Diabetic Kidney Disease Mice Research Articles

POS-359 Potential effect of broad-spectrum antibiotic therapy on progression of diabetic kidney disease in mice

Associated with significant advances in analytical methods for microbiome, the relationship between changes in gut microbiome and various diseases such as metabolic and chronic inflammatory diseases has been increasingly reported, raising the possibility that the development and progression of diabetic kidney disease might be associated with dysbiosis. The present study aimed to elucidate the association between gut microbiome and progression of kidney disease in diabetic mice administered broad-spectrum antibiotics.

Coagulation, Protease-Activated Receptors, and Diabetic Kidney Disease: Lessons from eNOS-Deficient Mice.

Endothelial nitric oxide synthase (eNOS) dysfunction is known to exacerbate the progression and prognosis of diabetic kidney disease (DKD). One of the mechanisms through which this is achieved is that low eNOS levels are associated with hypercoagulability, which promotes kidney injury. In the extrinsic coagulation cascade, the tissue factor (factor III) and downstream coagulation factors, such as active factor X (FXa), exacerbate inflammation through activation of the protease-activated receptors (PARs). Recently, it has been shown that the lack of or reduced eNOS expression in diabetic mice, as a model of advanced DKD, increases renal tissue factor levels and PAR1 and 2 expression in their kidneys. Furthermore, pharmaceutical inhibition or genetic deletion of coagulation factors or PARs ameliorated inflammation in DKD in mice lacking eNOS. In this review, we summarize the relationship between eNOS, coagulation, and PARs and propose a novel therapeutic option for the management of patients with DKD.

Latent TGF-β1 protects against diabetic kidney disease via Arkadia/Smad7 signaling.

TGF-β1 has long been considered as a key mediator in diabetic kidney disease (DKD) but anti-TGF-β1 treatment fails clinically, suggesting a diverse role for TGF-β1 in DKD. In the present study, we examined a novel hypothesis that latent TGF-β1 may be protective in DKD mice overexpressing human latent TGF-β1. Streptozotocin-induced Type 1 diabetes was induced in latent TGF-β1 transgenic (Tg) and wild-type (WT) mice. Surprisingly, compared to WT diabetic mice, mice overexpressing latent TGF-β1 were protected from the development of DKD as demonstrated by lowing microalbuminuria and inhibiting renal fibrosis and inflammation, although blood glucose levels were not altered. Mechanistically, the renal protective effects of latent TGF-β1 on DKD were associated with inactivation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathways. These protective effects were associated with the prevention of renal Smad7 from the Arkadia-induced ubiquitin proteasomal degradation in the diabetic kidney, suggesting protection of renal Smad7 from Arkadia-mediated degradation may be a key mechanism through which latent TGF-β1 inhibits DKD. This was further confirmed in vitro in mesangial cells that knockdown of Arkadia failed but overexpression of Arkadia reversed the protective effects of latent TGF-β1 on high glucose-treated mesangial cells. Latent TGF-β1 may protect kidneys from TGF-β1/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in diabetes through inhibiting Arkadia-mediated Smad7 ubiquitin degradation.

Low expression of HIV genes in podocytes accelerates the progression of diabetic kidney disease in mice

With the widespread use combination antiretroviral therapy, there has been a dramatic decrease in HIV-associated nephropathy. However, although the patients living with HIV have low or undetectable viral load, the prevalence of chronic kidney disease (CKD) in this population remains high. Additionally, improved survival is associated with aging-related comorbidities such as diabetes and cardiovascular disease. A faster progression of CKD is associated with concurrent HIV infection and diabetes than with HIV infection or diabetes alone. To explore the potential pathogenic mechanisms that synergistically drive CKD progression by diabetes and HIV infection, we generated a new mouse model with a relatively low expression of HIV-1 proviral genes specifically in podocytes (pod-HIV mice) to better mimic the setting of kidney injury in patients living with HIV. While no apparent kidney phenotypes were observed at baseline in pod-HIV mice, the induction of mild diabetic kidney disease with streptozotocin led to significant worsening of albuminuria, glomerular injury, podocyte loss, and kidney dysfunction as compared to the mice with diabetes alone. Mechanistically, diabetes and HIV-1 synergistically increased the glomerular expression of microRNA-34a (miR-34a), thereby reducing the expression of Sirtuin-1 (SIRT1) deacetylase. These changes were also associated with increased acetylation and activation of p53 and p65 NF-κB and with enhanced expression of senescence and inflammatory markers. The treatment of diabetic pod-HIV mice with the specific Sirtuin-1 agonist BF175 significantly attenuated albuminuria and glomerulopathy. Thus, our study highlights the reduction in Sirtuin-1 as a major basis of CKD progression in diabetic patients living with HIV and suggests Sirtuin-1 agonists as a potential therapy.

