Publications by authors named "Andrew Advani"

80 Publications

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

PLoS One 2021 24;16(6):e0252711. Epub 2021 Jun 24.

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

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252711PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224913PMC
June 2021

Empagliflozin Disrupts a Tnfrsf12a-Mediated Feed Forward Loop That Promotes Left Ventricular Hypertrophy.

Cardiovasc Drugs Ther 2021 Apr 22. Epub 2021 Apr 22.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael's Hospital, 209 Victoria Street, Toronto, Ontario, M5B 1T8, Canada.

Purpose: Although the cardioprotective benefits of sodium-glucose cotransporter 2 (SGLT2) inhibitors are now widely appreciated, the mechanisms underlying these benefits remain unresolved. Tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) is a receptor for tumor necrosis factor superfamily member 12 (Tnfsf12). Tnfrsf12a is highly inducible and plays a key role in the development of cardiac hypertrophy and heart failure. Here we set out to determine if SGLT2 inhibition affects the Tnfsf12/Tnfrsf12a system in the stressed myocardium.

Methods: C57BL/6N mice that had undergone sham or transverse aortic constriction (TAC) surgery were treated with either the SGLT2 inhibitor empagliflozin (400 mg/kg diet; 60-65 mg/kg/day) or standard chow alone and were followed for 8 weeks. Tnfrsf12a expression in mouse hearts was assessed by in situ hybridization, qRT-PCR, and immunoblotting.

Results: Left ventricular (LV) mass, end-systolic volume, and end-diastolic volume were all increased in TAC mice and were significantly lower with empagliflozin. Myocyte hypertrophy and interstitial fibrosis in TAC hearts were similarly attenuated with empagliflozin. Tnfrsf12a expression was upregulated in mouse hearts following TAC surgery but not in the hearts of empagliflozin-treated mice. In cultured cardiomyocytes, Tnfrsf12a antagonism attenuated the increase in cardiomyocyte size that was induced by phenylephrine.

Conclusion: Empagliflozin attenuates LV enlargement in mice with hypertrophic heart failure. This effect may be mediated, at least in part, by a reduction in loading conditions which limits upregulation of the inducible, proinflammatory, and prohypertrophic TNF superfamily receptor, Tnfrsf12a. Disruption of the Tnfsf12/Tnfrsf12a feed forward system may contribute to the cardioprotective benefits of SGLT2 inhibition.
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http://dx.doi.org/10.1007/s10557-021-07190-2DOI Listing
April 2021

The study of single cells in diabetic kidney disease.

J Nephrol 2021 Jan 21. Epub 2021 Jan 21.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael's Hospital, 6-151 61 Queen Street East, Toronto, ON, M5C 2T2, Canada.

In the past few years there has been a rapid expansion of interest in the study of single cells, especially through the new techniques that involve single-cell RNA sequencing (scRNA-seq). Recently, these techniques have provided new insights into kidney health and disease, including insights into diabetic kidney disease (DKD). However, despite the interest and the technological advances, the study of individual cells in DKD is not a new concept. Many clinicians and researchers who work within the DKD space may be familiar with experimental techniques that actually involve the study of individual cells, but may be unfamiliar with newer scRNA-seq technology. Here, with the goal of improving accessibility to the single-cell field, we provide a primer on single-cell studies with a focus on DKD. We situate the technology in its historical context and provide a brief explanation of the common aspects of the different technologies available. Then we review some of the most important recent studies of kidney (patho)biology that have taken advantage of scRNA-seq techniques, before emphasizing the new insights into the molecular pathogenesis of DKD gleaned with these techniques. Finally, we highlight common pitfalls and limitations of scRNA-seq methods and we look toward the future to how single-cell experiments may be incorporated into the study of DKD and how to interpret the findings of these experiments.
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http://dx.doi.org/10.1007/s40620-020-00964-1DOI Listing
January 2021

Lung and Kidney ACE2 and TMPRSS2 in Renin-Angiotensin System Blocker-Treated Comorbid Diabetic Mice Mimicking Host Factors That Have Been Linked to Severe COVID-19.

Diabetes 2021 03 11;70(3):759-771. Epub 2020 Dec 11.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada

The causes of the increased risk of severe coronavirus disease 2019 (COVID-19) in people with diabetes are unclear. It has been speculated that renin-angiotensin system (RAS) blockers may promote COVID-19 by increasing ACE2, which severe acute respiratory syndrome coronavirus 2 uses to enter host cells, along with the host protease TMPRSS2. Taking a reverse translational approach and by combining in situ hybridization, primary cell isolation, immunoblotting, quantitative RT-PCR, and liquid chromatography-tandem mass spectrometry, we studied lung and kidney ACE2 and TMPRSS2 in diabetic mice mimicking host factors linked to severe COVID-19. In healthy young mice, neither the ACE inhibitor ramipril nor the AT1 receptor blocker telmisartan affected lung or kidney ACE2 or TMPRSS2, except for a small increase in kidney ACE2 protein with ramipril. In contrast, mice with comorbid diabetes (aging, high-fat diet, and streptozotocin-induced diabetes) had heightened lung ACE2 and TMPRSS2 protein levels and increased lung ACE2 activity. None of these parameters were affected by RAS blockade. ACE2 was similarly upregulated in the kidneys of mice with comorbid diabetes compared with aged controls, whereas TMPRSS2 (primarily distal nephron) was highest in telmisartan-treated animals. Upregulation of lung ACE2 activity in comorbid diabetes may contribute to an increased risk of severe COVID-19. This upregulation is driven by comorbidity and not by RAS blockade.
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http://dx.doi.org/10.2337/db20-0765DOI Listing
March 2021

Acute Kidney Injury: A Bona Fide Complication of Diabetes.

Authors:
Andrew Advani

Diabetes 2020 11;69(11):2229-2237

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada

The landscape of kidney disease in diabetes has shifted. The classical dogma of "diabetic nephropathy" progressing through stages of albuminuria, leading to decline in glomerular filtration rate and end-stage kidney disease (ESKD), has been replaced by a more nuanced understanding of the complex and heterogeneous nature of kidney disease in diabetes. Paralleling this evolution, standardized definitions have resulted in a growing appreciation that acute kidney injury (AKI) is increasing in its incidence rapidly and that people with diabetes are much more likely to develop AKI than people without diabetes. Here, I propose that AKI should be considered a complication of diabetes alongside other complications that similarly do not fit neatly into the historical microvascular/macrovascular paradigm. In this article, we take a look at the evidence indicating that diabetes is a major risk factor for AKI and we review the causes of this increased risk. We consider the long-term implications of AKI in diabetes and its potential contribution to the future development of chronic kidney disease, ESKD, and mortality. Finally, we look toward the future at strategies to better identify people at risk for AKI and to develop new approaches to improve AKI outcomes. Recognizing AKI as a bona fide complication of diabetes should open up new avenues for investigation that may ultimately improve the outlook for people living with diabetes and at risk for kidney disease.
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http://dx.doi.org/10.2337/db20-0604DOI Listing
November 2020

Experiences and perspectives of the parents of emerging adults living with type 1 diabetes.

