Proximal Tubular Cannabinoid-1 Receptor Regulates Obesity-Induced CKD

Abstract

Obesity-related structural and functional changes in the kidney develop early in the course of obesity and occur independently of hypertension, diabetes, and dyslipidemia. Activating the renal cannabinoid-1 receptor (CB₁R) induces nephropathy, whereas CB₁R blockade improves kidney function. Whether these effects are mediated via a specific cell type within the kidney remains unknown. Here, we show that specific deletion of CB₁R in the renal proximal tubule cells did not protect the mice from obesity, but markedly attenuated the obesity-induced lipid accumulation in the kidney and renal dysfunction, injury, inflammation, and fibrosis. These effects associated with increased activation of liver kinase B1 and the energy sensor AMP-activated protein kinase, as well as enhanced fatty acid β-oxidation. Collectively, these findings indicate that renal proximal tubule cell CB₁R contributes to the pathogenesis of obesity-induced renal lipotoxicity and nephropathy by regulating the liver kinase B1/AMP-activated protein kinase signaling pathway.

Keywords: AMPK; CB1 receptor; Endocannabinoids; Renal Proximal Tubular Cell; chronic kidney disease; obesity.

Graphical abstract

Figure 1.

High-fat diet-induced obesity results in increased renal eCB tone. CB₁R gene locus containing flox sites (red triangles) with a forward G50 primer and reverse G51 and G53 primers (black arrows). Cre recombination results in a 600 bp product, whereas no recombination results in a 500 bp product (A). Cre recombination is shown only in RPTCs isolated from RPTC-CB₁R^−/− mice and not in cells extracted from wild-type mice or in tissues such as liver, brain, muscle, inguinal, and retroperitoneal fat pads and pancreas. (B). Loss of CB₁R protein expression specifically in RPTCs (black arrows), with its normal expression in the distal tubule and glomerulus in RPTC-CB₁R^−/− mice compared with their wild-type littermate control animals (C). Whole renal CB₁R mRNA expression levels were greatly reduced in RPTC-CB₁R^−/− mice compared with their wild-type littermate controls (D). HFD feeding induced a parallel increase in renal arachidonoyl ethanolamide and 2-arachidonoylglycerol levels in both mouse strains (E). CB₁R mRNA expression levels were greatly reduced in RPTC-CB₁R^−/− mice under both diets (F). The HFD-induced increase in CB₁R protein expression levels in wild-type control animals was limited to the RPTCs (black arrows) and not to distal tubules or glomerulus (G). Note the absence of HFD-induced upregulation in CB₁R protein expression in the RPTC-CB₁R^−/− mice. Scale bar, 20 µm. Data represent the mean±SEM from 8 to 10 mice per group. *P<0.05 relative to animals on STD of the same genotype; ^#P<0.05 relative to wild-type animals on the same diet. HET, heterozygous; NC, negative control; PC, positive control.

Figure 2.

RPTC-CB₁R^−/− mice are sensitive to HFD-induced obesity and have similar metabolic abnormalities. RPTC-CB₁R^−/− mice gained a similar body weight (A and B) and displayed equal fat (C) and lean (D) body masses as their wild-type littermate controls. Loss of CB₁R in RPTCs did not alter glucose homeostasis, as measured by fasting blood glucose (E), serum insulin levels (F), and the glucose tolerance test (G). An insulin sensitivity test revealed a significant improvement in the null mice on HFD (H). The absence of CB₁R in RPTCs did not change the lipid profile, as determined by the serum cholesterol levels (I) as well as the HDL/LDL cholesterol ratio (J). RPTC-CB₁R^−/− mice exhibited a similar liver dysfunction on HFD, as measured by the serum levels of ALT (K) and AST (L). Data represent the mean±SEM from 8 to 10 mice per group. *P<0.05 relative to animals on STD of the same genotype.

Figure 3.

