Diabetes Metab J.  2025 Mar;49(2):194-209. 10.4093/dmj.2023.0397.

Kidney Gastrin/CCKBR Attenuates Type 2 Diabetes Mellitus by Inhibiting SGLT2-Mediated Glucose Reabsorption through Erk/NF-κB Signaling Pathway

• Zhang X
• Zhang Y
• Shi Y
• Shi D
• Niu M
• Liu X
• Liu X
• Yang Z
• Wu X

• KMID: 2565344
• DOI: http://doi.org/10.4093/dmj.2023.0397

Abstract

Background
Both sodium-glucose cotransporters (SGLTs) and Na+/H+ exchangers (NHEs) rely on a favorable Na-electrochemical gradient. Gastrin, through the cholecystokinin B receptor (CCKBR), can induce natriuresis and diuresis by inhibiting renal NHEs activity. The present study aims to unveil the role of renal CCKBR in diabetes through SGLT2-mediated glucose reabsorption.
Methods
Renal tubule-specific Cckbr-knockout (Cckbr^CKO) mice and wild-type (WT) mice were utilized to investigate the effect of renal CCKBR on SGLT2 and systemic glucose homeostasis under normal diet, high-fat diet (HFD), and HFD with a subsequent injection of a low dose of streptozotocin. The regulation of SGLT2 expression by gastrin/CCKBR and the underlying mechanism was explored using human kidney (HK)-2 cells.
Results
CCKBR was downregulated in kidneys of diabetic mice. Compared with WT mice, Cckbr^CKO mice exhibited a greater susceptibility to obesity and diabetes when subjected to HFD. In vitro experiments using HK-2 cells revealed an upregulation of glucose transporters after incubation with high glucose, a response that was significantly attenuated following gastrin intervention. The glucose uptake from the culture medium of cells was altered accordingly. Moreover, gastrin administration effectively mitigated hyperglycemia in WT diabetic mice by inhibition of SGLT2 mediated glucose reabsorption, but this effect was compromised in the absence of CCKBR, as seen in Cckbr^CKO mice. Mechanistically, gastrin/CCKBR substantially reduced SGLT2 expression in HK-2 cells exposed to high glucose, via modulating Erk/nuclear factor-kappa B (NF-κB) pathway.
Conclusion
Our study underscores the crucial role of renal gastrin/CCKBR in SGLT2 regulation and glucose reabsorption, and renal gastrin/CCKBR can be a promising therapeutic target for diabetes.

Keyword

Diabetes mellitus; Gastrins; Glucose transporter type 2; Receptor, cholecystokinin B; Sodium-glucose transporter 2

Figure

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  Fig. 1. Renal tubule-specific cholecystokinin B receptor (Cckbr)-silenced mice do not develop diabetes under normal diet. (A) Reduced CCKBR mRNA level in the kidney of Cckbr-knockout (CckbrCKO) mice (n=4). (B) Decreased CCKBR protein level in the kidney of CckbrCKO mice (n=10–12). (C) Immunofluorescence staining for characterization of CCKBR deficiency in renal proximal tubule. Lotus tetragonolobus lectin (LTL; a proximal tubular marker, green), peanut agglutinin (PNA; a distal tubular marker, red), aquaporin 2 (AQP2; a collecting tubular marker, red). Circles indicate glomeruli. In wild-type (WT) mice, CCKBR positive signal was observed in glomeruli and proximal tubules, but was undetectable in the distal and collecting tubules. In CckbrCKO mice, CCKBR protein was nearly absent in the kidney. Scale bar=100 μm. (D, E, F) No significant difference in body weight or blood glucose between WT mice (n=12) and CckbrCKO mice (n=10) on normal diet. (G) Oral glucose tolerance test: blood glucose levels were determined at the indicated time points after loading glucose (2 g/kg body weight) with calculation of area under curve (AUC) (n=10–12). (H) mRNA expression levels of sodium-glucose cotransporter 2 (SGLT2), glucose transporter 1 (GLUT1), and GLUT2 in the two groups of mice. (I, J) Protein expression levels of SGLT2, GLUT1, and GLUT2 in the two groups of mice (n=7–12). NS, not significant. aP<0.05, bP<0.001, cP<0.0001.

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  Fig. 2. Renal tubule cholecystokinin B receptor (Cckbr)-silenced mice fed with high-fat diet (HFD) are prone to develop type 2 diabetes mellitus. (A, B) Decreased expression of CCKBR mRNA and protein level in the diabetic kidneys induced by stereptozotocin (STZ)+HFD (n=3–6). (C) Reduced renal CCKBR protein level in mice fed with HFD (n=3–6). (D) Deficiency of CCKBR protein in the kidney of Cckbr-knockout (CckbrCKO) mice fed with HFD (n=4). (E, F) CckbrCKO mice gained more body weight during HFD induction than wild-type (WT) mice (n=4). (G) Higher postprandial blood glucose in CckbrCKO mice (n=4). (H) Oral glucose tolerance test with quantification of area under curve (AUC) (n=4). (I) Plasm levels of postprandial insulin and C-peptide were significantly decreased in CckbrCKO mice fed with HFD (n=3–4). (J) Fasting plasma insulin level in the two groups of mice (n=3–4). (K) Homeostasis model assessment of insulin resistance (HOMA-IR) index in the two groups of mice (n=3–4). (L) Pancreatic insulin level in the two groups of mice (n=3–4). (M) Elevated plasma levels of low-density lipoprotein cholesterol (LDL-C), total cholesterol, and triglyceride in CckbrCKO mice than WT mice (n=4). (N, O) Significantly increased mRNA and protein levels of sodium-glucose cotransporter 2 (SGLT2), glucose transporter 1 (GLUT1), and GLUT2 in the kidneys of CckbrCKO mice fed with HFD (n=3–4). ND, normal diet; NS, not significant. aP<0.05, bP<0.01, cP<0.001, dP<0.0001.