The sodium-glucose cotransporter 2 inhibitor tofogliflozin prevents diabetic kidney disease progression in type 2 diabetic mice.

Trials on cardiovascular and renal outcomes in patients with type 2 diabetes have consistently demonstrated that sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of diabetic kidney disease (DKD) progression. However, their renal protective mechanisms have yet to be completely understood and the effect on albuminuria reduction in animal models is controversial. We investigated these issues using KK and KK-Ay mice as a control (CTRL) and as a model for type 2 diabetes (DKD), respectively. KK-Ay mice were treated with 0.015% tofogliflozin, which is an SGLT2 inhibitor, starting at seven weeks of age for eight weeks. Compared with the CTRL mice, the DKD mice had higher HbA1c levels and albuminuria. Although tofogliflozin treatment significantly lowered HbA1c levels, it did not reverse albuminuria. Tofogliflozin treatment enhanced damage in both the glomerular (i.e., enlarged mesangial area, increased foot process effacement rate, and decreased number of WT-1-positive cells) and tubulointerstitial (increased protein levels of KIM-1 and MCP-1, increased number of macrophages, and abnormal mitochondrial morphology) areas. Our results suggest that tofogliflozin may prevent glomerular and tubulointerstitial damage, partly by ameliorating hyperglycemia, renal inflammation, and abnormalmitochondrial morphology.

Connectivity Mapping Identifies BI-2536 as a Potential Drug to Treat Diabetic Kidney Disease.

Diabetic kidney disease (DKD) remains the most common cause of kidney failure, and the treatment options are insufficient. Here, we used a connectivity mapping approach to first collect 15 gene expression signatures from 11 DKD-related published independent studies. Then, by querying the Library of Integrated Network-based Cellular Signatures (LINCS) L1000 data set, we identified drugs and other bioactive small molecules that are predicted to reverse these gene signatures in the diabetic kidney. Among the top consensus candidates, we selected a PLK1 inhibitor (BI-2536) for further experimental validation. We found that PLK1 expression was increased in the glomeruli of both human and mouse diabetic kidneys and localized largely in mesangial cells. We also found that BI-2536 inhibited mesangial cell proliferation and extracellular matrix in vitro and ameliorated proteinuria and kidney injury in DKD mice. Further pathway analysis of the genes predicted to be reversed by the PLK1 inhibitor was of members of the TNF-α/NF-κB, JAK/STAT, and TGF-β/Smad3 pathways. In vitro, either BI-2536 treatment or knockdown of PLK1 dampened the NF-κB and Smad3 signal transduction and transcriptional activation. Together, these results suggest that the PLK1 inhibitor BI-2536 should be further investigated as a novel therapy for DKD.

Podocyte-Specific Induction of KLF6 Attenuates Diabetic Kidney Disease in Mice

Podocyte-Specific Induction of KLF6 Attenuates Diabetic Kidney Disease in Mice

Polysaccharides from Armillariella tabescens mycelia ameliorate renal damage in type 2 diabetic mice

Diabetic kidney disease (DKD), accompanied by chronic low-grade inflammation, is one of the most common complications of diabetes. Armillariella tabescens has potent anti-inflammatory and immunomodulatory properties. The purpose of the present study was to investigate the effects of polysaccharides from Armillariella tabescens mycelia (AT) on the kidney in type 2 diabetic mice and explore the underlying mechanism. The mice were randomized into 4 groups: normal control (NC), diabetic control (DC), DC + 200 mg/kg AT (LAT), and DC + 400 mg/kg AT (HAT). The results showed that compared with the NC group, the levels of fasting blood glucose, renal function-related indices, and serum pro-inflammatory mediators including lipopolysaccharide (LPS), interleukin (IL)-1β, and IL-18 were elevated; the renal morphopathological alterations, oxidative stress, and nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome-mediated inflammation and renal fibrosis were aggravated; the intestinal microbiota dysbiosis and colonic inflammation and barrier dysfunction were deteriorated in the DC group. After supplementation with AT, the aforementioned indices were ameliorated in the AT treatment groups, especially in the HAT group. In conclusion, these results demonstrated that modulating the intestinal microbiota and inflammatory reaction was implicated in the effects of AT against DKD in mice.

Decreased secretion and profibrotic activity of tubular exosomes in diabetic kidney disease.

