Features Of Diabetic Kidney Disease Research Articles

Activation of MST1 protects filtration barrier integrity of diabetic kidney disease in mice through restoring the tight junctions of glomerular endothelial cells.

As a pathological feature of diabetic kidney disease (DKD), dysregulated glomerular filtration barrier function could lead to the increased levels of proteinuria. The integrity of tight junctions (TJs) of glomerular endothelial cells (GECs) is a guarantee of physiological function of glomerular filtration barrier. Mammalian sterile 20-like kinase (MST1) is a key regulatory protein in the blood-brain barrier (BBB), and it regulates the expression of TJs-related proteins in cerebral vascular endothelial cells. Our previous study showed that MST1 was involved in renal tubulointerstitial fibrosis of DKD. In the present study we investigated the role of MST1 in barrier function of GECs of DKD, and explored its regulatory mechanisms. In kidney tissue section of DKD patients and db/db mice, and high glucose (HG)-cultured mouse glomerular endothelial cells (mGECs), we showed that MST1 was inactivated in the GECs of DKD accompanied by disrupted glomerular endothelial barrier. In db/db mice and HG-cultured mGECs, knockdown of MST1 increased proteinuria levels, and disrupted glomerular endothelial barrier through decreasing TJs-related proteins, whereas MST1 overexpression restored glomerular endothelial barrier through regaining TJs-related proteins. In db/db mice and HG-cultured mGECs, we demonstrated that MST1 inhibition induced TJs's disruption of GECs via activating YAP1/TEAD signaling. Verteporfin (an inhibitor of YAP1-TEAD interaction) and PY-60 (a YAP1 agonist) were used to verify the role of YAP1/TEAD signaling in the regulation effect of MST1 on barrier function of mGECs. In conclusion, MST1 activation recovers glomerular endothelial barrier of DKD by regaining TJs-related proteins via inhibiting YAP1/TEAD signaling. This study highlights the multiple regulation of MST1 activation on kidney injury.

RXRα/MR signaling promotes diabetic kidney disease by facilitating renal tubular epithelial cells senescence and metabolic reprogramming

Cell senescence and metabolic reprogramming are significant features of diabetic kidney disease (DKD). However, the underlying mechanisms between cell senescence and metabolic reprogramming are poorly defined. Here, we report that retinoid X receptor α (RXRα), a key nuclear receptor transcription factor, regulates cell senescence and metabolic reprogramming in DKD. Through high-throughput sequencing, bioinformatic analysis and experimental validation, we confirmed the critical role of RXRα in promoting cell senescence and metabolic dysregulation in renal tubular epithelial cells (RTECs) induced by lipid overload. In vivo, in situ injection of AAV9-shRxra into the kidney reduced proteinuria, RTECs senescence and insulin resistance in DKD mice. In vitro, knockdown of RXRα markedly improved G2/M phase arrest and suppressed the expression of senescence-associated secretory phenotypes (SASPs). Protein-protein interaction (PPI) analysis and unbiased bioinformatics were employed to identify the direct interactions between RXRα and the mineralocorticoid receptor (MR), which were subsequently validated through coimmunoprecipitation. Gene network analysis revealed the collaborative regulatory role of RXRα and MR in RTECs senescence. In an accelerated aging mouse model, treatment with a MR antagonist has been shown to inhibite the RXRα/MR signaling, improve RTECs senescence, and reduce interstitial fibrosis and lipid deposition in the kidneys. These findings indicate that inhibition of RXRα/MR signaling could alleviate cell senescence during metabolic disorders. Thus, our study revealed that RXRα/MR signaling serves as a critical regulatory factor mediating the crosstalk between cell senescence and metabolic reprogramming, shedding light on a novel mechanism for targeting cell senescence and metabolic dysregulation in DKD.

The role of PCSK9 in glomerular lipid accumulation and renal injury in diabetic kidney disease.

