Models Of Diabetic Kidney Disease Research Articles

Acute Kidney Injury in COVID-19: Emerging Evidence of a Distinct Pathophysiology.

Acute Kidney Injury in COVID-19: Emerging Evidence of a Distinct Pathophysiology.

Effect of the Novel Compound SMTP‐27 in a Mouse Model of Diabetic Kidney Disease

ObjectiveDiabetic kidney disease (DKD) is the most common complication of diabetes mellitus and one of the primary causes of end‐stage renal disease in many countries. Although several candidate substances have shown efficacy against DKD, no effective clinical treatment methods have been established for preventing the progression of DKD. It was recently noted that the occurrence of oxidative stress, one of the contributors to the pathogenesis of DKD, is closely related to inflammatory cell mobilization, a process that increases inflammation by inducing the production of cytokines such as interleukin‐1β and tumor necrosis factor‐α. The present study aimed to evaluate the effect of Stachybotrys microspora triprenyl phenol‐27 (SMTP‐27), which is reported to have an anti‐inflammatory effect, in a mouse model of DKD.MethodsThe right kidneys of male type 2 diabetic db/db mice (6 weeks) were removed through small flank incisions under isoflurane anesthesia to induce DKD. Male C57BL/6J mice of the same age, subjected to the same surgery, were used as the control group. DKD mice are randomly assigned to the vehicle‐treated group, SMTP‐27‐treated group (30 mg/kg), or metformin‐treated group (300 mg/kg). They were treated from six to sixteen weeks of age: SMTP‐27 injections were administered intraperitoneally once every two days, and metformin was orally administered daily. Urinary albumin (Ualb), serum creatinine (Scr), and creatinine clearance (Ccr) were measured to evaluate renal function. In addition, histological examination of an excised kidney was performed by hematoxylin‐eosin staining and periodic acid–Schiff staining to evaluate tubular regeneration and glomerular sclerosis.ResultsUalb and Scr levels were significantly higher and Ccr levels were significantly lower in type 2 diabetic db/db mice than in C57BL/6J mice. Moreover, tubular regeneration and glomerular sclerosis were significantly higher in db/db mice than in C57BL/6J mice. In the type 2 diabetes model, metformin only improved Ualb with causing antihyperglycemic effects; in contrast, SMTP‐27 significantly improved Ualb, Scr, and Ccr without inducing antihyperglycemic effects. In addition, SMTP‐27 reduced tubular regeneration, whereas metformin did not. Neither drug reduced glomerular sclerosis.ConclusionOur results showed that SMTP‐27 improved DKD without inducing antihyperglycemic effects. Thus, SMTP‐27 is a potential novel therapeutic agent for the treatment of DKD. In future studies, we plan to investigate the mechanism of action of SMTP‐27 against DKD.Support or Funding InformationThis study was supported in part by the Japan Society for the Promotion of Science KAKENHI (Grant Number 18K14958, awarded to KS; Grant Number 26460346, awarded to KN). SMTP‐27 was generously donated by TMS Co., Ltd. (Tokyo, Japan).

BCc1 Nanomedicine Therapeutic Effects in Streptozotocin and High-Fat Diet Induced Diabetic Kidney Disease.

BackgroundOne common feature of chronic diseases, such as cancer, diabetes and chronic kidney disease (CKD), is the disruption of iron metabolism and increase in labile iron pool, which can result in excessive production of harmful oxidative stress. The proper management of iron metabolism in this situation can be a valuable tool to ameliorate pathological events.Materials and MethodsIn the previous studies, the anti-neoplastic effects of BCc1, a nanochelating-based nanomedicine with iron-chelating property, were demonstrated in cell culture, animal models and clinical trials. In the present study, the therapeutic effects of BCc1 in animal model of diabetic kidney disease (DKD), induced by streptozotocin injection (35 mg/kg) and high-fat diet consumption, were evaluated.ResultsThe results showed that BCc1 significantly decreased HOMA-IR index, uric acid, blood urea nitrogen, malondialdehyde and 8-isoprostane. In addition, it reduced urinary albumin excretion rate and albumin-to-creatinine ratio in comparison to DKD control rats. This nanomedicine had no negative impact on liver iron content, hemoglobin level, red blood cell count, hematocrit and mean corpuscular volume, while it significantly decreased aspartate aminotransferase and alanine aminotransferase compared to DKD control group. Moreover, the histopathological assessment indicated that lesser glomerular basement membrane and wrinkling, mesangial matrix expansion and pathological changes in proximal cortical tubules were seen in the kidney samples of BCc1-treated rats.ConclusionIn conclusion, BCc1 as an iron-chelating agent shows promising impacts in DKD animal model, which can ameliorate biochemical and pathological events of this disease.

