The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function

Patrick Meagher; Robert Civitarese; Xavier Lee; Mark Gordon; Antoinette Bugyei-Twum; Jean-Francois Desjardins; Golam Kabir; Yanling Zhang; Hari Kosanam; Aylin Visram; Howard Leong-Poi; Andrew Advani; Kim A Connelly

doi:10.1371/journal.pone.0252711

. 2021 Jun 24;16(6):e0252711. doi: 10.1371/journal.pone.0252711

The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function

Patrick Meagher ^1,^2,^‡, Robert Civitarese ^1,^‡, Xavier Lee ^1,², Mark Gordon ¹, Antoinette Bugyei-Twum ^1,², Jean-Francois Desjardins ¹, Golam Kabir ¹, Yanling Zhang ¹, Hari Kosanam ¹, Aylin Visram ^1,², Howard Leong-Poi ^1,³, Andrew Advani ^1,³, Kim A Connelly ^1,^2,^3,^*

Editor: Vincenzo Lionetti⁴

¹St. Michael’s Hospital, Keenan Research Centre, Li Ka Shing Knowledge Institute, Toronto, Canada

²Department of Physiology, University of Toronto, Toronto, Canada

³Institute of Medical Science, University of Toronto, Toronto, Canada

⁴Scuola Superiore Sant’Anna, ITALY

Competing Interests: The authors have read the journal’s policy and have the following competing interests: KAC has received research grants to his institution from Astra Zeneca and Boehringer Ingelheim, received support for travel to scientific meeting from Boehringer Ingelheim, and received honoraria for speaking engagements and ad hoc participation in advisory boards from Astra Zeneca, Boehringer Ingelheim, and Janssen. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

‡ These authors are co-first authors on this work.

^✉

* E-mail: ConnellyK@smh.ca

Roles

Patrick Meagher: Data curation, Formal analysis, Supervision, Visualization, Writing – original draft, Writing – review & editing

Robert Civitarese: Data curation, Formal analysis, Visualization, Writing – original draft

Xavier Lee: Formal analysis, Writing – original draft

Mark Gordon: Data curation, Formal analysis

Antoinette Bugyei-Twum: Data curation, Formal analysis, Project administration, Supervision

Jean-Francois Desjardins: Formal analysis, Methodology, Software, Writing – review & editing

Golam Kabir: Investigation, Methodology

Yanling Zhang: Investigation, Methodology

Hari Kosanam: Investigation, Methodology, Software

Aylin Visram: Data curation, Formal analysis

Howard Leong-Poi: Conceptualization, Investigation, Writing – review & editing

Andrew Advani: Conceptualization, Methodology, Writing – review & editing

Kim A Connelly: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing

Vincenzo Lionetti: Editor

PMCID: PMC8224913 PMID: 34166385

Abstract

Background

Patients with diabetes are at a high risk for developing cardiac dysfunction in the absence of coronary artery disease or hypertension, a condition known as diabetic cardiomyopathy. Contributing to heart failure is the presence of diabetic kidney disease. The Goto-Kakizaki (GK) rat is a non-obese, non-hypertensive model of type 2 diabetes that, like humans, shares a susceptibility locus on chromosome 10. Herein, we perform a detailed analysis of cardio-renal remodeling and response to renin angiotensin system blockade in GK rats to ascertain the validity of this model for further insights into disease pathogenesis.

Methods

Study 1: Male GK rats along with age matched Wistar control animals underwent longitudinal assessment of cardiac and renal function for 32 weeks (total age 48 weeks). Animals underwent regular echocardiography every 4 weeks and at sacrifice, early (~24 weeks) and late (~48 weeks) timepoints, along with pressure volume loop analysis. Histological and molecular characteristics were determined using standard techniques. Study 2: the effect of renin angiotensin system (RAS) blockade upon cardiac and renal function was assessed in GK rats. Finally, proteomic studies were conducted in vivo and in vitro to identify novel pathways involved in remodeling responses.

Results

GK rats developed hyperglycaemia by 12 weeks of age (p<0.01 c/w Wistar controls). Echocardiographic assessment of cardiac function demonstrated preserved systolic function by 48 weeks of age. Invasive studies demonstrated left ventricular hypertrophy, pulmonary congestion and impaired diastolic function. Renal function was preserved with evidence of hyperfiltration. Cardiac histological analysis demonstrated myocyte hypertrophy (p<0.05) with evidence of significant interstitial fibrosis (p<0.05). RT qPCR demonstrated activation of the fetal gene program, consistent with cellular hypertrophy. RAS blockade resulted in a reduction blood pressure(P<0.05) cardiac interstitial fibrosis (p<0.05) and activation of fetal gene program. No significant change on either systolic or diastolic function was observed, along with minimal impact upon renal structure or function. Proteomic studies demonstrated significant changes in proteins involved in oxidative phosp4horylation, mitochondrial dysfunction, beta-oxidation, and PI3K/Akt signalling (all p<0.05). Further, similar changes were observed in both LV samples from GK rats and H9C2 cells incubated in high glucose media.

Conclusion

By 48 weeks of age, the diabetic GK rat demonstrates evidence of preserved systolic function and impaired relaxation, along with cardiac hypertrophy, in the presence of hyperfiltration and elevated protein excretion. These findings suggest the GK rat demonstrates some, but not all features of diabetes induced “cardiorenal” syndrome. This has implications for the use of this model to assess preclinical strategies to treat cardiorenal disease.

Introduction

Diabetes mellitus (DM) is a chronic heterogeneous metabolic disorder, characterised by hyperglycemia, which is estimated to effect 439 million adults worldwide by the year 2030 [1]. Type 2 DM (T2DM) is now disproportionately outweighing the number of type I diabetic patients (accounting for >90% of all cases), with unambiguous evidence from epidemiological and clinical data that, despite excellent glycemic control, T2DM patients have higher morbidity and mortality when compared to their non-diabetic counterparts in a range of complications such as heart failure, renal failure and death [2–4].

In particular, T2DM is associated with simultaneous impairment of both cardiac and renal function, referred to collectively as ‘cardiorenal syndrome’ [5]. T2DM induced changes such as impaired insulin signaling, hyperglycemia/glucotoxicity and lipotoxicity, are thought to contribute, along with hemodynamic changes, activation of the renin-angiotensin-aldosterone system (RAAS), endothelial dysfunction, inflammation and oxidative stress [6–9]. Indeed, approximately 20–40% of patients with T2D develop diabetic kidney disease, the most common cause of end stage renal failure [10].

Diabetic kidney disease is characterized by progressive glomerulosclerosis and interstitial fibrosis, in part due to over-activity of prosclerotic cytokines, such as the transforming growth factor β (TGF-β). In addition to effects on the kidneys, persons with T2DM are significant more likely to develop heart failure [11]. Despite being different organ systems, diabetic patients with worsening kidney function, as evident by reduced glomerular filtration rate and proteinuria, have a two-to-tenfold more rapid progression of cardiac related diseases, such as coronary artery disease and atherosclerosis, highlighting the interconnected nature of the cardiorenal system.

Unfortunately, despite the seminal discovery of insulin by Banting and Best in 1921, targeted treatment to prevent cardiorenal disease in T2DM individuals remain extremely limited. Until recently ACE inhibitor treatment has been the most effective treatment that improves morbidity and mortality but, the introduction of Sodium Glucose Co-Transporter 2 inhibitors (SGLT-2i) has demonstrated benefit in cardiovascular outcomes and all-cause mortality in T2DM patients [12–14]. Further, the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial and the Dapagliflozin and Prevention of Adverse Outcomes in Chronic Kidney Disease (DAPA-CKD) trial has recently demonstrated effectiveness to prevent kidney failure and mortality in DM patients with nephropathy [15, 16].