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  Fig. 3. Renal tubule cholecystokinin B receptor (Cckbr) knockout mice fed with high-fat diet (HFD) exhibit target renal damage to some extent. (A) Increased protein levels of matrix metalloproteinase-9 (mmp9), mmp2, and tissue inhibitor matrix metalloproteinase 1 (Timp1) in kidneys of Cckbr-knockout (CckbrCKO) mice fed with HFD (n=4). (B, C) Renal histology was assessed by hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and Masson staining, with quantification of collagen and mesangial matrix areas (n=4). (D) The serum concentration of creatinine and uric acid was increased in the kidneys of CckbrCKO mice fed with HFD (n=4), and the serum level of blood urea nitrogen was not significantly different between two groups. NS, not significant. aP<0.05, bP<0.01, cP<0.001.

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  Fig. 4. The kidney gastrin (SG)/cholecystokinin B receptor (CCKBR) is a negative regulator of glucose transporters expression and activity. (A) Representative images of sodium-glucose cotransporter 2 (SGLT2), glucose transporter 1 (GLUT1), GLUT2, and CCKBR visualized by fluorescence microscopy. Scale bar=250 μm. (B) Quantitative analysis of CCKBR colocalization with SGLT2, GULT1, and GLUT2. (C) Western blot analysis of CCKBR, GLUT1, and SGLT2 in human kidney 2 (HK-2) cells after treatment with different concentrations of SG (10–5, 10–6, 10–7, 10–8, 10–9 mM) (n=3–4). (D, E) SG (10–9 mM) reduced high glucose (HG)-induced glucose transporters expression at both protein and mRNA levels (n=5–6). (F) A 24-hour SG treatment (LG) decreased glucose absorption of HK-2 cells in the presence of HG, as indicated by reduced intracellular glucose content, with no change observed after 2 hours of SG incubation (n=6). aP<0.05, bP<0.01 vs. SG (0 mM); cP<0.05, dP<0.01, eP<0.001 vs. normal glucose (NG); fP<0.05, gP<0.001 vs. HG.

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  Fig. 5. The effects of cholecystokinin B receptor (CCKBR) deficiency and gastrin treatment on hyperglycemia. (A, B) Gastrin administration decreased fasting blood glucose level and enhanced urinary glucose excretion in wild-type (WT) diabetic mice induced by stereptozotocin (STZ) and high-fat diet (HFD), but this effect was blocked in Cckbr-knockout (CckbrCKO) diabetic mice (n=3–5). (C, D) Oral glucose tolerance test (OGTT) showed that gastrin administration ameliorated the impaired glucose tolerance and decreased area under curve (AUC) for glucose in WT diabetic mice, with these benefits negated in CckbrCKO diabetic mice (n=3–5). (E) Western blot analysis showed that gastrin administration reduced renal sodium-glucose cotransporter 2 (SGLT2) protein level, an effect reversed by renal tubule CCKBR deficiency (n=4–6). (F) CCKBR silencing and gastrin treatment in human kidney 2 (HK-2) cells lead to the alteration of SGLT2 expression (n=3–5). (G, H) CCKBR silencing resulted in decreased intracellular glucose content, and increased glucose content in cell medium, but this effect was abrogated when silencing CCKBR (n=4–6). NS, not significant; DM, diabetes mellitus; SiNC, negative control siRNA; SiCCKBR, CCKBR siRNA. aP<0.05, bP<0.01, cP<0.001.

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  Fig. 6. Kidney gastrin/cholecystokinin B receptor (CCKBR) suppresses sodium-glucose cotransporter 2 (SGLT2) expression through P44/42/nuclear factor-kappa B (NF-κB) signaling pathway. (A)Western blot results indicated significant alterations in the levels of p-p44/42, p-IκB-α, and p-p65 proteins by gastrin treatment in the presence of high glucose (HG) (n=4–6). (B) Western blotting illustrated the expression of SGLT2, p-p44/42, p-IκB-α, and p-p65 proteins in human kidney 2 (HK-2) cells pretreated with Erk agonist Honokil (5 μM) (n=4–5). (C) Western blotting depicted the expression of SGLT2, p-p44/42, p-IκB-α, and p-p65 proteins in HK-2 cells pretreated with NF-κB agonist Betulinic acid (1 μM) (n=4–6). aP<0.05, bP<0.01, cP<0.001 vs. normal glucose (NG); dP<0.05, eP<0.01, fP<0.001 vs. HG; gP<0.01, hP<0.001 vs. HG+gastrin.

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