BMJ Open Diabetes Res Care 2020 10;8(1)

Applied Health Research Centre, Li ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Ontario, Canada.

Introduction: Whereas it is widely recognized that emerging adulthood can be a difficult time in the life of an individual living with type 1 diabetes, relatively little is known about the experiences of their parents or guardians. These individuals once shouldered much of the burden for their child's diabetes 'self'-management, yet their contribution is often overlooked by the adult healthcare system. Here, we set out to gain an understanding of the perspectives of parents of emerging adults living with type 1 diabetes.

Research Design And Methods: Semi-structured interviews were performed with a purposeful sample of parents of emerging adults with type 1 diabetes recruited from two urban young adult diabetes clinics and through a national diabetes charity. Thematic coding was derived using a constant comparative approach.

Results: Analysis of interviews with 16 parents of emerging adults with type 1 diabetes identified three themes: parental experiences of the transition to adult care; negotiating parent-child roles, responsibilities and relationships; and new and evolving fears. Parents spoke in detail about the time surrounding their child's diagnosis of type 1 diabetes to emphasize the complexity of diabetes care and the need to establish a 'new normal' for the family. In turn, adolescence and emerging adulthood required a renegotiation of roles and responsibilities, with many parents continuing to play a role in high-level diabetes management. Several parents of emerging adults with type 1 diabetes (particularly those of young men) vocalized worries about their child's readiness to assume responsibility for their self-care, and some expressed frustration with the apparent dichotomy in the role expectations of parents between the pediatric and adult care settings.

Conclusions: Adult healthcare providers should recognize both the ongoing involvement of parents in the 'self'-management of emerging adults with type 1 diabetes and the unique aspects of the caregiver burden that they experience.
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http://dx.doi.org/10.1136/bmjdrc-2019-001125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534673PMC
October 2020

The Dipeptidyl Peptidase-4 Inhibitor Linagliptin Directly Enhances the Contractile Recovery of Mouse Hearts at a Concentration Equivalent to that Achieved with Standard Dosing in Humans.

Int J Mol Sci 2020 Aug 11;21(16). Epub 2020 Aug 11.

Keenan Research Center for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, ON M5B 1T8, Canada.

Despite a similar mechanism of action underlying their glucose-lowering effects in type 2 diabetes, dipeptidyl peptidase-4 (DPP-4) inhibitors have diverse molecular structures, raising the prospect of agent-specific, glucose-independent actions. To explore the issue of possible DPP-4 inhibitor cardiac heterogeneity, we perfused different DPP-4 inhibitors to beating mouse hearts ex vivo, at concentrations equivalent to peak plasma levels achieved in humans with standard dosing. We studied male and female mice, young non-diabetic mice, and aged diabetic high fat diet-fed mice and observed that linagliptin enhanced recovery after ischemia-reperfusion, whereas sitagliptin, alogliptin, and saxagliptin did not. DPP-4 transcripts were not detected in adult mouse cardiomyocytes by RNA sequencing and the addition of linagliptin caused ≤0.2% of cardiomyocyte genes to be differentially expressed. In contrast, incubation of C166 endothelial cells with linagliptin induced cell signaling characterized by phosphorylation of Akt and endothelial nitric oxide synthase, whereas the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine increased serine 16 phosphorylation of the calcium regulatory protein, phospholamban in cardiomyocytes. Furthermore, linagliptin increased cardiomyocyte cGMP when cells were co-cultured with C166 endothelial cells, but not when cardiomyocytes were cultured alone. Thus, at a concentration comparable to that achieved in patients, linagliptin has direct effects on mouse hearts. The effects of linagliptin on cardiomyocytes are likely to be either off-target or indirect, mediated through NO generation by the adjacent cardiac endothelium.
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http://dx.doi.org/10.3390/ijms21165756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460951PMC
August 2020

A common glomerular transcriptomic signature distinguishes diabetic kidney disease from other kidney diseases in humans and mice.

Curr Res Transl Med 2020 11 15;68(4):225-236. Epub 2020 May 15.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada. Electronic address:

Background: Current uncertainties about the similarity between human diseases and their experimental models are hampering the development of new therapies. This is especially the case for diabetic kidney disease (DKD), the most common cause of end-stage kidney disease. To better understand the nature of the commonality between humans and their mouse models, we posed the question: in diabetic kidney disease are transcriptional profiles primarily disease-specific or species-specific?

Methods: We performed a meta-comparison of the glomerular transcriptomic characteristics of 133 human and 66 mouse samples including five human kidney diseases and five mouse models, validating expression patterns of a central node by immunohistochemistry.

Findings: Principal component analysis controlled for mouse background, revealed that gene expression changes in glomeruli from humans with DKD are more similar to those of diabetic mice than they are to other human glomerular diseases. This similarity enabled the construction of a discriminatory classifier that distinguishes diabetic glomeruli from other glomerular phenotypes regardless of their species of origin. To identify where the commonality between mice and humans with diabetes lies, networks of maximally perturbed protein interactions were examined, identifying a central role for the epidermal growth factor receptor (EGFR). By immunohistochemical staining, we found EGFR to be approximately doubled in its glomerular expression in both humans and mice.

Interpretation: These findings indicate that diabetic mouse models do mimic some of the features of human kidney disease, at least with respect to their glomerular transcriptomic signatures, and they identify EGFR as being a central player in this inter-species overlap.
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http://dx.doi.org/10.1016/j.retram.2020.05.001DOI Listing
November 2020

Screening and Treatment Outcomes in Adults and Children With Type 1 Diabetes and Asymptomatic Celiac Disease: The CD-DIET Study.

Diabetes Care 2020 07 28;43(7):1553-1556. Epub 2020 Apr 28.

Division of Endocrinology, Markham-Stouffville Hospital, Markham, Ontario, Canada.

Objective: To describe celiac disease (CD) screening rates and glycemic outcomes of a gluten-free diet (GFD) in patients with type 1 diabetes who are asymptomatic for CD.

Research Design And Methods: Asymptomatic patients (8-45 years) were screened for CD. Biopsy-confirmed CD participants were randomized to GFD or gluten-containing diet (GCD) to assess changes in HbA and continuous glucose monitoring over 12 months.