The major obstacle to treating T2DM in an attempt prevent cardio-renal morbidity and mortality is the lack of detailed understanding of the mechanism(s), which has significantly hindered drug development. SGLT2 inhibitors are an example of such deficits in the understanding of the mechanisms of cardiovascular complications in T2DM patients. While there has been such a significant amount of study into its action within the heart and kidney, there is still no unified hypothesis to account for the protective effects of SGLT2 inhibitors [14, 15, 17].

In order to gain a better understanding of the mechanisms via which diabetic drugs elicit their effects, a robust pre-clinical model that expresses similar cardiorenal complications as seen in T2DM patients is imperative. The Goto-Kakizaki (GK) rat is a non-obese, non-hypertensive rodent model of T2DM, which by 4–5 weeks develop glucose intolerance and later peripheral insulin resistance. Further, GK rats have demonstrated vascular smooth cell dysfunction a hallmark of T2DM [18]. The GK rat genetically recapitulates human diabetes, in that it is a polygenic model of disease and shares a susceptibility locus corresponding to a region on human chromosome 10 [19–21]. However, the detailed cardiorenal characterization of the GK rat has been poorly documented. Thus, in this study, we performed functional, structural and molecular characterization of the GK rat at early and late time points, in order to determine whether the GK rat is an appropriate model of cardiorenal diabetic complications. In addition, in order to evaluate the effectiveness of this model for the study of human therapeutics, we examined the therapeutic effect of perindopril, an Angiotensin Converting Enzyme inhibitor (ACEi). Finally, as multiple studies use cell culture assays to help explore and determine mechanisms, we looked to identify whether changes in the LV proteome of GK rats would be recapitulated in high glucose (HG) treated H9c2 cardiomyoblasts.

Our data demonstrates that the GK rodent model shows some similar characteristics to human T2DM, which was subsequently improved following treatment with an ACEi, albeit demonstrating a mild disease phenotype and response. However, a major concern for this model is the time it takes the GK animals to develop significant cardiac and renal dysfunction. As such, the applicability of using the GK rat as a model of T2DM remains limited and highlights the need for the development of alternate pre-clinical models.

Methods

Study 1: Characterization of the Goto Kakizaki rat at early and late timepoints

Age-matched, male Wistar and GK rats were studied across time and assessed at an early and late time point. Animals sacrificed at ~28 weeks of age were classified as the ‘early’, whereas animals sacrificed at ~48 weeks of age were classified as the ‘late’ time point. Animals were housed at constant room temperature (21 ± 1°C) with a 12-hour light/dark cycle and were fed standard rat chow, formulated rodent diet designed to support growth, and water ad libitum. All animal studies were approved by the animal ethics committee at St Michael’s Hospital, Toronto, Ontario Canada in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996).

Diabetes was confirmed at 8 weeks of age in the GK rats. Each week, rats were weighed, and blood glucose was determined using Accu-check Advantage (Roche, Mississauga, ON, Canada). At 28 weeks (Early) and 48 weeks (Late) of age, all animals also underwent metabolic caging and glycated hemoglobin (HbA1c) assessment for determination/confirmation of diabetes status. HbA1c was measured using A1cNow+ (Bayer, Sunnyvale, CA). Additionally, following metabolic caging urine protein: creatine levels were measured by the Department of Pathology, Toronto General Hospital, Toronto ON, Canada. Animals were then euthanized, and their heart, lungs and kidneys were excised, for physiological, histopathological and molecular analyses.

Echocardiography

Animals underwent echocardiography at 8 weeks of age, and subsequently every 4 weeks. All assessments were done using the Vevo^® 2100 system and a MS-250 probe (VisualSonics, Ontario, CA). In brief, all animals were placed on a heating pad to maintain a body temperature of 37 ± 1°C and secured in the supine position. Two dimensional long-axis images of the LV in parasternal long- and short-axis views at mid-papillary muscle level were acquired as previously described [22].

Cardiac catheterization

Cardiac catheterization was performed, as previously described [23]. In brief, animals were placed on a warming pad (42°C), intubated and ventilated using positive pressure. Rats were secured in a recumbent position and the right jugular vein was cannulated. Pressure was calibrated after warming the catheter (Model SPR-838 Millar instruments, Houston, TX, USA) in 0.9% NaCl at 37°C for 30 min. The right internal carotid was then identified and ligated cranially. A 2F miniaturized combined conductance catheter-micro-manometer was inserted into the carotid artery to obtain aortic blood pressure, then advanced into the left ventricle until stable PV loops were obtained. Using the pressure conductance data, a range of functional parameters were then calculated (Millar analysis software LabChart 8.1).

Heart histopathology

Paraffin embedded sections of heart, each 4 μm thick, were examined. The accumulation of matrix was quantified on picrosirius red stained heart sections using computer-assisted image analysis (Halo, indica labs, Albuquerque, New Mexico, USA) in a blinded fashion, as previously reported [24]. The extent of cardiac myocyte hypertrophy was determined on haematoxylin and eosin stained sections, as adapted from the methods described by Kai and colleagues [25], and as we previously described [26].

Analysis of cardiac gene expression

Genes associated with cardiac dysfunction were assessed. In short, total RNA was isolated from homogenized cardiac tissue using trizol reagent. Total RNA (2 μg) was converted to cDNA (for a 20 μL reaction) using the high-Capacity cDNA Reverse Transcription Kit and stored at −20°C until further analysis. Measurement of the cardiac genes was relative to either RPL13a (study 1) or RPL32 (study 2) using MicroAmp^® Optical 384-Well Reaction Plates with a ViiA^™ 7 Real-Time polymerase chain reaction system, as previously reported [27]. Experiments were performed in triplicates, and data analysis was performed using Applied Biosystem’s Comparative C_T method.

Kidney histopathology and function

Paraffin embedded kidney section, each 4 μm thick were assessed to determine the extent of glomerulosclerosis from Periodic acid–Schiff (PAS) stained sections, as previously reported [28].

Study 2: Impact of ACEi upon renal and cardiac structure and function

Age-matched, male Wistar and GK rats were studied. Animals were housed in the same conditions as per study 1.

As in Study 1, diabetes was confirmed at 8 weeks of age in the GK rats and, at 28 weeks of age once diabetes complications were established, animals were randomized to receive vehicle or perindopril erbumine (0.2mg/kg per day in drinking water) for 12 weeks. All animals also underwent metabolic caging and glycated hemoglobin (HbA1c) assessment at 28 and 48 weeks.

After 12 weeks of vehicle or perindopril erbumine (aged 48 weeks), animals underwent functional analysis, echocardiography every 4 weeks and cardiac catheterisation as outlined in study one. Following cardiac catheterization, animals were euthanized, and heart, lungs and kidneys were excised, for histopathological (PSR, H & E and PAS) analyses, and QT-PCR as described above.

