nephropathy (E, upper and lower panel) and IgA nephropathy (F, upper and lower panel).

Fig. 2

NOX4 expression in normal renal tubular cells and in tubulo-interstitial injury. A-B: Representative images of NOX4 immunohistochemical analysis of normal human kidney. A marked cytoplasmic NOX4 immunostaining (brown) is observed in proximal tubules, whereas expression is much lower in distal segments of the nephron (lower magnification of the image shown in Fig. 1D) (A). NOX4 and Na, K-ATPase (red) double immunostaining. NOX4 (in brown) appears predominant at the basolateral side of proximal tubular cells and colocalizes with basolateral sodium-potassium pump Na, K-ATPase (B). C–D: Representative images of NOX4 immunohistochemical analysis in chronic tubulo-interstitial injury. NOX4 expression is decreased and almost absent in atrophic tubules in diabetes nephropathy (C). Periodic acid Schiff staining (D) showing area of fibrosis. Analysis of NOX4, GSTA1, NOX2 and TGFβ1 gene expression in the Affymetrix microarray expression dataset generated by the European Renal cDNA Kröner-Fresenius Biopsy bank demonstrating a marked decrease in NOX4 mRNA expression correlating with the different stages of CKD (E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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.)

Fig. 4

Pro-survival, antioxidant and angiogenesis pathways are activated in Nox4^KI mice at baseline. Gene expression analysis of pro-survival (Nrf2, Bcl2) and antioxidant pathway (Gstα, Nqo1) genes at baseline. The gene expression level for each gene is normalized to the housekeeping gene Rplp0 (A). Representative Western blot images of proteins involved in the cell survival, angiogenesis and fibrosis processes. Quantitation of Western blot experiments are shown in (B).

Fig. 5

Gene expression changes in Wt and Nox4^KI animals subjected to 3 days and 10 days of UUO. A-B: Gene expression analysis on Wt and Nox4^KI kidneys harvested 3 days (A) and 10 days (B) after subjecting the animals to UUO. The non-obstructed kidneys of the same animals were used as controls. Gene expression levels of Nox4, Nox2, pro-survival pathway genes (Bcl2, Nrf2) and antioxidant genes (Gstα, Nqo1) are shown. The gene expression level for each gene is normalized to the housekeeping gene Rplp0.

Fig. 6

Renal NOX4 protein expression is decreased in a severe fibrotic renal disease mouse model but not in NOX4^KI mice. A–B: Western blot analysis on Wt and Nox4^KI kidneys harvested 3 days (A) and 10 days (B) after subjecting to UUO. The non-obstructed kidneys were used as controls. Protein expression levels of NOX4, and proteins implicated in cell survival pathways (NRF2, BCL2) and fibrosis (Collagen 1α, Fibronectin and α-SMA) and the quantification are shown.

Fig. 7

Constitutive Nox4 expression in renal tubular cells does not prevent fibrotic disease progression. A, B, E: Representative images of Sirius red staining and quantification on the sections of Wt and Nox4^KI kidneys harvested 3 days (A, E) and 10 days (B, E) after subjecting to UUO. Endomucin immunostaining and quantification on the sections of Wt and Nox4^KI kidneys harvested 3 days (C, F) and 10 days (D, F) after subjecting to UUO are presented. The non-obstructed kidneys were used as controls. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)