Glomerular lipid accumulation is a defining feature of diabetic kidney disease (DKD); however, the precise underlying mechanism requires further elucidation. Recent evidence suggests a role for proprotein convertase subtilisin/kexin type 9 (PCSK9) in intracellular lipid homeostasis. Although PCSK9 is present in kidneys, its role within kidney cells and relevance to renal diseases remain largely unexplored. Therefore, we investigated the role of intracellular PCSK9 in regulating lipid accumulation and homeostasis in the glomeruli and podocytes under diabetic conditions. Furthermore, we aimed to identify the pathophysiological mechanisms responsible for the podocyte injury that is associated with intracellular PCSK9-induced lipid accumulation in DKD. In this study, glomeruli were isolated from human kidney biopsy tissues, and glomerular gene-expression analysis was performed. Also, db/db and db/m mice were used to perform glomerular gene-expression profiling. We generated DKD models using a high-fat diet and low-dose intraperitoneal streptozocin injection in C57BL/6 and Pcsk9 knockout (KO) mice. We analysed cholesterol and triacylglycerol levels within the kidney cortex. Lipid droplets were evaluated using BODIPY staining. We induced upregulation and downregulation of PCSK9 expression in conditionally immortalised mouse podocytes using lentivirus and siRNA transfection techniques, respectively, under diabetic conditions. A significant reduction in transcription level of PCSK9 was observed in glomeruli of individuals with DKD. PCSK9 expression was also reduced in podocytes of animals under diabetic conditions. We observed significantly higher lipid accumulation in kidney tissues of Pcsk9 KO DKD mice compared with wild-type (WT) DKD mice. Additionally, Pcsk9 KO mouse models of DKD exhibited a significant reduction in mitochondria number vs WT models, coupled with a significant increase in mitochondrial size. Moreover, albuminuria and podocyte foot process effacement were observed in WT and Pcsk9 KO DKD mice, with KO DKD mice displaying more pronounced manifestations. Immortalised mouse podocytes exposed to diabetic stimuli exhibited heightened intracellular lipid accumulation, mitochondrial injury and apoptosis, which were ameliorated by Pcsk9 overexpression and aggravated by Pcsk9 knockdown in mouse podocytes. The downregulation of PCSK9 in podocytes is associated with lipid accumulation, which leads to mitochondrial dysfunction, cell apoptosis and renal injury. This study sheds new light on the potential involvement of PCSK9 in the pathophysiology of glomerular lipid accumulation and podocyte injury in DKD.

Spectral computed tomography parameters could be surrogate imaging markers to detect early perfusion changes in diabetic kidneys.

Kidney microvasculopathy is the baseline pathophysiological feature of diabetic kidney disease (DKD). We aimed to evaluate the spectral computed tomography (CT) parameters for detecting renal perfusion changes among diabetic patients. From August 2020 to June 2022, 34 patients (age, 57.7±10.7 years; male, 20) clinically diagnosed with type 2 diabetes mellitus (DM) and 19 DM-free individuals (age, 48.1±16.9 years; male, 12) were selected for analysis. The series participants formed the DM group and control group, respectively. Spectral parameters, including effective atomic number (Zeff), iodine density (ID), normalized iodine density (NID) and the slope of the energy spectrum curves (λ), between the 2 groups were analyzed using independent samples t-test. Receiver operator characteristic (ROC) curves were used to evaluate the diagnostic performance of spectral parameters for detecting renal perfusion changes. The results indicate that in both cortical and medullary phases, the values of Zeff, ID, NID, and λ40-70 for the renal cortex of the DM group were significantly higher than those in the control group (P<0.05). In the cortex phase, the diagnostic efficacy of cortical spectral CT parameters discriminating DM patients from controls was as follows: the area under ROC curve (AUC) of ID value was 0.816 [95% confidence interval (CI): 0.679-0.921] at the optimal cutoff value 4.14, the AUC of Zeff value was 0.800 (95% CI: 0.668-0.901) at the optimal cutoff value 9.26, the AUC of λ40-70 value was 0.822 (95% CI: 0.675-0.918) at the optimal cutoff value 8.26, and the AUC of NID value was 0.851 (95% CI: 0.684-0.926) at the optimal cutoff value 0.37. In medullary phase: the AUC of ID value was 0.769 (95% CI: 0.617-0.846) at the optimal cutoff value 5.08, the AUC of Zeff value was 0.763 (95% CI: 0.614-0.837) at the optimal cutoff value 9.58, the AUC of λ40-70 value was 0.766 (95% CI: 0.617-0.839) at the optimal cutoff value 10.07, and the AUC of NID value was 0.79 (95% CI: 0.623-0.855) at the optimal cutoff value 1.37. Spectral CT could serve as an alternative protocol for the early identification of kidney injury in diabetic patients.