Study 3: Proteomics comparison of in-vivo and in-vitro models

Rat cardiomyoblast H9c2 cells were cultured in 5.5 mM glucose (NG) or 25 mM glucose (HG). After 48h, cells were harvested and separated into nuclear and cytoplasmic fractions. Left ventricular tissue was excised from 40 weeks old age matched male Wistar and GK rats. Nuclear and cytoplasmic fractions of tissue homogenates were obtained. Proteins from cell and tissue extracts were precipitated and subjected to reduction and alkylation, followed by trypsin digestion. Tryptic peptides were desalted and injected onto a nano-liquid chromatography system connected online to an orbitrap mass spectrometer. Peptides were resolved using gradient reverse phase liquid chromatography. To identify proteins, the mass spectrometry data was searched against rat and human international protein index databases using MASCOT and GPM algorithms. MASCOT identifications required at least X! Tandem identifications. Label-free quantitation was performed using spectral counting tools embedded in scaffold (Scaffold version 4.10.0, Proteome Software Inc., Portland, OR) software. Scaffold was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm [29]. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [30]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.

The proteomic datasets for uniquely upregulated proteins in the HG H9c2 cell lysates, and GK rats were uploaded to Ingenuity Pathway Analysis (IPA) for core analysis of protein functional networks, interactions, and pathways (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) [31].

Statistical analysis

Data are expressed as means ± SEM unless otherwise specified. Between group differences were analyzed by t-test or two-way ANOVA with Holm-Sidak post hoc test. All statistics were performed using GraphPad Prism 6 for Mac OS X (GraphPad Software Inc., San Diego, CA). A Fisher’s exact test was conducted to determine the probability that pathways, biological functions, and diseases were over-represented in the protein dataset. A p value of <0.05 was regarded as statistically significant.

Results

Study 1: Characterisation of GK rats

Analysis of mean hemoglobin A1c (HbA1c, Table 1) demonstrated GK rats had a significantly higher HbA1c than Wistar rats at both time points and increased over time (p<0.0001). Body Weight (Table 1) was significantly reduced in GK rats compared to Wistar rats at both the early and late time points. There was a time dependent increase in body weight in both Wistar and GK rats (p = 0.0003). Tibial length (TL, Table 1) was significantly smaller in GK rats compared to Wistar rats at both time points. However, in keeping with growth the TL increased in both Wistar and GK rats’ (p<0.0001) over time. Heart weight indexed to body weight (HW; BW, Table 1) was significantly increased in GK rats (p<0.0001) at both time points compared with Wistar rats, and there was a significant time dependent increase in the GK rats HW:BW (p = 0.0012). Lung weight indexed to body weight (LW:BW, Table 1) was significantly increased in GK rats compared to Wistar rats at the late time point (p<0.01) and significantly increased over time in GK rats (p = 0.0308). Kidney Weight indexed to body weight (KW:BW, Table 1) significantly increased in GK rats at both early (p<0.05) and late (p<0.001) time points compared to Wistar rats and there was a time dependent increase in both Wistar and GK rats (p<0.0001).

Table 1. Morphometric data of the GK and Wistar rats at early and late timepoints.

	Early		Late
	Wistar n = 6	GK n = 4	Wistar n = 6	GK n = 7
HbA1C (%)	4.77 ± 0.15	6.68 ± 0.88^***	4.62 ± 0.34^ϕϕϕ	9.36± 0.73^****,^####,^ϕϕϕϕ
Body Weight (BW; g)	681.00 ± 32.44	449.75± 18.64^****	951.83± 104.68^****,^ϕϕϕϕ	392.57± 12.18^****,^####
Tibia Length (mm)	42.17 ± 0.75	38.13 ± 0.85^****	45.67± 0.52^****,^ϕϕϕϕ	40.64± 0.63^***,^ϕϕϕϕ,^####
Heart weight (HW): BW (mg/g)	2.20 ± 0.13	2.70 ± 0.15^**	1.91 ± 0.22^*,^ϕϕϕϕ	2.94 ± 0.16^#,^ϕϕ
Lung weight (LW): BW (mg/g)	3.11 ± 0.33	3.18 ± 0.18	2.38 ± 0.36^*,^ϕϕ	3.40 ± 0.21^##
Left Kidney weight (KW): BW (mg/g)	2.41 ± 0.23	3.12 ± 0.31^*	2.26 ± 0.52^ϕϕ	5.10± 0.35^****,^ϕϕϕϕ,^###

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HbA1C (%) Hemoglobin A1C, Body Beight (BW/g), Tibal length (TL/mm), heart weight/body weight ratio (mg/g), lung weight/body weight ratio (mg/g), LKW/BW left kidney weight/body weight (mg/g) ratio. Where early = 28 weeks Late = 48 weeks GK = Goto Kakizaki rats, Wistar = Wistar rats.

* = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001 when compared to early Wistar;

^# = p<0.05, ^## = p<0.01, ^### = p<0.001 and ^#### = p<0.0001 when compared to late Wistar;

^ϕ = p<0.05, ^ϕϕ = p<0.01, ^ϕϕϕ = p<0.001 and ^ϕϕϕϕ = p<0.0001 when compared to early GK;

A Two-Way ANOVA with multiple comparisons Holm-Sidak test was used to compare groups across different time points. Data is expressed as mean±SEM N = 4–8 per group.

Echocardiography

Echocardiographic analysis of cardiac function demonstrated a small reduction in cardiac function in the GK rats at each time point relative to Wistar rats, which progressed with age (all p<0.005). Left ventricular diameter in diastole was significantly reduced in GK rats compared to Wistar rats (P = 0.0332), which did not change over time. Left ventricular systolic diameter significantly increased over time (P = 0.0004) but was not significantly different between GK and Wistar rats (Fig 1).

Fig 1 — A-D represents echocardiographic analysis taken from short axis M-mode view demonstrating (A) cardiac output, (B) ejection fraction, (C) diameter in systole and (D) diameter in diastole. E-I represent analysis of cardiac catherization (E) systolic pressure, (F) end diastolic pressure, (G) Dp/dt max, (H) Dp/dt min, and (I) Tau. Where early = 28 weeks, Late = 48 weeks GK = Goto Kakizaki rats, Wistar = Wistar rats. * = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001 when comparing groups. A Two-Way ANOVA with multiple comparisons Holm-Sidak test was used to compare groups across different time points. Yellow = Wistar and Blue = GK. Data is expressed as mean±SEM N = 4–8 per group.

Cardiac catheterization

Cardiac catheterization with a high-fidelity LV pressure manometer demonstrated that GK rats had higher systolic blood pressure compared to Wistar rats (P = 0.0495). dP/dt max was increased in GK rats compared to Wistar rats at the early time point (P<0.0001); an effect that was not observed at the late time point. Further, dP/dt min was significantly increased with age (P = 0.0001) and this effect was seen to be most pronounced in GK rats (P = 0.0326). Diastolic function assessment demonstrated that Tau significantly increased over time (P = 0.0002), this increase was seen to be most significant in aged GK rats (P = 0.0118, Fig 1).

Heart histopathology

Myocyte hypertrophy was significantly increased in GK rats (p = 0.025) compared to that of Wistar rats, but no effect was seen over time. Picrosirius red staining demonstrated that collagen content as measured by the portion of red stained pixels was increased in GK rats compared to that of Wistar rats (GK rats, p = 0.0144) but there was no significant effect over time (all Fig 2).

Fig 2 — Representative hematoxylin and eosin-stained sections and PicroSirius red stained sections. Where hearts of Wistar (A,C,F,H) rats and Goto Kakizaki (B,D,G,I) rats at early(28 weeks; A,B,F,G) and late (48 weeks; C,D,H,I) time points had myocyte hypertrophy (E) and fibrosis (J, positive PSR stained pixels) measured. * = p<0.05 when comparing groups; A Two-Way ANOVA with multiple comparisons Holm-Sidak test used to compare groups. Yellow = Wistar and Blue = GK Data is expressed as mean±SEM N = 4–8 per group. Scale bars (A-D) 50μm and (F-I) 200 μm.