Results: Adults had higher CD-seropositivity rates than children (6.8% [95% CI 4.9-8.2%, = 1,298] vs. 4.7% [95% CI 3.4-5.9%, = 1,089], = 0.035) with lower rates of prior CD screening (6.9% vs. 44.2%, < 0.0001). Fifty-one participants were randomized to a GFD ( = 27) or GCD ( = 24). No HbA differences were seen between the groups (+0.14%, 1.5 mmol/mol; 95% CI -0.79 to 1.08; = 0.76), although greater postprandial glucose increases (4-h +1.5 mmol/L; 95% CI 0.4-2.7; = 0.014) emerged with a GFD.

Conclusions: CD is frequently observed in asymptomatic patients with type 1 diabetes, and clinical vigilance is warranted with initiation of a GFD.
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http://dx.doi.org/10.2337/dc19-1944DOI Listing
July 2020

The Endothelial Barrier Is not Rate-limiting to Insulin Action in the Myocardium of Male Mice.

Endocrinology 2020 04;161(4)

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

To act on tissues, circulating insulin must perfuse the relevant organ and then leave the bloodstream by crossing the endothelium-a process known as insulin delivery. It has been postulated that the continuous endothelium is a rate-limiting barrier to insulin delivery but existing data are contradictory. This conflict is in part due to the limitations of current models, including the inability to maintain a constant blood pressure in animals and the absence of shear stress in cultured cells. We developed a murine cardiac ex vivo perfusion model that delivers insulin to the heart in situ at a constant flow. We hypothesized that if the endothelial barrier were rate-limiting to insulin delivery, increasing endothelial permeability would accelerate insulin action. The kinetics of myocardial insulin action were determined in the presence or absence of agents that increased endothelial permeability. Permeability was measured using Evans Blue, which binds with high affinity to albumin. During our experiments, the myocardium remained sensitive to insulin and the vasculature retained barrier integrity. Perfusion with insulin induced Akt phosphorylation in myocytes but not in the endothelium. Infusion of platelet-activating factor or vascular endothelial growth factor significantly increased permeability to albumin without altering insulin action. Amiloride, an inhibitor of fluid-phase uptake, also did not alter insulin action. These data suggest that the endothelial barrier is not rate limiting to insulin's action in the heart; its passage out of the coronary circulation is consistent with diffusion or convection. Modulation of transendothelial transport to overcome insulin resistance is unlikely to be a viable therapeutic strategy.
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http://dx.doi.org/10.1210/endocr/bqaa029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069687PMC
April 2020

Load-independent effects of empagliflozin contribute to improved cardiac function in experimental heart failure with reduced ejection fraction.

Cardiovasc Diabetol 2020 02 8;19(1):13. Epub 2020 Feb 8.

Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, 61 Queen Street East, Toronto, M5C 2T2, ON, Canada.

Background And Aims: Sodium-glucose linked cotransporter-2 (SGLT2) inhibitors reduce the likelihood of hospitalization for heart failure and cardiovascular death in both diabetic and non-diabetic individuals with reduced ejection fraction heart failure. Because SGLT2 inhibitors lead to volume contraction with reductions in both preload and afterload, these load-dependent factors are thought to be major contributors to the cardioprotective effects of the drug class. Beyond these effects, we hypothesized that SGLT2 inhibitors may also improve intrinsic cardiac function, independent of loading conditions.

Methods: Pressure-volume (P-V) relationship analysis was used to elucidate changes in intrinsic cardiac function, independent of alterations in loading conditions in animals with experimental myocardial infarction, a well-established model of HFrEF. Ten-week old, non-diabetic Fischer F344 rats underwent ligation of the left anterior descending (LAD) coronary artery to induce myocardial infarction (MI) of the left ventricle (LV). Following confirmation of infarct size with echocardiography 1-week post MI, animals were randomized to receive vehicle, or the SGLT2 inhibitor, empagliflozin. Cardiac function was assessed by conductance catheterization just prior to termination 6 weeks later.

Results: The circumferential extent of MI in animals that were subsequently randomized to vehicle or empagliflozin groups was similar. Empagliflozin did not affect fractional shortening (FS) as assessed by echocardiography. In contrast, load-insensitive measures of cardiac function were substantially improved with empagliflozin. Load-independent measures of cardiac contractility, preload recruitable stroke work (PRSW) and end-systolic pressure volume relationship (ESPVR) were higher in rats that had received empagliflozin. Consistent with enhanced cardiac performance in the heart failure setting, systolic blood pressure (SBP) was higher in rats that had received empagliflozin despite its diuretic effects. A trend to improved diastolic function, as evidenced by reduction in left ventricular end-diastolic pressure (LVEDP) was also seen with empagliflozin. MI animals treated with vehicle demonstrated myocyte hypertrophy, interstitial fibrosis and evidence for changes in key calcium handling proteins (all p < 0.05) that were not affected by empagliflozin therapy.

Conclusion: Empagliflozin therapy improves cardiac function independent of loading conditions. These findings suggest that its salutary effects are, at least in part, due to actions beyond a direct effect of reduced preload and afterload.
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http://dx.doi.org/10.1186/s12933-020-0994-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007658PMC
February 2020

Positioning time in range in diabetes management.

Authors:
Andrew Advani

Diabetologia 2020 02 7;63(2):242-252. Epub 2019 Nov 7.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St Michael's Hospital, 209 Victoria Street, Toronto, ON, M5B 1T8, Canada.

Recent upswings in the use of continuous glucose monitoring (CGM) technologies have given people with diabetes and healthcare professionals unprecedented access to a range of new indicators of glucose control. Some of these metrics are useful research tools and others have been welcomed by patient groups for providing insights into the quality of glucose control not captured by conventional laboratory testing. Among the latter, time in range (TIR) is an intuitive metric that denotes the proportion of time that a person's glucose level is within a desired target range (usually 3.9-10.0 mmol/l [3.5-7.8 mmol/l in pregnancy]). For individuals choosing to use CGM technology, TIR is now often part of the expected conversation between patient and healthcare professional, and consensus recommendations have recently been produced to facilitate the adoption of standardised TIR targets. At a regulatory level, emerging evidence linking TIR to risk of complications may see TIR being more widely accepted as a valid endpoint in future clinical trials. However, given the skewed distribution of possible glucose values outside of the target range, TIR (on its own) is a poor indicator of the frequency or severity of hypoglycaemia. Here, the state-of-the-art linking TIR with complications risk in diabetes and the inverse association between TIR and HbA are reviewed. Moreover, the importance of including the amount and severity of time below range (TBR) in any discussions around TIR and, by inference, time above range (TAR) is discussed. This review also summarises recent guidance in setting 'time in ranges' goals for individuals with diabetes who wish to make use of these metrics. For most people with type 1 or type 2 diabetes, a TIR >70%, a TBR <3.9 mmol/l of <4%, and a TBR <3.0 mmol/l of <1% are recommended targets, with less stringent targets for older or high-risk individuals and for those under 25 years of age. As always though, glycaemic targets should be individualised and rarely is that more applicable than in the personal use of CGM and the data it provides.
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http://dx.doi.org/10.1007/s00125-019-05027-0DOI Listing
February 2020

Dysregulated expression but redundant function of the long non-coding RNA HOTAIR in diabetic kidney disease.

