Cardiovascular and renal complications are the predominant causes of morbidity and mortality amongst patients with diabetes. Development of novel treatments have been hampered by the lack of available animal models recapitulating the human disease. We hypothesized that experimental diabetes in rats combined with a cardiac or renal stressor, would mimic diabetic cardiomyopathy and nephropathy, respectively. Diabetes was surgically induced in male Sprague Dawley rats by 90% pancreatectomy (Px). Isoprenaline (Iso, 1 mg/kg, sc., 10 days) was administered 5 weeks after Px with the aim of inducing cardiomyopathy, and cardiac function and remodeling was assessed by echocardiography 10 weeks after surgery. Left ventricular (LV) fibrosis was quantified by Picro Sirius Red and gene expression analysis. Nephropathy was induced by Px combined with uninephrectomy (Px-UNx). Kidney function was assessed by measurement of glomerular filtration rate (GFR) and urine albumin excretion, and kidney injury was evaluated by histopathology and gene expression analysis. Px resulted in stable hyperglycemia, hypoinsulinemia, decreased C-peptide, and increased glycated hemoglobin (HbA1c) compared with sham-operated controls. Moreover, Px increased heart and LV weights and dimensions and caused a shift from α-myosin heavy chain (MHC) to β-MHC gene expression. Isoprenaline treatment, but not Px, decreased ejection fraction and induced LV fibrosis. There was no apparent interaction between Px and Iso treatment. The superimposition of Px and UNx increased GFR, indicating hyperfiltration. Compared with sham-operated controls, Px-UNx induced albuminuria and increased urine markers of kidney injury, including neutrophil gelatinase-associated lipocalin (NGAL) and podocalyxin, concomitant with upregulated renal gene expression of NGAL and kidney injury molecule 1 (KIM-1). Whereas Px and isoprenaline separately produced clinical endpoints related to diabetic cardiomyopathy, the combination of the two did not accentuate disease development. Conversely, Px in combination with UNx resulted in several clinical hallmarks of diabetic nephropathy indicative of early disease development.

Background Cardiovascular and renal complications are the predominant causes of morbidity and mortality amongst patients with diabetes. Development of novel treatments have been hampered by the lack of available animal models recapitulating the human disease. We hypothesized that experimental diabetes in rats combined with a cardiac or renal stressor, would mimic diabetic cardiomyopathy and nephropathy, respectively. Methods Diabetes was surgically induced in male Sprague Dawley rats by 90% pancreatectomy (Px). Isoprenaline (Iso, 1 mg/kg, sc., 10 days) was administered five weeks after Px with the aim of inducing cardiomyopathy, and cardiac function and remodeling was assessed by echocardiography ten weeks after surgery. Left ventricular (LV) fibrosis was quantified by Picro Sirius Red and gene expression analysis. Nephropathy was induced by Px combined with uninephrectomy (Px-UNx). Kidney function was assessed by measurement of glomerular filtration rate (GFR) and urine albumin excretion, and kidney injury was evaluated by histopathology and gene expression analysis. Results Px resulted in stable hyperglycemia, hypoinsulinemia, decreased C-peptide, and increased glycated hemoglobin (HbA1c) compared with sham-operated controls. Moreover, Px increased LV mass and dimensions, and caused a shift from α-myosin heavy chain (MHC) to β-MHC gene expression. Isoprenaline treatment, but not Px, decreased ejection fraction and induced LV fibrosis. There was no apparent interaction between Px and Iso treatment. The superimposition of Px and UNx increased GFR, indicating hyperfiltration. Compared with sham-operated controls, Px-UNx induced albuminuria and increased urine markers of kidney injury, including neutrophil gelatinase-associated lipocalin (NGAL) and podocalyxin, concomitant with upregulated renal gene expression of NGAL and kidney injury molecule 1 (KIM-1). Conclusion Whereas Px and isoprenaline separately produced clinical endpoints related to diabetic cardiomyopathy, the combination of the two did not accentuate disease development. Conversely, Px in combination with UNx resulted in several clinical hallmarks of diabetic nephropathy indicative of early disease development.

