Urotensin II Receptor Deletion Mitigates Kidney Injury and Renal Lipid Remodeling in Mice Fed a Long‐Term High‐Fat Diet

Figures

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  FIGURE 1. High‐fat diet caused significant increases in (A) body weight, (B) fasting blood glucose, and (C) plasma insulin levels in both WT and UT KO. Fasting blood glucose levels at 21 weeks were not significantly elevated by HFD despite a significant increase observed in body weight. (D) Normalized urine albumin excretion was significantly elevated in only WT HFD but not KO HFD, suggesting a probable protective effect of UT KO. (E) Glucagon levels were unaltered. (mean ± SEM; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001, One‐way or two‐way ANOVA with Tukey's multiple comparisons test). A high‐fat diet increased (F) UII and (G) UT mRNA expression in kidney tissue. (mean ± SEM; n = 5; *p < 0.05, Mann Whitney test).

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  FIGURE 2. UT KO alleviates high‐fat diet‐induced interstitial fibrosis, and glomerular and tubular injury. A high‐fat diet significantly increased total kidney and intraglomerular collagen IV and fibronectin content. (A), (B) and (C) show an increase in collagen IV staining (brown) in WT HFD mice, which UT KO reversed. (D), (E) and (F) show increased fibronectin stain intensity in WT HFD, which was reversed in KO HFD. (G) Pathological scoring of Periodic Acid–Schiff (PAS) stained kidney sections showed significantly higher combined injury score in WT HFD, with injury mitigation in UT KO. Each panel shows glomeruli on the left and tubules on the right. WT HFD images exhibit moderate distention of the glomerular interstitium by a PAS‐positive material (yellow arrow—indicating glomerulonephritis/membranoproliferative changes) and well‐demarcated cytoplasmic vacuolation (white arrow), mainly of the epithelial cells of the proximal tubules. Glomerular distension with mesangial expansion was notably absent in KO HFD. (H) Combined injury scores (comprising inflammatory cell infiltrates, tubular casts, epithelial vacuolization, and mesangial expansion; n = 5; One‐way ANOVA with Tukey's multiple…

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  FIGURE 3. Transmission electron microscopic imaging of kidney glomeruli. (A) Representative images of mouse glomeruli showing glomerular basement membrane (GBM) and podocyte foot processes. Yellow arrowheads indicate areas of significant podocyte foot process effacement. White arrowheads show examples of normal foot processes. Yellow parallel lines indicate thickening of the GBM, white parallel lines show normal GBM. Scale bars: 2 μM. WT HFD showed (B) podocyte foot process effacement, and (C) thickening of GBM, which were mitigated by UT KO. Foot process width was calculated using the formula: FPW =π.4. Total length ofGBMnumber of foot processes, where the correction factor π/4 accounts for the random orientation of glomeruli in tissue sections (n = 22 glomerular sections from 5 mice; One‐way ANOVA with Tukey's multiple comparisons test; *p < 0.05, **p < 0.01, ****p < 0.0001).

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  FIGURE 4. Ut is expressed by podocytes in glomerular explants and in culture. Colocalization of (A) UT (red) and nephrin (green), and (B) UT (red) and podocin (green) in glomerular explants. (C) A nested PCR gel blot image of an immortalized podocyte cell line shows UT expression. WT‐1 is a podocyte marker. (D) Immunofluorescence images of cultured podocytes show UT (green) and F‐Actin (red).