Tubular changes contribute to the development of renal pathologies in diabetic kidney disease (DKD), including interstitial fibrosis. It is unclear how tubular cells relay signals to interstitial fibroblasts. Recently, exosomes have been recognized as crucial mediators of intercellular communication. We hypothesized that exosomes secreted from tubular cells may stimulate fibroblasts for interstitial fibrosis in DKD. In this study, we isolated and purified exosomes from the renal cortex of DKD mice and high glucose-treated mouse proximal tubular cells. Compared with nondiabetic mice, exosome secretion in kidney tissues decreased in DKD mice. Likewise, high glucose incubation reduced exosome secretion in mouse kidney proximal tubular BUMPT cells. To study the effect of tubular cell exosomes on fibroblasts, exosomes from BUMPT cells were added to renal fibroblast NRK-49F cell cultures. Notably, exosomes from high glucose conditioned BUMPT cells induced higher proliferation, significant morphological change, and substantial production of fibronectin, α-smooth muscle actin, and collagen type Ι in NRK-49F fibroblasts. Proteomics analysis was further performed to profile the proteins within tubular cell exosomes. Interestingly, 22 proteins were found to be differentially expressed between tubular exosomes derived from high glucose conditioned cells and those from normal glucose conditioned cells. Cytoscape analysis suggested the existence of two protein-protein interaction networks in these exosomal differentially expressed proteins. While one of the protein-protein interaction networks comprised enolase 1 (Eno1), heat shock protein family A member 8 (Hspa8), thioredoxin 1 (Txn1), peptidylprolyl isomerase A (Ppia), phosphoglycerate kinase 1 (Pgk1), DNA topoisomerase II-β (Top2b), and β-actin (Actb), the other had the family proteins of human leucocyte antigen F (Ywhag), a component of the ND10 nuclear body (Ywhae), interferon regulatory factor-8 (Ywhaq), and human leucocyte antigen A (Ywhaz). Gene expression analysis via Nephroseq showed a correlation of Eno1 expression with DKD clinical manifestation. In conclusion, DKD is associated with a decrease in exosome secretion in renal tubular cells. Exosomes from high glucose conditioned tubular cells may regulate the proliferation and activation of fibroblasts, contributing to the paracrine signaling mechanism responsible for the pathological onset of renal interstitial fibrosis in DKD.

Berberine protects against diabetic kidney disease via promoting PGC-1α-regulated mitochondrial energy homeostasis.

Background and PurposeDisordered lipid metabolism and disturbed mitochondrial bioenergetics play pivotal roles in the initiation and development of diabetic kidney disease (DKD). Berberine is a plant alkaloid, used in Chinese herbal medicine. It has multiple therapeutic actions on diabetes mellitus and its complications, including regulation of glucose and lipid metabolism, improvement of insulin sensitivity, and alleviation of oxidative damage. Here, we investigated the reno‐protective effects of berberine.Experimental ApproachWe used samples from DKD patients and experiments with models of DKD (db/db mice) and cultured podocytes, to characterize energy metabolism profiles using metabolomics. Molecular targets and mechanisms involved in the regulation of mitochondrial function and bioenergetics by berberine were investigated, along with its effects on metabolic alterations in DKD mice.Key ResultsMetabolomic analysis suggested altered mitochondrial fuel usage and generalized mitochondrial dysfunction in patients with DKD. In db/db mice, berberine treatment reversed the disordered metabolism, podocyte damage and glomerulosclerosis. Lipid accumulation, excessive generation of mitochondrial ROS, mitochondrial dysfunction, and deficient fatty acid oxidation in DKD mouse models and in cultured podocytes were suppressed by berberine. These protective effects of berberine were accompanied by activation of the peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) signalling pathway, which promoted mitochondrial energy homeostasis and fatty acid oxidation in podocytes.Conclusion and ImplicationsPGC‐1α‐mediated mitochondrial bioenergetics could play a key role in lipid disorder‐induced podocyte damage and development of DKD in mice. Restoration of PGC‐1α activity and the energy homeostasis by berberine might be a potential therapeutic strategy against DKD.

MYDGF attenuates podocyte injury and proteinuria by activating Akt/BAD signal pathway in mice with diabetic kidney disease.