Design and methodology of the PRIMETIME 1 cohort study: PRecIsion MEdicine based on kidney TIssue Molecular interrogation in diabetic nEphropathy.

Clinical features of diabetic kidney disease alone cannot differentiate between the histopathology that defines diabetic nephropathy (DN) and non-diabetic nephropathy (NDN). A kidney biopsy is necessary to make the definitive diagnosis of DN. However, there is no consensus on when to perform a kidney biopsy in individuals with diabetes and kidney disease. Furthermore, the implications of NDN versus DN for management, morbidity and kidney prognosis are unclear. To address the gap in knowledge, we aimed to create a national retrospective cohort of people with diabetes and a performed kidney biopsy. Adults diagnosed with diabetes in Denmark between 1996 and 2020 who had a kidney biopsy performed were included. The cohort was established by linking a nationwide diabetes registry with the Danish Pathology Registry. Data from 11 national registries and databases were compiled. The type of kidney disease was classified using a three-step analysis of Systematized Nomenclature of Medicine codes reported in relation to the histopathological examinations of kidney tissue. The final cohort and classification of kidney disease was as follows: out of 485 989 individuals with diabetes 2586 were included, 2259 of whom had type 2 diabetes. We were able to classify 599 (26.5%) with DN, 703 (31.1%) with NDN and 165 (7.3%) with mixed disease in individuals with type 2 diabetes. In individuals with type 1 diabetes, 132 (40.4%) had DN, 73 (22.3%) NDN and 39 (11.9%) mixed disease. The remaining could not be classified or had normal histology. The overall median (Q1-Q3) follow-up time was 3.8 (1.6-7.2) years. This cohort is a novel platform based on high-quality registry data for important longitudinal studies of the impact of kidney disease diagnosis on prognosis. With regular updates of data from the Danish registries, the presented follow-up will increase over time and is only limited by emigration or death.

Cover Image.

The cover image is based on the Research Article Type 2 diabetes albuminuric and non-albuminuric phenotypes have different morphological and functional ultrasound features of diabetic kidney disease by Monia Garofolo et al., https://doi.org/10.1002/dmrr.3585.

Type 2 diabetes albuminuric and non-albuminuric phenotypes have different morphological and functional ultrasound features of diabetic kidney disease.

Whether different diabetic kidney disease (DKD) phenotypes recognise differences in morphological and vascular properties of the kidney is still unexplored. We evaluated the potential role of kidney ultrasonography in differentiating DKD phenotypes in subjects with type 2 diabetes. This is a cross-sectional, single-centre study. Total (TRV) and parenchymal renal volumes (PRV) were calculated by applying the ellipsoid formula for conventional (2D) ultrasonography and with manual segmentation for 3D ultrasonography, and then adjusted for body surface area (aTRV, aPRV). Renal resistive index (RI) was contextually determined. DKD phenotypes have been defined based on increased urinary albumin-to-creatinine ratio (ACR >30mg/g) and/or reduced eGFR (<60ml/min/1.73m2 ). Recruitment was planned to have groups of the same size. Among 256 subjects, 26.2% had No-DKD, 24.6% increased albuminuria only (Alb+ ), 24.2% non-albuminuric DKD (Alb- DKD), and 25.0% albuminuric DKD (Alb+ DKD). Compared to No-DKD, RI was significantly higher in all DKD phenotypes, being the highest in Alb+ DKD, and with a significant trend of RI>0.70 to increase across phenotypes. In comparison with No-DKD, both 2D and 3D volumes were increased in Alb+ and significantly reduced in Alb- DKD as well as in Alb+ DKD, with significantly lower volumes in Alb- DKD as compared to Alb+ DKD at the same reduced levels of eGFR. In adjusted regressions, compared to No-DKD, RI was associated with Alb+ ; both RI and aPRV3D were associated with Alb+ DKD; only aPRV3D with Alb- DKD. Compared to No-DKD, Receiver Operating Characteristic curve analyses, designed taking into account conventional risk factors, showed that US parameters did not ameliorate the characterisation of Alb+ and Alb+ DKD, while aPRV3D significantly improved the phenotyping of Alb- DKD. As a novel information, we reported that, in type 2 diabetes, the emerging normoalbuminuric DKD phenotype showed reduced TRVs and PRVs even when compared, at similarly reduced eGFR levels, with Alb+ DKD opening. In perspective, these findings suggest a possible role of imaging for better discrimination of DKD phenotypes in clinical practice.

