Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

Abstract

Kidney is one of the most important organs in maintaining the normal life activities. With the high abundance of mitochondria, renal tubular cell plays the vital role in functioning in the reabsorption and secretion of kidney. Reports have shown that mitochondrial dysfunction is of great importance to renal tubular cell senescence and subsequent kidney ageing. However, the underlying mechanisms are not elucidated. Cannabinoid receptor 2 is one of the two receptors responsible for the activation of endocannabinoid system. CB2 is primarily upregulated in renal tubular cells in chronic kidney diseases and mediates fibrogenesis. However, the role of CB2 in tubular mitochondrial dysfunction and kidney ageing has not been clarified. In this study, we found that CB2 was upregulated in kidneys in 24-month-old mice and d-galactose (d-gal)-induced accelerated ageing mice, accompanied by the decrease in mitochondrial mass. Furthermore, gene deletion of CB2 in d-gal-treated mice could greatly inhibit the activation of β-catenin signalling and restore the mitochondrial integrity and Adenosine triphosphate (ATP) production. In CB2 knockout mice, renal tubular cell senescence and kidney fibrosis were also significantly inhibited. CB2 overexpression or activation by the agonist AM1241 could sufficiently induce the decrease in PGC-1α and a variety of mitochondria-related proteins and trigger cellular senescence in cultured human renal proximal tubular cells. CB2-activated mitochondrial dysfunction and cellular senescence could be blocked by ICG-001, a blocker for β-catenin signalling. These results show CB2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing. The intrinsic mechanism may be related to its activation in β-catenin signalling.

Keywords: CB2; kidney ageing; mitochondrial dysfunction; tubular cell; β-catenin.

FIGURE 1

CB2 is upregulated in aged kidneys. (A) Representative micrographs showing CB2 expression in kidneys from 2‐month‐old and 24‐month‐old mice. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2. Arrow indicates positive staining. scale bar, 25 μm. (B‐E) Representative Western blot and quantitative data showing renal expression of CB2 from 2‐month‐old and 24‐month‐old mice (B and C) or mice which were administered subcutaneous injections of d‐gal at 150mg/kg/day for 6 weeks (D and E). Numbers (1–5) indicate each individual animal in a given group. **p < 0.01 versus 2‐month‐old mice group or the sham control group (n = 5). (F) Representative images showing renal expression of CB2 in d‐gal‐treated mice. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2. Arrow indicates positive staining. scale bar, 25 μm. (G) Representative micrographs showing the colocalization of CB2 and various segment‐specific tubular markers in kidneys. Frozen kidney sections were stained for CB2 (red) using FISH and various segment‐specific tubular markers (green) by immunofluorescence. The following segment‐specific tubular markers were used: proximal tubule, lotus tetragonolobus lectin (LTL); distal tubule, peanut agglutinin (PNA); arrows indicate positive tubules with colocalization of CB2 and specific tubular markers. Scale bar, 25 μm. (H) Representative micrographs showing the expression of CB2 and TOMM20 in tubules in 2‐month‐old and 24‐month‐old mice. Cryosections were subjected to FISH staining of CB2 (red) and stained with TOMM20 (green) antibody by immunofluorescence. Arrows indicate positive staining. Scale bar, 25μm

FIGURE 2

CB2 gene ablation does not affect kidney ageing or mitochondrial function in young mice. (A) RT‐PCR analyses showing renal expression of CB2 in wild‐type mice (WT) and CB2 knockout mice (KO). Numbers (1–4) indicate each individual animal in a given group. (B‐F) Quantitative real‐time PCR results showing renal expression of CB2, fibronectin, α‐SMA, CollagenⅠa1 and CollagenⅢa1 in WT and KO mice. **p < 0.01, n.s. versus WT mice group (n = 4). n.s.: no significance. (G) Representative micrographs showing Periodic acid‐Schiff (PAS) staining, Sirius red staining, senescence‐associated β‐galactosidase activity (SA‐β‐gal) staining and the expression of TOMM20. Paraffin‐embedded kidney sections were subjected to PAS and Sirius red staining. Frozen kidney sections were stained for SA‐β‐gal activity and TOMM20. Scale bar, 50 μm. (H‐K) Representative Western blot and quantitative data showing renal expression of PGC‐1α, TOMM20 and TFAM in WT and KO mice. Numbers (1–4) indicate each individual animal in a given group. n.s. versus WT mice group (n=4). n.s.: no significance. (L‐M) Quantitative real‐time PCR results showing renal expression of p16^INK4A and γH2AX in WT and KO mice. n.s. versus WT mice group (n = 4). n.s.: no significance. (N‐R) Representative Western blot and quantitative data showing renal expression of β‐catenin, MMP7, Snail1 and AT1 in WT and KO mice. Numbers (1–4) indicate each individual animal in a given group. n.s. versus WT mice group (n = 4). n.s.: no significance