Myeloid-derived growth factor (MYDGF), mainly secreted by bone marrow-derived cells, has been known to promote glucagon-like peptide-1 production and improve glucose/lipid metabolism in mouse models of diabetes, but little is known about the functions of MYDGF in diabetic kidney disease (DKD). Here, we investigated whether MYDGF can prevent the progression of DKD. In vivo experiments, both loss- and gain-of-function strategies were used to evaluate the effect of MYDGF on albuminuria and pathological glomerular lesions. We used streptozotocin-treated Mydgf knockout and wild-type mice on high fat diets to induce a model of DKD. Then, albuminuria, glomerular lesions and podocyte injury were evaluated in Mydgf knockout and wild-type DKD mice treated with adeno-associated virus-mediated Mydgf gene transfer. In vitro and ex vivo experiments, the expression of slit diaphragm protein nephrin and podocyte apoptosis were evaluated in conditionally immortalised mouse podocytes and isolated glomeruli from non-diabetic wild-type mice treated with recombinant MYDGF. MYDGF deficiency caused more severe podocyte injury in DKD mice, including the disruption of slit diaphragm proteins (nephrin and podocin) and an increase in desmin expression and podocyte apoptosis, and subsequently caused more severe glomerular injury and increased albuminuria by 39.6% compared with those of wild-type DKD mice (p < 0.01). Inversely, MYDGF replenishment attenuated podocyte and glomerular injury in both wild-type and Mydgf knockout DKD mice and then decreased albuminuria by 36.7% in wild-type DKD mice (p < 0.01) and 34.9% in Mydgf knockout DKD mice (p < 0.01). Moreover, recombinant MYDGF preserved nephrin expression and inhibited podocyte apoptosis in vitro and ex vivo. Mechanistically, the renoprotection of MYDGF was attributed to the activation of the Akt/Bcl-2-associated death promoter (BAD) pathway. The study demonstrates that MYDGF protects podocytes from injury and prevents the progression of DKD, providing a novel strategy for the treatment of DKD. Graphical abstract.

Drug Testing for Residual Progression of Diabetic Kidney Disease in Mice Beyond Therapy with Metformin, Ramipril, and Empagliflozin.

Progression of CKD in type 2 diabetes, despite dual inhibition of sodium-glucose transporter-2 and the renin-angiotensin system, remains a concern. Bromoindirubin-3'-oxime (BIO), previously reported to promote podocyte survival and regeneration, is a candidate additional drug to elicit renoprotective effects beyond therapy with metformin, ramipril, and empagliflozin (MRE). Evaluating a drug with standard therapeutics more closely mimics the clinical setting than evaluating the drug alone. Uninephrectomized BKS-Lepr-/- (db/db) mice treated with or without MRE served as a model of progressive CKD in type 2 diabetes. Mice on or off MRE were randomized to only 4 weeks of add-on BIO or vehicle. The primary end point was slope of GFR (ΔGFR). Four weeks of MRE treatment alone did not affect ΔGFR, but significantly attenuated hyperglycemia, albuminuria, and glomerulosclerosis and increased podocyte filtration slit density, as assessed by STED super-resolution microscopy upon tissue clearing. BIO alone improved albuminuria, podocyte density in superficial and juxtamedullary nephrons, and podocyte filtration slit density. MRE+BIO combination therapy had additive protective effects on ΔGFR, glomerulosclerosis, podocyte density in juxtamedullary nephrons, and filtration slit density. Add-on treatment with BIO for only 4 weeks attenuates progression of CKD beyond MRE therapy in mice with type 2 diabetes. Additional drug combinations may help to further delay ESKD in type 2 diabetes.

Aspartic acid supplementation ameliorates symptoms of diabetic kidney disease in mice.

Diabetic kidney disease (DKD) is among the most common and serious complications of both type 1 and type 2 diabetes. In this study, we used KK/Ta‐Ins2Akita (KK‐Akita) mice as a model of DKD and KK/Ta (KK) mice as controls to identify novel factors related to the development/progression of DKD. Capillary electrophoresis coupled with mass spectrometry analysis revealed that circulating Asp (l‐aspartic acid) levels in diabetic KK‐Akita mice tend to be lower than those in control KK mice. Therefore, we evaluated the effect of Asp supplementation to prevent the progression of DKD in KK‐Akita mice. Mice were divided into three groups: (a) untreated KK mice (Control group), (b) untreated KK‐Akita mice (DKD group), and (c) treated (double‐volume Asp diet) KK‐Akita mice (Tx group). Kidney sections were stained with fluorescein isothiocyanate‐labeled lectins, wheat germ agglutinin (WGA), and anti‐endothelial nitric oxide synthase (eNOS) antibody for evaluation of endothelial surface layer (ESL) and NO synthesis. The mesangial area and glomerular size in the DKD group were significantly larger than those in the Control group; however, there was no significant difference in those between the DKD and Tx groups. Albuminuria, the ratio of foot process effacement, and thickness of glomerular basement membrane in the Tx group were significantly lower than those in the DKD group. Furthermore, the expression levels of glomerular WGA and microvascular eNOS in the Tx group improved significantly and approached the level in the Control group. In conclusion, the improvement of albuminuria in the Tx group may be caused by the reduction of oxidative stress in the kidneys, which may lead to the subsequent improvement of glomerular ESL.