Insulin Resistance is Associated with Clinical Manifestations of Diabetic Kidney Disease (Glomerular Hyperfiltration, Albuminuria, and Kidney Function Decline).

Clinical features of diabetic kidney disease include glomerular hyperfiltration, albuminuria, and kidney function decline towards End-Stage Kidney Disease (ESKD). There are presently neither specific markers of kidney involvement in patients with diabetes nor strong predictors of rapid progression to ESKD. Serum-creatinine-based equations used to estimate glomerular filtration rate are notoriously unreliable in patients with diabetes. Early kidney function decline, reduced glomerular filtration rate, and proteinuria contribute to identifying diabetic patients at higher risk for rapid kidney function decline. Unlike proteinuria, the elevation of urinary albumin excretion in the range of microalbuminuria is frequently transient in patients with diabetes and does not always predict progression towards ESKD. Although the rate of progression of kidney function decline is usually accelerated in the presence of proteinuria, histological lesions of diabetes and ESKD may occur with normal urinary albumin excretion. No substantial reduction in the rate of ESKD associated with diabetes has been observed during the last decades despite intensified glycemic control and reno-protective strategies, indicating that existing therapies do not target underlying pathogenic mechanisms of kidney function decline. Very long-term effects of sodium-glucose transporters- 2 inhibitors and glucagon-like peptide-1 analogs remain to be defined. In patients with diabetes, glucagon secretion is typically elevated and induces insulin resistance. Insulin resistance is consistently and strongly associated with clinical manifestations of diabetic kidney disease, suggesting that reduced insulin sensitivity participates in the pathogenesis of the disease and may represent a therapeutic objective. Amelioration of insulin sensitivity in patients with diabetes is associated with cardioprotective and kidney-protective effects.

GABA alleviates high glucose-induced podocyte injury through dynamically altering the expression of macrophage M1/M2-derived exosomal miR-21a-5p/miR-25-3p

Podocyte injury is the main clinical pathological feature of diabetic kidney disease (DKD). The studies showed that DKD is associated with the polarization of macrophages to different types (M1 or M2) and the inflammatory processes which they mediate. It is a hot research topic in the treatment of DKD that find and intervene genes which related to M1/M2 phenotype. The potential anti-inflammatory effects on γ-aminobutyric acid (GABA) have been shown to be related to the regulation of the polarization direction of macrophages. However, it remains unknown that whether GABA can alleviate DKD. The purpose of current study was to explore the role of GABA involved in high glucose (HG)-induced podocyte injury by regulating the M1/M2 phenotype. In the beginning, our results revealed that exogenous GABA treatment altered the direction of HG-induced macrophage polarization and suppressed the inflammatory response. Subsequently, macrophage-derived exosomes under HG treatment were found to be involved in aggravating HG-induced podocyte injury (decreased the proliferation capacity and increased apoptosis rate of cells). Furthermore, GABA treatment blocked the promotion of macrophage-mediated exosomes on podocyte injury under HG. Then, we found that GABA alleviated the promoting effect of macrophages on podocyte injury by regulating the expression of exosomal miR-21a-5p/miR-25-3p which mediated by macrophages. Finally, it was elucidated that Tnpo1/ATXN3 were the targets of miR-21a-5p/miR-25-3p, respectively, and mediated the promotion of podocyte injury by macrophage-derived exosomes under HG. This research suggested that GABA alleviated podocyte injury by reversing the M1/M2 polarization direction of macrophages under HG and regulating the miR-21a-5p-Tnpo1/miR-25-3p-ATXN3 signal axis of macrophage-derived exosomes.

Research progress of endothelial-mesenchymal transition in diabetic kidney disease.

