Western diet exacerbates a murine model of Balkan nephropathy

Abstract

Aristolochic acid (AA) ingestion causes Balkan nephropathy, characterized by tubular injury and progression to chronic kidney disease (CKD). AA is taken up by proximal tubule cells via organic anion transport and induces p21-mediated DNA damage response, but little is known about dietary modulating factors. Western diet (WD) is rich in saturated fats and sugars and can promote metabolic disorders and CKD progression. Here, we determined the impact of WD on AA-induced kidney injury. Five-week-old male C57BL/6J mice were fed WD or normal chow (NC) for 8 wk, followed by administration of AA every 3 days for 3 wk. Measurements were performed after the last injection and following a 3-wk recovery. Independent of dosing AA by body weight (3 mg/kg/day) or same dose/mouse (0.1125 mg/day), the AA-induced increase in plasma creatinine and reduction of hematocrit were greater in WD versus NC. This was associated with increased kidney gene expression in WD vs. NC of markers of DNA damage (p21), injury (Kim1 and Ngal), and inflammation (Tnfa) and kidney fibrosis staining. WD alone increased fractional excretion of indoxyl sulfate by 7.5-fold, indicating enhanced kidney organic anion transport. Kidney proteomics identified further WD-induced changes that could increase kidney sensitivity to AA and contribute to the altered response to AA including weakening of energy metabolism, potentiation of immune and infection pathways, and disruption in RNA regulation. In conclusion, WD can increase the susceptibility of mice to Balkan nephropathy, possibly in part through facilitating kidney uptake of the organic anion AA.NEW & NOTEWORTHY This study shows that a Western diet (WD) aggravates a murine model of Balkan nephropathy induced by the application of the organic anion and nephrotoxin aristolochic acid (AA). Mechanistically, this may involve WD-induced kidney organic anion secretion, which can facilitate the AA uptake into proximal tubular cells and thereby contribute to the injury. Kidney proteomics identified further changes induced by feeding a WD that could have increased the sensitivity of the kidney to stress and injury.

Keywords: Western diet; aristolochic acid; obesity; organic anion transporter; proximal tubule.

Graphical abstract

Figure 1.

Experimental timeline. C57BL/6J mice (5-wk-old) were treated with a Western diet or regular chow for 8 wk, then given an intraperitoneal injection of aristolochic acid (AA; 3 mg/kg or fixed dose, 0.1125 mg) or vehicle (shown by arrowheads) every 3 days for 3 wk and were then left for another 3 wk to recover. Before the first injection, measurements were taken to obtain basal values (“Basal”). Further measurements were taken 3 days after the last injection to examine the active phase (“After injections”), and 3 wk after the last injection, when mice were euthanized and kidney, heart, and liver samples were harvested to examine the recovery/chronic phase (“harvest”). Urine and blood samples were paired with body weight measurements to obtain basal values and after injection and at harvest.

Figure 2.

Body and organs weights. A: basal body weight (wt) (n = 18–20 mice pooled). B: total dose of applied AA. C: changes of body weight after aristolochic acid (AA) injections (After Inj) and at harvest compared with basal values. D: weights of kidney, liver, and heart. Values are means ± SE and dots show individual mouse data. Student’s t test was used for comparison between Western diet (WD) and control diet groups treated with vehicle. Two-way ANOVA on AA groups to probe for a significant effect of diet (P_WD), how AA was dosed (dosing per body weight vs. fixed amount) (P_{AA mode}), and their interaction (P_inter). If the interaction was statistically significant, then a pair-wise multiple comparison procedure (Holm–Sidak method) identified the significant effects. *P < 0.05 vs. control diet. #P < 0.05 vs. AA (3 mg/kg). n = 4 mice for vehicle groups, n = 7–8 mice for AA groups.

Figure 3.

Western diet (WD) aggravated the aristolochic acid (AA)-induced fall in hematocrit and rise in plasma creatinine. A: hematocrit at basal (n = 19–20 mice pooled), and changes of hematocrit after AA injections (After Inj) and at harvest as compared with basal values. B: plasma creatinine (Pcrea) after AA injections and at harvest. C: urinary albumin-to-creatinine ratio (UACR) after AA injections and at harvest. D: kidney mRNA expression of nephrin (Nphs1) and megalin (Lrp2). mRNA expression was normalized to ribosomal protein L19 (Rpl19) gene and vehicle group. Values are means ± SE and dots show individual mouse data. Student’s t test was used for comparison between WD and control diet groups treated with vehicle. Two-way ANOVA on AA groups to probe for a significant effect of diet (P_WD), how AA was dosed (dosing per body weight vs. fixed amount) (P_{AA mode}), and their interaction (P_inter). If the interaction was statistically significant, then a pair-wise multiple comparison procedure (Holm–Sidak method) identified the significant effects. *P < 0.05 vs. control diet. #P < 0.05 vs. AA (3 mg/kg). n = 4 mice for vehicle groups, n = 7–8 mice for AA groups.