Dual blockade of protease-activated receptor 1 and 2 additively ameliorates diabetic kidney disease.

Protease-activated receptors (PARs) are coagulation protease targets, and they increase expression of inflammatory cytokines and chemokines in various diseases. Of all PARs, previous reports have shown that PAR1 or PAR2 inhibition is protective against diabetic glomerular injury. However, how PAR1 and PAR2 cooperatively contribute to diabetic kidney disease (DKD) pathogenesis and whether dual blockade of PARs is more effective in DKD remain elusive. To address this issue, male type I diabetic Akita mice heterozygous for endothelial nitric oxide synthase were used as a model of DKD. Mice (4 mo old) were divided into four treatment groups and administered vehicle, PAR1 antagonist (E5555, 60 mg·kg-1·day-1), PAR2 antagonist (FSLLRY, 3 mg·kg-1·day-1), or E5555 + FSLLRY for 4 wk. The results showed that the urinary albumin creatinine ratio was significantly reduced when both PAR1 and PAR2 were blocked with E5555 + FSLLRY compared with the vehicle-treated group. Dual blockade of PAR1 and PAR2 by E5555 + FSLLRY additively ameliorated histological injury, including mesangial expansion, glomerular macrophage infiltration, and collagen type IV deposition. Marked reduction of inflammation- and fibrosis-related gene expression in the kidney was also observed. In vitro, PAR1 and PAR2 agonists additively increased mRNA expression of macrophage chemoattractant protein 1 or plasminogen activator inhibitor-1 in human endothelial cells. Changes induced by the PAR1 agonist were blocked by a NF-κB inhibitor, whereas those of the PAR2 agonist were blocked by MAPK and/or NF-κB inhibitors. These findings suggest that PAR1 and PAR2 additively contribute to DKD pathogenesis and that dual blockade of both could be a novel therapeutic option for treatment of patients with DKD.

Targeting angiogenesis and lymphangiogenesis in kidney disease

The kidney is permeated by a highly complex vascular system with glomerular and peritubular capillary networks that are essential for maintaining the normal functions of glomerular and tubular epithelial cells. The integrity of the renal vascular network depends on a balance of proangiogenic and antiangiogenic factors, and disruption of this balance has been identified in various kidney diseases. Decreased levels of the predominant proangiogenic factor, vascular endothelial growth factor A (VEGFA), can result in glomerular microangiopathy and contribute to the onset of preeclampsia, whereas upregulation of VEGFA has roles in diabetic kidney disease (DKD) and polycystic kidney disease (PKD). Other factors that regulate angiogenesis, such as angiopoietin 1 and vasohibin 1, have been shown to be protective in animal models of DKD and renal fibrosis. The renal lymphatic system is important for fluid homeostasis in the kidney, as well as the transport of immune cells and antigens. Experimental studies suggest that the lymphangiogenic factor VEGFC might have protective effects in PKD, DKD and renal fibrosis. Understanding the physiological and pathological roles of factors that regulate angiogenesis and lymphangiogenesis in the kidney has led to the development of novel therapeutic strategies for kidney diseases.

DESI-MSI and METASPACE indicates lipid abnormalities and altered mitochondrial membrane components in diabetic renal proximal tubules.