Renal fibrosis is an important pathological feature of diabetic kidney disease (DKD), manifested as tubular interstitial fibrosis, tubular atrophy, glomerulosclerosis and damage to the normal structure of the kidney. Renal fibrosis can eventually develop into renal failure. A better understanding of renal fibrosis in DKD is needed due to clinical limitations of current anti‐fibrotic drugs in terms of effectiveness, cost‐effectiveness and side effects. Fibrosis is characterized by local excessive deposition of extracellular matrix, which is derived from activated myofibroblasts to increase its production or specific tissue inhibitors of metalloproteinases to reduce its degradation. In recent years, endothelial‐mesenchymal transition (EndMT) has gradually integrated into the pathogenesis of fibrosis. In animal models of diabetic kidney disease, it has been found that EndMT is involved in the formation of renal fibrosis and multiple signalling pathways such as TGF‐β signalling pathway, Wnt signalling pathway and non‐coding RNA network participate in the regulation of EndMT during fibrosis. Here, we mainly review EndMT regulation and targeted therapy of renal fibrosis in DKD.

Non-diabetic Kidney Disease in Diabetic Population: A Single-Center Study From South India.

Introduction: Diabetic kidney disease (DKD) is the commonest cause of chronic kidney disease and end-stage kidney disease worldwide, consequently it has become an important productive implication to the healthcare system. This study was conducted to assess the prevalence of non-DKD (NDKD) in diabetic patients from south India.Objective: To assess the prevalence of NDKD in type 2 diabetes mellitus patients presenting to a tertiary care hospital from south India and also to analyze clinical clues to establish a diagnosis of NDKD.Patient and methods: It is a retrospective observational study of analyzing patient characteristics and renal biopsies. All Diabetic patients with a clinical suspicion of non-diabetic kidney disease who underwent renal biopsy during the study period between January 2012 and June 2017 were included. Based on the biopsy findings, the patients were classified into three groups (isolated diabetic nephropathy, isolated NDKD, and NDKD with underlying diabetic nephropathy) and patients’ characteristics were compared between the groups for analysis.Results: A total of 236 renal biopsies were analyzed for the study. Of that, 114 had features of DKD, 78 NDKD with diabetic nephropathy (DN) and 44 had isolated NDKD. Acute interstitial nephritis was the most common cause of NDKD.Conclusion: From the current study, the long duration of diabetes mellitus beyond five years and hypertension beyond two years reasonably predict DKD.

The HDAC2/SP1/miR-205 feedback loop contributes to tubular epithelial cell extracellular matrix production in diabetic kidney disease.

Extracellular matrix (ECM) accumulation is considered an important pathological feature of diabetic kidney disease (DKD). Histone deacetylase (HDAC) inhibitors protect against kidney injury. However, the potential mechanisms of HDACs in DKD are still largely unknown. Here, we describe a novel feedback loop composed of HDAC2 and miR-205 that regulates ECM production in tubular epithelial cells in individuals with DKD. We found that HDAC2 mRNA expression in peripheral blood was markedly higher in patients with DKD than in patients with diabetes. Nuclear HDAC2 protein expression was increased in TGFβ1-stimulated tubular epithelial cells and db/db mice. We also found that miR-205 was regulated by HDAC2 and down-regulated in TGFβ1-treated HK2 cells and db/db mice. In addition, HDAC2 reduced histone H3K9 acetylation in the miR-205 promoter region to inhibit its promoter activity and subsequently suppressed miR-205 expression through an SP1-mediated pathway. Furthermore, miR-205 directly targeted HDAC2 and inhibited HDAC2 expression. Intriguingly, miR-205 also regulated its own transcription by inhibiting HDAC2 and increasing histone H3K9 acetylation in its promoter, forming a feedback regulatory loop. Additionally, the miR-205 agonist attenuated ECM production in HK2 cells and renal interstitial fibrosis in db/db mice. In conclusion, the HDAC2/SP1/miR-205 feedback loop may be crucial for the pathogenesis of DKD.

