Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Expressing, Fluorescence, In Situ Hybridization, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Expressing, Fluorescence, In Situ Hybridization, Staining, Western Blot, Control, Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Knock-Out, Real-time Polymerase Chain Reaction, Staining, Activity Assay, Western Blot

Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Expressing, Staining, Western Blot, Transmission Assay, Electron Microscopy

Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Expressing, Activity Assay, Staining, Western Blot

Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Western Blot, Expressing, Staining

Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: In Vitro, Western Blot, Expressing, Transfection, Plasmid Preparation, Control

Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Western Blot, Expressing, Control, Transfection, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

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

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: 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)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Western Blot, Expressing, Control, Activity Assay, Staining