Diabetologia 2019 11 9;62(11):2129-2142. Epub 2019 Aug 9.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St Michael's Hospital, 209 Victoria Street, Toronto, ON, M5C 1T8, Canada.

Aims/hypothesis: Long non-coding RNAs (lncRNAs) are garnering increasing attention for their putative roles in the pathogenesis of chronic diseases, including diabetic kidney disease (DKD). However, much about in vivo lncRNA functionality in the adult organism remains unclear. To better understand lncRNA regulation and function in DKD, we explored the effects of the modular scaffold lncRNA HOTAIR (HOX antisense intergenic RNA), which approximates chromatin modifying complexes to their target sites on the genome.

Methods: Experiments were performed in human kidney tissue, in mice with streptozotocin-induced diabetes, the db/db mouse model of type 2 diabetes, podocyte-specific Hotair knockout mice and conditionally immortalised mouse podocytes.

Results: HOTAIR was observed to be expressed by several kidney cell-types, including glomerular podocytes, in both human and mouse kidneys. However, knockout of Hotair from podocytes had almost no effect on kidney structure, function or ultrastructure. Glomerular HOTAIR expression was found to be increased in human DKD, in the kidneys of mice with streptozotocin-induced diabetes and in the kidneys of db/db mice. Likewise, exposure of cultured mouse podocytes to high glucose caused upregulation of Hotair expression, which occurred in a p65-dependent manner. Although HOTAIR expression was upregulated in DKD and in high glucose-exposed podocytes, its knockout did not alter the development of kidney damage in diabetic mice. Rather, in a bioinformatic analysis of human kidney tissue, HOTAIR expression closely paralleled the expression of its genic neighbour, HOXC11, which is important to developmental patterning but which has an uncertain role in the adult kidney.

Conclusions/interpretation: Many lncRNAs have been found to bind to the same chromatin modifying complexes. Thus, there is likely to exist sufficient redundancy in the system that the biological effects of dysregulated lncRNAs in kidney disease may often be inconsequential. The example of the archetypal scaffold lncRNA, HOTAIR, illustrates how lncRNA dysregulation may be a bystander in DKD without necessarily contributing to the pathogenesis of the condition. In the absence of in vivo validation, caution should be taken before ascribing major functional roles to single lncRNAs in the pathogenesis of chronic diseases.
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http://dx.doi.org/10.1007/s00125-019-4967-1DOI Listing
November 2019

The 3i Conceptual Framework for Recognizing Patient Perspectives of Type 1 Diabetes During Emerging Adulthood.

JAMA Netw Open 2019 07 3;2(7):e196944. Epub 2019 Jul 3.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St Michael's Hospital, Toronto, Ontario, Canada.

Importance: Early emerging adulthood, the developmental life stage roughly spanning between the ages of 18 and 24 years, can be a challenging time for individuals living with type 1 diabetes, being associated with an increased risk of acute complications, loss to follow-up, and the emergence of long-term diabetes complications. Few evidence-based interventions exist, and practice guidelines instead emphasize the importance of delivering individualized, developmentally appropriate care.

Objective: To inform the provision of tailored care and education by illuminating the individual lived experiences of emerging adults with type 1 diabetes.

Design, Setting, And Participants: This qualitative study used in-depth interviews conducted with 33 emerging adults living with type 1 diabetes between October 14, 2016, and May 16, 2017. Participants were recruited from 2 urban Young Adult Diabetes Clinics in the city of Toronto, Ontario, Canada, using a convenience sampling approach. Narrative analysis of participants' accounts was conducted inductively to define story typology.

Main Outcomes And Measures: Participant accounts of type 1 diabetes during childhood, adolescence, and emerging adulthood.

Results: Interviews were conducted with 33 participants aged 18 to 24 years (mean [SD] age at interview, 20.6 [1.7] years); 17 (51%) were women. Duration of diabetes ranged from 3 to 20 years (mean [SD] duration, 12.0 [4.8] years). Narrative analysis identified 3 distinct story types, or lenses, termed ingrained (n = 14), intrusive (n = 12), and inconspicuous (n = 7). Ingrained narratives portrayed an active integration of diabetes self-management into daily life. Intrusive narratives were characterized by stories of struggles for acceptance and striving for control. Inconspicuous narratives were differentiated by descriptions of a willingness to ignore diabetes and its attendant daily tasks with the goals of preserving normalcy and limiting distress. Mean levels of hemoglobin A1c were higher in participants conveying an intrusive lens narrative (mean [SD], 9.3% [1.6%]) than those conveying an ingrained lens (mean [SD], 7.4% [0.7%]) (difference between intrusive and ingrained, 1.9%; 95% CI, 0.8%-2.9%; P = .007) or an inconspicuous lens (mean [SD], 8.6% [1.5%]).

Conclusions And Relevance: Although every person is unique, there are commonalities in how emerging adults perceive and depict their experiences living with type 1 diabetes. Among the emerging adults interviewed in the present study, the 3i conceptual framework of ingrained, intrusive, and inconspicuous lenses encapsulated these commonalities. Recognition of the different psychosocial contexts surrounding type 1 diabetes during emerging adulthood may help health care professionals individualize their care accordingly.
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http://dx.doi.org/10.1001/jamanetworkopen.2019.6944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628591PMC
July 2019

Empagliflozin Improves Diastolic Function in a Nondiabetic Rodent Model of Heart Failure With Preserved Ejection Fraction.

JACC Basic Transl Sci 2019 Feb 25;4(1):27-37. Epub 2019 Feb 25.

Keenan Research Centre, Li Ka Shying Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada.