当院通院2型糖尿病患者の糖尿病性腎臓病（DKD）合併状況；最近10年間の区分変化と経過観察結果

当院通院2型糖尿病患者の糖尿病性腎臓病（DKD）合併状況；最近10年間の区分変化と経過観察結果

Rotten to the Cortex: Ceramide-Mediated Lipotoxicity in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is a prevalent and progressive comorbidity of diabetes mellitus that increases one’s risk of developing renal failure. Progress toward development of better DKD therapeutics is limited by an incomplete understanding of forces driving and connecting the various features of DKD, which include renal steatosis, fibrosis, and microvascular dysfunction. Herein we review the literature supporting roles for bioactive ceramides as inducers of local and systemic DKD pathology. In rodent models of DKD, renal ceramides are elevated, and genetic and pharmacological ceramide-lowering interventions improve kidney function and ameliorate DKD histopathology. In humans, circulating sphingolipid profiles distinguish human DKD patients from diabetic controls. These studies highlight the potential for ceramide to serve as a central and therapeutically tractable lipid mediator of DKD.

MiR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1

Diabetic kidney disease (DKD) is a major complication of diabetes. Expression of members of the microRNA (miRNA) miR-379 cluster is increased in DKD. miR-379, the most upstream 5′-miRNA in the cluster, functions in endoplasmic reticulum (ER) stress by targeting EDEM3. However, the in vivo functions of miR-379 remain unclear. We created miR-379 knockout (KO) mice using CRISPR-Cas9 nickase and dual guide RNA technique and characterized their phenotype in diabetes. We screened for miR-379 targets in renal mesangial cells from WT vs. miR-379KO mice using AGO2-immunopreciptation and CLASH (cross-linking, ligation, sequencing hybrids) and identified the redox protein thioredoxin and mitochondrial fission-1 protein. miR-379KO mice were protected from features of DKD as well as body weight loss associated with mitochondrial dysfunction, ER- and oxidative stress. These results reveal a role for miR-379 in DKD and metabolic processes via reducing adaptive mitophagy. Strategies targeting miR-379 could offer therapeutic options for DKD.

AGE-Induced Suppression of EZH2 Mediates Injury of Podocytes by Reducing H3K27me3

Background: Chronic hyperglycemia, a pivotal feature of diabetes mellitus (DM), initiates the formation of advanced glycation end products (AGEs) and the dysregulation of epigenetic mechanisms, which may cause injury to renal podocytes, a central feature of diabetic kidney disease (DKD). Previous data of our group showed that AGEs significantly reduce the expression of NIPP1 (nuclear inhibitor of protein phosphatase 1) in podocytes in vitro as well as in human and murine DKD. NIPP1 was shown by others to interact with enhancer of zeste homolog 2 (EZH2), which catalyzes the repressive methylation of H3K27me3 on histone 3. Therefore, we hypothesized that AGEs can directly induce epigenetic changes in podocytes. Methods: We analyzed the relevance of AGEs on EZH2 expression and activity in a murine podocyte cell line. Cells were treated with 5 mg/mL glycated BSA for 24 h. To determine the meaning of EZH2 suppression, EZH2 activity was inhibited by incubating the cells with the pharmacological methyltransferase inhibitor 3-deazaneplanocin A; EZH2 expression was repressed with siRNA. mRNA expression was analyzed with real-time PCR, and protein expression with Western blot. EZH2 expression and level of H3K27 trimethylation in podocytes of diabetic db/db mice, a mouse model for type 2 DM, were analyzed using immunofluorescence. Results: Our data demonstrated that AGEs decrease EZH2 expression in podocytes and consequently reduce H3K27me3. This suppression of EZH2 mimicked the AGE effects and caused an upregulated expression of pathological factors that contribute to podocyte injury in DKD. In addition, analyses of db/db mice showed significantly reduced H3K27me3 and EZH2 expression in podocytes. Moreover, the suppression of NIPP1 and EZH2 showed similar effects regarding podocyte injury. Conclusions: Our studies provide a novel pathway how AGEs contribute to podocyte injury and the formation of the so-called metabolic memory in DKD.

Inhibitor of growth 2 regulates the high glucose-induced cell cycle arrest and epithelial-to-mesenchymal transition in renal proximal tubular cells.