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  FIGURE 5. HFD‐induced ER stress is attenuated by UT knockout in the mouse kidney, including the glomeruli. (A) Immunofluorescence imaging of kidney sections showed a significant increase in total kidney and glomerular staining of ER stress markers, GRP78/BiP (B, C) and CHOP (D, E) in WT HFD, the effects reversed by UT KO (n = 15 data points from 5 mice; One‐way ANOVA with Tukey's multiple comparisons test). A high‐fat diet (F) increased kidney tissue mRNA expression of ER stress marker GRP94 (n = 5; Kruskal–Wallis test) and (G) elevated plasma levels of the soluble ER stress marker CRELD2 (n = 5; One‐way ANOVA with Tukey's multiple comparisons test). These effects were alleviated by UT KO. (H) UII (5 μM) increased [Ca2+]I, and (I) depletion of the ER Ca2+ store with 10 μM thapsigargin prevented the action of UII on subsequent exposure. (J) UII‐induced ER Ca2+ release blocked by thapsigargin (mean ± SEM; n = 6–7, unpaired t‐test); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  FIGURE 6. UII stimulates TRPC6‐mediated SOCE and cation currents in podocytes. (A) UII‐induced SOCE in cultured mouse podocytes was significantly reduced by TRPC6 inhibition (SAR 7443), but not by TRPC5 inhibition (AC 1903). (B) Plot of Δ ratios depicting the SOCE‐associated increase in [Ca2+]i with UII, TRPC5, and TRPC6 inhibition. (mean ± SEM; n = 6–8; One‐way ANOVA with Tukey's multiple comparisons test). Whole‐cell patch‐clamp recordings of cation currents from cultured mouse podocytes show (C) lack of inhibition of UII‐induced cation currents by TRPC5 inhibitor (20 μM AC 1903). (D) TRPC6 inhibition (200 nM SAR 7443) significantly reduced UII‐induced cation currents. (mean ± SEM; n = 7–8; One‐way repeated measures ANOVA with Tukey's multiple comparisons test); *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 7. UII‐induced increase in podocyte ER stress is reversed by UT and TRPC6 inhibition. (A) UII (5 μM) increased ER stress in cultured mouse podocytes as indicated by the increase in fluorescence of GRP78 (Green) and CHOP (Red). Inhibition of UT (GSK1562590) and TRPC6 (SAR 7443) reversed this effect. Quantification of normalized fluorescent densities of (B) GRP78 (BiP) and (C) CHOP. (mean ± SEM; n = 25–26; One‐way ANOVA with Tukey's multiple comparisons test). UII treatment of cultured podocytes significantly elevated the concentrations of soluble ER stress markers (D) CRELD2 and (E) DNAJB11 in podocyte culture media. These were reversed by inhibition of UT (GSK) and TRPC6 (SAR) (mean ± SEM; n = 6–10; One‐way ANOVA with Tukey's multiple comparisons test); *p < 0.05, **p < 0.01, ****p < 0.0001.

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  FIGURE 8. Super‐resolution microscopy of cultured mouse podocytes shows significant fragmentation of Actin filaments with UII, an effect reversed by inhibition of UT and TRPC6. (A) Representative images of podocyte Actin filaments (Green) in control (DMSO), UII (5 μM), GSK (1 μM) + UII, and SAR (200 nM) + UII. (B–D) Activation of UT caused a significant increase in the fragmentation of Actin filaments as indicated by a decrease in the percentage of long filaments (151–200 and 101–150 a.u.) and an increase in short filaments (51–100 a.u.; mean ± SEM; n = 10–13; One‐way ANOVA with Tukey's multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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  FIGURE 9. UT KO mitigated HFD‐induced increase in kidney injury biomarkers. Plasma neutrophil gelatinase‐associated lipocalin (A) and cystatin C (B), and urinary retinol‐binding protein 4 (C) and α1‐macroglobulin (D) were elevated in WT HFD, but not in KO HFD mice (n = 5; One‐way ANOVA with Tukey's multiple comparisons test). (E) Immunofluorescence imaging of kidney slices shows increased expression of kidney injury biomarkers in WT HFD. Both total kidney and glomerular staining for tissue inhibitor of metalloproteinase 2 (F, G) and insulin‐like growth factor‐binding protein 7 (H, I) were significantly increased in WT HFD. This effect was reversed by UT KO (n = 15–25 data points from 5 mice; One‐way ANOVA with Tukey's multiple comparisons test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  FIGURE 10. UT deletion attenuated HFD‐induced alterations in the renal lipid profile. Volcano plots show differentially regulated lipid species in kidney samples from KO LFD, WT HFD, and KO HFD mice relative to the WT LFD group. No lipid species were significantly altered in the KO LFD group (FDR > 0.05). In contrast, 52 lipid species were differentially regulated in the WT HFD group at FDR < 0.05, whereas only 11 of these species remained significantly altered in the KO HFD group. The Venn diagram summarizes the number of unique and shared significantly altered lipid species among the three groups relative to the WT LFD group. Of the 52 lipid species altered in WT HFD mice, 41 were unique to the WT HFD group and 11 were shared with the KO HFD group. No significantly altered lipid species were unique to the KO LFD or KO HFD groups, and no shared lipid species involved the KO LFD group. These findings indicate that UT deletion attenuated the HFD‐induced differential regulation of 41 of the 52 lipid species identified in WT mice.