Cardiac gene expression

Expression of genes known to be important in cardiac remodelling were assessed in both Wistar and GK rats. Atrial natriuretic peptide (ANP) mRNA was significantly increased in GK rats (p = 0.009) compared to Wistar rats and was significantly increased over time (p = 0.028). Beta myosin heavy chain (β-MHC) expression was significantly increased in GK rats (P = 0.0064) compared to Wistar rats but this effect was seen to be most prominent in the aged GK rats (p<0.0001). Alpha myosin heavy chain (⍺-MHC) expression was significantly increased in GK rats (p = 0.014) compared to Wistar rats and, ⍺-MHC expression significantly increased with age (p<0.0001, Fig 3).

Fig 3 — (A) Atrial natriuretic Factor, (B) beta-Myosin Heavy Chain (βMHC), (C) alpha-myosin heavy chain (⍺MHC). Where Early = 28 weeks and Late = 48 weeks. * = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001. A Two-Way ANOVA with multiple comparisons Holm-Sidak test was used to compare groups across different time points. Yellow = Wistar and Blue = GK. Data is expressed as mean±SEM N = 4–8 per group.

Kidney histopathology and function

The glomerulus score index (GSI) of 40 weeks old rats was significantly increased in GK rats compared to Wistar rats (1.57±0.29 vs 0.35±0.12, p<0.001, Fig 4C). Moreover, the protein:creatinine ratio was significantly increased in GK rats compared to Wistar rats (0.4±0.22 vs 0.076±0.054, p<0.05) measured at 40 weeks of age. Further, Immunohistochemical staining of Collagen IV demonstrated Collagen IV content was significantly increased in GK rats compared to Wistar rats (P<0.05) at 48 weeks (S1 Fig).

Study 2: The effect of RAS blockade in the Goto-Kakizaki rat

Morphology

When GK rats were compared to aged matched GK rats treated with the ACEi perindopril, there was no significant impact upon HbA1C, body weight (BW), tibial length, heart or kidney weight (Table 2). However, the HW:BW ratio was reduced in GK rats treated with perindopril (P<0.001) compared to vehicle treated GK rats (Table 2).

Table 2. Morphometric data of GK and GK rats following treatment with Angiotensin Converting enzyme inhibitor perindopril.

	GK control (n = 4)	GK + Perindopril (n = 5)
HbA1C (%)	6.03 ± 1.51	6.72 ± 2.43
Body Weight (BW; g)	490.25 ± 7.27	472.40 ± 22.40
Tibia Length (mm)	41.00 ± 0.00	41.00 ± 0.00
Heart weight (HW): BW (mg/g)	2.79 ± 0.10	2.50 ± 0.03***
Lung weight (LW): BW (mg/g)	3.14 ± 0.13	2.99 ± 0.25
Left Kidney weight (KW): BW (mg/g)	2.90 ± 0.23	2.98 ± 0.26

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HbA1C (%); Hemoglobin A1C, Body weight;(BW;g), Tibal length;(TL/mm), HW/BW; heart weight/body weight ratio, LW/BW; lung weight/body weight ratio, LKW/BW; left kidney weight/body weight ratio (mg/g) Where *** = <0.001 and when compared to GK rats (student’s t-test was used to compare groups). Data is expressed as mean ± SEM N = 4–5 per group

Echocardiography

Following treatment with perindopril, echocardiography demonstrated a significant increase in cardiac output in GK rats treated with perindopril c/w vehicle treated animals (p<0.01). Further, ejection fraction was significantly increased by perindopril treatment (p<0.05). Chamber size as assessed by internal diameter in systole or diastole was not altered by perindopril treatment (Fig 5).

Fig 5 — A-D represents echocardiographic analysis taken from short axis M-mode view demonstrating (A) cardiac output, (B) ejection fraction, (C) diameter in systole and (D) diameter in diastole. E-I represent analysis of cardiac catherization (E) systolic pressure, (F) end diastolic pressure (G) Dp/dt max, (H) Dp/dt min and (I) Tau. All parameters were measured in Goto Kakizaki rats either treated with saline (GK) or ACE inhibitor perindopril (GK + perindopril). Where * = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001 when compared to GK rats; (student’s t-test was used to compare groups). Data is expressed as mean±SEM N = 4–5 per group.

Cardiac catheterization

Cardiac catheterization demonstrated that end systolic pressure was significantly decreased by perindopril (p<0.0001) treatment versus control GK rats. Finally, end diastolic pressure (EDP), dP/dt max, dP/dt min and Tau did not significantly change following perindopril treatment (p = N.S, Fig 5).

Heart histopathology

Perindopril treatment did not result in any differences in myocyte hypertrophy. Fibrosis as measured from positive PSR staining was significantly decreased following treatment with perindopril when compared to control GK rats (p<0.05, Fig 6).

Fig 6 — Representative hematoxylin and eosin-stained sections and PicroSirius red stained sections. Hearts of Goto Kakizaki (A,D) rats and Goto Kakizaki rats treated with Angiotensin Converting enzyme inhibitor perindopril (B,E) had myocyte hypertrophy (C) and fibrosis (F, positive PSR stained pixels) measured. Parameters were measured in Goto Kakizaki rats either treated with saline (GK) or ACE inhibitor perindopril (GK + perindopril). * = p<0.05, when compared to GK rats (student’s t-test was used to compare groups). Data is expressed as mean±SEM N = 4–5 per group. Scale bars (A-B) 50μm and (C-D) 300 μm.

Cardiac gene expression

Following treatment with perindopril mRNA expression of ANF (p<0.05), β-MHC (p<0.001), ⍺-MHC(p<0.001) was significantly reduced compared to control GK rats (p<0.05, Fig 7).

Fig 7 — (A) Atrial natriuretic Factor, (B) beta-Myosin Heavy Chain (β-MHC), (C) alpha-myosin heavy chain (⍺-MHC). Where hearts tissue of Goto Kakizaki rats either treated with saline (GK) or ACE inhibitor perindopril (GK + perindopril) were analysed. Where * = p<0.05, when compared to GK (students t-test was used to assess statistical significance).

Kidney histopathology and function

12 weeks of perindopril treatment had no impact upon GSI and protein; creatinine when compared to control GK rats (all p = N.S, Fig 8).

Study 3: Proteomics comparison of in-vivo and in-vitro models

A total of 1191 common proteins were identified in both H9C2 cell lysates incubated in normal and high glucose media. Of these proteins, 896 were common to both HG and NG H9C2 cells. However, 128 proteins where uniquely overexpressed in HG cultures compared to that of the NG Cultures (p<0.05, Fig 9A).

Fig 9 — (a) Commonly and exclusively quantified proteins in H9C2 cells fed either Normal Glucose or High Glucose concentration. (b) Commonly and exclusively quantified proteins in cardiac tissue from GK or Wistar rats.

Analysis of proteins from Wistar and GK rats at both the early and late time points identified 1071 common proteins. However, 253 of these identified proteins where significantly overexpressed in the GK rats compared to Wistar rats (p<0.05) (Fig 9B).