Diabetic kidney disease (DKD) is the most prevalent complication in diabetic patients, which contributes to high morbidity and mortality. Urine and plasma metabolomics studies have been demonstrated to provide valuable insights for DKD. However, limited information on spatial distributions of metabolites in kidney tissues have been reported. In this work, we employed an ambient desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) coupled to a novel bioinformatics platform (METASPACE) to characterize the metabolome in a mouse model of DKD. DESI-MSI was performed for spatial untargeted metabolomics analysis in kidneys of mouse models (F1 C57BL/6J-Ins2Akita male mice at 17weeks of age) of type 1 diabetes (T1D, n = 5) and heathy controls (n = 6). Multivariate analyses (i.e., PCA and PLS-DA (a 2000 permutation test: P < 0.001)) showed clearly separated clusters for the two groups of mice on the basis of 878 measured m/z's in kidney cortical tissues. Specifically, mice with T1D had increased relative abundances of pseudouridine, accumulation of free polyunsaturated fatty acids (PUFAs), and decreased relative abundances of cardiolipins in cortical proximal tubules when compared with healthy controls. Results from the current study support potential key roles of pseudouridine and cardiolipins for maintaining normal RNA structure and normal mitochondrial function, respectively, in cortical proximal tubules with DKD. DESI-MSI technology coupled with METASPACE could serve as powerful new tools to provide insight on fundamental pathways in DKD.

Taurine Supplementation Reverses Diabetes-Induced Podocytes Injury via Modulation of the CSE/TRPC6 Axis and Improvement of Mitochondrial Function

Background: The protective effects of taurine supplementation on diabetic kidney disease (DKD) have been defined, but the mechanisms are not quite clear yet. TRPC6 has been shown to function in the homeostasis of podocytes, but whether TRPC6-modulated mitochondrial dysfunctions participating in taurine-induced renal protection during diabetes are unclear. Methods: A DKD model was constructed using streptozocin (STZ), and an immortalized mouse podocytes cell line MPC-5 was used. Renal histology and western blot were used to analyze the expression levels of certain proteins. Cell proliferation assays, apoptosis assays, calcium influx, and mitochondrial functions were evaluated. Results: In this study, taurine intervention improved STZ-induced DKD injuries, while it decreased both 24-h urinary protein and podocytes apoptosis. In detail, this study showed that taurine treatment decreased mitochondrial ROS productions by suppressing calcium overload and improving mitochondrial respiratory functions. Furthermore, the upregulation of TRPC6 is partially responsible for the calcium overload during high glucose treatment, whereas taurine treatment inhibited TRPC6 expression and partially attenuated high glucose-induced podocytes injuries. In addition, we demonstrated that taurine could upregulate CSE expression and inhibits TRPC6 expression via promoting the synthesis of H₂S. Conclusion: Our study reveals that taurine intervention could partially attenuate the lesions of DKD by modulating the CSE/TRPC6 axis.

Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression, and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK−2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1β, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD.

SGLT-2 inhibitors in Diabetic Kidney Disease: What Lies Behind their Renoprotective Properties?

Despite optimal management of diabetic kidney disease (DKD) with intensive glycemic control and administration of agents blocking the renin-angiotensinaldosterone- system, the residual risk for nephropathy progression to end-stage-renal-disease (ESRD) remains high. Sodium-glucose co-transporter type 2 (SGLT-2)-inhibitors represent a newly-introduced anti-diabetic drug class with pleiotropic actions extending above their glucose-lowering efficacy. Herein, we provide an overview of preclinical and clinical-trial evidence supporting a protective effect of SGLT-2 inhibitors on DKD. A systematic literature search of bibliographic databases was conducted to identify preclinical studies and randomized trials evaluating the effects SGLT-2 inhibitors on DKD. Preclinical studies performed in animal models of DKD support the renoprotective action of SGLT-2 inhibitors showing that these agents exert albuminuria-lowering effects and reverse glomerulosclerosis. The renoprotective action of SGLT-2 inhibitors is strongly supported by human studies showing that these agents prevent the progression of albuminuria and retard nephropathy progression to ESRD. This beneficial effect of SGLT-2 inhibitors is not fully explained by their glucose-lowering properties. Attenuation of glomerular hyperfiltration and improvement in a number of surrogate risk factors, including associated reduction in systemic blood pressure, body weight, and serum uric acid levels may represent plausible mechanistic explanations for the cardio-renal protection offered by SGLT-2 inhibitors. Furthermore, the tubular cell metabolism seems to be altered towards a ketone-prone pathway with protective activities. SGLT-2 inhibition emerges as a novel therapeutic approach of diabetic with anticipated benefits towards cardio-renal risk reduction. Additional research efforts are clearly warranted to elucidate this favorable effect in patients with overt DKD.