The epithelial-to-mesenchymal transition (EMT)-based tubulointerstitial fibrosis is the major pathological feature of diabetic kidney disease (DKD). While several studies have linked cell cycle dysregulation to various kidney injuries in recent years, its involvement in fibrosis of DKD is far from being clarified. ING2 (inhibitor of growth 2) is the second member of the inhibitor of growth family and participates in the regulation of many cellular processes. So far the role of ING2 in DKD remains largely unknown. In the present study, ING2 expression was detected by western blotting and immunofluorescent staining both in vitro high glucose-stimulated human proximal tubular epithelial cells (HK-2) and in vivo streptozotocin-induced diabetic mice. Cell proliferation was analyzed by CCK-8 and EdU assay, and cell cycle arrest was measured by flow cytometry. Quantitative polymerase chain reaction (qPCR) and western blotting were used to detect the EMT markers, and the p53 signaling activation was evaluated by chromatin immunoprecipitation (ChIP), qPCR, and western blotting. We found that the proliferation of the cells was reduced upon high glucose stimulation, which was accompanied by cell cycle arrest. The expression of ING2 was increased in hyperglycemia conditions both in vivo and in vitro. ING2 suppression ameliorated the reduced proliferation and cell cycle arrest induced by high glucose in HK-2 cells. Moreover, ING2 knockdown suppressed p21 expression by reducing p53 acetylation and finally alleviated the EMT progress in the high glucose-stimulated HK-2 cells. Our study demonstrated that cell cycle regulation is bound up with the kidney fibrosis in DKD, suggesting a novel function of ING2 as a potential therapeutic strategy targeting cell cycle arrest for DKD.

Interplay between RNA-binding protein HuR and Nox4 as a novel therapeutic target in diabetic kidney disease

ObjectiveGlomerular injury is a prominent pathological feature of diabetic kidney disease (DKD). Constitutively active NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species that mediates hyperglycemia-induced mesangial cell (MC) fibrotic injury. However, the mechanism that Nox4 utilizes to achieve its biological outcome remains elusive, and the signaling pathways that regulate this isoform oxidase are not well understood. Here, our goal is to study the detailed mechanism by which NAPDH oxidase 4 (Nox4) is post-transcriptionally regulated in MC during diabetic pathology. MethodsWe studied the protein expression of HuR, Nox4 and matrix proteins by western blotting, while we assessed the mRNA stability of Nox4 by RT-PCR and polysomal assay, examined in vitro cultured glomerular mesangial cells treated by high glucose (HG) and diabetic animal induced by STZ. The binding assay between HuR and the Nox4 promoter was done by immuno-precipiating with HuR antibody and detecting the presence of Nox4 mRNA, or by pull-down by using biotinlyated labeled Nox4 promoter RNA and detecting the presence of the HuR protein. The binding was also confirmed in MCs where Nox4 promoter-containing luciferage constructs were transfected. ROS levels were measured with DHE/DCF dyes in cells, or lucigenin chemiluminescence for Nox enzymatic levels, or HPLC assay for superoxide. HuR protein was inhibited by antisense oligo that utilized osmotic pumps for continuous delivery in animal models. The H1bAc1 ratio was measured by an ELISA kit for mice. ResultsWe demonstrate that in MCs, high glucose (HG) elicits a rapid upregulation of Nox4 protein via translational mechanisms. Nox4 mRNA 3′ untranslated region (3′-UTR) contains numerous AU-rich elements (AREs) that are potential binding sites for the RNA-binding protein human antigen R (HuR). We show that HG promotes HuR activation/expression and that HuR is required for HG-induced Nox4 protein expression/mRNA translation, ROS generation, and subsequent MC fibrotic injury. Through a series of invitro RNA-binding assays, we demonstrate that HuR acts via binding to AREs in Nox4 3′-UTR in response to HG. The invivo relevance of these observations is confirmed by the findings that increased Nox4 is accompanied by the binding of HuR to Nox4 mRNA in kidneys from type 1 diabetic animals, and further suppressing HuR expression showed a reno-protective role in a type 1 diabetic mouse model via reducing MC injury, along with the improvement of hyperglycemia and renal function. ConclusionsWe established for the first time that HuR-mediated translational regulation of Nox4 contributes to the pathogenesis of fibrosis of the glomerular microvascular bed. Thus therapeutic interventions affecting the interplay between Nox4 and HuR could be exploited as valuable tools in designing treatments for DKD.