To further investigate the molecular interactions of the identified proteins, we submitted our proteomic datasets for the unique H9c2-HG and GK rat proteins for an IPA-based protein network analysis. The top-enriched networks based on a high percentage of focus proteins in our datasets included pathways linked to cardiovascular disease. cardiac hypertrophy, cancer, cell death and survival with upregulation of proteins such as MYH7, MYL2, APOA1 and phospholamban (Fig 9, Table of Top enriched networks from core analysis of both H9c2 and GK overexpressed proteins S1 Table). Commonly upregulated proteins in both H9c2 cell lysates and GK hearts were involved pathways that regulated oxidative phosphorylation, Kreb’s cycle, mitochondrial dysfunction, beta-oxidation, and PI3K/Akt signalling (all p<0.05).

Discussion

Given that cardiac and renal dysfunction remain the most common presentation of diabetes induced complications [19, 32], the structural, functional and molecular underpinnings of such derangements require elucidation. Herein we describe the time course of development of cardiovascular complications in a rodent model of spontaneous, non-obese type 2 DM, the Goto Kakizaki (GK) rat. We show that GK rats develop hyperglycaemia by 12 weeks of age, then progressive mild hypertension leading to left ventricular hypertrophy over 40 weeks, with evidence of impaired diastolic function, interstitial fibrosis and gene derangements over a 28-week time period, along with the development of proteinuria. Furthermore, the GK rats demonstrate progressive and significant changes in cardiac protein expression, when compared to Wistar control rats, broadly involving alterations in contractile proteins, metabolism, hypertrophy and fibrosis.

The GK strain was developed by the successive inbreeding of Wistar rats [19, 32]. Importantly, this is a model of spontaneous, non-obese diabetes, therefore removing the confounding effects of excess adiposity upon the development of diabetes complications. Furthermore, this model shares a susceptibility locus on chromosome 10 [19] with T2 DM in humans, thus providing a genetic basis for the development of diabetes. Of note, the development of relatively mild complications is analogous to the development of complications in humans with T2DM, as this model takes 40 weeks before the development of diastolic dysfunction, and elevated urinary protein [17]. Although, it should be noted that this model is not age representative of the development of cardiorenal syndrome. In other words, it cannot be directly age matched.

Cardiac dysfunction

Despite evidence for early left ventricular hypertrophy, systolic function was preserved, albeit at a lower number, along with evidence for abnormalities of early, active energy dependent relaxation. In order to assess systolic and diastolic function, we utilized echocardiographic parameters along with the assessment of invasive measurements of pressure. Between the early and late time points, there was a small, but significant reduction in systolic function, ventricular volumes and LVH increased and GK rats developed an increase in lung weight. The increase in lung weight coincided with a small increase in end diastolic pressure, suggesting “pulmonary” congestion. Left ventricular hypertrophy remains a powerful predictor of adverse CV outcomes [33], with studies consistently showing regression with the use of ACEi and SGLT2i associate with improved outcomes [17, 34]. Furthermore, the findings of pulmonary congestion, subtly reduced systolic function, elevated filling pressures and LVH suggest this model, at the late stage, has features consistent with the syndrome of heart failure with preserved ejection fraction (HFpEF) [7, 35–37]. This is particularly important given the lack of evidence-based therapies to treat HFpEF.

Importantly, these functional findings were mirrored by the development of myocyte hypertrophy and interstitial fibrosis. Furthermore, a proteomic analysis demonstrated broad reprogramming which occurred in a time dependent fashion. Key reprogramming was seen in proteins involved in cardiac muscle contraction, glycolysis, oxidation phosphorylation, cell matrix adhesion and protein folding/sorting. This is consistent with human biopsy studies [38] whereby changes in myocyte stiffness and extracellular matrix appear responsible for some, but not all changes in cardiac function parameters. Indeed, the presence of LVH, interstitial fibrosis and altered metabolism is a prominent feature of so called diabetes induced cardiac dysfunction [39]. Of note, rodents remain resistant to the development of coronary artery disease (CAD) [34, 40] hence these findings represent the direct impact on ventricular structure and function independent on CAD.

Importantly, these findings fulfill the criteria as set out by the AMDCC [41], recapitulating many of the features of humans with T2DM, thus providing relevance. Furthermore, our findings are consistent with the reported literature. Al Kury et al provide a detailed review of cardiac function, along with identified molecular derangements in the GK rats across multiple time points [42]. The results demonstrated that at 6 months of age, GK rats had significantly lower CO and EF compared to controls. This corresponds with our evidence that GK rats had reduced cardiac function when compared to control Wistar rats. However, the GK rats in our study exhibited milder cardiac dysfunction than in the study by Al Kury et al. [42].

Diabetic kidney disease

We demonstrated glomerulosclerosis and fibrosis accompanied by the development of mild proteinuria. Elevated urinary protein is a key diagnostic feature of the development of diabetic kidney disease, and a powerful marker for adverse CV outcome [43]. Of note, the changes were relatively mild by this late time point. The elevated urinary protein was accompanied by the typical change of diabetic nephropathy, as evidence by elevated glomerulosclerotic index. This index assesses the presence of excess extracellular matrix and mesangial expansion. Importantly, these findings occurred in the absence of significant hypertension, confirming a direct impact of hyperglycemia upon renal structure and function. Our current observation is in line with other observation within the literature which, suggest the GK rat has relatively mild phenotype of diabetic kidney disease (DKD) [44, 45]. The development of the T2DN rat a sub-strain of the GK rat has shown much promise as it exhibits many of the hallmarks of clinical human DKD. However, the GK rat may be of use to understand the early changes which result in DKD and potentially contribute to cardio-renal syndrome.

Impact of ACEi

Blockade of the renin angiotensin system has been a key therapeutic strategy in the treatment of human cardiac and renal disease, and is widely utilized [46, 47]. We therefore assessed the impact of a perindopril, an ACEi upon cardiac and renal outcomes. Importantly, we commenced RAS blockade at 28 weeks, once disease was progressive, and followed until the late time point. RAS blockade reduced BP and LVH as well as ANF, βMHC and ⍺MHC expression with no significant impact upon cardiac systolic or diastolic function. These findings are in keeping with the response in humans [48]. Intriguingly, we saw little response in diabetic glomerulosclerosis at the late time point in perindopril treated GK rats, suggesting that the increase in GSI was an effect of hyperglycemia, as opposed to blood pressure related mechanisms.

Proteomic comparison

In-vitro studies using H9c2 cells incubated in both normal and high concentrations is a model that is widely used to investigate molecular mechanisms associated with diabetes and cardiovascular complications [49–51]. The H9c2 cell line is an immortalized rat cardiomyoblast which, has been demonstrated to mimic the responses of primary cardiomyocytes [52]. Therefore, we employed proteomics to investigate if the H9c2 cell model would be an appropriate in-vitro model to investigate molecular mechanisms that are involved in cardiovascular complications in the GK rat. Our results demonstrate that pathways that modulate oxidative phosphorylation, Kreb’s cycle, mitochondrial dysfunction, beta-oxidation, and PI3K/Akt signalling were similarly upregulated in cardiac tissue from GK rats and HG fed H9c2 cell. These results would suggest that the high glucose fed H9c2 cells are an appropriate in-vitro model to investigate molecular mechanisms eluded from the GK rat. Importantly, we provide access to the entire data set to encourage others to identify new mechanisms of diabetes-induced complications.