Angiotensin-(1-7) for diabetic kidney disease: better than an angiotensin-converting enzyme inhibitor alone?

In this commentary we emphasize the renoprotective effect of cyclicangiotensin-(1-7) described by Cassis etal. in a mouse modelofdiabetic kidney disease. The importance of the study is thatthis peptide was even more protective than the angiotensin-converting enzyme inhibitor lisinopril administered alone and that when the 2 componds were combined, the renoprotective action wasadditive.

A new, easily generated mouse model of diabetic kidney fibrosis

Our understanding of diabetic kidney disease pathogenesis has been hampered by the lack of easily generated pre-clinical animal models that faithfully recapitulate critical features of human disease. While most standard animal models develop manifestations of early stage diabetic injury such as hyperfiltration and mesangial matrix expansion, only a select few develop key late stage features such as interstitial fibrosis and reduced glomerular filtration rate. An underlying theme in these late stage disease models has been the addition of renin-angiotensin system hyperactivation, an important contributor to human disease pathogenesis. Widespread use of these models has been limited, however, as they are either labour intensive to generate, or have been developed in the rat, preventing the use of the many powerful genetic tools developed for mice. Here we describe the Akita+/− Ren+/− mouse, a new, easily generated murine model of diabetic kidney disease that develops many features of late stage human injury, including not only hyperglycemia, hypertension, and albuminuria, but also reduced glomerular filtration rate, glomerulosclerosis, and interstitial fibrosis.

The brown fat-secreted adipokine neuregulin 4 is decreased in human and murine chronic kidney disease.

Neuregulin 4 (NRG4) has recently been introduced as a novel brown adipose tissue (BAT)-secreted adipokine with beneficial metabolic effects in mice. However, regulation of Nrg4 in end-stage kidney disease (ESKD) and type 2 diabetes mellitus (T2DM) has not been elucidated, so far. Serum NRG4 levels were quantified by ELISA in 60 subjects with ESKD on chronic hemodialysis as compared to 60 subjects with an estimated glomerular filtration rate >50 mL/min/1.73 m2 in a cross-sectional cohort. Within both groups, about half of the patients had a T2DM. Furthermore, mRNA expression of Nrg4 was determined in two mouse models of diabetic kidney disease (DKD) as compared to two different groups of non-diabetic control mice. Moreover, mRNA expression of Nrg4 was investigated in cultured, differentiated mouse brown and white adipocytes, as well as hepatocytes, after treatment with the uremic toxin indoxyl sulfate. Median serum NRG4 was significantly lower in patients with ESKD compared to controls and the adipokine was independently associated with a beneficial renal, glucose and lipid profile. In mice with DKD, Nrg4 mRNA expression was decreased in all adipose tissue depots compared to control mice. The uremic toxin indoxyl sulfate did not significantly alter Nrg4 mRNA expression in adipocytes and hepatocytes, in vitro. Circulating NRG4 is independently associated with a preserved renal function and mRNA expression of -Nrg4 is reduced in adipose tissue depots of mice with DKD. The BAT-secreted adipokine is further associated with a beneficial glucose and lipid profile supporting NRG4 as potential treatment target in metabolic and renal disease states.

