Rat pancreatectomy combined with isoprenaline or uninephrectomy as models of diabetic cardiomyopathy or nephropathy

Rat pancreatectomy combined with isoprenaline or uninephrectomy as models of diabetic cardiomyopathy or nephropathy

AnimalsMale Sprague–Dawley rats (NTac:SD) (8–10 weeks old, 210–280 g, Taconic Biosciences) were single-housed in a controlled environment (20–22 °C, humidity 40–60%) with a 12 h light/dark cycle. The animals were acclimatized for at least 1 week before surgery. All animals had ad libitum access to regular chow diet (Altromin 1324, Brogaarden) and tap water throughout the study period. The studies were approved by The Danish Animal Experiments Inspectorate (license no. 2019-15-0201-01648, 2017-15-0201-01183, 2017-15-0201-01286) and conformed to the European Parliament Directive on the Protection of Animals Used for Scientific Purposes (2010/63/EU).Experimental designThree separate experiments were conducted in three cohorts of rats (Fig. 1). Experiment 1 involved metabolic characterization of the Px rat model and included sham surgery (n = 10), 60% Px (n = 10), and 90% Px (n = 10). Experiment 2 evaluated the 90% Px and isoprenaline-treated rat as a model of DbCM, and included sham-vehicle (Sham-Veh, n = 10), Px-vehicle (Px-Veh, n = 15), sham-isoprenaline (Sham-Iso, n = 12), and Px-isoprenaline (Px-Iso, n = 18) groups. Experiment 3 investigated the renal phenotype of the 90% Px and uninephrectomized rat model of DN in sham-operated (n = 11) and Px and uninephrectomized rats (Px-UNx, n = 12). In experiments 2 and 3, only Px rats exhibiting fasted or fed blood glucose above 10 or 12 mmol/L, respectively, 2 weeks post-Px were included.Figure 1Study design of experiment 1–3. GFR: glomerular filtration rate, Iso: isoprenaline, OGTT: oral glucose tolerance test, Px: pancreatectomy, Veh: vehicle, UNx: uninephrectomy.PancreatectomyRats were fasted overnight and received subcutaneous injections of atropine (0.1 mg/kg), enrofloxacin (50 mg/kg), carprofen (50 mg/kg) and saline (20 ml/kg) prior to surgery. All surgical interventions were performed under isoflurane anesthesia (2–3%). The rats were fasted until the next day, whereupon 5 g of chow was administered to each animal. Two days after surgery, the animals were fed ad libitum.PancreatectomyThrough a midline abdominal incision, the pancreatic tissue was removed by gentle abrasion with disposable micro-brushes (Plandent). All major blood vessels were left intact. For 60% Px, the tail and body of the pancreas were removed whereas the pancreatic tissue within the duodenal loop, comprising the entire head of the pancreatic tissue, was preserved. For 90% Px, only the pancreatic tissue between the common bile duct extending to the first loop of the duodenum was preserved.Sham surgeryThrough a midline abdominal incision, the tail and body of the pancreatic tissue was disengaged from the mesentery and gently manipulated before being repositioned to the abdominal cavity.Isoprenaline treatmentIsoprenaline hydrochloride (Sigma-Aldrich) was dissolved in saline immediately prior to dosing and administered subcutaneously (1 mg/kg) for ten consecutive days beginning 5 weeks after sham surgery or 90% Px.UninephrectomyAnimals were subjected to uninephrectomy at the time of 90% Px. The right ureter, renal artery and vein were identified and ligated. Subsequently, the right kidney was removed. In sham-operated animals, the right kidney was exposed and gently manipulated.Blood and plasma analysesBlood glucoseTail vein blood was collected once weekly from non-fasted or fasted (4 h) rats into heparinized glass capillary tubes and immediately suspended in glucose/lactate system solution buffer (EKF-diagnostics). The glucose concentration was measured immediately using a BIOSEN c-Line glucometer (EKF-diagnostics) according to the manufacturer’s instructions.Oral glucose tolerance test (OGTT)In experiment 1, animals were fasted for 4 h prior to oral glucose administration (2 g/kg body weight, 500 mg glucose/mL, Fresenius Kabi), and blood glucose was measured at 6 timepoints over the course of 240 min post-injection.Blood and plasma markersPlasma insulin and C-peptide were measured in duplicate using AlphaLISA platform (Perkin Elmer) according to the manufacturer’s instructions. Glycated haemoglobin (HbA1c), plasma creatinine, and urea were measured using commercial assays (Roche Diagnostics) on the Cobas C-501 autoanalyzer, and plasma cystatin C was measured using a commercial assay (R&D Systems) as per manufacturers’ instructions.Urine collection and biochemical analysesRats were single-housed in metabolic cages (Techniplast) with free access to powdered chow and