GRAPHICAL ABSTRACT

Highlights

• Renal CCKBR expression was downregulated in diabetic mice.

• Renal tubule-specific Cckbr-knockout mice showed higher obesity and diabetes risk with HFD.

• Gastrin/CCKBR reduced SGLT2 in HK-2 cells under high glucose via Erk/NF-κB modulation.

INTRODUCTION

According to the 10th edition of the Diabetes Atlas released by International Diabetes Federation, there are currently 537 million adults aged 20 to 79 suffering from diabetes, encompassing 10.5% of the global population in this age group. The total number is expected to rise to 643 million (11.3%) by 2030 and 783 million (12.2%) by 2045, and almost 90% of people with undiagnosed diabetes live in low- and middle-income countries [1]. Type 2 diabetes mellitus (T2DM) is a prevalent chronic condition, intricately linked to a spectrum of macrovascular and microvascular complications [2]. As such, strategic prevention and optimal management of T2DM are essential for reducing the associated health burden [3].

Gastrin, synthesized and secreted by G cells from the gastric antrum, is renowned for its role in regulating gastric acid secretion through binding to cholecystokinin B receptor (CCKBR) [4], predominantly expressed in the mid-glandular region of the fundic mucosa [5]. Both gastrin and its receptor CCKBR exhibit widespread expression beyond the digestive tract [6]. Gastrin is abundant in fetal and neonatal islets [7], while human islet cells express gastrin/CCKBR receptors [8]. It has further been reported that gastrin delivery via syngeneic mesenchymal stem cells can protect non-obese diabetic (NOD) mice from developing type 1 diabetes mellitus [9], and gastrin can stimulate islet β-cell neogenesis and augment insulin secretion partially by acting on newly expressed CCKBR [10], indicating the beneficial role of gastrin/CCKBR in diabetes. In the kidney, CCKBR transcripts are detectable in selected regions (most highly in the tubules, followed by the glomeruli and interstitium), and accordingly renal CCKBR exerts its regulatory functions under various conditions [11], However, to date, no studies yet investigated whether gastrin exerts its hypoglycemic function by interacting with renal CCKBR.

In the present study, we employed renal tubule-specific Cckbr-knockout (Cckbr^CKO) mice and human renal cortex proximal tubule epithelial cells (human kidney 2 [HK-2]) to delineate the role and mechanism of renal gastrin/CCKBR in the pathogenesis of diabetes. Overall, our data demonstrated the indispensable function of gastrin/CCKBR in regulating proximal tubular glucose reabsorption and in maintaining the blood glucose within the normal range by regulating sodium-glucose cotransporter 2 (SGLT2) expression, providing novel insights into the clinical application of gastrin, specifically targeting renal CCKBR, in the management of T2DM.

METHODS

Animals

Cckbr floxed (Cckbr^fl/fl, wild-type [WT]) mice on C57BL/6J background were generated by View Solid Biotechnology Co. Ltd. (Beijing, China). Tubule-specific Pax8-Cre mice on C57BL/6J background (Cat. no. NM-KI-200151) were purchased from Shanghai Model Organisms Center Inc. (Shanghai, China). C57BL/6J mice with Cckbr^CKO along the entire tubular system (Cckbr^fl/fl Pax8-Cre, CKO) were generated by mating Pax8-Cre mice with Cckbr^fl/fl mice. Pax8-Cre, Cckbr^fl/fl (Cckbr^CKO) mice, and the littermate Cckbr^fl/fl (WT) mice were used in this study. All mice were housed under standard conditions (temperature, 22°C±1°C; humidity, 55%–60%; 12-hour light-dark cycle), with free access to water and food. After a short acclimatization period (7 days), the mice were divided into normal diet (containing 18% fat, 58% carbohydrate, 24% protein) and high-fat diet (HFD; containing 60% fat, 20% carbohydrate, 20% protein) group. These mice were handled according to the approval of Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Peking Union Medical College, China (approved number: YZW19005), and the experimental protocols conformed to the ethical guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

To evaluate the effect of gastrin on T2DM both in WT mice and Cckbr^CKO mice, mice were intraperitoneally injected with stereptozotocin (STZ) when fed with HFD to induce T2DM. Briefly, the 8- to 10-week-old WT mice and Cckbr^CKO mice were fed with HFD for 4 weeks and then injected with STZ (50 mg/kg/day in 0.1 M citrate buffer, pH4.2 to 4.5) for 5 consecutive days. Concurrently, gastrin-17 treatment (260 μg/kg/day, HY-P1806A, MCE, Shanghai, China) was initiated, preceding STZ injection, via subcutaneous osmotic minipumps over 60 days. Body weight and 12-hour fasting glucose levels were monitored monthly. A 24-hour urine samples were collected using metabolic cage, with urine volume and the urinary glucose concentration determined by glucose assay kit (Solarbio BC2505, Solarbio, Beijing, China). Urine glucose excretion (UGE) was calculated: UGE (μmol/hr)=Urine glucose (μmol/mL)×Urine excretion rate (mL/hr). At the end of experiment, mice were anesthetized and sacrificed in a CO₂ chamber, followed by collection of blood and tissue samples.