SUN-302 The Relative Roles of Pro-oxidant Ezymes Nox4 versus Nox5 in Diabetic Kidney Disease

Renal oxidative stress plays an important role in mediating kidney injury in diabetes. There is increasing evidences that recently discovered pro-oxidant enzyme, Nox5 plays a significant role in human diabetic kidney disease (DKD). Nox5 is present in humans and rabbits but not in mice or rats. Thus, there is a paucity of information about Nox5 in conventional mice/rats models of DKD. We examined the role of Nox5 in human diabetic nephropathy, in human renal cell populations as well as in a high fat fed rabbit model of kidney disease. In addition, in vivo, we also examined the contribution of Nox5 in the pathogenesis of DN in the absence of Nox4 expression. Protein expression of Nox4 and Nox5 and their localization in glomerular (podocytes and mesangial cells) and tubular cells were examined by immunostaining in human kidney biopsies obtained from non-diabetic and diabetic individuals. In vitro, human mesangial cells, podocytes and proximal tubules were exposed to high glucose, TGF-β and AngII and Nox4 and Nox5 were knockdown in these renal cells. Cell morphology, gene and protein expression of markers of fibrosis and inflammation as well as putative signalling pathways and the level of ROS were assessed in these human renal cells. In addition, we have developed a novel mode of diabetic complications which is more similar to the human context, the New Zealand White rabbit which expresses Nox5 and the other Nox isoforms as in humans. We have exposed the rabbits to high fat feeding (as a model of type 2 diabetes) as well as inducing insulin deficient diabetes (type 1 diabetes) using alloxan. As a world first, we have generated a Nox5 knockout rabbit using CRISP/Cas9.We have also generated a unique humanised Nox5 transgenic mouse with concomitant Nox4 KO with the aim to definitively delineate the relative effects of Nox4 versus Nox5 in diabetic complications. Expression of Nox5 was increased in both glomerular and tubular compartments of kidney biopsies obtained from diabetic individuals when compared to non-diabetic individuals. Nox5 is the Nox isoform which shows the highest upregulation in response to high glucose. Silencing of Nox5 reduces ROS formation and expression of proinflammatory and profibrotic cytokines and growth factors as well as putative elements that are implicated in DKD. Our data also suggest that Nox5 is upstream of Nox5 and that Nox5 inhibition also downregulates Nox4, but not vice versa. High fat feeding and alloxan induced diabetes rabbits develop increased mesangial area, increased Nox5 expression in renal cortex and upregulation of a range of pro-inflammatory (MCP-1) and pro-fibrotic genes (CTGF) as analysed by RNAseq and confirmed by RT-PCR. In addition, over expression of Nox5 in mesangial cells of Nox4 KO diabetic mice demonstrated 30% increase in albuminuria, mesangial expansion, increased renal injury and inflammation via enhanced ROS production. These studies will for the first time explore in a comprehensive manner the role of Nox5 in recently generated rabbit models of diabetes (type 1 and type 2) as well as delineate the relative role of Nox4 versus Nox5 in diabetic complications. These studies will provide evidence if Nox5 blockade alone or combined with Nox4/Nox5 blockade is the optimal approach to treat and prevent diabetic complications, in particular nephropathy.

Long noncoding RNA: an emerging player in diabetes and diabetic kidney disease.

Diabetic kidney disease (DKD) is among the most common complications of diabetes mellitus (DM), and remains the leading cause of end-stage renal diseases (ESRDs) in developed countries, with no definitive therapy yet available. It is imperative to decipher the exact mechanisms underlying DKD and identify novel therapeutic targets. Burgeoning evidence indicates that long non-coding RNAs (lncRNAs) are essential for diverse biological processes. However, their roles and the mechanisms of action remain to be defined in disease conditions like diabetes and DKD. The pathogenesis of DKD is twofold, so is the principle of treatments. As the underlying disease, diabetes per se is the root cause of DKD and thus a primary focus of therapy. Meanwhile, aberrant molecular signaling in kidney parenchymal cells and inflammatory cells may directly contribute to DKD. Evidence suggests that a number of lncRNAs are centrally involved in development and progression of DKD either via direct pathogenic roles or as indirect mediators of some nephropathic pathways, like TGF-β1, NF-κB, STAT3 and GSK-3β signaling. Some lncRNAs are thus likely to serve as biomarkers for early diagnosis or prognosis of DKD or as therapeutic targets for slowing progression or even inducing regression of established DKD. Here, we elaborated the latest evidence in support of lncRNAs as a key player in DKD. In an attempt to strengthen our understanding of the pathogenesis of DKD, and to envisage novel therapeutic strategies based on targeting lncRNAs, we also delineated the potential mechanisms of action as well as the efficacy of targeting lncRNA in preclinical models of DKD.

