Cinacalcet-mediated activation of the CaMKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy

Abstract

Apoptosis and autophagy are harmoniously regulated biological processes for maintaining tissue homeostasis. AMP-activated protein kinase (AMPK) functions as a metabolic sensor to coordinate cellular survival and function in various organs, including the kidney. We investigated the renoprotective effects of cinacalcet in high-glucose treated human glomerular endothelial cells (HGECs), murine podocytes and C57BLKS/J-db/db mice. In cultured HGECs and podocytes, cinacalcet decreased oxidative stress and apoptosis and increased autophagy that were attributed to the increment of intracellular Ca²⁺ concentration and the phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinaseβ (CaMKKβ)-Liver kinase B1 (LKB1)-AMPK and their downstream signals including the phosphorylation of endothelial nitric oxide synthase (eNOS) and increases in superoxide dismutases and B cell leukemia/lymphoma 2/BCL-2-associated X protein expression. Interestingly, intracellular chelator BAPTA-AM reversed cinacalcet-induced CaMKKβ elevation and LKB1 phosphorylation. Cinacalcet reduced albuminuria without influencing either blood glucose or Ca²⁺ concentration and ameliorated diabetes-induced renal damage, which were related to the increased expression of calcium-sensing receptor and the phosphorylation of CaMKKβ-LKB1. Subsequent activation of AMPK was followed by the activation of peroxisome proliferator-activated receptor γ coactivator-1α and phospho-Ser¹¹⁷⁷eNOS-nitric oxide, resulting in a decrease in apoptosis and oxidative stress as well as an increase in autophagy.Our results suggest that cinacalcet increases intracellular Ca²⁺ followed by an activation of CaMKKβ-LKB1-AMPK signaling in GECs and podocytes in the kidney, which provides a novel therapeutic means for type 2 diabetic nephropathy by modulation of apoptosis and autophagy.

Fig. 1. The changes of [Ca²⁺]i and intracellular signaling in HGECs exposed to cinacalcet and high-glucose media.

a The changes of [Ca²⁺]i in HGECs exposed to cinacalcet and high-glucose media. To determine whether the addition of cinacalcet might modulate [Ca²⁺]i in HGECs, FURA-2AM-loaded HGECs were stimulated using different concentrations (15, 100 nM) of cinacalcet in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) media. The area under curve (AUC) was estimated from the baseline of normalized data (at the point of injection) to a fluorescence level and between time points of injection (0 min) and 10 min. The peak of the curve was measured as highest value of the curve. The peak amplitude and AUC of [Ca²⁺]i were significantly increased by cinacalcet in dose-dependent manners in both LG and HG media. In Fig. 1a, the arrow denotes the administration of cinacalcet (15 and 100 nM, respectively) (n = 6 independent experiments in each experiments). *p < 0.05; **p < 0.01 compared with LG and HG. b The changes of intracellular signaling in HGECs exposed to cinacalcet and high-glucose media. Representative immunofluorescent (n = 6 independent experiments in each experiments) and western blot analyses (n = 4 independent experiments in each experiments) of CaSR, CaMKKα/β, phospho-Ser⁴²⁸ LKB1, and phospho-Thr¹⁷² AMPK in the cultured HGECs in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (15 nM) and the quantitative analyses of the results are shown. *P < 0.05; **P < 0.01 and #P < 0.001 compared with other groups

Fig. 2

The effect of cinacalcet on intracellular signaling for AMPK-eNOS oxidative stress and apoptosis in the HGECs cultured in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (1, 5, 15 nM) (a–d). Representative Western blot analyses and quantitative analyses of total AMPK, phosphor-Thr¹⁷² AMPK, total eNOS, phospho-Ser¹¹⁷⁷ eNOS (a, *P < 0.05 and **P < 0.01 compared with LG control), SOD1 and SOD2 (b, *P < 0.05 compared with other groups), dihydroethidium expression (as an oxidative stress marker; c, *P < 0.05 and #P < 0.001 compared with other groups), Bcl-2, Bax, and TUNEL-positive HGECs (e, *P < 0.05 and **P < 0.01 compared with other groups), and β-actin levels in the cultured HGECs and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). d The effect of BAPTA-AM (25 μM) on cinacalcet-indueced in the HGECs cultured in low-glucose or high-glucose (HG; 30 mmol/l D-glucose) with or without cinacalcet treatment (15 nM). Representative Western blot analyses and quantitative analyses of CaMKKβ, phospho-LKB1, and total LKB1 (n = 4 independent experiments in each experiments). *P < 0.05 and **P < 0.01 compared with LG control. f The changes of intracellular signaling related to autophagy in HGECs exposed to cinacalcet and high-glucose media. Representative Western blot analyses and quantitative analyses of beclin-1, LC3-II/LC3-I ratio, and β-actin levels in the cultured HGECs and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). Representative immunofluorescent analyses of LC3 punctae in HGECs and the quantitative analyses of the results are shown (n = 6 independent experiments in each experiments). **P < 0.01 compared with other groups