Oral glucose tolerance test

After an overnight fast, the mice were administered D-glucose (0.4 g/mL) by gavage at 2 g/kg body weight, and blood glucose was measured before and at 30, 60, 90, and 120 minutes after glucose administration using a handheld glucometer (One Touch Ultra 2, LifeScan Inc., Malvern, PA, USA).

Insulin measurements and homeostasis model assessment of insulin resistance calculation

The fasting plasma glucose (FPG) was determined by a glucometer (Yuwell, Jiangsu, China). The pancreatic insulin content, fasting plasma insulin (FINS) and postprandial plasma insulin were measured radio-immunochemically. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated: HOMA-IR=FPG×FINS/22.5.

Blood biochemistry

Routine biochemical parameters, including low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), blood urea nitrogen, creatinine, and uric acid (UA) were measured by Mindray BS-360 automatic biochemical analyzer (Mindray, Anshan, China).

Cell culture and gastrin treatment

HK-2 were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China), and were cultured in RPMI medium supplemented with 2.05 mM L-glutamine, 10% fetal bovine serum, 100 μg/mL penicillin, and 10 μg/mL streptomycin. Cells were maintained at 37°C in a humidified cell culture incubator supplied with 5% CO₂ and 95% O₂. HK-2 cells with low passage numbers (<15) were used for this study. The cells underwent gastrin pretreatment for two distinct durations: 2 hours (short-duration gastrin treatment group [SG]) and 24 hours (long-duration gastrin treatment group [LG]), preceding a 24-hour exposure to high glucose conditions. To confirm that the effect of gastrin was dependent on CCKBR, CCKBR siRNA and the negative control were synthesized by Beijing Tsingke Biotech Co. Ltd. (Beijing, China), and were transfected into HK-2 cells using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) for 24-hour prior to gastrin treatment. Post-treatment, the glucose levels in the cells and supernatant were detected using glucose assay kit (Solarbio BC2505).

Quantitative real-time polymerase chain reaction

Total RNA from kidneys and cells were extracted using Trizol reagents (Invitrogen). The extracted RNA was transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. The obtained cDNA underwent quantitative real-time polymerase chain reaction (qRT-PCR) analysis, illuminated by SYBR green, on an ABI7500 qRT-PCR system. All the primers used in this study are provided in Supplementary Table 1. Data were analyzed using the 2^–∆∆Ct method with β-actin as an internal control.

Western blot

Total protein was extracted from cells and kidney tissues using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor and phosphatase inhibitor, and protein concentrations were determined by bicinchoninic acid Protein Assay Kit (Solarbio). Equal amounts of protein samples were loaded on to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies against CCKBR (sc166690, Santa Cruz Biotechnology, Santa Cruz, CA, USA), glucose transporter 2 (GLUT2; sc-518022, Santa Cruz Biotechnology), P-IκB-α (c-8404, Santa Cruz Biotechnology), SGLT2 (PA5-101893, Thermo Fisher Scientific, Waltham, MA, USA), GLUT1 (ab115730, Abcam, Cambridge, UK), p-p65 (3033p, CST, Danvers, MA, USA), p-p44/42 (4370s, CST), matrix metalloproteinase-9 (Mmp9; 10375-2-AP, Proteintech, Rosemont, IL, USA,), Mmp2 (23394-I-AP, Proteintech), tissue inhibitor matrix metalloproteinase 1 (Timp1; ab38978, Abcam), and β-actin (Gene protein link, P03S01S) at 4°C overnight, followed by incubation with secondary antibodies on the next day. Protein bands were imaged by Tanon 5500 Chemiluminescent Imaging System (Tanon, Shanghai, China).