Implications

Whilst the GK rat develops changes similar to patients who suffer from HFpEF, these remain relatively mild. Importantly, the model responded well to a guideline indicated therapy for cardiorenal disease, blockade of the renin-angiotensin system. These findings suggest that the GK rat is a reasonable model for testing novel therapeutic strategies in diabetes induced HFpEF. While these changes are mild, they are similar to that observed in patients with HFpEF [16, 45]. However, a number of limitations exist in this model. Firstly, the relatively long-time course and mild changes remains costly to study and do not indicate the impact of therapeutic strategies in more advanced disease. Secondly, the current study did not address hyperglycemia. The HbA1c was significantly elevated at ~9%, hence the impact of improving glucose control was not addressed in the current study. However, recent large clinical trials [53–55] demonstrate that despite aggressive therapy to improve plasma glucose, HbA1c remain poorly controlled around ~7.5–8% in the majority of patients, thus our findings remain generalizable. Thirdly, we did not study older GK rats (i.e. ~60 weeks of age), hence whether they develop significant LV dysfunction after 50 weeks was not determined. Finally, the Wistar controls developed excess weight gain (almost double GK rats). Future studies using “pair wise” feeding may be necessary to regulate weight gain in the control animals and therefore provide a more appropriate control.

Conclusions

By 48 weeks of age, the diabetic GK rat demonstrates evidence of preserved systolic function and impaired relaxation, along with cardiac hypertrophy, in the presence of hyperfiltration and elevated protein excretion. These findings suggest the GK rat demonstrates some, but not all features of diabetes induced “cardiorenal syndrome”. This has implications for the use of this model to assess preclinical strategies to treat cardiorenal disease and may provide a model that best replicates the early complications of cardiorenal syndrome.

Supporting information

S1 Fig. Kidney fibrosis in Wistar and GK at the late time point.

Representative Collagen IV stained Wistar (A) and GK (B) kidneys at 48 weeks, respectively. Percent positivity staining (C) * = p<0.05 when compared to Wistar rats; (students t-test was used to assess statistical significance). Yellow = Wistar and Blue = GK. Data is expressed as mean±SEM N = 4–8 per group. Scale bars 100 μm.

(TIFF)

Click here for additional data file.^{(3.6MB, tiff)}

S1 Table. Top five functional networks associated with overexpressed proteins identified in GK nuclear and cytosol proteomes.

(DOCX)

Click here for additional data file.^{(228.4KB, docx)}

S2 Table. David pathway analysis demonstrating unique pathways significantly different in the GK rats Compare to Wistar rats.

(DOCX)

Click here for additional data file.^{(49.9KB, docx)}

S1 Datasets. Proteomic datasets of GK v’s Wistar rats and normal glucose and high glucose fed H9C2 cell line.

(ZIP)

Click here for additional data file.^{(336.8KB, zip)}

Acknowledgments

We would like to thank Suzanne Advani for her help with the acquisition of representative images of GSI images for study 2.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This study was funded in part by the Heart and Stroke Foundation of Canada in the form of a grant (G-15-0009282) and by St. Michael’s Hospital Foundation “SCAR WARS” program in the form of funds, both to KAC. The Department of Medicine, University of Toronto also provided support in the form of a Merit Award for KAC. The specific roles of these authors are articulated in the ‘author contributions’ section.

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The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function

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The authors should discuss their data in light of recent study demonstrating that ADAMTS 13 deficiency may underlie the onset of lethal arrhythmias in diabetes affecting lifespan (Diabetes. 2018 Oct;67(10):2069-2083.).

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"KAC has received research grants to his institution from Astra Zeneca and Boehringer Ingelheim, received support for travel to scientific meeting from Boehringer Ingelheim and honoraria for speaking engagements and ad hoc participation in advisory boards from Astra Zeneca, Boehringer Ingelheim and Janssen. All other authors declare that they have no competing interests"

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8. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://www.onlinecjc.ca/article/S0828-282X(11)01034-8/fulltext

The text that needs to be addressed involves the Abstract.

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: In this study, Meagher and colleagues performed throughout analysis of diabetes progression and cardio-renal remodeling in GK rat, a non-obese, non-hypertensive model of type 2 diabetes. Both cardiac and renal phenotypes are assessed. Furthermore, the authors tested response to renin-angiotensin system blockade in GK rats and performed proteomic studies in rat cardiomyoblast H9c2 cells and Left ventricular tissue. While the current research does not test a novel hypothesis, it, nevertheless, makes an important report of disease progression in GK rats. The paper is well written and very readable with nice results. I have a few suggestions that I think will improve these studies.

Major:

1. One of the main conclusions of the manuscript is that the GK rat is not a good model to study cardiorenal phenotype. However, as described by the authors and others, this strain has a number of futures of both cardiac and renal disease progression. Therefore, it is suggested to focus on the discussion of the data and lessen this conclusion about the inapplicability of this model for this type of study.

2. Renal function, as described in Figure 4 and the text, was tested at 40 weeks age. However, as described for protocol 1, animals were sacrificed at ~48 weeks. Which one is correct?

3. It appears that kidney sections are available, and kidney weights to body weights were significantly different between groups. It would be nice if the authors check fibrosis.

4. Proteomic analysis: there is some inconsistency in the description of figure 9 and the numbers shown in the figure. As mentioned in the text, 163 proteins were uniquely overexpressed in HG cultures (Figure 9A), and 253 proteins are significantly overexpressed in GK rats (figure 9B), but boxes in the figure indicate 128 and 253 proteins, respectively. Furthermore, it should be important to compare the data in cell culture and in GK/Wistar rats to show overlap, if any. Please also describe key proteins/pathways identified by proteomic analysis rather than reference to supplemental table, which is also incomplete.

5. It would be great if the authors perform proteomic analysis of renal tissue as well. While it might be outside of the scope of this manuscript, this data will be important considering that the authors attempt to study cardiorenal interaction in GK rats.

6. The authors describe in the text (including abstract) that rats are age and sex-matched. While this is correct, it is slightly misleading since only male rats are studied. This is especially critical since there is a significant difference between the development of diabetes between male and female.

7. Progression of kidney disease of GK (compared to Wistar and T2DN rats, which were developed from GK rats) was recently described (PMID: 31566426). Earlier studies also described renal disease in GK and T2DN rats (PMID: 24319624). Consider comparing the findings and discuss if T2DN rats might be a better model to study cardiorenal phenotype.

8. Tables 1 and 2 – Include a number of rats for each group in the table.

9. Figure 2 legend (page 14). It appears that letters referencing to wrong images “Where hearts of Wistar (A,C,E,G) rats and Goto Kakizaki (B,D,F,H) rats at early(28 weeks; A,B,E,F) and late (48 weeks;C,D,G,H)….”. The same comment about figure 6 (figures 6F-J).

Minor:

1. Figure 2: A-D – Scales (lines/font) should be bigger. E and J: Describe a color for Wistar/GK rats.

2. Figure 2J. Is it statistical difference between Wistar and GK rats at 28 weeks?

3. Figure 4A and B – add scales.

4. Methods: Please describe what is a standard chow.

5. Please mention that 40-48 weeks old rat is not aged (40-50 years old???) when compared to human.

6. Introduction, second paragraph, reference #10. Please consider citing more updated statistics.

7. Page 11. SGLT trials (CREDENCE and DAPA-CKD) should not be bold.

Reviewer #2: In this study Meagher et al. have investigated whether the Goto-Kakizaki (GK) rat is a valid model to study the pathogenesis of diabetic cardiomyopathy. The authors find that by 48 weeks of age, the diabetic GK rat demonstrates evidence of preserved systolic function and impaired relaxation, along with cardiac hypertrophy, in the presence of hyperfiltration and elevated protein excretion. This important piece of evidence highlights how the GK rat partially demonstrates features that occur in human diabetes, including “cardiorenal syndrome”. The implications of this study are important as pre-clinical therapeutic strategies can be implemented on the GK rat to assess efficacy.