Recent studies send an unambiguous signal that the class of agents known as sodium-glucose-linked co-transporter-2 inhibitors (SGLT2i) prevent heart failure hospitalization in patients with type 2 diabetes. However, the mechanisms remain unclear. Herein the authors utilize a rodent model of heart failure with preserved ejection fraction (HFpEF), and demonstrate that treatment with the SGLT2i empagliflozin, reduces left ventricular mass, improving both wall stress and diastolic function. These findings extend the observation that the main mechanism of action of empagliflozin involves improved hemodynamics (i.e., reduction in preload and afterload) and provide a rationale for upcoming trials in patients with HFpEF irrespective of glycemic status.
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http://dx.doi.org/10.1016/j.jacbts.2018.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390677PMC
February 2019

Renal Hemodynamic Function and RAAS Activation Over the Natural History of Type 1 Diabetes.

Am J Kidney Dis 2019 06 22;73(6):786-796. Epub 2019 Feb 22.

Division of Nephrology, Department of Medicine, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada.

Rationale & Objective: The renin-angiotensin-aldosterone system (RAAS) is associated with renal and cardiovascular disease in diabetes. Unfortunately, early RAAS blockade in patients with type 1 diabetes mellitus (T1DM) does not prevent the development of complications. We sought to examine the role of hyperfiltration and RAAS activation across a wide range of T1DM duration to better understand renal hemodynamic status in patients with T1DM.

Study Design: Post hoc analysis of blood samples.

Setting & Participants: 148 Canadian patients with T1DM: 28 adolescents (aged 16.2±2.0 years), 54 young adults (25.4±5.6 years), and 66 older adults (65.7±7.5 years) studied in a clinical investigation unit.

Exposure: Angiotensin II infusion (1ng/kg/min; a measure of RAAS activation) during a euglycemic clamp.

Outcomes: Glomerular filtration rate measured using inulin clearance, effective renal plasma flow measured using para-aminohippurate, afferent (R) and efferent (R) arteriolar resistances, and glomerular hydrostatic pressure estimated using the Gomez equations.

Results: In a stepwise fashion, glomerular filtration rate, effective renal plasma flow, and glomerular hydrostatic pressure were higher, while renal vascular resistance and R were lower in adolescents versus young adults versus older adults. R was similar in adolescents versus young adults but was higher in older adults. Angiotensin II resulted in blunted renal hemodynamic responses in older adults (renal vascular resistance increase of 3.3% ± 1.6% vs 4.9% ± 1.9% in adolescents; P<0.001), suggesting a state of enhanced RAAS activation.

Limitations: Homogeneous study participants limit the generalizability of findings to other populations. Studying older adult participants with T1DM may be associated with a survivorship bias.

Conclusions: A state of relatively low RAAS activity and predominant afferent dilation rather than efferent constriction characterize early adolescents and young adults with T1DM. This state of endogenous RAAS inactivity in early T1DM may explain why pharmacologic blockade of this neurohormonal system is often ineffective in reducing kidney disease progression in this setting. Older adults with long-standing T1DM who have predominant afferent constriction and RAAS activation may experience renoprotection from therapies that target the afferent arteriole. Further work is required to understand the potential role of non-RAAS pharmacologic agents that target R in patients with early and long-standing T1DM.
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http://dx.doi.org/10.1053/j.ajkd.2018.12.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082148PMC
June 2019

Association between uric acid, renal haemodynamics and arterial stiffness over the natural history of type 1 diabetes.

Diabetes Obes Metab 2019 06 28;21(6):1388-1398. Epub 2019 Mar 28.

Department of Medicine, Division of Nephrology, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada.

Aims: To examine the relationship between normal plasma uric acid (PUA) levels, renal haemodynamic function, arterial stiffness and plasma renin and aldosterone over a wide range of type 1 diabetes (T1D) durations in adolescents, young adults and older adults.

Materials And Methods: PUA, glomerular filtration rate (GFR), effective renal plasma flow (ERPF), vascular stiffness parameters (aortic augmentation index [AIx], carotid AIx, carotid femoral pulse wave velocity [cfPWV]), and plasma renin and aldosterone were measured during a euglycaemic clamp in people with T1D: 27 adolescents (mean ± SD age 16.8 ± 1.9 years), 52 young adults (mean ± SD age 25.6 ± 5.5 years) and 66 older adults (mean ± SD age 65.7 ± 7.5 years).

Results: PUA was highest in patients with the longest T1D duration: 197 ± 44 μmol/L in adolescents versus 264 ± 82 μmol/L in older adults (P < 0.001). Higher PUA correlated with lower GFR only in older adults, even after correcting for age, glycated haemoglobin and sex (β = -2.12 ± 0.56; P = 0.0003), but not in adolescents or young adults. Higher PUA correlated with lower carotid AIx (β = -1.90, P = 0.02) in adolescents. In contrast, PUA correlated with higher cfPWV (P = 0.02) and higher plasma renin (P = 0.01) in older adults with T1D.

Conclusions: The relationship between higher PUA with lower GFR, increased arterial stiffness and renin angiotensin aldosterone system (RAAS) activation was observed only in older adults with longstanding T1D. T1D duration may modify the association between PUA, renal haemodynamic function and RAAS activation, leading to renal vasoconstriction and ischaemia. Further work must determine whether pharmacological PUA-lowering prevents or reverses injurious haemodynamic and neurohormonal sequelae of longstanding T1D, thereby improving clinical outcomes.
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http://dx.doi.org/10.1111/dom.13665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504604PMC
June 2019

Histone H3 Serine 10 Phosphorylation Facilitates Endothelial Activation in Diabetic Kidney Disease.

Diabetes 2018 12 13;67(12):2668-2681. Epub 2018 Sep 13.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada

The posttranslational histone modifications that epigenetically affect gene transcription extend beyond conventionally studied methylation and acetylation patterns. By examining the means by which podocytes influence the glomerular endothelial phenotype, we identified a role for phosphorylation of histone H3 on serine residue 10 (phospho-histone H3Ser10) in mediating endothelial activation in diabetes. Culture media conditioned by podocytes exposed to high glucose caused glomerular endothelial vascular cell adhesion protein 1 (VCAM-1) upregulation and was enriched for the chemokine CCL2. A neutralizing anti-CCL2 antibody prevented VCAM-1 upregulation in cultured glomerular endothelial cells, and knockout of the CCL2 receptor CCR2 diminished glomerular VCAM-1 upregulation in diabetic mice. CCL2/CCR2 signaling induced glomerular endothelial VCAM-1 upregulation through a pathway regulated by p38 mitogen-activated protein kinase, mitogen- and stress-activated protein kinases 1/2 (MSK1/2), and phosphorylation of H3Ser10, whereas MSK1/2 inhibition decreased H3Ser10 phosphorylation at the promoter. Finally, increased phospho-histone H3Ser10 levels were observed in the kidneys of diabetic endothelial nitric oxide synthase knockout mice and in the glomeruli of humans with diabetic kidney disease. These findings demonstrate the influence that histone protein phosphorylation may have on gene activation in diabetic kidney disease. Histone protein phosphorylation should be borne in mind when considering epigenetic targets amenable to therapeutic manipulation in diabetes.
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http://dx.doi.org/10.2337/db18-0124DOI Listing
December 2018

Histone Deacetylase Inhibitors and Diabetic Kidney Disease.