Immunofluorescence

Thin sections (3 μm) of formalin-fixed, paraffin-embedded mouse kidneys were deparaffinized in xylene and rehydrated with step-down concentrations of ethanol, and then penetrated by 0.6% Triton X-100 for 1 hour, followed by blocking with goat serum. Subsequently, the tissue sections were incubated with the following antibodies: mouse anti-CCKBR (1:100, sc-166690, Santa Cruz Biotechnology), rabbit anti-GLUT2 (1:100, ab54460, Abcam), rabbit anti-SGLT2 antibody (1:100, ab37296, Abcam), rabbit anti-GLUT1 (1:200, 66290-1-Ig, Proteintech), anti-aquaporin 2 (AQP2) polyclonal antibody (1:200, 29386-1-AP, Proteintech). On the subsequent day, the sections were incubated with the following secondary antibodies: Alexa Fluor 568 Goat Anti-Mouse IgG H&L (1:500, ab175473, Abcam), Alexa Fluor 568 Donkey Anti-Rabbit IgG H&L (1:500, ab175470, Abcam), Alexa Fluor 488 Goat Anti-Rabbit IgG H&L (1:500, ab150077, Abcam), Alexa Fluor 488 Goat Anti-Mouse IgG H&L (1:500, ab150113, Abcam) and fluorochrome including lotus tetragonolobus lectin (LTL), fluorescein (1:100, L32480, Invitrogen), peanut agglutinin (PNA)-Alexa Fluor 594 (1:100, L32459, Invitrogen). After the nuclei were labeled with 4´,6-diamidino-2-phenylindole (DAPI), the sections were observed and photographed by laser scanning confocal microscopy (Leica Microsystems GmbH, Wetzlar, Germany). Colocalization of CCKBR and SGLT2/GLUT1/GLUT2 was identified by a yellow color in the merged images. The immunofluorescent images were quantitatively analyzed using ImageJ software (http://imagej.nih.gov/ij/); provided in the public domain by the National Institutes of Health, Bethesda, MD, USA. The colocalization of CCKBR/SGLT2, CCKBR/GLUT1, CCKBR/GLUT2 was determined by measuring the Manders’ coefficients M1 and M2 using the JACoP plugin in Image J [12].

Renal histopathology

Histological assessments on paraffin-embedded kidney tissue sections were performed using hematoxylin and eosin (HE), periodic acid-Schiff (PAS) (G1281, Solarbio) and Masson staining (G1346, Solarbio) according to the manufacturer’s instruction respectively. The sections after staining were captured with NDP view 2 (Hamamatsu software, Iwata, Japan). For PAS and Masson staining sections, positive collagen area and mesangial matrix fraction were calculated using Image J software.

Statistical analysis

Data were expressed as mean±standard error of the mean, and were analyzed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). Differences were determined by Student’s t-test for comparison between two groups, or by one-way analysis of variance (ANOVA) followed by a NewmanKeuls test for comparison among more than two groups. P<0.05 was considered significantly different.

RESULTS

Renal tubule-specific Cckbr-silenced mice do not develop diabetes under normal diet

We first studied whether CCKBR deficiency in renal tubules would affect body weight and blood glucose at baseline. Utilizing a Pax8-Cre/Flox recombination strategy, we generated renal tubule CCKBR conditional knockout mice (Cckbr^CKO), exhibiting approximately a 70% reduction in CCKBR protein expression and a 90% reduction in CCKBR mRNA levels in the kidney relative to the Cckbr^f/f (WT) mice (Fig. 1A and B). Immunofluorescence colocalization showed that CCKBR was abundantly expressed in glomeruli and proximal tubules, with minimal expression in distal tubules and collecting ducts in WT mice (Fig. 1C). In Cckbr^CKO mice, CCKBR positive staining was confined to a few glomeruli, absent in renal tubules, thereby validating the specific deletion of CCKBR in renal tubules (Fig. 1C).

Then, we conducted a comparison of the fundamental phenotypes between Cckbr^CKO and WT mice under standard dietary conditions, and the results showed that there were no differences of body weight and blood glucose levels between the two groups (Fig. 1D-F). Oral glucose tolerance test (OGTT) revealed that Cckbr^CKO mice showed similar glucose tolerance to WT mice (Fig. 1G). Additionally, qPCR analysis exposed no variance in the mRNA levels of SGLT2, GLUT1, and GLUT2 between Cckbr^CKO and WT mice (Fig. 1H). Nonetheless, Western blot results indicated a marked increase of SGLT2 levels in the kidney of Cckbr^CKO mice, contrasting with the unaltered levels of GLUT1 and GLUT2 (Fig. 1I and J).