Figure 4.

Western diet (WD) enhanced aristolochic acid (AA)-induced markers of tubular injury and inflammation. A: kidney mRNA expression of p53 and p21. B: kidney mRNA of hepatitis a virus cellular receptor 1 [Harvc1, kidney injury molecule-1 (KIM-1)] and Lipocalin-2 [Lcn2, NGAL, neutrophil gelatinase-associated lipocalin]. C: kidney mRNA expression of tumor necrosis factor alfa (Tnfa) and monocyte chemoattractant protein-1 (MCP-1) (Ccl2). mRNA expression was normalized to ribosomal protein L19 (Rpl19) gene and vehicle group. Values are means ± SE and dots show individual mouse data. Two-way ANOVA on AA groups to probe for a significant effect of diet (P_WD), how AA was dosed (dosing per body weight vs. fixed amount) (P_{AA mode}), and their interaction (P_inter). If the interaction was statistically significant, then a pair-wise multiple comparison procedure (Holm–Sidak method) identified the significant effects. *P < 0.05 vs. control diet. #P < 0.05 vs. AA (3 mg/kg). n = 4 mice for vehicle groups, n = 7–8 mice for AA groups. The statistically lower Tnfa expression in the WD group on fixed (0.9 mg total AA) vs. body weight-adjusted (0.73 mg total AA) dosing of AA cannot be explained and could be by chance.

Figure 5.

Western diet (WD) exacerbates urinary tubular injury markers and renal fibrosis in aristolochic acid (AA)-induced nephropathy. A: Urinary kidney injury molecule-1 (KIM1) and neutrophil gelatinase-associated lipocalin (NGAL) to creatinine ratios after AA injections and at harvest from mice treated with vehicle or fixed dose AA. Pairwise Student’s t tests were performed with Bonferroni correction for multiple comparisons. B: Masson’s trichrome staining of cortex and medulla from mice treated with vehicle or fixed dose AA. Scale bars = 100 µm. C: quantification of the fibrotic area according to Masson’s trichrome staining with investigator blinded to study groups. D: Western blotting for kidney α-smooth muscle actin (α-SMA) protein expression, which was normalized to β-tubulin. Two-way ANOVA was used to probe for a significant effect of diet (P_WD), AA (P_AA), and their interaction (P_inter). If the interaction was statistically significant, then a pair-wise multiple comparison procedure (Holm–Sidak method) identified the significant effects. *P < 0.05 vs. control diet. #P < 0.05 vs. vehicle. n = 4 mice for vehicle groups, n = 7–8 mice for AA groups, except for western blotting: n = 4.

Figure 6.

Western diet (WD) alters aristolochic acid (AA)-induced kidney proteomics response. A: differentially expressed (DE) proteins among the groups. B: principal component analysis (PCA). C: heatmap of DE proteins shows three main clusters when comparing the response to AA in mice on a WD versus normal chow (NC). Cluster 3 can be further divided into three subclusters.

Figure 7.

Western diet (WD) upregulates Slc22a8 (organic anion transporter 3, OAT3) mRNA expression and renal indoxyl sulfate (IS) excretion. A: kidney mRNA expression of Slc22a6 (OAT1) and Slc22a8 (OAT3) at harvest. mRNA expression was normalized to ribosomal protein L19 (Rpl19) gene and vehicle group. B: plasma and urine indoxyl sulfate (IS) and fractional excretion of IS (FE IS) before injections (n = 9–10). Student’s t test was used for comparison between WD and normal chow (NC) groups treated with vehicle or comparison of IS values before injections. Two-way ANOVA was used to probe for a significant effect of diet (P_WD), how AA was dosed (dosing per body weight vs. fixed amount) (P_{AA mode}), and their interaction (P_inter). *P < 0.05 vs. control diet. n = 4 mice for vehicle groups, n = 7–8 mice for AA groups.

Figure 8.

Western diet (WD) aggravates aristolochic acid (AA)-induced acute kidney injury (AKI) and subsequent chronic kidney disease (CKD). A WD aggravated the AA-induced increase in markers of tubular injury and plasma creatinine and the decrease in hematocrit in the acute and chronic phases, as well as proinflammatory TNFa levels and the renal fibrotic response. A WD increased kidney organic anion secretion, maybe to compensate for impaired liver metabolism. This can facilitate AA uptake into proximal tubular cells and thereby contribute to injury. Kidney proteomics identified multiple changes induced by feeding a WD that could have increased the sensitivity of the kidney to stress and injury (see discussion), and contributed to the observed altered proteomic response to AA including weakening of energy metabolism and potentiating immune and infection pathways associated with a breakdown of RNA regulating pathways.