Fig. 2

The effect of cinacalcet on intracellular signaling for AMPK-eNOS oxidative stress and apoptosis in the HGECs cultured in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (1, 5, 15 nM) (a–d). Representative Western blot analyses and quantitative analyses of total AMPK, phosphor-Thr¹⁷² AMPK, total eNOS, phospho-Ser¹¹⁷⁷ eNOS (a, *P < 0.05 and **P < 0.01 compared with LG control), SOD1 and SOD2 (b, *P < 0.05 compared with other groups), dihydroethidium expression (as an oxidative stress marker; c, *P < 0.05 and #P < 0.001 compared with other groups), Bcl-2, Bax, and TUNEL-positive HGECs (e, *P < 0.05 and **P < 0.01 compared with other groups), and β-actin levels in the cultured HGECs and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). d The effect of BAPTA-AM (25 μM) on cinacalcet-indueced in the HGECs cultured in low-glucose or high-glucose (HG; 30 mmol/l D-glucose) with or without cinacalcet treatment (15 nM). Representative Western blot analyses and quantitative analyses of CaMKKβ, phospho-LKB1, and total LKB1 (n = 4 independent experiments in each experiments). *P < 0.05 and **P < 0.01 compared with LG control. f The changes of intracellular signaling related to autophagy in HGECs exposed to cinacalcet and high-glucose media. Representative Western blot analyses and quantitative analyses of beclin-1, LC3-II/LC3-I ratio, and β-actin levels in the cultured HGECs and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). Representative immunofluorescent analyses of LC3 punctae in HGECs and the quantitative analyses of the results are shown (n = 6 independent experiments in each experiments). **P < 0.01 compared with other groups

Fig. 3

Immunoblot for CaMKKβ, LKB1, phospho-AMPK, SIRT1 and phospho-Ser¹¹⁷⁷ eNOS in AMPKα1 siRNA, AMPKα2 siRNA, or SIRT1 siRNA knock-down HGECs in a high-glucose environment with cinacalcet treatment (a and b). The cultured HGECs were transfected with a final concentration of 50 nM CaMKKβ and LKB1, α1 and α2-AMPK, SIRT1 siRNAs for 24-h by transfection reagent and treated with cinacalcet (15 nM) in high-glucose media. Representative Western blot analyses of CaMKKβ and phospho-Ser⁴²⁸ LKB1 (a), as well as phospho-Thr¹⁷² AMPK, total AMPK, SIRT1, phospho-Ser¹¹⁷⁷ eNOS (b) and β-actin levels and the quantitative analyses of the results are also shown (a and b, respectively) (n = 4 independent experiments in each experiments).*P < 0.05, **P < 0.01 compared with control siRNA with HG

Fig. 4. The changes of [Ca²⁺]i and intracellular signaling in podocytes exposed to cinacalcet and high-glucose media.

a The changes of [Ca²⁺]i in podocytes exposed to cinacalcet and high-glucose media. To determine whether the addition of cinacalcet might modulate [Ca²⁺]i in podocytes, FURA-2AM-loaded podocytes were stimulated using different concentrations (15, 100 nM) of cinacalcet in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) media. The AUC was estimated from the baseline of normalized data (at the point of injection) to a fluorescence level and between time points of injection (0 min) and 10 min. The peak of the curve was measured as highest value of the curve. The peak amplitude and AUC of [Ca²⁺]i were significantly increased by cinacalcet in dose-dependent manners in both LG and HG media. In Fig. 4a, the arrow denotes the administration of cinacalcet (15 and 100 nM, respectively) (n = 6 independent experiments in each experiments). **P < 0.01 compared with LG and HG and *P < 0.05 compared with LG + 15 and HG + 15. b The changes of intracellular signaling in podocytes exposed to cinacalcet and high-glucose media. Representative immunofluorescent (n = 6 independent experiments in each experiments) and western blot analyses (n = 4 independent experiments in each experiments) of CaSR, CaMKKβ, phosphor-Ser⁴²⁸ LKB1, and phospho-Thr¹⁷² AMPK in the cultured podocytes in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (15 nM) and the quantitative analyses of the results are shown (n = 6 independent experiments in each experiments). *P < 0.05; **P < 0.01 and #P < 0.001 compared with other groups