Int J Mol Sci 2018 Sep 5;19(9). Epub 2018 Sep 5.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, ON M5B 1W8, Canada.

Despite recent clinical trial advances and improvements in clinical care, kidney disease due to diabetes remains the most common cause of chronic kidney failure worldwide. In the search for new treatments, recent attentions have turned to drug repurposing opportunities, including study of the histone deacetylase (HDAC) inhibitor class of agents. HDACs are a group of enzymes that remove functional acetyl groups from histone and non-histone proteins and they can affect cellular function through both epigenetic and non-epigenetic means. Over the past decade, several HDAC inhibitors have been adopted into clinical practice, primarily for the treatment of hematological malignancy, whereas other existing therapies (for instance valproate) have been found to have HDAC inhibitory effects. Here we review the current HDAC inhibitors in the clinic and under development; the literature evidence supporting the renoprotective effects of HDAC inhibitors in experimental diabetic kidney disease; and the adverse effect profiles that may prevent existing therapies from entering the clinic for this indication. Whereas recent research efforts have shed light on the fundamental actions of HDACs in the diabetic kidney, whether these efforts will translate into novel therapies for patients will require more specific and better-tolerated therapies.
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http://dx.doi.org/10.3390/ijms19092630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165182PMC
September 2018

The Dipeptidyl Peptidase 4 Substrate CXCL12 Has Opposing Cardiac Effects in Young Mice and Aged Diabetic Mice Mediated by Ca Flux and Phosphoinositide 3-Kinase γ.

Diabetes 2018 11 27;67(11):2443-2455. Epub 2018 Aug 27.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada

Blood glucose-lowering therapies can positively or negatively affect heart function in type 2 diabetes, or they can have neutral effects. Dipeptidyl peptidase 4 (DPP-4) inhibitors lower blood glucose by preventing the proteolytic inactivation of glucagon-like peptide 1 (GLP-1). However, GLP-1 is not the only peptide substrate of DPP-4. Here, we investigated the GLP-1-independent cardiac effects of DPP-4 substrates. Pointing to GLP-1 receptor (GLP-1R)-independent actions, DPP-4 inhibition prevented systolic dysfunction equally in pressure-overloaded wild-type and GLP-1R knockout mice. Likewise, DPP-4 inhibition or the DPP-4 substrates substance P or C-X-C motif chemokine ligand 12 (CXCL12) improved contractile recovery after no-flow ischemia in the hearts of otherwise healthy young adult mice. Either DPP-4 inhibition or CXCL12 increased phosphorylation of the Ca regulatory protein phospholamban (PLN), and CXCL12 directly enhanced cardiomyocyte Ca flux. In contrast, hearts of aged obese diabetic mice (which may better mimic the comorbid patient population) had diminished levels of PLN phosphorylation. In this setting, CXCL12 paradoxically impaired cardiac contractility in a phosphoinositide 3-kinase γ-dependent manner. These findings indicate that the cardiac effects of DPP-4 inhibition primarily occur through GLP-1R-independent processes and that ostensibly beneficial DPP-4 substrates can paradoxically worsen heart function in the presence of comorbid diabetes.
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http://dx.doi.org/10.2337/db18-0410DOI Listing
November 2018

Histones and heart failure in diabetes.

Cell Mol Life Sci 2018 Sep 22;75(17):3193-3213. Epub 2018 Jun 22.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, 6-151, 61 Queen Street East, Toronto, ON, M5C 2T2, Canada.

Although heart failure is now accepted as being a major long-term complication of diabetes, many of the recent advances in our understanding of the pathobiology of diabetes complications have come about through the study of more traditional microvascular or macrovascular diseases. This has been the case, for example, in the evolving field of the epigenetics of diabetes complications and, in particular, the post-translational modification of histone proteins. However, histone modifications also occur in human heart failure and their perturbation also occurs in diabetic hearts. Here, we review the principal histone modifications and their enzymatic writers and erasers that have been studied to date; we discuss what is currently known about their roles in heart failure and in the diabetic heart; we draw on lessons learned from the studies of microvascular and macrovascular complications; and we speculate that therapeutically manipulating histone modifications may alter the natural history of heart failure in diabetes.
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http://dx.doi.org/10.1007/s00018-018-2857-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063320PMC
September 2018

Sirtuin 1 activation attenuates cardiac fibrosis in a rodent pressure overload model by modifying Smad2/3 transactivation.

Cardiovasc Res 2018 10;114(12):1629-1641

Division of Cardiology, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, 209 Victoria Street, Toronto, Ontario, Canada.

Aims: Transforming growth factor β1 (TGF-β1) is a prosclerotic cytokine involved in cardiac remodelling leading to heart failure (HF). Acetylation/de-acetylation of specific lysine residues in Smad2/3 has been shown to regulate TGF-β signalling by altering its transcriptional activity. Recently, the lysine de-acetylase sirtuin 1 (SIRT1) has been shown to have a cardioprotective effect; however, SIRT1 expression and activity are paradoxically reduced in HF. Herein, we investigate whether pharmacological activation of SIRT1 would induce cardioprotection in a pressure overload model and assess the impact of SIRT1 activation on TGF-β signalling and the fibrotic response.

Methods And Results: Eight weeks old male C57BL/6 mice were randomized to undergo sham surgery or transverse aortic constriction (TAC) to induce pressure overload. Post-surgery, animals were further randomized to receive SRT1720 or vehicle treatment. Echocardiography, pressure-volume loops, and histological analysis revealed an impairment in cardiac function and deleterious left ventricular remodelling in TAC-operated animals that was improved with SRT1720 treatment. Genetic ablation and cell culture studies using a Smad-binding response element revealed SIRT1 to be a specific target of SRT1720 and identified Smad2/3 as a SIRT1 specific substrate.

Conclusion: Overall, our data demonstrate that Smad2/3 is a specific SIRT1 target and suggests that pharmacological activation of SIRT1 may be a novel therapeutic strategy to prevent/reverse HF via modifying Smad activity.
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http://dx.doi.org/10.1093/cvr/cvy131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148332PMC
October 2018

HDAC6 Inhibition Promotes Transcription Factor EB Activation and Is Protective in Experimental Kidney Disease.