1929-P: Role of miR379 in Promoting Skeletal Muscle Atrophy in Type 1 Diabetes (T1D)

T1D can lead to skeletal muscle atrophy which decreases quality of life and increases mortality. Non-coding RNAs such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are increasingly implicated in various pathological conditions. Recently, our lab demonstrated a unique megacluster of ∼ 40 miRNAs including miR379 and their host transcript lnc-MGC (a lncRNA) could induce early features of diabetic kidney disease. Based on new data showing that the expression of miR379 is increased in the atrophied skeletal muscle of T1D mice, in the current study, we investigated the in vivo role of miR379 in promoting skeletal muscle atrophy in vivo in T1D. We used miR379 knockout (KO) mice generated by CRISPR-Cas9 technology. C57BL/6 (WT) and miR379 KO male mice were injected with streptozotocin (STZ 50mg/kg) for 5 consecutive days to induce T1D. miR379 target genes and muscle atrophy markers in the skeletal muscle were analyzed using qPCR. Body composition analysis was performed using Echo-MRI. Fasting blood glucose levels one week after diabetes development, were significantly lower in in miR379 KO mice compared to WT-mice. Moreover, miR379 KO mice showed significant protection from T1D-associated loss in body weight, muscle and fat mass relative to WT mice. Toward interrogating the underlying mechanism, the expression of atrophy-associated genes and miR379 target genes were assessed in skeletal muscle samples. In WT mice, T1D increased the expression of muscle atrophy-associated genes (myostatin and Atrogin1), and decreased FIS1 and EDEM3, miR379 target proteins involved in regulation of mitochondrial health and ER stress respectively. Intriguingly, miR379 KO mice depicted significant protection from these T1D-induced changes. These results suggest miR-379 promotes skeletal muscle atrophy in T1D via upregulating genes involved in muscle wasting and downregulating genes involved in mitochondrial health, thus uncovering novel non-coding RNA dependent mechanisms of skeletal muscle dysfunction in T1D. Disclosure R. Tunduguru: None. M. Kato: None. M. Abdollahi: None. L.L. Lanting: None. R. Natarajan: None. Funding National Institutes of Health

491-P: Obesity-Induced Kidney Damage Is Attenuated by Genetic Deletion of miR-379 in Mice

Obesity is significantly associated with diabetes and related complications like kidney disease. Evidence shows dysregulation of certain non-coding RNAs including microRNAs can accelerate diabetic kidney disease (DKD). Recently, we found that miR-379 and its host transcript long non-coding RNA, lncMGC, were regulated by endoplasmic reticulum (ER) stress in glomerular cells, and lncMGC could promote features of DKD in mice. In this study, we examined the in vivo role of miR-379 in obesity-induced kidney disease by testing miR-379-knockout (KO) mice generated by CRISPR-Cas9 editing. Male (M) and female (F) mice were fed with control diet, or high fat diet (HFD) for 24 weeks to induce obesity in wild type (WT) and miR-379KO mice. Kidney function was examined by measuring urinary albumin and creatinine. Expression of miR-379 target genes (Edem3, Fis1), Leptin receptor (LepR) and profibrotic genes (Tgf-β1, Ctgf, Col4a1, Col1a2) was measured in isolated glomerular cells. Cortical sections were stained for histopathology. Blood glucose levels were similar in miR379KO-HFD and WT-HFD. miR379KO-HFD-F mice exhibited significantly lower body weight and body fat composition versus WT-HFD-F. Hyperinsulinemia observed in WT-HFD-F mice was attenuated in miR379KO-HFD-F. LepR expression was significantly decreased in WT-HFD-F but not miR379KO-HFD-F mice relative to controls. Increased expression of profibrotic genes, glomerular hypertrophy and interstitial fibrosis observed in WT-HFD-F were significantly attenuated in miR379KO-HFD-F mice. Similar to females, male miR-379KO-HFD-M mice showed significant reductions in glomerular hypertrophy, fibrosis and ER stress markers vs. WT-HFD-M. However, unlike females, miR379KO-HFD-M mice did not display reduced body weight compared to WT-HFD-M. These results suggest miR-379 deficiency protects mice from HFD-induced obesity and kidney injury, with some gender differences. miR-379 inhibition may be a novel therapy for HFD-induced metabolic disorders. Disclosure M. Abdollahi: None. M. Kato: None. R. Tunduguru: None. L.L. Lanting: None. R. Natarajan: None. Funding National Institutes of Health