water. The excreted urine was collected for 16 h. Urine creatinine was quantified using a CREP2 assay (Roche Diagnostics) on the Cobas C-501 autoanalyzer as per manufacturer’s instructions. Urine albumin was measured using a rat albumin ELISA (Bethyl Laboratories). Urine neutrophil gelatinase-associated lipocalin (NGAL), soluble tumor necrosis factor receptors (sTNFR) I and II were measured using ELISA assays (R&D Systems), while urine nephrin and podocalyxin were quantified using ELISA assays from LSBio and CusaBio, respectively. All ELISA assays were run as per manufacturers’ instructions and levels of analytes were reported as the analyte-to-creatinine ratio (ACR) in urine. Urine ACR values were log10-transformed before statistical analyses.EchocardiographyEchocardiographic assessments were performed under isoflurane anesthesia (2–3%). After chest hair removal, the rats were positioned in supine position on a heated pad. Electrocardiogram electrodes (lead II configuration, 3M) were placed and the rectal temperature was measured before and after examination. All examinations were performed using an ultrasound device (Philips, iE33) with a 12 MHz sector array probe. Function and dimensions of the LV were assessed from parasternal short and apical long axis views. The LV internal diameter (LVID), anterior and posterior wall thickness (LVAW, LVPW) were assessed in systole (LVIDs, LVAWs and LVPWs, respectively) and diastole (LVIDd, LVAWd and LVPWd, respectively) using 2D-guided M-mode in a short axis view at the level of the papillary muscles, according to the leading edge to leading edge principle34. The ejection fraction (EF) was calculated using the Teichholz formula35. To correct for the difference in LV size, all short axis measurements were normalized to body weight. LV filling (E and A velocity and deceleration time) was measured in the apical 4-chamber long axis view with the sample volume placed at the tip of the mitral valve leaflets using pulsed wave Doppler. Echocardiographic analyses were performed offline using Q-station software (version 3.8.5, Philips). Image acquisitions and analyses were performed by one operator blinded towards identity of the animals.Glomerular filtration rate measurementGFR was assessed by a fluorescein isothiocyanate (FITC)-inulin test. Briefly, a 5% solution of FITC-inulin (TdB Consultancy) was prepared in 0.85% saline by heating and overnight dialysis using a Spectra/Por 6 dialysis tube (1 kDa molecular cut-off; Spectrum Labs). The FITC-inulin solution was sterile filtered (0.2 µm syringe filter) before tail vein injections in conscious rats (100 mg/kg). Sublingual blood was collected in heparinized tubes at 8 timepoints over the course of 75 min post-injection. Plasma FITC-inulin concentrations were measured using a plate-reader (CLARIOstar, BMG LABTECH) and GFR was calculated using a two-compartment model from the rate of decay in plasma FITC-inulin as described previously36 and normalized to body weight.Histology and stereologyPancreasAt termination, pancreatic samples from sham, 60% and 90% Px rats were removed en-bloc and incubated in 10% neutral buffered formalin until further processing. Pancreatic tissue was carefully dissected, weighed, and processed as previously described37. Briefly, pancreas was rolled tightly into strips of gauze before infiltration with paraffin in an automated tissue processor (VIP5, Sakura). The pancreas was then cut into 7–9 systematic uniform random tissue slabs with a razor blade fractionator and embedded in paraffin blocks with the cut surface down. Subsequently, 5 µm thick sections were cut from each block on a microtome and collected on microscope slides. Immunohistochemistry against beta cells and non-beta cells were performed as a double staining using standard procedures. Briefly, after deparaffinization and microwave oven pretreatment in Tris-EGTA buffer (pH 9), sections were stained for non-beta cells using an antibody cocktail consisting of rabbit anti-glucagon (Phoenix, H-028–02, 1:5000), rabbit anti-somatostatin (Dako, 0566, 1:7500), and rabbit anti-pancreatic polypeptide (Europroxima, B32-1, 1:5000). The antibody cocktail was detected using Envision + anti-rabbit HRP-coupled polymer system (Dako, K4002) and developed with DAB-Nickel as chromogen. Next, beta cells were stained using a guinea pig anti-insulin antibody (Dako, A0564, 1:6000) followed by a biotinylated secondary donkey anti-guinea pig antibody (Jackson ImmunoResearch 706-065-148, 1:2000). Amplification and development of the insulin staining was performed using the Vectastain ABC elite kit (Vector Laboratories PK6100) and Impact NovaRed (Vector Laboratories, SK-4805). Finally, sections