FIGURE 3

β‐catenin signalling is inhibited by CB2 gene ablation in d‐gal‐treated mice. (A) Experimental design. Black bar indicated that mice were administered subcutaneous injections of d‐gal at 150mg/kg/day for 6 weeks after surgery for 1 week. UNX: unilateral nephrectomy. (B) Representative micrographs showing renal expression of CB2 in different groups. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2. Arrow indicates positive staining. scale bar, 25 μm. (C) Quantitative real‐time PCR results showing renal expression of CB2. *p < 0.05 versus WT mice group alone; ^## p < 0.01 versus the d‐gal‐treated WT mice group alone (n = 5–6). (D and E) Representative Western blot and quantitative data showing renal expression of β‐catenin. Numbers (1–3) indicate each individual animal in a given group. ***p < 0.001 versus WT mice group alone; ^### p < 0.001 versus the d‐gal‐treated WT mice group alone (n = 5–6). (F and G) Quantitative real‐time PCR results showing renal expression of MMP7 and AT1. *p < 0.05 and ***p < 0.001 versus WT mice group alone; ^# p < 0.05 and ^## p < 0.01 versus the d‐gal‐treated WT mice group alone (n = 5–6). (H) Representative micrographs showing the expression of active β‐catenin. Frozen kidney sections were stained with an antibody against active β‐catenin. Arrow indicates positive staining. Scale bar, 75μm. (I) Quantitative data showing quantification of positive staining. *p < 0.05 versus WT mice group alone; ^# p < 0.05 versus the d‐gal‐treated WT mice group alone (n = 5–6)

FIGURE 4

CB2 deficiency protects renal mitochondrial homeostasis in the accelerated ageing mice. (A) Representative micrographs showing renal expression of PGC‐1α and TOMM20 in different groups. Paraffin‐embedded kidney sections were immunostained with an antibody against PGC‐1α or TOMM20. Arrows indicate positive staining. Scale bar, 50 μm. (B‐C) Quantitative data showing quantification of positive staining. *p < 0.05, ***p < 0.001 versus WT mice group alone; ^# p < 0.05, ^### p < 0.001 versus the d‐gal‐treated WT mice group alone (n = 5–6). (D) Representative graph showing the production of adenosine triphosphate (ATP) in different groups. *p < 0.05 versus WT mice group alone; ^## p < 0.01 versus the d‐gal‐treated WT mice group alone (n = 5–6). (E–H) Representative Western blot and quantitative data showing renal expression of PGC‐1α, TOMM20 and Cytb. Numbers (1–3) indicate each individual animal in a given group. *p < 0.05, **p < 0.01, ***p < 0.001 versus the WT mice group alone; ^# p < 0.05, ^### p < 0.001 versus the d‐gal‐treated WT mice group alone (n = 5–6). (I) Representative transmission electron microscopy graphs showing mitochondrial ultrastructure of renal tubular cells in different groups. Arrows indicate damaged and abnormal‐shaped mitochondria. Scale bar, 1μm

FIGURE 5

CB2 gene ablation ameliorates kidney ageing. (A) Representative micrographs showing renal expression of γH2AX and SA‐β‐gal activity in different groups. Paraffin‐embedded kidney sections were immunostained with an antibody against γH2AX (top). Frozen kidney sections were stained for SA‐β‐gal activity (bottom). Arrows indicate positive staining. Scale bar, 50 μm. (B‐C) Quantitative data showing quantification of positive staining. **p < 0.01 versus WT mice group alone; ^## p < 0.01 versus the d‐gal‐treated WT mice group alone (n = 5–6). (D–G) Representative Western blot and quantitative data showing renal expression of p16^INK4A, γH2AX and p19^ARF in different groups. Numbers (1–3) indicate each individual animal in a given group. **p < 0.01, ***p < 0.001 versus the WT mice group alone; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus the d‐gal‐treated WT mice group alone (n = 5–6). (H and I) Representative micrographs showing renal expression of klotho in different groups (H). Paraffin‐embedded kidney sections were immunostained with an antibody against klotho. Arrows indicate positive staining. Scale bar, 50 μm. (I) Quantitative data showing quantification of positive staining. ***p < 0.001 versus the WT mice group alone; ^### p < 0.001 versus the d‐gal‐treated WT mice group alone (n = 5–6)