The reviewer offers the following minor critiques:

A recent study demonstrated that accelerated cerebral vascular injury in diabetes is associated with vascular smooth muscle cell dysfunction (PMID: 32166556). Is it known if these features are exhibited by the GK rat? Similarly, can the authors comment on the plasma levels of mitokines FGF21, GDF15, and Humanin in the GK rat? Evidence has suggested those mitokines are elevated in type II diabetes and Alzheimer's disease in comparison with healthy aging (PMID: 33131010)

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Jun 24;16(6):e0252711. doi: 10.1371/journal.pone.0252711.r002

Author response to Decision Letter 0

13 May 2021

Dear Dr. Lionetti,

Thank you for giving us the opportunity to submit a revised draft of the manuscript “The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function.” for publication in PLOS ONE. We greatly appreciate the time and effort that you and the reviewers committed to providing us with feedback. The critiques provided helped us immensely to improve the quality of our manuscript. We have incorporated the suggestions made by yourself and the reviewers. All changes and alterations are highlighted in the manuscript with track changes. Please see below a point-by-point response to the reviewers’ comments and recommended revisions, with the line numbers as to their location where applicable. Further we have uploaded additional supplementary material for the proteomics study.

Sincerely,

Patrick Meagher and Kim Connelly

Additional Editor Comments:

We thank the editor for this important comment. We politely ask that whilst this is important, it is outside the scope of the current paper as we did not explore the development of development of arrythmias but the onset of cardio-renal syndrome. We therefore humbly request that this not be included in the revised version of our paper.

Reviewer 1

Major:

Thank-you for this important insight. This was not our intention and we have altered our main conclusion – see page 26, line 957-963.

By 48 weeks of age, the diabetic GK rat demonstrates evidence of preserved systolic function and impaired relaxation, along with cardiac hypertrophy, in the presence of hyperfiltration and elevated protein excretion. These findings suggest the GK rat demonstrates some, but not all features of diabetes induced “cardiorenal syndrome”. This has implications for the use of this model to assess preclinical strategies to treat cardiorenal disease and may provide a model that best replicates the early complications of cardiorenal syndrome.

2. Renal function, as described in Figure 4 and the text, was tested at 40 weeks age. However, as described for protocol 1, animals were sacrificed at ~48 weeks. Which one is correct?

Thank-you we have corrected this to 48 weeks . Page 10 Line 229

3. It appears that kidney sections are available, and kidney weights to body weights were significantly different between groups. It would be nice if the authors check fibrosis.

This is an excellent suggestion; we have analysed Collagen IV content in paraffin embedded kidney sections. We observed an increase in Collagen IV content in GK rats compared to Wistar rats. This has been incorporated in the text, page 16, line 85 and supplemental figure 1:

Further, immunohistochemical staining of Collagen IV demonstrated Collagen IV content was significantly increased in GK rats compared to Wistar rats (P<0.05) at 48 weeks (supplemental figure 1).

Apologies we have rectified this in the abstract please see page 20 line 823

However, 128 proteins where uniquely overexpressed in HG cultures compared to that of the NG Cultures (p<0.05, Figure 9A).

Please also see page 20 line 831-836

The top-enriched networks based on a high percentage of focus proteins in our datasets included pathways linked to cardiovascular disease. cardiac hypertrophy, cancer, cell death and survival with upregulation of proteins such as MYH7, MYL2, APOA1 and Phospholamban (Figure 9, Table of Top enriched networks from core analysis of both H9c2 and GK overexpressed proteins Supplemental Table 1).

Finally, we did compare the HG H9C2 cells and GK rats to see if there was overlap in pathways that were commonly upregulated between them. Please the additional supplementary files which have our proteomics analysis within them.

Please see page 20 line 836-838

Commonly upregulated proteins in both H9c2 cell lysates and GK hearts were involved pathways that regulated oxidative phosphorylation, Kreb’s cycle, mitochondrial dysfunction, beta-oxidation, and PI3K/Akt signalling (all p<0.05).

This is a pertinent comment. The exploration of the kidney proteomics is outside the scope of the current study. Further, simple exploration of proteomics in kidney samples would likely have confounding results due to the differential protein expression between the medulla and cortex, owing to the complex nature of renal anatomy. Therefore, exploration of protein expression within these sections of the kidney would be extremely difficult to examine in whole kidney samples. We will pursue this in future studies, however.

6. The authors describe in the text (including abstract) that rats are age and sex matched. While this is correct, it is slightly misleading since only male rats are studied. This is especially critical since there is a significant difference between the development of diabetes between male and female.

Apologies, we have amended this to make it clear we are studying male sex.

See Abstract page 2 Line 33 and page 7 line 148

Thank-you for the suggestion. We have made a comment on the T2DN rat, and provided appropriate reference to these two papers. see page 23 Line 591

Our current observation is in line with other observation within the literature which, suggest the GK rat has relatively mild phenotype of diabetic kidney disease (DKD) (PMID: 24319624, PMID: 31566426). The development of the T2DN rat, a sub-strain of the GK rat has shown much promise as it exhibits many of the hallmarks of clinical human DKD. However, the GK rat may be of use to understand the early changes which result in DKD and potentially contribute to cardio-renal syndrome.

8. Tables 1 and 2 – Include a number of rats for each group in the table.

Apologies. This has been corrected. See Table 1 - Page 12, Table 2 Page 16.

Apologies. This has been corrected. Page 15 Line 348-353 & Page 18 Line 427-429

Minor:

1. Figure 2: A-D – Scales (lines/font) should be bigger. E and J: Describe a color for Wistar/GK rats.

Apologies. This has been corrected. Scale bars have been altered see new uploaded figure 2. The addition of a colour description has been added to figure legend.

2. Figure 2J. Is it statistical difference between Wistar and GK rats at 28 weeks?

Unfortunately, there was no significance between these groups.

3. Figure 4A and B – add scales.

Apologies. This has been corrected. Please see new figure 4.

4. Methods: Please describe what is a standard chow.

Apologies. This has been corrected. see methods, page 7 line 153.

For more info on the rodent diet, the vivarium orders 18% protein diet which can be found here: https://www.envigo.com/rodent-natural-ingredient-2018-diets

5. Please mention that 40-48 weeks old rat is not aged (40-50 years old???) when compared to human.

Apologies. This has been corrected. see page 21 line 529.

Although, it should be noted that this model is not age representative of the development of cardiorenal syndrome. In other words, it cannot be directly age-matched.

6. Introduction, second paragraph, reference #10. Please consider citing more updated statistics.

Apologies. This has been corrected. see line 81 in introduction

Reference: Roy et al. Working Group of the Endocrine Society of Bengal. Kidney Disease in Type 2 Diabetes Mellitus and Benefits of Sodium-Glucose Cotransporter 2 Inhibitors: A Consensus Statement. Diabetes Ther. 2020 Dec;11(12):2791-2827. doi: 10.1007/s13300-020-00921-y. Epub 2020 Oct 6. PMID: 33025397; PMCID: PMC7644753.

