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. 2015 May;87(5):948-62.
doi: 10.1038/ki.2014.386. Epub 2015 Jan 7.

p47(phox) contributes to albuminuria and kidney fibrosis in mice

Hongtao Wang  1 , Xiwu Chen  1 , Yan Su  1 , Paisit Paueksakon  2 , Wen Hu  1 , Ming-Zhi Zhang  1 , Raymond C Harris  3 , Timothy S Blackwell  4 , Roy Zent  3 , Ambra Pozzi  3
Affiliations

p47(phox) contributes to albuminuria and kidney fibrosis in mice

Hongtao Wang et al. Kidney Int. 2015 May.

Abstract

Reactive oxygen species (ROS) have an important pathogenic role in the development of many diseases, including kidney disease. Major ROS generators in the glomerulus of the kidney are the p47(phox)-containing NAPDH oxidases NOX1 and NOX2. The cytosolic p47(phox) subunit is a key regulator of the assembly and function of NOX1 and NOX2 and its expression and phosphorylation are upregulated in the course of renal injury, and have been shown to exacerbate diabetic nephropathy. However, its role in nondiabetic-mediated glomerular injury is unclear. To address this, we subjected p47(phox)-null mice to either adriamycin-mediated or partial renal ablation-mediated glomerular injury. Deletion of p47(phox) protected the mice from albuminuria and glomerulosclerosis in both injury models. Integrin α1-null mice develop more severe glomerulosclerosis compared with wild-type mice in response to glomerular injury mainly due to increased production of ROS. Interestingly, the protective effects of p47(phox) knockout were more profound in p47(phox)/integrin α1 double knockout mice. In vitro analysis of primary mesangial cells showed that deletion of p47(phox) led to reduced basal levels of superoxide and collagen IV production. Thus, p47(phox)-dependent NADPH oxidases are a major glomerular source of ROS, contribute to kidney injury, and are potential targets for antioxidant therapy in fibrotic disease.