RPTC-CB₁R^−/− mice are protected from obesity-induced morphologic changes in the kidney, renal dysfunction, and injury. The HFD-induced morphologic changes in the kidney, such as glomerular enlargement (A and G), an increased Bowman’s space area (B and G), and a mesangial expansion (C and G) were attenuated in obese RPTC-CB₁R^−/− mice. Similarly, the HFD-induced kidney dysfunction, as manifested by the increased albumin-to-creatinine ratio (D), creatinine clearance (E), and BUN (F), was markedly attenuated in the obese RPTC-CB₁R^−/− mice. The HFD-induced upregulation in renal mRNA and protein expression levels as well as the urine excretion levels of the kidney injury markers TIMP-1 (H and J–L), and KIM-1 (I and M–O) were attenuated or normalized in RPTC-CB₁R^−/− mice on the same diet. Scale bar, 20 µm. Original magnification, ×3 in G, H, and I. Data represent the mean±SEM from 8 to 10 mice per group. *P<0.05 relative to animals on STD of the same genotype; ^#P<0.05 relative to wild-type animals on the same diet.

Figure 4.

RPTC-CB₁R^−/− mice are protected from obesity-induced morphologic changes in the kidney, renal dysfunction and injury even after a very prolonged (43 weeks) duration of HFD feeding. The HFD-induced morphologic changes in the kidney, such as glomerular enlargement (A and B), an increased Bowman’s space area (A and C), and mesangial expansion (A and D) were attenuated in obese RPTC-CB₁R^−/− mice. The HFD-induced kidney dysfunction, as manifested by the increased albumin-to-creatinine ratio (E), creatinine clearance (F), and increased BUN (G) was significantly attenuated in the obese RPTC-CB₁R^−/− mice. The HFD-induced upregulation in the renal mRNA expression levels of the kidney injury markers Timp-1 (H), Lcn2 (I), and Clu (J), as well as in the urinary excretion levels of TIMP-1 (L) and KIM-1 (M) were normalized in RPTC-CB₁R^−/− mice on the same diet. Similarly, the HFD-induced renal fibrosis was attenuated in RPTC-CB₁R^−/− mice, as ascertained by measuring the renal mRNA expression levels of the profibrotic markers Col1 (N), Col3 (O), αSma (P), and Fn1 (Q), as well as by quantifying collagen deposition by Sirius Red staining (K and R). Scale bar, 20 µm. Original magnification, ×3 in A and K. Data represent the mean±SEM from 5 to 10 mice per group. *P<0.05 relative to animals on STD of the same genotype; ^#P<0.05 relative to wild-type animals on the same diet.

Figure 5.

RPTC-CB₁R^−/− mice are protected from obesity-induced tubulointerstitial inflammation and fibrosis. The HFD-induced upregulation in the renal mRNA and the protein expression levels of the inflammatory markers IL-18 (A, B, and C), TNFα (A, B, and D), iNOS (A, B, and E), and MCP-1 (A, B, and F) were attenuated or normalized in the RPTC-CB₁R^−/− mice on the same diet. The HFD-induced upregulation in the renal mRNA and protein expression levels of the fibrogenic markers collagen-1 (G, H, and J), collagen-3 (G, H, and K), and αSMA (G, H, and L) were attenuated or normalized in the RPTC-CB₁R^−/− mice on the same diet. Similarly, an elevated collagen deposition, measured by Sirius Red staining, was evident in the HFD-fed wild-type controls compared with the RPTC-CB₁R^−/− mice on the same diet (I and M). Scale bar, 20 µm. Original magnification, ×3 for all inserts. Data represent the mean±SEM from 8 to 10 mice per group. *P<0.05 relative to animals on STD of the same genotype; ^#P<0.05 relative to wild-type animals on the same diet.

Figure 6.