Renal tubule-specific Cckbr-knockout mice are more susceptible to develop diabetes under high-fat diet

To further explore the role of gastrin/CCKBR in diabetes, we detected CCKBR expression in kidneys of diabetic mice induced by HFD feeding with a subsequent injection of multiple low dose STZ injections. The results indicated a significant reduction in both CCKBR mRNA and protein levels in diabetic kidneys (Fig. 2A and B). Additionally, a notable decrease of CCKBR protein levels were observed in kidneys of mice fed with HFD for 4 months (Fig. 2C). Next, we subjected Cckbr^CKO mice to 4 months of HFD. Fig. 2D verified Cckbr deficiency in the kidneys. Upon HFD feeding, Cckbr^CKO mice exhibited a marked increase in body weight compared to the mice in WT group (Fig. 2E and F). Further results showed that Cckbr^CKO mice presented markedly higher postprandial blood glucose levels (Fig. 2G), impaired glucose tolerance (Fig. 2H) and reduced postprandial plasma insulin and C-peptide levels (Fig. 2I), in contrast to their HFD-fed WT counterparts. No significant difference was noted in fasting insulin level and HOMA-IR between the two groups (Fig. 2J and K). Pancreatic insulin was reduced in Cckbr^CKO mice fed with HFD, but this reduction was not significantly different (Fig. 2L). Serum levels of LDL-C, TC, and TG were significantly increased in Cckbr^CKO mice fed with HFD (Fig. 2M). Moreover, both qPCR and Western blot analysis demonstrated a substantial upregulation of renal glucose transporter expression, including SGLT2, GLUT1, and GLUT2, in Cckbr^CKO mice when compared to WT mice following HFD induction (Fig. 2N and O). These data collectively imply that mice with renal tubule-specific Cckbr deficiency are at an increased risk of developing obesity and diabetes under HFD, potentially linked to the heightened expression of SGLT2.

Renal tubule-specific Cckbr-knockout mice show kidney damage under high-fat diet

Previous studies have indicated that renal gastrin/CCKBR protects the kidney against ischemia/reperfusion injury [13] and hypertensive injury [14]. We wondered whether renal tubule-specific Cckbr deficiency has an effect on kidney and thus examined proteins indicative of renal damage, including Mmp2, Mmp9, Timp1 and alongside conducting a histopathological review. Western blot analysis showed that the expression of Mmp2 and Mmp9 in kidney were significantly upregulated in Cckbr^CKO mice (Fig. 3A). Furthermore, the fractions of collagen deposition and mesangial area were considerably greater in the kidneys of Cckbr^CKO mice compared to those of WT mice fed with HFD (Fig. 3B and C). In addition, serum levels of creatinine and UA were markedly elevated in the kidneys of Cckbr^CKO mice (Fig. 3D). These findings support the notion that the kidneys of Cckbr^CKO mice are more vulnerable to damage when subjected to an HFD induction.

Gastrin/CCKBR effectively modulates glucose transporter expression and regulates glucose uptake in HK-2 cell

Additionally, to substantiate the link between glucose transporters and CCKBR, we conducted immunofluorescence staining to observe the colocalization of SGLT2, GLUT2, and GLUT1 with CCKBR in kidneys of mice. Our findings revealed a substantial colocalization of CCKBR with SGLT2, whereas CCKBR exhibited minimal colocalization with GLUT1 and GLUT2 (Fig. 4A). Further quantitative colocalization analysis also demonstrated greatest overlap of CCKBR and SGLT2 staining in the renal cortex (Fig. 4B). These observations suggest a potential interaction between CCKBR and SGLT2, either directly or indirectly. Consequently, our study primarily concentrated on SGLT2.

To delineate the regulatory relationship between glucose transporters and CCKBR, we used gastrin to stimulate CCKBR in HK-2 cells. The expression of CCKBR showed an upward trend following varying concentrations of gastrin. Notably, gastrin significantly reduced SGLT2 expression at the concentration of 10^–9 mM (Fig. 4C). Hence, we selected 10^–9 mM for gastrin for subsequent experiments. Western blot analysis demonstrated that the expression of SGLT2, along with GLUT1 and GLUT2, was increased under high glucose conditions, while gastrin remarkably reversed this effect (Fig. 4D). The qPCR results further verified this phenomenon (Fig. 4E). Next, we detected the effect of gastrin/CCKBR on glucose uptake in HK-2 cells. The glucose absorption in LG group was significantly reduced, whereas there was no difference in the SG group (Fig. 4F), indicating the regulatory effect of gastrin/CCKBR on glucose uptake.

Gastrin alleviates hyperglycemia by inhibiting glucose reabsorption through renal CCKBR

To further ascertain the role of renal tubule CCKBR in diabetes and in the regulation of SGLT2, both WT mice and Cckbr^CKO mice were administrated with gastrin for 2 months during diabetes induction by STZ and HFD. We found that gastrin treatment led to a reduction in fasting blood glucose level (Fig. 5A), an elevation in UGE (Fig. 5B), and an improvement in the impaired glucose tolerance test in WT mice (Fig. 5C and D), while the protective effects of gastrin were abolished in renal tubule CCKBR-deficient mice. Additionally, Western blot analysis showed that gastrin administration significantly reduced the expression of SGLT2, an effect that was counteracted by renal CCKBR deficiency (Fig. 5E).