Endothelial or vascular smooth muscle cell-specific expression of human NOX5 exacerbates renal inflammation, fibrosis and albuminuria in the Akita mouse.

Excessive production of reactive oxygen species (ROS) plays a detrimental role in the progression of diabetic kidney disease (DKD). Renal oxidative stress activates proinflammatory cytokines, chemokines and profibrotic factors in DKD. Increased expression of the prooxidant enzyme NADPH oxidase (NOX) 5 in kidneys of diabetic individuals has been hypothesised to correlate with renal injury and progression of DKD. Since the gene encoding NOX5 is not expressed in the mouse genome, we examined the effect of inducible human NOX5 expression in renal cells, selectively in either endothelial cells or vascular smooth muscle cells (VSMCs)/mesangial cells in a model of insulin-deficient diabetes, the Akita mouse. Renal structural injury, including glomerulosclerosis, mesangial expansion and extracellular matrix protein accumulation, as well as renal inflammation, ROS formation and albuminuria, were examined in the NOX5 transgenic Akita mouse model of DKD. Expression of NOX5 in either endothelial cells or VSMCs/mesangial cells in diabetic Akita mice was associated with increased renal inflammation (monocyte chemoattractant protein-1, NF-κB and toll-like receptor-4) and glomerulosclerosis, as well as upregulation of protein kinase C-α and increased expression of extracellular matrix genes (encoding collagen III, fibronectin and α-smooth muscle actin) and proteins (collagen IV), most likely mediated via enhanced renal ROS production. The effect of VSMC/mesangial cell-specific NOX5 expression resulted in more pronounced renal fibrosis in comparison with endothelial cell-specific NOX5 expression in diabetic mice. In addition, albuminuria was significantly increased in diabetic VEcad+NOX5+ mice (1192 ± 194μg/24h) when compared with diabetic VEcad+NOX5- mice (770 ± 98μg/24h). Furthermore, the regulatory components of NOX5 activation, including heat shock protein 90 and transient receptor potential cation channel subfamily C member 6, were upregulated only in the presence of both NOX5 and diabetes. The findings from this study highlight the importance of NOX5 in promoting diabetes-related renal injury and provide the rationale for the development of a selective NOX5 inhibitor for the prevention and/or treatment of DKD.

499-P: The Relative Roles of Pro-oxidant Enzymes Nox4 and Nox5 in Diabetic Kidney Disease

Background: Renal oxidative stress plays an important role in mediating kidney injury in diabetes. There is increasing evidences that recently discovered pro-oxidant enzyme, Nox5 plays a significant role in human diabetic kidney disease (DKD). We examined the role of Nox5 in human DKD, in human renal cell populations as well as in a high fat fed and alloxan rabbit model of DKD. In addition, we also examined the contribution of Nox5 in the in DKD in the absence of Nox4 expression using renal cell specific Nox5Tg mice. Results: Expression of Nox5 was increased in both glomerular and tubular compartments of kidney biopsies obtained from diabetic individuals. Nox5 is the Nox isoform which shows the highest upregulation in response to high glucose. Silencing of Nox5 reduces ROS formation and expression of proinflammatory and profibrotic cytokines and growth factors as well as putative elements that are implicated in DKD. Our data also suggest that Nox5 is upstream of Nox4 and that Nox5 inhibition downregulates Nox4, but not vice versa. High fat feeding and alloxan induced diabetes rabbits showed upregulation of Nox5 and increased ROS in association with increased mesangial area, ECM protein accumulation and upregulation of a range of pro-inflammatory (MCP-1) and pro-fibrotic genes (CTGF) as analysed by RNAseq and confirmed by RT-PCR. In addition, over expression of Nox5 in mesangial cells of Nox4 KO diabetic mice demonstrated 30% increase in albuminuria, mesangial expansion, increased renal injury and inflammation via enhanced ROS production. Conclusions: These studies will for the first time explore in a comprehensive manner the role of Nox5 in recently generated Nox5KO rabbit models in the context of type 1 and type 2 diabetes as well as delineate the relative role of Nox4 versus Nox5 in diabetic complications. These studies will provide evidence if Nox5 blockade alone or combined with Nox4/Nox5 blockade is the optimal approach to treat and prevent diabetic complications, in particular nephropathy. Disclosure J.C. Jha: None. M.E. Cooper: Advisory Panel; Self; AstraZeneca, Lilly Diabetes. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Servier. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Novo Nordisk A/S. Speaker's Bureau; Self; Bayer AG, Merck Sharp &amp; Dohme Corp., Novartis AG. C. Kennedy: Research Support; Self; Prometic Life Sciences Inc. K. Jandeleit-Dahm: Advisory Panel; Self; Australian Diabetes Society, Genkyotex. Consultant; Self; Metavention.