Front Pharmacol 2018 1;9:34. Epub 2018 Feb 1.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, ON, Canada.

To contend with the deleterious effects of accumulating misfolded protein aggregates or damaged organelles cells rely on a system of quality control processes, among them the autophagy-lysosome pathway. This pathway is itself controlled by a master regulator transcription factor termed transcription factor EB (TFEB). When TFEB localizes to the cell nucleus it promotes the expression of a number of genes involved in protein clearance. Here, we set out to determine (1) whether TFEB expression is altered in chronic kidney disease (CKD); (2) whether inhibition of the cytosolic deacetylase histone deacetylase 6 (HDAC6) affects TFEB acetylation and nuclear localization; and (3) whether HDAC6 inhibition, in turn, alters the natural history of experimental CKD. TFEB mRNA and protein levels were observed to be diminished in the kidneys of humans with diabetic kidney disease, accompanied by accumulation of the protein aggregate adaptor protein p62 in tubule epithelial cells. In cultured NRK-52E cells, HDAC6 inhibition with the small molecule inhibitor Tubastatin A acetylated TFEB, increasing TFEB localization to the nucleus and attenuating cell death. In a rat model of CKD, Tubastatin A prevented the accumulation of misfolded protein aggregates in tubule epithelial cells, attenuated proteinuria progression, limited tubule cell death and diminished tubulointerstitial collagenous matrix deposition. These findings point to the common occurrence of dysregulated quality control processes in CKD and they suggest that TFEB downregulation may contribute to tubule injury in CKD. They also identify a regulatory relationship between HDAC6 and TFEB. HDAC6 inhibitors and TFEB activators both warrant further investigation as treatments for CKD.
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http://dx.doi.org/10.3389/fphar.2018.00034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5799228PMC
February 2018

Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease.

J Clin Invest 2018 01 11;128(1):483-499. Epub 2017 Dec 11.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada.

Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain-containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways.
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http://dx.doi.org/10.1172/JCI95946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749498PMC
January 2018

Dapagliflozin in focal segmental glomerulosclerosis: a combined human-rodent pilot study.

Am J Physiol Renal Physiol 2018 03 15;314(3):F412-F422. Epub 2017 Nov 15.

Department of Medicine, Division of Nephrology, University Health Network, University of Toronto , Toronto, Ontario , Canada.

Focal segmental glomerulosclerosis (FSGS) is an important cause of nondiabetic chronic kidney disease (CKD). Sodium-glucose cotransporter 2 inhibition (SGLT2i) therapy attenuates the progression of diabetic nephropathy, but it remains unclear whether SGLT2i provides renoprotection in nondiabetic CKD such as FSGS. The primary aim of this pilot study was to determine the effect of 8 wk of dapagliflozin on glomerular filtration rate (GFR) in humans and in experimental FSGS. Secondary end points were related to changes in renal hemodynamic function, proteinuria, and blood pressure (BP). GFR (inulin) and renal plasma flow (para-aminohippurate), proteinuria, and BP were measured in patients with FSGS ( n = 10), and similar parameters were measured in subtotally nephrectomized (SNx) rats. In response to dapagliflozin, changes in GFR, renal plasma flow, and 24-h urine protein excretion were not statistically significant in humans or rats. Systolic BP (SBP) decreased in SNx rats (196 ± 26 vs. 165 ± 33 mmHg; P < 0.001), whereas changes were not statistically significant in humans (SBP 112.7 ± 8.5 to 112.8 ± 11.2 mmHg, diastolic BP 71.8 ± 6.5 to 69.6 ± 8.4 mmHg; P = not significant), although hematocrit increased (0.40 ± 0.05 to 0.42 ± 0.05%; P = 0.03). In archival kidney tissue from a separate patient cohort, renal parenchymal SGLT2 mRNA expression was decreased in individuals with FSGS compared with controls. Short-term treatment with the SGLT2i dapagliflozin did not modify renal hemodynamic function or attenuate proteinuria in humans or in experimental FSGS. This may be related to downregulation of renal SGLT2 expression. Studies examining the impact of SGLT2i on markers of kidney disease in patients with other causes of nondiabetic CKD are needed.
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http://dx.doi.org/10.1152/ajprenal.00445.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899226PMC
March 2018

EP4 inhibition attenuates the development of diabetic and non-diabetic experimental kidney disease.

Sci Rep 2017 06 13;7(1):3442. Epub 2017 Jun 13.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada.

The therapeutic targeting of prostanoid subtype receptors may slow the development of chronic kidney disease (CKD) through mechanisms that are distinct from those of upstream COX inhibition. Here, employing multiple experimental models of CKD, we studied the effects of inhibition of the EP4 receptor, one of four receptor subtypes for the prostanoid prostaglandin E. In streptozotocin-diabetic endothelial nitric oxide synthase knockout mice, EP4 inhibition attenuated the development of albuminuria, whereas the COX inhibitor indomethacin did not. In Type 2 diabetic db/db mice, EP4 inhibition lowered albuminuria to a level comparable with that of the ACE inhibitor captopril. However, unlike captopril, EP4 inhibition had no effect on blood pressure or hyperfiltration although it did attenuate mesangial matrix accumulation. Indicating a glucose-independent mechanism of action, EP4 inhibition also attenuated proteinuria development and glomerular scarring in non-diabetic rats subjected to surgical renal mass ablation. Finally, in vitro, EP4 inhibition prevented transforming growth factor-ß1 induced dedifferentiation of glomerular podocytes. In rodent models of diabetic and non-diabetic CKD, EP4 inhibition attenuated renal injury through mechanisms that were distinct from either broadspectrum COX inhibition or "standard of care" renin angiotensin system blockade. EP4 inhibition may represent a viable repurposing opportunity for the treatment of CKD.
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http://dx.doi.org/10.1038/s41598-017-03237-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469816PMC
June 2017

Janus Kinase 2 Regulates Transcription Factor EB Expression and Autophagy Completion in Glomerular Podocytes.

