Nephroprotective Effects of Semaglutide as Mono- and Combination Treatment with Lisinopril in a Mouse Model of Hypertension-Accelerated Diabetic Kidney Disease

Background: Obesity, hyperglycemia and hypertension are critical risk factors for development of diabetic kidney disease (DKD). Emerging evidence suggests that glucagon-like peptide-1 receptor (GLP-1R) agonists improve cardiovascular and renal outcomes in type 2 diabetes patients. Here, we characterized the effect of the long-acting GLP-1R agonist semaglutide alone and in combination with an ACE inhibitor (lisinopril) in a model of hypertension-accelerated, advanced DKD facilitated by adeno-associated virus-mediated renin overexpression (ReninAAV) in uninephrectomized (UNx) female diabetic db/db mice. Methods: Female db/db mice received a single intravenous injection of ReninAAV 1 week prior to UNx. Six weeks post-nephrectomy, db/db UNx-ReninAAV mice were administered (q.d.) vehicle, semaglutide (30 nmol/kg, s.c.) or semaglutide (30 nmol/kg, s.c.) + lisinopril (30 mg/kg, p.o.) for 11 weeks. Endpoints included blood pressure, plasma/urine biochemistry, kidney histopathology and RNA sequencing. Results: Vehicle-dosed db/db UNx-ReninAAV mice developed hallmarks of DKD characterized by severe albuminuria and advanced glomerulosclerosis. Semaglutide robustly reduced hyperglycemia, hypertension and albuminuria concurrent with notable improvements in glomerulosclerosis severity, podocyte filtration slit density, urine/renal kidney injury molecule-1 (KIM-1) levels and gene expression markers of inflammation and fibrogenesis in db/db UNx-ReninAAV mice. Co-administration of lisinopril further ameliorated hypertension and glomerulosclerosis. Conclusions: Semaglutide improves disease hallmarks in the db/db UNx-ReninAAV mouse model of advanced DKD. Further benefits on renal outcomes were obtained by adjunctive antihypertensive standard of care. Collectively, our study supports the development of semaglutide for management of DKD

Characterisation of age-dependent beta cell dynamics in the male db/db mice.

AIM: To characterise changes in pancreatic beta cell mass during the development of diabetes in untreated male C57BLKS/J db/db mice. METHODS: Blood samples were collected from a total of 72 untreated male db/db mice aged 5, 6, 8, 10, 12, 14, 18, 24 and 34 weeks, for measurement of terminal blood glucose, HbA1c, plasma insulin, and C-peptide. Pancreata were removed for quantification of beta cell mass, islet numbers as well as proliferation and apoptosis by immunohistochemistry and stereology. RESULTS: Total pancreatic beta cell mass increased significantly from 2.1 ± 0.3 mg in mice aged 5 weeks to a peak value of 4.84 ± 0.26 mg (P < 0.05) in 12-week-old mice, then gradually decreased to 3.27 ± 0.44 mg in mice aged 34 weeks. Analysis of islets in the 5-, 10-, and 24-week age groups showed increased beta cell proliferation in the 10-week-old animals whereas a low proliferation is seen in older animals. The expansion in beta cell mass was driven by an increase in mean islet mass as the total number of islets was unchanged in the three groups. CONCLUSIONS/INTERPRETATION: The age-dependent beta cell dynamics in male db/db mice has been described from 5-34 weeks of age and at the same time alterations in insulin/glucose homeostasis were assessed. High beta cell proliferation and increased beta cell mass occur in young animals followed by a gradual decline characterised by a low beta cell proliferation in older animals. The expansion of beta cell mass was caused by an increase in mean islet mass and not islet number

Impaired behavioural pain responses in hph-1 mice with inherited deficiency in GTP cyclohydrolase 1 in models of inflammatory pain

BACKGROUND: GTP cyclohydrolase 1 (GTP-CH1), the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4), encoded by the GCH1 gene, has been implicated in the development and maintenance of inflammatory pain in rats. In humans, homozygous carriers of a “pain-protective” (PP) haplotype of the GCH1 gene have been identified exhibiting lower pain sensitivity, but only following pain sensitisation. Ex vivo, the PP GCH1 haplotype is associated with decreased induction of GCH1 after stimulation, whereas the baseline BH4 production is not affected. Contrary, loss of function mutations in the GCH1 gene results in decreased basal GCH1 expression, and is associated with DOPA-responsive dystonia (DRD). So far it is unknown if such mutations affect acute and inflammatory pain. RESULTS: In the current study, we examined the involvement of the GCH1 gene in pain models using the hyperphenylalaninemia 1 (hph-1) mouse, a genetic model for DRD, with only 10% basal GTP-CH1 activity compared to wild type mice. The study included assays for determination of acute nociception as well as models for pain after sensitisation. Pain behavioural analysis of the hph-1 mice showed reduced pain-like responses following intraplantar injection of CFA, formalin and capsaicin; whereas decreased basal level of GTP-CH1 activity had no influence in naïve hph-1 mice on acute mechanical and heat pain thresholds. Moreover, the hph-1 mice showed no signs of motor impairment or dystonia-like symptoms. CONCLUSIONS: In this study, we demonstrate novel evidence that genetic mutations in the GCH1 gene modulate pain-like hypersensitivity. Together, the present data suggest that BH4 is not important for basal heat and mechanical pain, but they support the hypothesis that BH4 plays a role in inflammation-induced hypersensitivity. Our studies suggest that the BH4 pathway could be a therapeutic target for the treatment of inflammatory pain conditions. Moreover, the hph-1 mice provide a valid model to study the consequence of congenital deficiency of GCH1 in painful conditions