Genetic characterization of early renal changes in a novel mouse model of diabetic kidney disease

Genetic factors influence susceptibility to diabetic kidney disease. Here we mapped genes mediating renal hypertrophic changes in response to diabetes. A survey of 15 mouse strains identified variation in diabetic kidney hypertrophy. Strains with greater (FVB/N(FVB)) and lesser (C57BL/6 (B6)) responses were crossed and diabetic F2 progeny were characterized. Kidney weights of diabetic F2 mice were broadly distributed. Quantitative trait locus analyses revealed diabetic mice with kidney weights in the upper quartile shared alleles on chromosomes (chr) 6 and 12; these loci were designated as Diabetic kidney hypertrophy (Dkh)-1 and -2. To confirm these loci, reciprocal congenic mice were generated with defined FVB chromosome segments on the B6 strain background (B6.Dkh1/2f) or vice versa (FVB.Dkh1/2b). Diabetic mice of the B6.Dkh1/2f congenic strain developed diabetic kidney hypertrophy, while the reciprocal FVB.Dkh1/2b congenic strain was protected. The chr6 locus contained the candidate gene; Ark1b3, coding aldose reductase; the FVB allele has a missense mutation in this gene. Microarray analysis identified differentially expressed genes between diabetic B6 and FVB mice. Thus, since the two loci identified by quantitative trait locus mapping are syntenic with regions identified for human diabetic kidney disease, the congenic strains we describe provide a valuable new resource to study diabetic kidney disease and test agents that may prevent it.

Effect of liraglutide on the Janus kinase/signal transducer and transcription activator (JAK/STAT) pathway in diabetic kidney disease in db/db mice and in cultured endothelial cells.

Emerging evidence demonstrates the involvement of Janus tyrosine kinase/signal transducer and transcription activator (JAK/STAT) proteins in the pathophysiology of diabetic kidney disease (DKD). The JAK/STAT pathway is involved in the inflammatory response and endothelial cell dysfunction observed in DKD. The glucagon-like peptide-1 (GLP-1) analog liraglutide is an effective treatment for type 2 diabetes because it improves the inflammatory changes observed in experimental models of DKD. This study used db/db mice and endothelial cells (ECs) to determine the effect of diabetic environment on the JAK/STAT pathway and to assess the potential effect of liraglutide (200 μg/kg) in both models. Diabetic db/db mice (12 weeks old) were treated with liraglutide for 14 weeks. The kidneys were then perfused with saline and removed for mRNA, protein, and immunohistochemical analyses. Endothelial cells were stimulated advanced glycation end products (AGEs) (200 μg/μL) glucose (200 mg/dL) and liraglutide (100 nM) for 24 hours. Total RNA and protein were extracted and analyzed for expression of JAK/STAT signaling. Phosphorylated (p-) STAT3 was significantly upregulated in db/db mice compared with non-diabetic mice. Liraglutide significantly downregulated p-STAT3 protein expression in db/db mice. In db/db mice, p-STAT3 was primarily expressed in the glomeruli, whereas p-JAK2 was also expressed in kidney tubules. In ECs, liraglutide treatment prevented increased expression of p-STAT3 and p-JAK2. Liraglutide inhibited the target gene suppressor of cytokine signaling 3 (SOCS3) and sirtuin 1 (SIRT1) in db/db mice and in cultured EC. This study suggests that the GLP-1 analog liraglutide inhibits the JAK/STAT pathway, which participates in intracellular processes in experimental models of diabetes.

