Obesity-induced kidney injury is attenuated by amelioration of aberrant PHD2 activation in proximal tubules

Abstract

The involvement of tissue ischemia in obesity-induced kidney injury remains to be elucidated. Compared with low fat diet (LFD)-mice, high fat diet (HFD)-fed mice became obese with tubular enlargement, glomerulomegaly and peritubular capillary rarefaction, and exhibited both tubular and glomerular damages. In HFD-fed mice, despite the increase in renal pimonidazole-positive areas, the expressions of the hypoxia-responsive genes such as Prolyl-hydroxylase PHD2, a dominant oxygen sensor, and VEGFA were unchanged indicating impaired hypoxic response. Tamoxifen inducible proximal tubules (PT)-specific Phd2 knockout (Phd2-cKO) mice and their littermate control mice (Control) were created and fed HFD or LFD. Control mice on HFD (Control HFD) exhibited renal damages and renal ischemia with impaired hypoxic response compared with those on LFD. After tamoxifen treatment, HFD-fed knockout mice (Phd2-cKO HFD) had increased peritubular capillaries and the increased expressions of hypoxia responsive genes compared to Control HFD mice. Phd2-cKO HFD also exhibited the mitigation of tubular damages, albuminuria and glomerulomegaly. In human PT cells, the increased expressions of hypoxia-inducible genes in hypoxic condition were attenuated by free fatty acids. Thus, aberrant hypoxic responses due to dysfunction of PHD2 caused both glomerular and tubular damages in HFD-induced obese mice. Phd2-inactivation provides a novel strategy against obesity-induced kidney injury.

Figure 1. Renal injury and histological changes in obese mice.

(A) Urinary excretion of albumin (u-Alb, left panel), neutrophil gelatinase-associated lipocalin (u-NGAL, middle panel) and cystatin C (right panel). (B) Representative pathology of kidneys of mice fed high fat diet (HFD) and low fat diet (LFD). Scale bar is 50 μm. Average areas of proximal tubule (PT) cells and glomeruli are shown in the right panel. (C) Immunostaining for CD34 (upper panel) in a low power field (LPF, upper pictures) and a high power field that focused on CD34+ cells (HPF, lower pictures). Lower bar graph shows the counts of CD34-positive cells reflecting the density of peritubular capillaries. Scale bar is 50 μm. (D) Immunostaining for pimonidazole (upper panel) and measurement of pimonidazole-positive areas, indicating a hypoxic state (lower panel). Scale bar is 50 μm. *p < 0.05 vs. LFD-fed mice, n = 8.

Figure 2. Lack of a hypoxic response in kidneys of HFD-fed mice.

(A) mRNA expression of PHD2 in high fat diet (HFD) and low fat diet (LFD) fed mice. Bar graph represents the quantification of immunostained areas. Scale bar indicates 50 μm. (B) mRNA expression of Vegfa, Slc2a1, Pgk1, and Ldha in HFD and LFD-fed mice. Bar graph represents the quantification of immunostained areas. Scale bar is 50 μm.

Figure 3. Tamoxifen-inducible proximal tubule-specific PHD2 knockout mice.

(A) Representative results of genotyping of Phd2 allele (upper panel) and Ndrg1-Cre (lower panel) mice. PCR products are indicated by arrows. (B) Real-time RT-PCR analysis of mRNA expression of Phd2 in kidney samples from each experimental group. Groups 1–4 represent low fat diet (LFD) and groups 5–8 represent high fat diet (HFD)-fed mice. Groups 2, 4, 6 and 8 represent mice harboring both Phd2^F/F and Ndrg1-Cre genes. Groups 3, 4, 7 and 8 represent mice treated with tamoxifen (Tam). *p < 0.05 vs. group 1, ^#p < 0.05 vs. group 5. (C) Representative immunostaining for PHD2 (left panel) and quantitation of immunostained areas (right panel) in Control or Phd2-cKO mice fed with LFD or HFD. (D) mRNA expression of Vegfa, Slc2a1, Pgk1, and Ldha in Control or Phd2-cKO mice fed with LFD or HFD. (E) Representative immunostaining for VEGF-A (left panel) and quantitation of immunostained areas (right panel). *p < 0.05 vs. Control LFD, ^#p < 0.05 vs. Control HFD. Scale bar indicates 50 μm.

Figure 4. Phenotypes of PHD2 conditional knockout mice fed HFD or LFD.

Body weight (A) serum concentrations of free fatty acids (B, FFA) in the four experimental groups. Phd2-cKO; tamoxifen (Tam)-inducible PT-specific Phd2 knockout mice, Control; littermate control mice, LFD; low fat diet, HFD; high fat diet. (C) Urinary excretion of albumin (u-alb), neutrophil gelatinase-associated lipocalin (u-NGAL) and cystatin C in the four experimental groups. *p < 0.05 vs. Control LFD; ^#p < 0.05 vs. Control HFD, n = 8. (D) CD34 immunostaining to assess peritubular capillary density. Bar graph shows the number of CD34-positive cells in a high power field (HPF). Scale bar is 50 μm. *p < 0.05 vs. Control LFD; ^#p < 0.05 vs. Control HFD, n = 8. (E) Immunostaining for pimonidazole to detect hypoxic tissue. Bar graph shows the quantitation of pimonidazole-positive areas. Scale bar is 50 μm. *p < 0.05 vs. Control LFD; ^#p < 0.05 vs. Control HFD, n = 8. (F) Representative findings of PTC in EM. In HFD mice, endothelial cells of PTC was enlarged, basement membrane was thickening (*), slit structures collapsed (black arrows), and lumen of PTC was narrowing, which were ameliorated in Phd2-cKO HFD mice. (G) Representative pathology of the kidneys, including glomeruli and tubulointerstitial lesions, from each experimental group. Bar graph shows the average areas of proximal tubule cells and glomeruli. *p < 0.05 vs. Control LFD; ^#p < 0.05 vs. Control HFD, n = 8.

Figure 5. Effects of insulin and free fatty acids on the hypoxic response of HK-2 cells.

The mRNA expression of SLC2A1 (Glut1: glucose transporter1) (A) and VEGFA (B). HK-2 cells were subjected to normoxic (normoxia) or hypoxic (hypoxia) culture conditions and pretreated with vehicle (groups 1 and 2), insulin (groups 3 and 4) or free fatty acids (FFA) (groups 5 and 6). Each bar graph represents the results of six experiments. *p < 0.05 vs. group 1, ^#p < 0.05 vs. group 3, n = 8.