Moreover, we conducted in vitro experiments to illustrate the role of CCKBR in mediating the effect of gastrin. We used siRNA to silence CCKBR in HK-2 cells, and found that gastrin-induced downregulation of SGLT2 was reversed after silencing CCKBR (Fig. 5F), and accordingly the decreased glucose uptake by gastrin was also abrogated (Fig. 5G and H).

Gastrin/CCKBR regulates the expression of SGLT-2 via Erk and NF-κB pathway

Previous studies have illuminated the regulation of SGLT2 via the protein kinase C (PKC), activator of phosphoinositide-3 kinase (PI3K/Akt), mitogen-activated protein kinases (MAPKs), and nuclear factor-kappa B (NF-κB) pathways [15,16]. Our results showed that high glucose increased the phosphorylation of p65, IκB-α, and p44/42, effects which are attenuated by gastrin (Fig. 6A). To verify the involvement of Erk and NF-κB in gastrin-mediated downregulation of SGLT2 in HK-2 cells, honokiol (an Erk agonist) and betulinic acid (a NF-κB agonist) were used. Western blot analysis showed that honokiol reversed the downregulation of SGLT2 and the inhibited phosphorylation of p44/42, IκB-α, and p65 induced by gastrin (Fig. 6B), whereas betulinic acid also inhibited the downregulation of SGLT2, without influence on the phosphorylation of p44/42 (Fig. 6C), implying that MAPK is upstream of NF-κB. These results suggested that gastrin regulates SGLT2 expression through the Erk-NF-κB signaling pathway in HK-2 cells.

DISCUSSION

In this study, we elucidated previously unrecognized function of renal CCKBR in T2DM, showing that CCKBR expression was significantly decreased in diabetic kidneys, while renal tubule CCKBR-deficient mice fed with HFD exhibited impaired glucose tolerance, reduced postprandial plasma insulin and C-peptide levels, concurrent with upregulation of renal SGLT2. Furthermore, gastrin administration effectively alleviated hyperglycemia by curbing glucose reabsorption in diabetic WT mice; however, this effect was abrogated in diabetic Cckbr^CKO mice. In vitro, gastrin/CCKBR prevented high glucose-induced upregulation and activation of SGLT2 through Erk/NF-κB signaling pathway. Taken together, these findings suggest that targeting renal gastrin/CCKBR is a promising strategy for T2DM.

The role of gastrin extends beyond its well-known function in gastric acid secretion. Gastrin functions by acting on CCKBR receptors in different organs. While predominantly expressed in the nervous system and gastrointestinal tract [17], CCKBR has also been identified in the kidney [11]. Studies have indicated that gastrin improves salt-sensitive hypertension via intestinal CCKBR receptors [18,19]. In kidneys, gastrin/CCKBR mediates renal potassium and sodium absorption [20,21], and can attenuate kidney ischemia/reperfusion injury via a PI3K/Akt/Bad-mediated anti-apoptosis signaling [13]. In the context of diabetes, gastrin fosters the regeneration and functional recovery of pancreatic β-cells [22], and enhances insulin secretion [23]. Notably, combination of gastrin and glucagon-like peptide-1, more effectively than either alone, has been shown to restore normoglycemia in diabetic NOD mice [24]. Our study introduces a novel perspective by demonstrating that gastrin, through action on renal CCKBR, regulates SGLT2 expression and affects glucose reabsorption, thereby elucidating a new link between renal CCKBR and diabetes.

To elucidate the role of renal CCKBR in gastrin-induced hypoglycemia, we constructed renal tubule-specific Cckbr^CKO mice. Subsequent experiments showed that gastrin could lower blood glucose and promote urinary glucose excretion in diabetic WT mice, and those effects were absent in Cckbr^CKO mice, underscoring the importance of renal CCKBR. Notably, renal tubule-specific Cckbr^CKO did not alter body weight or blood glucose levels in mice on a normal diet, despite the upregulation of SGLT2, but could promote obesity and impaired glucose tolerance under HFD conditions. Compared to SGLT2, the relative contribution of SGLT1 to renal glucose reabsorption may be greater under hypoglycemic or normoglycemic conditions [25]. Thus, even though we observed upregulation of SGLT2 in Cckbr^CKO mice under normal diet, SGLT1 or other glucose transporters may compensate for the increase in SGLT2, thereby maintaining normal glucose reabsorption and blood glucose levels. Under HFD, owing to excessive fat intake, the body may not be able to effectively compensate for the CCKBR missing, leading to metabolic disorders and impaired glucose tolerance. On the other hand, the body weight gain in Cckbr^CKO mice fed with HFD can not be solely attributed to glucose reabsorption inhibition, because we also observed increased plasma levels of LDL-C, TC, and TG in Cckbr^CKO mice, hinting at disrupted lipid metabolism. Lipids metabolism disorder contributes to the development of obesity; thus, elevated lipids levels may contribute to body weight gain in Cckbr^CKO mice. The weight gain in these mice on HFD may also involve changes in food intake, energy expenditure, and fat mass, which merit further research.