were counterstained with Mayer’s hematoxylin (Sigma-Aldrich).Heart and kidneyOne half of the sagittally-divided LV or left kidney were fixed in 10% neutral buffered formalin for 24 h at room temperature. The fixed tissues were cut into 5–7 systematic uniform random tissue slabs with a razor blade fractionator and embedded in paraffin blocks with the cut surface down. For Picro Sirius Red staining, a set of 3 µm sections were cut on a microtome from each block and collected on microscope slides. For Periodic acid-Schiff (PAS) staining, another set of sections were cut in pairs with a dissector distance of 30 µm. Picro Sirius Red staining: After deparaffinization, sections were incubated in Wiegert’s iron hematoxylin (Sigma-Aldrich) and then stained in Picro Sirius Red (Sigma-Aldrich) before they were cover slipped with Pertex. PAS staining: sections were deparaffinized and oxidized with 0.5% periodic acid solution followed by incubation with Schiff’s reagent. Sections were counterstained in Mayer’s hematoxylin and cover slipped with Pertex. Podocin/type IV collagen double fluorescent immunohistochemical staining of kidney sections was performed as previously described22.Image analysis and stereological quantificationStereological quantification of beta cell and non-beta cell mass was calculated as an area fraction (area of beta cells versus total pancreatic area) multiplied by the dissected pancreas mass as previously described37. Heart and kidney collagen content was determined as Picro Sirius Red area fraction using image analysis. Stained slides were scanned under a 20 × objective in an Aperio Scanscope AT slide scanner and imported into an image analysis module in Visiopharm Integrator System (Visiopharm). A Bayesian classifier was trained to detect Picro Sirius Red positive collagen versus other tissue components. The collagen area fraction was calculated as the Picro Sirius Red area divided by the total tissue area. Intra-glomerular collagen type 4 content and stereological estimation of kidney compartmental volumes were determined as previously described22. The mean glomerular volume was calculated as total glomerular volume divided by the mean number of glomeruli estimated using the physical dissector on serially cut reference and look-up sections with a dissector distance of 30 µm38.RNA sequencingTranscriptome analysis was performed by sequencing of RNA extracts from LV tissue and renal cortex. RNA was purified from homogenized tissue using the NucleoSpin RNA Plus kit (Macherey-Nagel GmbH). The RNA quantity was measured using Qubit (Thermo Scientific, Eugene, OR) and RNA quality was determined using a bioanalyzer with RNA 6000 Nano kit (Agilent). Purified RNA from each sample was used to generate a single end cDNA library using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA). cDNA libraries were sequenced on a NextSeq 500 using NextSeq 500/550 High Output Kit V2 (Illumina) for 75 cycles to a depth of 14–28 million reads per sample. Reads were aligned to the Ensembl Rnor_6.0 release 89 Rattus norvegicus genome using STAR v.2.5.2a with default parameters39.Statistical analysisExcept for the gene expression data set, which was analysed using DEseq2 package for R with default parameters40, all data were analyzed using GraphPad Prism software (version 8.2). Normal distribution of data was assessed by inspection of QQ plots and by using the Shapiro–Wilk test. Normally distributed data are shown as mean ± standard error of mean (SEM). In experiment 1, repeated measures two-way analyses of variance (ANOVA) with Bonferroni’s post-hoc test were used for time-course data and one-way ANOVA with Tukey’s post-hoc test was used for insulin and beta cell mass data. In experiment 2, two-way ANOVA was applied and in addition to evaluating the main effects of Px and isoprenaline treatment, the interaction between the two was included to evaluate a potential synergistic effect. Main effects of Px or isoprenaline were reported when no interaction between these two was found. Unpaired t-test and repeated measurements two-way ANOVA with Bonferroni’s Multiple Comparisons test were used in experiment 3. A p value < 0.05 was considered statistically significant.Ethics approval and consent to participateThe animal experiments were approved by The Danish Animal Experiments Inspectorate (license no. 2019-15-0201-01648, 2017-15-0201-01183, 2017-15-0201-01286) and conformed to the European Parliament Directive on the Protection of Animals Used for Scientific Purposes (2010/63/EU).Consent for publicationAll authors have declared their consent for this publication.

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