A Hamster Model of Diet-Induced Obesity for Preclinical Evaluation of Anti-Obesity, Anti-Diabetic and Lipid Modulating Agents

<div><p>Aim</p><p>Unlike rats and mice, hamsters develop hypercholesterolemia, and hypertriglyceridemia when fed a cholesterol-rich diet. Because hyperlipidemia is a hallmark of human obesity, we aimed to develop and characterize a novel diet-induced obesity (DIO) and hypercholesterolemia Golden Syrian hamster model.</p><p>Methods and Results</p><p>Hamsters fed a highly palatable fat- and sugar-rich diet (HPFS) for 12 weeks showed significant body weight gain, body fat accumulation and impaired glucose tolerance. Cholesterol supplementation to the diet evoked additional hypercholesterolemia. Chronic treatment with the GLP-1 analogue, liraglutide (0.2 mg/kg, SC, BID, 27 days), normalized body weight and glucose tolerance, and lowered blood lipids in the DIO-hamster. The dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin (3.0 mg/kg, PO, QD) also improved glucose tolerance. Treatment with peptide YY_3-36 (PYY_3-36, 1.0 mg/kg/day) or neuromedin U (NMU, 1.5 mg/kg/day), continuously infused via a subcutaneous osmotic minipump for 14 days, reduced body weight and energy intake and changed food preference from HPFS diet towards chow. Co-treatment with liraglutide and PYY_3-36 evoked a pronounced synergistic decrease in body weight and food intake with no lower plateau established. Treatment with the cholesterol uptake inhibitor ezetimibe (10 mg/kg, PO, QD) for 14 days lowered plasma total cholesterol with a more marked reduction of LDL levels, as compared to HDL, indicating additional sensitivity to cholesterol modulating drugs in the hyperlipidemic DIO-hamster. In conclusion, the features of combined obesity, impaired glucose tolerance and hypercholesterolemia in the DIO-hamster make this animal model useful for preclinical evaluation of novel anti-obesity, anti-diabetic and lipid modulating agents.</p></div

Effect of ezetimibe on blood lipids.

<p>Semi-fasted blood lipid levels of obese hyperlipidemic hamsters treated for 14 days with vehicle (PO, QD) or Ezetimibe (10.0 mg/kg, PO, QD). Values are means ± SEM (n = 8 per group; P<0.01 (**); P<0.001 (***) vs. vehicle group).</p><p>Effect of ezetimibe on blood lipids.</p

Effect of NMU, PYY_3-36, liraglutide or co-treatment with liraglutide and PYY_3-36 on body weight and cumulative energy intake.

<p>(A) Body weight change (% of day 0), (B) Cumulative total energy intake (kJ), (C) cumulative intake of the HPFS diet with 0.5% cholesterol supplement (kJ), and (D) cumulative intake of regular chow (kJ) after 14 days treatment of obese hyperlipidemic hamsters with vehicle (SC, BID) + vehicle (osmotic pump), liraglutide (0.1 mg/kg, SC, BID) + vehicle (osmotic pump), vehicle (SC, BID) + PYY_3-36 (1.0 mg/kg/day, osmotic pump), the co-treatment group liraglutide (0.1 mg/kg, SC, BID) + PYY_3-36 (1.0 mg/kg/day, osmotic pump), or vehicle (SC, BID) + NMU (1.5 mg/kg/day, osmotic pump). Hamsters included in the study were fed a HPFS diet with 0.5% cholesterol supplemented for 12 weeks before initiation of the study. Data are given as mean ± SEM with n = 8/group. Statistical analysis: two-way ANOVA with Bonferronis post hoc test; ^a<i>P</i><0.05 for liraglutide vs. vehicle group, ^b<i>P</i><0.05 for PYY_3-36 vs. vehicle group, ^c<i>P</i><0.05 for PYY_3-36 + liraglutide vs. vehicle group, and ^d<i>P</i><0.05 for NMU vs. vehicle group.</p

Diet-induced obesity and hypercholesterolemia in the Golden Syrian hamster.

<p>Whole-body fat mass (g), free-fed blood lipid and insulin levels of hamsters fed high fat-high carbohydrate (HPFS) palatable diet with 0.5% cholesterol supplementation for 12 weeks. Values are means ± SEM (n = 6 per group; <i>P</i><0.05 (*), <i>P</i><0.001 (***) vs. chow-fed hamsters).</p><p>Diet-induced obesity and hypercholesterolemia in the Golden Syrian hamster.</p