Serum amyloid A and Janus kinase 2 in a mouse model of diabetic kidney disease.

BackgroundSerum amyloid A (SAA), a potent inflammatory mediator, and Janus kinase 2 (JAK2), an intracellular signaling kinase, are increased by diabetes. The aims were to elucidate: 1) a JAK2-mediated pathway for increased SAA in the kidneys of diabetic mice; 2) a JAK2-SAA pathway for inflammation in podocytes.MethodsAkita diabetic mice (129S6) with podocyte JAK2 overexpression and angiotensin II infusion (4 weeks) were given a JAK1,2 inhibitor (LY03103801, 3 mg/kg/day orally for the last two weeks). Kidneys were immunostained for SAA isoform 3 (SAA3). SAA3 knockout and control mouse podocytes were exposed to advanced glycation end products (AGE) or exogenous SAA with JAK2 inhibition (Tyrphostin AG 490, 50μM). JAK2 activity (phosphorylation, Western blot, 1 hour) and mRNA for SAA3 and associated inflammatory genes (Cxcl5, Ccl2, and Ccl5) were measured by RT-PCR (20 hours).ResultsSAA3 protein was present throughout the diabetic kidney, and podocyte JAK2 overexpression increased tubulointerstitial SAA3 compared to wild type diabetic controls, 43% versus 14% (p = 0.007); JAK1,2 inhibition attenuated the increase in SAA3 to 15% (p = 0.003). Urine albumin-to-creatinine ratio (r = 0.49, p = 0.03), mesangial index (r = 0.64, p = 0.001), and glomerulosclerosis score (r = 0.51, p = 0.02) were associated with SAA3 immunostaining scores across mouse groups. Exposing podocytes to AGE or exogenous SAA increased JAK2 activity within one hour and mRNA for associated inflammatory genes after 20 hours. JAK2 inhibition reduced SAA3 mRNA expression in podocytes exposed to AGE or SAA. SAA3 knockout podocytes had >85% lower AGE-induced inflammatory genes.ConclusionJAK1,2 inhibition reduced SAA and histological features of DKD in podocyte JAK2-overexpressing mice. In podocytes exposed to a diabetes-like condition, JAK2 inhibition reduced expression of SAA, while SAA knockout blocked expression of associated pro-inflammatory mediators. SAA may promote JAK2-dependent inflammation in the diabetic kidney.

Protease-activated receptor 2 exacerbates cisplatin-induced nephrotoxicity.

Acute kidney injury (AKI) is associated with hypercoagulability. Tissue factor/factor VIIa complex and factor Xa in the coagulation cascade activate protease-activated receptor 2 (PAR2). Previously, we have shown that PAR2-mediated inflammation aggravates kidney injury in models of diabetic kidney disease and adenine-induced renal fibrosis. However, the role of PAR2 in AKI remains unclear. To clarify the role of PAR2, we administered cisplatin, one of the most common causal factors of AKI, to wild-type and PAR2-deficient mice. The expression levels of tissue factor and PAR2 were significantly increased in the kidneys of mice that were administered cisplatin. A lack of PAR2 corrected the levels of plasma blood urea nitrogen and creatinine as well as ameliorated the acute tubular injury score in the kidney. A lack of PAR2 corrected the infiltration of neutrophils and the gene expression levels of proinflammatory cytokines/chemokines in these mouse kidneys. Similarly, apoptotic markers, such as cleaved caspase-3-positive area and Bax/Bcl2 ratio, were attenuated via PAR2 deletion. Thus, elevated PAR2 exacerbates cisplatin nephrotoxicity, and targeting PAR2 is a novel therapeutic option that aids in the treatment of patients with cisplatin-induced AKI.