Introduction: Diabetic nephropathy (DN) is a long-term complication that occurs in ∼40% of diabetes patients and is a leading cause of end-stage renal disease. In a newly established mouse model of progressive DN, we investigated the effects of hypertension on kidney injury. Methods: Female db/db mice were uninephrectomized (UNx) at 8 weeks of age and injected i.v. with a Renin adeno-associated virus (AAV) construct at different doses to induce hypertension, while a LacZAAV construct was used as negative control. Sham-operated db/+ mice served as healthy controls. Blood pressure and glomerular filtration rate (GFR) was measured at 22 weeks of age, while urine albumin to creatinine ratio (ACR) was measured before termination and kidney tissue sampling 24 weeks of age. Results: GFR measurements indicated hyperfiltration in all AAV-injected UNx db/db mice compared to db/+ mice, while ReninAAV tended to dose-dependently decrease GFR compared to LacZAAV in UNx db/db mice. Urine ACR was worsened by ReninAAV-induced hypertension compared to LacZAAV controls. 3D kidney imaging demonstrated increased glomerular volume in LacZAAV UNx db/db mice compared to db/+ mice with no further effect in ReninAAV groups. Automized AI-based glomerulosclerosis scoring showed ReninAAV dose-dependent increases in glomerulosclerosis compared to LacZAAV controls. RNA sequencing revealed upregulated gene expression markers of kidney fibrogenesis (Col1a1, Col4, Fn1) and tubular injury (Ngal and Kim-1), as well as downregulation of proximal tubular markers (Megalin, Aqp1) in ReninAAV UNx db/db mice compared to LacZAAV controls. Conclusion: ReninAAV-induced hypertension in female UNx db/db mice aggravates albuminuria and glomerulosclerosis paralleled by increased expression of genes associated with tubular injury renal fibrosis, thus confirming that ReninAAV UNx db/db mice is a reliable model of DN with features of late stage human disease. Disclosure M.V. Østergaard: None. I.R. Sørensen: None. A.A. Pedersen: None. T. Secher: Employee; Self; Gubra. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Gubra. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. F.E. Sembach: Employee; Self; Gubra. M.R. Madsen: None. K. Fosgerau: None. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. N. Vrang: Board Member; Self; Gubra. Employee; Self; Gubra. Stock/Shareholder; Self; Gubra.

Background and Aims Diabetic nephropathy (DN) is a long-term complication that occurs in ∼40% of diabetes patients and is a leading cause of end-stage renal disease. Despite recent emergence of SGLT2 inhibitors and GLP-1 receptor agonists for nephroprotection in diabetes patients, drug discovery has been halted by the lack of reliable rodent models exhibiting features of human DN. In a newly established mouse model of progressive DN, we investigate the effects of hypertension on kidney injury. Method Female db/db mice were uninephrectomized (UNx) at 8 weeks of age and injected i.v. with a Renin adeno-associated virus (AAV) construct at different doses to induce hypertension, while a LacZAAV construct was used as negative control. db/+ mice served as healthy controls. Hypertension was measured by tail cuff and glomerular filtration rate (GFR) transcutaneous recoding of FITC-sinistrin after i.v. bolus injection at 22 weeks of age. Urine ACR measured in spot urine samples collected before termination 24 weeks of age. Terminal kidney samples were collected for 3D image analyses, histopathological evaluation, and next generation sequencing for gene expression analyses. Results GFR measurements indicated hyperfiltration in all AAV-injected UNx db/db mice compared to db/+ mice, while ReninAAV tended to dose-dependently decrease GFR compared to LacZAAV in UNx db/db mice. Urine ACR was worsened by ReninAAV-induced hypertension compared to LacZAAV controls. Automized AI-based glomerulosclerosis scoring showed ReninAAV dose-dependent increases in glomerulosclerosis compared to LacZAAV controls. 3D kidney imaging demonstrated increased glomerular volume in LacZAAV UNx db/db mice compared to db/+ mice with no further effect in ReninAAV groups. RNA sequencing revealed upregulated gene expression markers of fibrogenesis (incl. Col1a1, Col3, Col4, Fn1, Lamc2 and Vim) and tubular injury markers (Ngal and Kim-1), as well as downregulation of proximal tubular markers (Megalin and Aqp1) in ReninAAV UNx db/db mice compare to LacZAAV controls. Conclusion ReninAAV-induced hypertension in female UNx db/db mice accelerates kidney injury in uninephrectomized db/db mice and aggravates GFR, albuminuria and glomerulosclerosis in parallel with increased expression of genes associated with tubular injury renal fibrosis. Together, these data confirm that ReninAAV UNx db/db mice is a reliable model of DN with features of late stage human disease.