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Figures

Figure 1
Figure 1. Loss of p47phox improves adriamycin-mediated albuminuria
Kidney injury was induced by a single tail vein injection of adriamycin (10 mg/Kg b.w.) in wild type (WT), integrin α1KO, p47phoxKO, integrin α1KO/p47phoxKO (DKO) mice and changes in body weight (A) and urine albuminuria (B, C) were examined over time. (*) indicates significant differences (p≤0.05) between α1KO and WT, or α1KO and p47phoxKO, or α1KO and DKO. Values are the mean ± SD of the number of mice indicated. (B) SimplyBlue staining of 1 μl urine showing elevated albuminuria in adriamycin-treated integrin α1KO mice compared to WT, p47phoxKO or DKO mice. Alb = mouse serum albumin (1.25 μg/lane). (C) Urine albumin excretion expressed as albumin-to-creatinine ratio (ACR) in untreated or adriamycin-treated WT, α1KO, p47phoxKO, and DKO mice. Values represent the mean ± SD of the mice indicated. Difference between injured WT and α1KO (*) or injured WT and p47phoxKO (**) or injured α1KO and DKO (δ) mice were significant (p≤0.05).
Figure 2
Figure 2. Loss of p47phox improves adriamycin-induced kidney injury
(A) Representative light micrographs of periodic-acid-Schiff (PAS) stained kidneys from uninjured (Control) or adriamycin-treated (4w-ADR) WT, α1KO, p47phoxKO, and DKO mice. Loss of p47phox rescues the severe glomerular and tubular damage observed in injured integrin α1KO mice. (B-D) Matrix mesangial expansion (MME) (B), global glomerulosclerosis index (GSI) (C), and interstitial fibrosis index (IFI) (D) were evaluated 4 weeks after adriamycin injection and scored as described in the Methods. Values represent the mean ± SD of the number of mice indicated.
Figure 3
Figure 3. Loss of p47phox improves adriamycin-induced matrix deposition
(A) Masson's Trichrome staining of kidneys from uninjured (Control) or adriamycin-treated (4w-ADR) WT, α1KO, p47phoxKO, and DKO mice. Note the presence of fibrillar collagen (blue) in both glomeruli and tubules of integrin α1KO mice which was reduced in kidneys of p47phoxKO and DKO mice. (B) Collagen IV staining of kidney sections from 4 weeks injured WT, α1KO, p47phoxKO and DKO mice. Note the high levels of glomerular collagen IV in integrin α1KO mice which were reduced in kidneys of p47phoxKO and DKO mice. (C) Equal amount of kidney lysates (20 μg/lane) from WT, α1KO, p47phoxKO, and DKO mice (n=3 shown) 4 weeks after adriamycin injection were analyzed by Western blot for levels of collagen IV and collagen I. (D) The collagen I, collagen IV and β-actin bands were analyzed by densitometry analysis and the levels of collagen IV are expressed as collagen I/β-actin or collagen IV/β-actin ratio. The values represent the mean ± SD of the number of kidneys indicated.
Figure 4
Figure 4. Loss of p47phox improves adriamycin-induced oxidative stress
(A) The levels of urinary F2-isoprostane in the mice indicated were analyzed by ELISA before or after adriamycin injection and were expressed F2-isoprostane/urine creatinine ratio. Values are the mean ± SD of the number of mice indicated. (*), (**) and (δ) are as in Fig. 1. (B) Paraffin sections of kidneys from uninjured (Control) or adriamycin-treated (1w-ADR and 4w-ADR) WT, α1KO, p47phoxKO, or DKO mice were stained with anti-nitrotyrosine (N-Tyr) antibodies to evaluate the degree of oxidative stress-induced tyrosine nitration (brown staining). Slides were counterstained with toluidine blue (blue staining). The high-magnification images on the right show nitrotyrosine staining in infiltrating cells (IC), mesangial cells (MC), podocytes (P), and proximal tubules (PT). (C) Equal amount of kidney lysates (20 μg/lane) from WT, α1KO, p47phoxKO, and DKO mice (n=3) 1 and 4 weeks after adriamycin injection were analyzed by Western blot for levels of nitro-tyrosine. (D) The nitro-tyrosine and β-actin bands were analyzed by densitometry analysis and the levels of tyrosine nitration are expressed as N-Tyr/β-actin ratio. The values represent the mean ± SD of 3 kidneys/genotype. (*), (**) and (δ) are as in Fig. 1.
Figure 5
Figure 5. Analysis of p47phox levels and localization in kidneys from uninjured and adriamycin-injured mice
(A) Equal amount of kidney lysates (20 μg/lane) from uninjured (Control) or adriamycin-treated (1w-ADR and 4w-ADR) WT, α1KO, p47phoxKO, and DKO mice (n=3 shown) were analyzed by Western blot for levels of p47phox and NOX4. (B) The p47phox, NOX4 and β-actin bands were analyzed by densitometry and the levels of p47phox and NOX4 are expressed as p47phox/β-actin or NOX4/β-actin ratio. Open circles represent values of individual kidneys, while the bar represents the mean value. (C) Equal amount of kidney lysates (Total) membrane enriched (Membrane) and membrane deprived (Cytosol) fractions (40 μg/lane) from uninjured (Control) or adriamycin-treated (4w-ADR) WT and integrin α1KO mice (n=3 shown) were analyzed by Western blot for p47phox localization. Membranes were re-blotted with anti-ERK, anti-N-cadherin, and anti β-actin antibodies to verify the purity of various fractions.
Figure 6
Figure 6. Loss of p47phox improves adriamycin-induced podocyte loss
(A) WT-1 staining in kidneys from uninjured (Control) or adriamycin-treated (1w-ADR and 4w-ADR) WT, α1KO, p47phoxKO, and DKO mice. Slides were counterstained with toluidine blue (blue staining). (B) The number of WT1 positive cells (arrowhead) was evaluated in 20 randomly chosen glomeruli per kidney. The values represent the mean ± SD of the kidneys indicated. (*) and (δ) are as in Fig. 1. Note the decreased number of podocytes in injured integrin α1KO mice which was reduced in kidneys of p47phoxKO and DKO mice.
Figure 7
Figure 7. Loss of p47phox improves adriamycin-induced macrophage infiltration
(A) F4/80 staining in kidneys from uninjured (Control) or adriamycin-treated (1w-ADR and 4w-ADR) WT, α1KO, p47phoxKO, and DKO mice. Slides were counterstained with toluidine blue (blue staining). (B) The number of macrophages (arrowhead) was evaluated in 20 randomly chosen field per kidney. The values represent the mean ± SD of the kidneys indicated. (*) and (δ) are as in Fig. 1. Note the increased number of macrophages in 4 week injured integrin α1KO mice which was reduced in kidneys of p47phoxKO and DKO mice.
Figure 8
Figure 8. Loss of p47phox improves adriamycin-mediated activation of pro-fibrotic pathways
(A) Equal amount of glomerular lysates (20 μg/lane) from 1 week adriamycin-treated WT, α1KO, p47phoxKO, and DKO mice were analyzed by Western blot for levels of phosphorylated and total EGF receptor (EGFR). (B) pEGFR and EGFR bands were quantified by densitometry analysis and the levels of activated EGFR are expressed as pEGFR/EGFR ratio. Values are the mean ± SD of 3-4 mice/genotype. (C) Primary mesangial cells isolated from the mice indicated were cultured in serum free medium. Twenty-four hours later, cells were incubated with 2 μM dihydro-rhodamine and 2 hours later ROS generation was determined by FACS as described in the Methods. The level of ROS was expressed as a ratio of fluorescence intensity of cells with dihydro-rhodamine vs. that of cells without dihydro-rhodamine. Data represent the mean ± SD of 3 samples/genotypes. The same experiment was repeated twice with similar result. (D) Equal amount of cell lysates (20 μg/lane) from the serum starved cells indicated were analyzed by Western blot for the phosphorylated and total EGFR, activated and total Rac, phosphorylated and total ERK, as well as collagen IV (CIV) levels. (E) The collagen IV and β-actin bands were analyzed and expressed as described in Fig. 3. The values represent the mean ± SD of 3 independent experiments.
Figure 9
Figure 9
Schematic reorientation of how loss of p47phox ameliorates the fibrotic response in integrin α1KO cells or mice. (A) Loss of integrin α1β1 leads to increased activation of EGFR (1) which in turn promotes p47phox membrane translocation and the assembly of the NADPH oxidase system by activating Rac1 (2) and/or ERK (3). NADPH-generated superoxide (4) can then exert a pro-fibrotic action by promoting collagen synthesis (5) and prolonging EGFR activation (6). (B) Loss of p47phox reduces this fibrotic response by dampening ROS production (4), collagen synthesis (5), and EGFR activation (6).
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