Fig. 5

The effect of cinacalcet on intracellular signaling for AMPK-eNOS, apoptosis, and oxidative stress in the podocytes cultured in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (1 nM, 5 nM, 15 nM) (a–d). Representative Western blot analyses and quantitative analyses of total AMPK, phosphor-Thr¹⁷² AMPK, total eNOS, phospho-Ser¹¹⁷⁷ eNOS (a, *P < 0.05 and **P < 0.01 compared with LG control), SOD1 and SOD2 (b, *P < 0.05 and **P < 0.01 compared with LG control), dihydroethidium expression (c, *P < 0.05 and #P < 0.001 compared with other groups), Bcl-2, Bax, and TUNEL-positive podocytes (e, *P < 0.05 and **P < 0.01 compared with LG control and #P < 0.001 compared with other groups), and β-actin levels in the cultured podocytes and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). d The effect of BAPTA-AM on cinacalcet-indueced in the podocytes cultured in low-glucose or high-glucose (HG; 30 mmol/l D-glucose) with or without cinacalcet treatment (15 nM). Representative Western blot analyses and quantitative analyses of CaMKKβ, phospho-LKB1, and total LKB1 (n = 4 independent experiments in each experiments). *P < 0.05 and **P < 0.01 compared with LG control. f The changes of intracellular signaling related to autophagy in podocytes exposed to cinacalcet and high-glucose media. Representative western blot analyses and quantitative analyses of beclin-1, LC3-II/LC3-I ratio, and β-actin levels in the cultured podocytes and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). Representative immunofluorescent analyses of LC3 punctae in podocytes and the quantitative analyses of the results are shown. **P < 0.01 and # P < 0.001 compared with other groups (n = 6 independent experiments in each experiments)

Fig. 5

The effect of cinacalcet on intracellular signaling for AMPK-eNOS, apoptosis, and oxidative stress in the podocytes cultured in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (1 nM, 5 nM, 15 nM) (a–d). Representative Western blot analyses and quantitative analyses of total AMPK, phosphor-Thr¹⁷² AMPK, total eNOS, phospho-Ser¹¹⁷⁷ eNOS (a, *P < 0.05 and **P < 0.01 compared with LG control), SOD1 and SOD2 (b, *P < 0.05 and **P < 0.01 compared with LG control), dihydroethidium expression (c, *P < 0.05 and #P < 0.001 compared with other groups), Bcl-2, Bax, and TUNEL-positive podocytes (e, *P < 0.05 and **P < 0.01 compared with LG control and #P < 0.001 compared with other groups), and β-actin levels in the cultured podocytes and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). d The effect of BAPTA-AM on cinacalcet-indueced in the podocytes cultured in low-glucose or high-glucose (HG; 30 mmol/l D-glucose) with or without cinacalcet treatment (15 nM). Representative Western blot analyses and quantitative analyses of CaMKKβ, phospho-LKB1, and total LKB1 (n = 4 independent experiments in each experiments). *P < 0.05 and **P < 0.01 compared with LG control. f The changes of intracellular signaling related to autophagy in podocytes exposed to cinacalcet and high-glucose media. Representative western blot analyses and quantitative analyses of beclin-1, LC3-II/LC3-I ratio, and β-actin levels in the cultured podocytes and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). Representative immunofluorescent analyses of LC3 punctae in podocytes and the quantitative analyses of the results are shown. **P < 0.01 and # P < 0.001 compared with other groups (n = 6 independent experiments in each experiments)

Fig. 6. Effects of cinacalcet on renal phenotypes in db/m and db/db mice at 20 weeks.

a Glomerular mesangial fractional area, TGF-β1 and type IV collagen (Col IV) expression, and F4/80-positive cell infiltration in the glomerulus in the cortical area of the db/m and db/db mice, with or without cinacalcet treatment. Representative sections stained with periodic acid-Schiff reagent and representative immunohistochemical staining for TGF-β1, Col IV, and F4/80-positive cells (dark brown) are shown (original magnification, x400). Quantitative analyses of the results for the mesangial fractional area (%), TGF-β1, COL IV, and F4/80-positive cells (fold) are shown (n = 8 in each groups). Representative Western blot analyses of the TNF-α, IL-1β, and β-actin expressions and their quantitative analyses of the results are shown (n = 4 independent experiments in each experiments). *P < 0.05 and **P < 0.01 compared with db/m cont and db/m + cina groups. b Effects of cinacalcet on podocyte phenotypes in db/m and db/db mice at 20 weeks. The thickened glomerular basement membrane and widened foot process width and narrowed slit diaphragm diameter in db/db mice compared with db/m mice were normalized after cinacalcet treatment (n = 8 in each groups). # P < 0.001 compared with db/m cont and db/m + cina groups