FIGURE 6

CB2 deficiency retards age‐related kidney fibrosis. (A and B) Quantitative data showing serum creatinine (Scr) and blood urea nitrogen (BUN) levels in different groups. n.s.: no significance. (C–E) Representative Western blot and quantitative data showing renal expression of fibronectin and α‐SMA in different groups. Numbers (1–3) indicate each individual animal in a given group. *p < 0.05 versus the WT mice group alone; ^# p < 0.05, ^## p < 0.01 versus the d‐gal‐treated WT mice group alone (n = 5–6). (F–H) Representative micrographs showing renal expression of fibronectin and Sirius red staining in different groups. Paraffin‐embedded kidney sections were stained with Sirius red and were immunostained with an antibody against fibronectin. Arrows indicate positive staining. Scale bar, 50 μm. Quantitative data showing quantification of positive staining of fibronectin (G) and fibrotic area (H). **p < 0.01, ***p < 0.001 versus the WT mice group alone; ^## p < 0.01, ^### p < 0.001versus the d‐gal‐treated WT mice group alone (n = 5–6)

FIGURE 7

CB2 induces mitochondrial dysfunction and cellular senescence in vitro. (A–I) Representative Western blot and quantitative data showing the expression of CB2, PGC‐1α, Cytb, TOMM20, COX1, COX2, p16^INK4A, γH2AX in HKC‐8 cells. HKC‐8 cells were transfected with CB2 expression plasmid (pCMV‐CB2) for 24 h. *p < 0.05, **p < 0.01 versus the pcDNA3 group (n = 3). (J–T) Representative Western blot and quantitative data showing the expression of CB2, PGC‐1α, Cytb, TOMM20, COX1, COX2, TFAM, p16^INK4A, γH2AX and p14^ARF in HKC‐8 cells. HKC‐8 cells were treated with AM1241 (10 μM) for 48h. *p < 0.05, **p < 0.01 versus the control group (n = 3)

FIGURE 8

β‐catenin plays a mediative role in CB2‐induced mitochondrial dysfunction and cellular senescence. (A–E) Representative Western blot (A, F) and quantitative data (C–E, G and H) showing the expression of COX1, TFAM, TOMM20, p14^ARF and γH2AX in HKC‐8 cells. HKC‐8 cells were treated with AM1241 (10 μM) for 48 h and pretreated with XL‐001 (10 μM) for 1 h. Quantitative data graph (B) showing the production of adenosine triphosphate (ATP) in HKC‐8 cells. *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group alone; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001versus the AM1241 group alone (n = 3). (I–N) Representative Western blot (I, M) and quantitative data (J–L, N) showing the expression of PGC‐1α, Cytb, TOMM20 and p16^INK4A in HKC‐8 cells. HKC‐8 cells were transfected with CB2 expression plasmid (pCMV‐CB2), followed by the stimulation of ICG‐001 at 10μM for 24 h *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group alone; ^# p < 0.05, ^## p < 0.01versus the pCMV‐CB2 group alone (n = 3)

FIGURE 9

CB2 plays a central role in the accelerated ageing in renal tubular cells. (A–H) Representative Western blot and quantitative data showing the expression of CB2, PGC‐1α, TOMM20, COX1, p16^INK4A, p14^ARF and β‐catenin in HKC‐8 cells. HKC‐8 cells were treated with D‐gal at 10mg/ml for 72h and pretreated with XL‐001 (10μM) for 1 h. *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group alone;^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus the d‐gal group alone (n = 3). (I and J) Representative micrographs and quantitative data showing SA‐β‐gal activity in different groups. Frozen kidney sections were stained for SA‐β‐gal activity. Arrows indicate positive staining. Scale bar, 20 μm. ***p < 0.001 versus the control group alone; ^### p < 0.001 versus the d‐gal group alone (n = 3). (K–N) Representative Western blot and quantitative data showing renal expression of PGC‐1α, TOMM20 and p14^ARF in HKC‐8 cells. HKC‐8 cells were treated with D‐gal at 10mg/ml or cotreated with AM1241 (10 μM) for 72 h and pretreated with ICG‐001 (10 μM) for 1 h. *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group alone; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus the d‐gal group alone; ^†† p < 0.01, ^††† p < 0.001 versus the d‐gal+AM1241 group alone (n = 3). (O and P) Representative micrographs and quantitative data showing SA‐β‐gal activity in different groups. Frozen kidney sections were stained for SA‐β‐gal activity. Arrows indicate positive staining. Scale bar, 20 μm. **p < 0.01 versus the control group alone; ^### p < 0.001 versus the d‐gal group alone; ^††† p < 0.001 versus the d‐gal+AM1241 group alone (n = 3)