J Am Soc Nephrol 2017 Sep 19;28(9):2641-2653. Epub 2017 Apr 19.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada;

The nonreceptor kinase Janus kinase 2 (JAK2) has garnered attention as a promising therapeutic target for the treatment of CKD. However, being ubiquitously expressed in the adult, JAK2 is also likely to be necessary for normal organ function. Here, we investigated the phenotypic effects of JAK2 deficiency. Mice in which JAK2 had been deleted from podocytes exhibited an elevation in urine albumin excretion that was accompanied by increased podocyte autophagosome fractional volume and p62 aggregation, which are indicative of impaired autophagy completion. In cultured podocytes, knockdown of JAK2 similarly impaired autophagy and led to downregulation in the expression of lysosomal genes and decreased activity of the lysosomal enzyme, cathepsin D. Because transcription factor EB (TFEB) has recently emerged as a master regulator of autophagosome-lysosome function, controlling the expression of several of the genes downregulated by JAK2 knockdown, we questioned whether TFEB is regulated by JAK2. In immortalized mouse podocytes, JAK2 knockdown decreased TFEB promoter activity, expression, and nuclear localization. analysis and chromatin immunoprecipitation assays revealed that the downstream mediator of JAK2 signaling STAT1 binds to the TFEB promoter. Finally, overexpression of TFEB in JAK2-deficient podocytes reversed lysosomal dysfunction and restored albumin permselectivity. Collectively, these observations highlight the homeostatic actions of JAK2 in podocytes and the importance of TFEB to autophagosome-lysosome function in these cells. These results also raise the possibility that therapeutically modulating TFEB activity may improve podocyte health in glomerular disease.
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http://dx.doi.org/10.1681/ASN.2016111208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576936PMC
September 2017

Renal and Vascular Effects of Uric Acid Lowering in Normouricemic Patients With Uncomplicated Type 1 Diabetes.

Diabetes 2017 07 13;66(7):1939-1949. Epub 2017 Apr 13.

Department of Medicine, Division of Nephrology, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada

Higher plasma uric acid (PUA) levels are associated with lower glomerular filtration rate (GFR) and higher blood pressure (BP) in patients with type 1 diabetes (T1D). Our aim was to determine the impact of PUA lowering on renal and vascular function in patients with uncomplicated T1D. T1D patients ( = 49) were studied under euglycemic and hyperglycemic conditions at baseline and after PUA lowering with febuxostat (FBX) for 8 weeks. Healthy control subjects were studied under normoglycemic conditions ( = 24). PUA, GFR (inulin), effective renal plasma flow (para-aminohippurate), BP, and hemodynamic responses to an infusion of angiotensin II (assessment of intrarenal renin-angiotensin-aldosterone system [RAAS]) were measured before and after FBX treatment. Arterial stiffness, flow-mediated dilation (FMD), nitroglycerin-mediated dilation (GMD), urinary nitric oxide (NO), and inflammatory markers were measured before and after FBX treatment. Gomez equations were used to estimate arteriolar afferent resistance, efferent resistance (R), and glomerular hydrostatic pressure (P). FBX had a modest systolic BP-lowering effect in T1D patients (112 ± 10 to 109 ± 9 mmHg, = 0.049) without impacting arterial stiffness, FMD, GMD, or NO. FBX enhanced the filtration fraction response to hyperglycemia in T1D patients through larger increases in R P, and interleukin-18 but without impacting the RAAS. FBX lowered systolic BP and modulated the renal R responses to hyperglycemia but without impacting the RAAS or NO levels, suggesting that PUA may augment other hemodynamic or inflammatory mechanisms that control the renal response to hyperglycemia at the efferent arteriole. Ongoing outcome trials will determine cardiorenal outcomes of PUA lowering in patients with T1D.
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http://dx.doi.org/10.2337/db17-0168DOI Listing
July 2017

Urinary adenosine excretion in type 1 diabetes.

Am J Physiol Renal Physiol 2017 Aug 5;313(2):F184-F191. Epub 2017 Apr 5.

Division of Nephrology, Department of Medicine, University Health Network, University of Toronto, Toronto, Ontario, Canada;

In experimental models of diabetes, augmented sodium-glucose cotransport-2 (SGLT2) activity diminishes sodium (Na) delivery at the macula densa. As a result, less vasoconstrictive adenosine is generated, leading to afferent arteriolar vasodilatation and hyperfiltration. The measurement and significance of urinary adenosine in humans has not been examined extensively in states of renal hemodynamic impairment like that of diabetes. Our aim was to validate a method for urine adenosine quantification in humans and perform an exploratory post hoc analysis to determine whether urinary adenosine levels change dynamically in response to natriuresis in patients with type 1 diabetes (T1D) before and after treatment with the SGLT2 inhibitor (SGLT2i) empagliflozin. We hypothesized that SGLT2i, which reduces renal hyperfiltration through increased Na delivery to the macula densa, would increase urinary adenosine excretion. Urine adenosine corrected for creatinine was measured using our validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in 40 healthy participants and 40 patients with T1D. In the T1D cohort, measurements were performed during clamped euglycemic and hyperglycemic conditions before and following 8 wk of SGLT2i therapy. Urinary adenosine was detectable in healthy subjects (0.32 ± 0.11 µmol/mmol Cr) and patients with T1D. In response to SGLT2i, urine adenosine increased during clamped hyperglycemia (0.40 ± 0.11 vs. 0.45 ± 0.12 µmol/mmol Cr, = 0.005). Similar trends were observed during clamped euglycemia ( = 0.08). In conclusion, SGLT2i increases urinary adenosine excretion under clamped hyperglycemic conditions in patients with T1D. The potentially protective role of SGLT2i against glomerular hyperfiltration and its mediation by adenosine in diabetes merits further study.
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http://dx.doi.org/10.1152/ajprenal.00043.2017DOI Listing
August 2017

VEGF and the diabetic kidney: More than too much of a good thing.

J Diabetes Complications 2017 01 22;31(1):273-279. Epub 2016 Oct 22.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada. Electronic address:

Over a decade and a half has passed since the publication of early reports hinting at a pathogenetic role for vascular endothelial growth factor ("VEGF") in the development of diabetic kidney disease. In diabetic rats, renal mRNA levels of the VEGF-A isoform were upregulated and administration of a VEGF-A neutralizing antibody attenuated albuminuria: VEGF was "bad" in diabetic nephropathy. Since that time, our understanding of the complexity of the renal VEGF system has advanced. Unlike its experimental counterpart, human diabetic nephropathy is associated with diminished VEGF-A levels and experience in the oncological setting has taught us that VEGF blocking therapy can cause adverse renal effects in patients. Correspondingly, investigational studies in cultured cells and rodent models have demonstrated that the biological effects of the VEGF system are dependent not only on the amount of VEGF, but also the type of VEGF, its sites of action and the prevailing milieu. Here we reflect back on the discoveries that have been made since those initial reports that shone the spotlight on the importance of the VEGF system in the diabetic kidney and we consider that the role of VEGF in diabetic nephropathy extends well beyond being "too much of a good thing".
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http://dx.doi.org/10.1016/j.jdiacomp.2016.10.020DOI Listing
January 2017
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