Unexpectedly, our results indicated the enhanced renal SGLT2 expression in HFD-fed Cckbr^CKO mice did not result in a significant increase of fasting blood glucose levels, but increased blood glucose levels following exogenous glucose loading. Likely, increased SGLT2 expression in the kidney may affect the filtration and reabsorption of glucose by the kidney, but overall blood glucose levels are also affected by other factors such as liver, muscle, and adipose tissue. In the fasting state, limited glucose availability results in limited glucose reabsorption, despite a significant increase in renal SGLT2 expression. However, during the OGTT, glucose loading triggers a temporary increase in blood glucose levels, during which mice with elevated SGLT2 levels facilitate more glucose reabsorption into circulation, manifesting as heightened postprandial glucose levels. Moreover, Cckbr^CKO mice showed significantly reduced postprandial plasma insulin levels, but not fasting insulin levels, reflecting that Cckbr^CKO mice cleared glucose at a slower rate than WT mice. As such, even though the fasting blood glucose of Cckbr^CKO mice did not significantly increase, postprandial blood glucose and glucose tolerance showed obvious abnormalities.

Previous studies have demonstrated that intestinal Cckbrspecific knockout mice develop salt-sensitive hypertension by regulating Na⁺/H⁺ exchanger 3 (NHE3) pathway [18,19]. Additionally, evidence indicates that enhanced tubular gluconeogenesis in NHE3-knockout mice contributes to the downregulation of SGLT2 mRNA and protein levels [26], implicating a link between CCKBR and SGLT2. In this study, renal tubule CCKBR deficiency led to significant upregulation of SGLT2, with immunofluorescence revealing clear colocalization between CCKBR and SGLT2. We further demonstrated that gastrin can regulate the SGLT2 expression through the Erk-NF-κB signaling pathway in vitro. Nonetheless, we cannot exclude the role of other glucose transporters. SGLT2 is a major determinant of renal glucose reabsorption, responsible for the bulk uptake of glucose across the apical membrane of the early proximal tubule [27]. After the apical uptake, glucose transporters GLUT2 and GLUT1 facilitate glucose’s basolateral exit [28]. Compared with GLUT1, GLUT2 plays a greater role for the basolateral exit of glucose in proximal tubules [29]. GLUT1 also mediates glucose uptake by the distal tubule segments for energy supply, and is implicated in diabetic nephropathy [30]. In this study, we found that Cckbr^CKO mice presented significantly elevated levels of both GLUT2 and GLUT1, in addition to SGLT2. This may be a maladaptive effect as it sustains hyperglycemia. The increase of GLUT2 and GLUT1 may further exacerbate the effects of renal Cckbr^CKO on diabetes and kidney injury. The changes in GLUT1 and GLUT2 might be an interlinked response to SGLT2 upregulation, as evidence suggests crosstalk between renal GLUT2 and SGLT2 in glucose homeostasis regulation [31]. Further research is warranted to clarify the specific roles of GLUT1 and GLUT2 in renal CCKBR-deficient mice.

Recent studies have highlighted the protective role of kidney gastrin/CCKBR against ischemia/reperfusion injury [13] and hypertensive injury [14] by using CCKBR antagonist and germline deletion of CCKBR in mice. Herein, we used renal tubule CCKBR-deficient mice to explore the specific role of renal CCKBR on kidney function. Similarly, we found that Cckbr^CKO mice exhibited pronounced kidney damage under HFD, characterized by elevated matrix metalloproteinases (MMPs) expression, increased collagen and glycogen deposition. Moreover, serum creatinine and UA levels were significantly increased in Cckbr^CKO mice, reflecting the susceptibility to kidney injury.

In conclusion, the present study demonstrates that gastrin can mitigate hyperglycemia partially through the inhibition of filtered glucose reabsorption by targeting renal CCKBR, positioning renal CCKBR as a potential therapeutic target for diabetes.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: X.W.

Acquisition, analysis, or interpretation of data: X.Z., Y.Z., Y.S., D.S., X.L., X.L., X.W.

Drafting the work or revising: Y.Z., Y.S., D.S., X.W.

Final approval of the manuscript: all authors.

FUNDING

This study was supported by National Key Research and Development Program of China (2022YFF0710600), Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CAMS, 2021-I2M-1-035, 2021-I2M-1-072).