Genetic deletion or pharmacologic blockade of CB₁R ameliorates lipid accumulation in RPTCs via a LKB1/AMPK/ACC signaling pathway. A high accumulation of lipid droplets was found in obese wild-type mice, and not in RPTC-CB₁R^−/− animals (A and D). Significant reductions in the expression of the activated/phosphorylated form of AMPK after both long (14 weeks; B and E) and short (7 days; G and I) periods of HFD feeding and in the nonactivated/phosphorylated form of ACC (14 weeks: C and F; 7 days: H and J) as well as in the activated/phosphorylated form of LKB1 (14 weeks: K and L; 7 days: M and N) were found in obese wild-type mice. Genetic deletion of CB₁R in RPTCs (B, C, E, F, K, and L) or the peripheral blockade of CB₁R by JD5037 (3 mg/kg, administered orally) (G–J, M, and N) significantly normalized the expression levels of pLKB1, pAMPK, and pACC. Scale bar, 20 µm. Original magnification, ×3 for all inserts. Data represent the mean±SEM from 8 to 10 mice per group. *P<0.05 relative to animals on STD of the same genotype; ^#P<0.05 relative to wild-type animals on the same diet.

Figure 7.

Peripheral CB₁R blockade reverses inflammation and lipid accumulation in RPTCs via AMPK signaling. Increased activity of the eCB system was reflected by the elevated levels of 2-arachidonoylglycerol and its endogenous precursor and degraded ligand AA, but not arachidonoyl ethanolamide (A) as well as the upregulation of mRNA (B) and the protein (C) expression levels of CB₁R in HK-2 cells exposed to 0.5 mM oleate/palmitate for 24 hours. This elevation was associated with upregulating the mRNA expression levels of several kidney injury and inflammatory markers, such as Tnfα, Il-1α, Il-6, Ip-10, Il-18, Lcn2, Tgf-β, and Timp-1 (D). Likewise, a direct activation of CB₁R by the synthetic agonist ACEA resulted in similar upregulation of these genes (E). Both effects were completely normalized by pretreating the cells with the peripherally restricted CB₁R inverse agonist JD5037 (100 nM). Similarly, JD5037 reversed the oleate/palmitate–induced downregulation in the phosphorylated forms of LKB1, AMPK, and ACC (F–I), as well as increased the phosphorylation of these proteins despite the presence of ACEA (F, and J–L). Pretreating HK-2 cells with JD5037 also reversed the accumulation of fat droplets in cells with oleate/palmitate or ACEA (M). Scale bar, 20 µm. Data represent the mean±SEM from three independent experiments. *P<0.05 relative to vehicle-treated cells under normal conditions; ^#P<0.05 relative to vehicle-treated cells under oleate/palmitate conditions or ACEA treatment.

Figure 8.

CB₁R modulates lipogenesis or fatty acid β-oxidation in RPTCs via the Gi-PKA axis. Pretreating HK-2 cells with 100 ng/ml pertussis toxin (PTX; a Gα_i/o blocker) with the CB₁R agonist ACEA significantly increased the phosphorylation of LKB1, AMPK, and ACC (A and C). JD5037 loses the ability to increase the phosphorylation of these proteins while incubating HK-2 cells with 500 nM H-89 (a PKA inhibitor) (B and D). Reduced mRNA expression levels of Pparα, Slc27a, and Fabp1 were documented in HFD-induced obese wild-type mice (E) as well as in HK-2 cells treated with oleate/palmitate (F) or ACEA (G). These reductions were normalized by either genetic ablation or pharmacologic blockade of CB₁R in RPTCs. Increased basal and maximal respiratory rates (H–J) as well as ATP production (K) were measured in HK-2 cells treated with JD5037 (100 nM) with oleate/palmitate. The suggested molecular mechanism by which CB₁R in RPTCs regulates renal lipotoxicity (L). In vitro data represent the mean±SEM from three independent experiments. *P<0.05 relative to vehicle-treated cells under normal conditions; ^#P<0.05 relative to vehicle-treated cells under oleate/palmitate conditions or ACEA treatment; ^&P<0.05 relative to JD5037-treated cells under oleate/palmitate conditions. In vivo data represent the mean±SEM from 8 to 10 mice per group. ^∧P<0.05 relative to animals on STD of the same genotype; ^&P<0.05 relative to wild-type animals on the same diet.