7. Page 11. SGLT trials (CREDENCE and DAPA-CKD) should not be bold.

Apologies. This has been corrected. Page 5 line 104-105

Reviewer 2:

This is outside the scope of the study. Unfortunately, in this study we did not investigate smooth muscle cell dysfunction. However, Sandu et al. (2000) demonstrated impaired vascular smooth muscle cell relaxation in GK rats (PMID: 11118023). There is no evidence that we are aware of that demonstrate accelerated cerebral vascular injury in the GK rat.

Similarly, can the authors comment on the plasma levels of mitokines FGF21, GDF15, and Humanin in the GK rat? Evidence has suggested those mitokines are elevated in type II diabetes and Alzheimer’s disease in comparison with healthy aging (PMID: 33131010)

This is outside the scope of our study. In this study we did not investigate the level of the mentioned mitokines within the plasma. Further, we did not see any significant changes in these mitokines within our proteomics analysis of the heart tissue. Finally, to our knowledge the plasma levels of the mentioned mitokines has not been investigated in the GK rat.

Grant requirements:

1) We note your current Financial Disclosure and Competing Interest read as follows:

“Studies were supported by research grants from the HSF Canada, and funds from the St Michaels Hospital Foundation “SCAR WARS” program. Dr Kim A Connelly is supported by a Merit Award from the Department of Medicine, University of Toronto.”

“KAC has received research grants to his institution from Astra Zeneca and Boehringer Ingelheim, received support for travel to scientific meeting from Boehringer Ingelheim and honoraria for speaking engagements and ad hoc participation in advisory boards from Astra Zeneca, Boehringer Ingelheim and Janssen. All other authors declare that they have no competing interests.”

We also note the following additional comments:

"Finally, Kim A. Connelly has received research grants to his institution from Astra Zeneca and

Boehringer Ingelheim, received support for travel to scientific meeting from Boehringer

Ingelheim and honoraria for speaking engagements and ad hoc participation in advisory boards

from Astra Zeneca, Boehringer Ingelheim and Janssen. However, this will have no-effect in

regard to data restriction and all pertinent data related to the study will be made available."

For the support regarding HSF Canada and St. Michael’s Hospital Foundation, please provide the following information:

a.) Any specific grant or award numbers associated with the support.

- The HSF grant has an award number which has been added. The other grants are generic and do not have a specific name or award number, hence this has been left blank.

b.) The names of the specific authors who received the support.

-the grant is to Dr Kim A Connelly. The other authors did not receive funding to support this particular project.

For the Merit Award from the Department of Medicine, University of Toronto, please provide the following information:

a.) Any specific grant or award numbers associated with the support.

-The Merit award does not have a specific name or award number, hence this has been left blank.

Please confirm whether there are any patents, products in development or marketed products associated with this research to declare.

Reply: There are no patents, products in development or marketed products associated with this research.

Please also confirm whether the support from Astra Zeneca, Boehringer Ingelheim, and Janssen, that is unrelated to the study, in any way alters your adherence to PLOS ONE policies on sharing data and materials.

Reply: There is no support from the aforementioned companies for this project. Dr Connelly has received monies for other projects. This is no way influences PLOS One policies and all data is freely available to be shared as there is no agreement with the above companies.

I have been in email contact with Anna Fodor - please see the email below in regards to disclosures.

PONE-D-20-39982R1

The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function

PLOS ONE

Dear Dr. Connelly,

Your confirmation via email is acceptable, funding information should not appear in any areas of the manuscript, we will only publish funding information present in the Funding Statement section of the online submission form, which I have already updated based on your confirmation, so please just send back the manuscript, and I will process it back to the academic editor for review.

Thanks a lot,

Anna Fodor

PLOS ONE

Attachment

Submitted filename: PLOS one comments.docx

Click here for additional data file.^{(21.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0252711.r003

Decision Letter 1

Vincenzo Lionetti

21 May 2021

The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function

PONE-D-20-39982R1

Dear Dr. Connelly,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Vincenzo Lionetti, M.D., PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: All concerns are addressed. I do not have any outstanding issues. Nice manuscript summarizing the use of GK rats for cardiorenal research.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. doi: 10.1371/journal.pone.0252711.r004

Acceptance letter

Vincenzo Lionetti

3 Jun 2021

PONE-D-20-39982R1

The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function.

Dear Dr. Connelly:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Kidney fibrosis in Wistar and GK at the late time point.

(TIFF)

Click here for additional data file.^{(3.6MB, tiff)}

S1 Table. Top five functional networks associated with overexpressed proteins identified in GK nuclear and cytosol proteomes.

(DOCX)

Click here for additional data file.^{(228.4KB, docx)}

S2 Table. David pathway analysis demonstrating unique pathways significantly different in the GK rats Compare to Wistar rats.

(DOCX)

Click here for additional data file.^{(49.9KB, docx)}

S1 Datasets. Proteomic datasets of GK v’s Wistar rats and normal glucose and high glucose fed H9C2 cell line.

(ZIP)

Click here for additional data file.^{(336.8KB, zip)}

Attachment

Submitted filename: PLOS one comments.docx

Click here for additional data file.^{(21.1KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

The Goto Kakizaki rat: Impact of age upon changes in cardiac and renal structure, function

Patrick Meagher

Robert Civitarese

Xavier Lee

Mark Gordon

Antoinette Bugyei-Twum

Jean-Francois Desjardins

Golam Kabir

Yanling Zhang

Hari Kosanam

Aylin Visram

Howard Leong-Poi

Andrew Advani

Kim A Connelly

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Methods

Study 1: Characterization of the Goto Kakizaki rat at early and late timepoints

Echocardiography

Cardiac catheterization

Heart histopathology

Analysis of cardiac gene expression

Kidney histopathology and function

Study 2: Impact of ACEi upon renal and cardiac structure and function

Study 3: Proteomics comparison of in-vivo and in-vitro models

Statistical analysis

Results

Study 1: Characterisation of GK rats

Table 1. Morphometric data of the GK and Wistar rats at early and late timepoints.

Echocardiography

Fig 1. Echocardiograph and cardiac catheterization parameters of Wistar and GK rats over time.

Cardiac catheterization

Heart histopathology

Fig 2. Cardiac Structure in Wistar and GK rats over time.

Cardiac gene expression

Fig 3. RT-qPCR analysis of cardiac genes in heart tissue from GK and Wistar rats over time.

Kidney histopathology and function

Fig 4. Kidney structure and function in Wistar and GK at the late time point.

Study 2: The effect of RAS blockade in the Goto-Kakizaki rat

Morphology

Table 2. Morphometric data of GK and GK rats following treatment with Angiotensin Converting enzyme inhibitor perindopril.

Echocardiography

Fig 5. Echocardiographic and cardiac catheterization parameters of GK rats and GK rat treated with Angiotensin Converting enzyme inhibitor perindopril.

Cardiac catheterization

Heart histopathology

Fig 6. Cardiac structure in control GK rats and GK rats treated perindopril.

Cardiac gene expression

Fig 7. RT-qPCR analysis of cardiac genes in control GK and GK rats treated with perindopril.

Kidney histopathology and function

Fig 8. Kidney Structure and function in control GK rats and GK rats treated with perindopril.

Study 3: Proteomics comparison of in-vivo and in-vitro models

Fig 9. Proteomic comparison of Wistar rats, GK rats and the H9C2 cell line.

Discussion

Cardiac dysfunction

Diabetic kidney disease

Impact of ACEi

Proteomic comparison

Implications

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Vincenzo Lionetti

Roles

Author response to Decision Letter 0

Decision Letter 1

Vincenzo Lionetti

Roles

Acceptance letter

Vincenzo Lionetti

Roles