Acknowledgements

None

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 (Cckbr^CKO) mice (n=4). (B) Decreased CCKBR protein level in the kidney of Cckbr^CKO 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 Cckbr^CKO 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 Cckbr^CKO 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.

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 (Cckbr^CKO) mice fed with HFD (n=4). (E, F) Cckbr^CKO mice gained more body weight during HFD induction than wild-type (WT) mice (n=4). (G) Higher postprandial blood glucose in Cckbr^CKO 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 Cckbr^CKO 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 Cckbr^CKO 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 Cckbr^CKO 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.

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 (Cckbr^CKO) 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 Cckbr^CKO 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.

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.

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 (Cckbr^CKO) 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 Cckbr^CKO 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.

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.

REFERENCES

• 1. Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, et al. IDF Diabetes Atlas: global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. Diabetes Res Clin Pract 2022;183:109119.ArticlePubMed
• 2. Zheng Y, Ley SH, Hu FB. Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nat Rev Endocrinol 2018;14:88-98.ArticlePubMedPDF
• 3. Lemieux I. Reversing type 2 diabetes: the time for lifestyle medicine has come! Nutrients 2020;12:1974.ArticlePubMedPMC
• 4. Dockray G, Dimaline R, Varro A. Gastrin: old hormone, new functions. Pflugers Arch 2005;449:344-55.ArticlePubMedPDF
• 5. Dufresne M, Seva C, Fourmy D. Cholecystokinin and gastrin receptors. Physiol Rev 2006;86:805-47.ArticlePubMed
• 6. Khan D, Vasu S, Moffett RC, Irwin N, Flatt PR. Expression of gastrin family peptides in pancreatic islets and their role in β-cell function and survival. Pancreas 2018;47:190-9.ArticlePubMed
• 7. Larsson LI, Rehfeld JF, Sundler F, Hakanson R. Pancreatic gastrin in foetal and neonatal rats. Nature 1976;262:609-10.ArticlePubMedPDF
• 8. Reubi JC, Waser B, Gugger M, Friess H, Kleeff J, Kayed H, et al. Distribution of CCK1 and CCK2 receptors in normal and diseased human pancreatic tissue. Gastroenterology 2003;125:98-106.ArticlePubMed
• 9. Gaudreau MC, Gudi RR, Li G, Johnson BM, Vasu C. Gastrin producing syngeneic mesenchymal stem cells protect non-obese diabetic mice from type 1 diabetes. Autoimmunity 2022;55:95-108.ArticlePubMed
• 10. Tellez N, Joanny G, Escoriza J, Vilaseca M, Montanya E. Gastrin treatment stimulates β-cell regeneration and improves glucose tolerance in 95% pancreatectomized rats. Endocrinology 2011;152:2580-8.ArticlePubMedPDF
• 11. de Weerth A, Jonas L, Schade R, Schoneberg T, Wolf G, Pace A, et al. Gastrin/cholecystokinin type B receptors in the kidney: molecular, pharmacological, functional characterization, and localization. Eur J Clin Invest 1998;28:592-601.ArticlePubMedPDF
• 12. Velez EJ, Schnebert S, Goguet M, Balbuena-Pecino S, Dias K, Beauclair L, et al. Chaperone-mediated autophagy protects against hyperglycemic stress. Autophagy 2024;20:752-68.ArticlePubMed
• 13. Liu C, Chen K, Wang H, Zhang Y, Duan X, Xue Y, et al. Gastrin attenuates renal ischemia/reperfusion injury by a PI3K/Akt/Bad-mediated anti-apoptosis signaling. Front Pharmacol 2020;11:540479.ArticlePubMedPMC
• 14. Gu D, Fang D, Zhang M, Guo J, Ren H, Li X, et al. Gastrin, via activation of PPARα, protects the kidney against hypertensive injury. Clin Sci (Lond) 2021;135:409-27.ArticlePubMedPMCPDF
• 15. Fu M, Yu J, Chen Z, Tang Y, Dong R, Yang Y, et al. Epoxyeicosatrienoic acids improve glucose homeostasis by preventing NF-κB-mediated transcription of SGLT2 in renal tubular epithelial cells. Mol Cell Endocrinol 2021;523:111149.PubMed