Fig. 7. Intra-renal expressions of the CaSR and CaMKKβ-LKB1-AMPK-eNOS signaling pathways in db/m and db/db mice without or with cinacalcet treatment.

a and b Representative Western blot analyses of the CaSR, CaMMKα/β, total LKB1, phospho-Ser⁴²⁸ LKB1, total AMPK, phospho-Thr¹⁷² AMPK, PGC-1α, total eNOS, phospho-Ser¹¹⁷⁷ eNOS and β-actin expressions. Quantitative analyses of the results are shown (n = 3 in each experiments). *P < 0.05 and **P < 0.01 compared with other groups. c Intra-renal expressions of the SOD1 and SOD2 and d 24-h urinary 8-hydroxy-deoxyguanosine (8-OH-dG) and isoprostane concentrations in db/m and db/db mice without or with cinacalcet treatment. Representative Western blot analyses of the SOD1 and SOD2 (n = 3 in each groups) and 24-h urinary 8-OH-dG and isoprostane concentrations of the results are shown (n = 8 in each experiments). *P < 0.05 and **P < 0.01 compared with other groups. e Intra-renal Bcl-2/Bax ratio and f TUNEL-and WT-1-positive cells in the glomerulus in db/m and db/db mice without or with cinacalcet treatment. Representative Western blot analysis of the proapoptotic Bax, antiapoptotic Bcl-2 and β-actin expressions (n = 3 in each experiments) and immunohistochemical staining for TUNEL-positive cells (dark brown) are shown (original magnification, x400) (n = 8 in each groups). The quantitative analyses of the results are shown. *P < 0.05; **P < 0.01, and #P < 0.001 compared with other groups. g Intra-renal beclin 1 and LC3-II/LC3-I ratio and h LC3-II-PECAM-1, and i LC3-II-nephrin-positive cell in the glomerulus of db/m and db/db mice without or with cinacalcet treatment. Representative Western blot analysis of beclin-1, LC-3-I, LC3-II, and β-actin expressions are shown. The quantitative analyses of the results are shown (n = 3 in each groups). *P < 0.05 and **P < 0.01 compared with other groups

Fig. 7. Intra-renal expressions of the CaSR and CaMKKβ-LKB1-AMPK-eNOS signaling pathways in db/m and db/db mice without or with cinacalcet treatment.

a and b Representative Western blot analyses of the CaSR, CaMMKα/β, total LKB1, phospho-Ser⁴²⁸ LKB1, total AMPK, phospho-Thr¹⁷² AMPK, PGC-1α, total eNOS, phospho-Ser¹¹⁷⁷ eNOS and β-actin expressions. Quantitative analyses of the results are shown (n = 3 in each experiments). *P < 0.05 and **P < 0.01 compared with other groups. c Intra-renal expressions of the SOD1 and SOD2 and d 24-h urinary 8-hydroxy-deoxyguanosine (8-OH-dG) and isoprostane concentrations in db/m and db/db mice without or with cinacalcet treatment. Representative Western blot analyses of the SOD1 and SOD2 (n = 3 in each groups) and 24-h urinary 8-OH-dG and isoprostane concentrations of the results are shown (n = 8 in each experiments). *P < 0.05 and **P < 0.01 compared with other groups. e Intra-renal Bcl-2/Bax ratio and f TUNEL-and WT-1-positive cells in the glomerulus in db/m and db/db mice without or with cinacalcet treatment. Representative Western blot analysis of the proapoptotic Bax, antiapoptotic Bcl-2 and β-actin expressions (n = 3 in each experiments) and immunohistochemical staining for TUNEL-positive cells (dark brown) are shown (original magnification, x400) (n = 8 in each groups). The quantitative analyses of the results are shown. *P < 0.05; **P < 0.01, and #P < 0.001 compared with other groups. g Intra-renal beclin 1 and LC3-II/LC3-I ratio and h LC3-II-PECAM-1, and i LC3-II-nephrin-positive cell in the glomerulus of db/m and db/db mice without or with cinacalcet treatment. Representative Western blot analysis of beclin-1, LC-3-I, LC3-II, and β-actin expressions are shown. The quantitative analyses of the results are shown (n = 3 in each groups). *P < 0.05 and **P < 0.01 compared with other groups

Fig. 8. The proposed role of cinacalcet in diabetic nephropathy and the interplay between cinacalcet and kidney, especially glomerular endothelial cells (GECs) and podocytes, in type 2 diabetes.

AMPK AMP-activated protein kinase, CaMKKβ Ca²⁺/calmodulin-dependent protein kinase kinaseβ, CaSR calcium-sensing receptor, LKB1 liver kinase B1, [Ca²⁺]i intracellular calcium, PGC-1α peroxisome proliferator-activated receptor γ coactivator 1-α