• 16. Kim EJ, Lee YJ, Lee JH, Han HJ. Effect of epinephrine on alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells. Cell Physiol Biochem 2004;14:395-406.ArticlePubMedPDF
• 17. Nishimura S, Bilguvar K, Ishigame K, Sestan N, Gunel M, Louvi A. Functional synergy between cholecystokinin receptors CCKAR and CCKBR in mammalian brain development. PLoS One 2015;10:e0124295.ArticlePubMedPMC
• 18. Jiang X, Liu Y, Zhang XY, Liu X, Liu X, Wu X, et al. Intestinal gastrin/CCKBR (cholecystokinin B receptor) ameliorates salt-sensitive hypertension by inhibiting intestinal Na+/H+ exchanger 3 activity through a PKC (protein kinase C)-mediated NHERF1 and NHERF2 pathway. Hypertension 2022;79:1668-79.ArticlePubMed
• 19. Zhang QY, Guo Y, Jiang XL, Liu X, Zhao SG, Zhou XL, et al. Intestinal Cckbr-specific knockout mouse as a novel model of salt-sensitive hypertension via sodium over-absorption. J Geriatr Cardiol 2023;20:538-47.ArticlePubMedPMC
• 20. von Schrenck T, Ahrens M, de Weerth A, Bobrowski C, Wolf G, Jonas L, et al. CCKB/gastrin receptors mediate changes in sodium and potassium absorption in the isolated perfused rat kidney. Kidney Int 2000;58:995-1003.Article
• 21. Chen Y, Asico LD, Zheng S, Villar VA, He D, Zhou L, et al. Gastrin and D1 dopamine receptor interact to induce natriuresis and diuresis. Hypertension 2013;62:927-33.PubMed
• 22. Brand SJ, Tagerud S, Lambert P, Magil SG, Tatarkiewicz K, Doiron K, et al. Pharmacological treatment of chronic diabetes by stimulating pancreatic beta-cell regeneration with systemic co-administration of EGF and gastrin. Pharmacol Toxicol 2002;91:414-20.PubMed
• 23. Rehfeld JF. CCK, gastrin and diabetes mellitus. Biomark Med 2016;10:1125-7.ArticlePubMed
• 24. Suarez-Pinzon WL, Power RF, Yan Y, Wasserfall C, Atkinson M, Rabinovitch A. Combination therapy with glucagon-like peptide-1 and gastrin restores normoglycemia in diabetic NOD mice. Diabetes 2008;57:3281-8.ArticlePubMedPMCPDF
• 25. Nagata T, Fukazawa M, Honda K, Yata T, Kawai M, Yamane M, et al. Selective SGLT2 inhibition by tofogliflozin reduces renal glucose reabsorption under hyperglycemic but not under hypo- or euglycemic conditions in rats. Am J Physiol Endocrinol Metab 2013;304:E414-23.ArticlePubMed
• 26. Onishi A, Fu Y, Darshi M, Crespo-Masip M, Huang W, Song P, et al. Effect of renal tubule-specific knockdown of the Na+/H+ exchanger NHE3 in Akita diabetic mice. Am J Physiol Renal Physiol 2019;317:F419-34.ArticlePubMedPMC
• 27. Vallon V, Platt KA, Cunard R, Schroth J, Whaley J, Thomson SC, et al. SGLT2 mediates glucose reabsorption in the early proximal tubule. J Am Soc Nephrol 2011;22:104-12.ArticlePubMedPMC
• 28. Chin E, Zhou J, Bondy C. Anatomical and developmental patterns of facilitative glucose transporter gene expression in the rat kidney. J Clin Invest 1993;91:1810-5.ArticlePubMedPMC
• 29. Vallon V, Nakagawa T. Renal tubular handling of glucose and fructose in health and disease. Compr Physiol 2021;12:2995-3044.ArticlePubMedPMCPDF
• 30. Wang Y, Heilig K, Saunders T, Minto A, Deb DK, Chang A, et al. Transgenic overexpression of GLUT1 in mouse glomeruli produces renal disease resembling diabetic glomerulosclerosis. Am J Physiol Renal Physiol 2010;299:F99-111.ArticlePubMedPMC
• 31. de Souza Cordeiro LM, Bainbridge L, Devisetty N, McDougal DH, Peters DJM, Chhabra KH. Loss of function of renal Glut2 reverses hyperglycaemia and normalises body weight in mouse models of diabetes and obesity. Diabetologia 2022;65:1032-47.ArticlePubMedPMCPDF

Figure & Data

References

Citations

Citations to this article as recorded by  

About this article

Zhang X, Zhang Y, Shi Y, Shi D, Niu M, Liu X, Liu X, Yang Z, Wu X. Kidney Gastrin/CCKBR Attenuates Type 2 Diabetes Mellitus by Inhibiting SGLT2-Mediated Glucose Reabsorption through Erk/NF-κB Signaling Pathway. Diabetes Metab J. 2024 Dec 24. doi: 10.4093/dmj.2023.0397. Epub ahead of print.

Received: Nov 06, 2023; Accepted: Sep 07, 2024

DOI: https://doi.org/10.4093/dmj.2023.0397.

Download citation ↓