Egr1 Knockdown Combined with an ACE Inhibitor Ameliorates Diabetic Kidney Disease in Mice: Blockade of Compensatory Renin Increase.

BackgroundIncreased compensatory intrarenal renin diminishes the efficacy of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) in the treatment of diabetic kidney disease (DKD). Early growth response-1 (Egr1) is a crucial transcriptional factor in the progress of DKD and is a potential transcription factor of intrarenal renin according to bioinformatic analysis. However, whether inhibition of Egr1 can suppress compensatory renin increase in DKD is unclear.MethodsWe generated a high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mouse model. The mice were treated with either enalapril (an ACEI) or enalapril combined with a shEgr1 plasmid, and age-matched DKD mice were used as controls. Urine microalbumin, urinary renin and kidney TGF-β1 were determined by enzyme-linked immunosorbent assay (ELISA). Hematoxylin and eosin (H&E) and Masson staining were used to determine renal pathological changes. Egr1, renin, TNF-α, and FN were measured by real-time quantitative PCR, Western blot, and immunohistochemistry. The SV40-MES13 murine mesangial cell line was transfected with pENTER-Egr1 plasmid and siEgr1.ResultsOur results showed that enalapril increased the renin level of urinary and renal in DKD mice, while shEgr1 attenuated this effect. In addition, enalapril treatment reduced the levels of urinary microalbumin, TNF-α, TGF-β1 and FN, and alleviated the pathological changes, while shEgr1 strengthened these effects. The protein and mRNA expression of renin in the SV40 MES13 cells was upregulated and downregulated following overexpression and silence of Egr1, respectively.ConclusionSilence of Egr1 could alleviate renal injury in DKD by downregulating intrarenal renin.

Long noncoding RNA NEAT1 is involved in the protective effect of Klotho on renal tubular epithelial cells in diabetic kidney disease through the ERK1/2 signaling pathway

Klotho, an antiaging protein, has been shown to play a protective role in renal tubular epithelial-mesenchymal transition (EMT) during the development of diabetic kidney disease (DKD). Long noncoding RNAs (lncRNAs) participate in the progression of EMT in many diseases. However, the effect of Klotho on lncRNAs during the development of DKD is still unknown. In this study, we found that Klotho overexpression in high-fat diet (HFD)- and streptozotocin (STZ)-induced DKD mice significantly inhibited the expression of lncRNA nuclear-enriched abundant transcript 1 (Neat1). We demonstrated that NEAT1 was significantly upregulated in both bovine serum albumin (BSA)-stimulated HK2 cells and mice with HFD- and STZ-induced diabetes. In addition, we observed that Klotho displays colocalization with NEAT1. Furthermore, overexpression of Klotho can inhibit the high expression of NEAT1 in BSA-stimulated HK2 cells, while silencing Klotho can further upregulate the expression of NEAT1. Silencing NEAT1 in HK2 cells resulted in inhibition of the EMT-related markers alpha smooth muscle actin (α-SMA) and vimentin (VIM) and the renal fibrosis-related markers transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF). The effect of NEAT1 on DKD was partly mediated by regulation of the ERK1/2 signaling pathway. Finally, we found that silencing NEAT1 can reverse the activation of EMT and fibrosis caused by Klotho silencing in a manner dependent on the ERK1/2 signaling pathway. These findings reveal a new regulatory pathway by which Klotho regulates ERK1/2 signaling via NEAT1 to protect against EMT and renal fibrosis, suggesting that NEAT1 is a potential therapeutic target for DKD.

LncRNA GAS5 exacerbates renal tubular epithelial fibrosis by acting as a competing endogenous RNA of miR-96-5p

Renal fibrosis is at the core of various renal diseases, including diabetic kidney disease (DKD). Long noncoding RNAs (lncRNAs) are known players in the regulation of renal fibrosis. However, their expression and function in DKD still need to be elucidated. The purpose of this study was to assess how lncRNA GAS5 regulates fibrosis and its mechanism in TGF-β1-treated renal proximal tubular cell.In this study, the lncRNA GAS5 was upregulated in both TGF-β1-treated HK-2 cells and the kidneys of HDF/STZ mice. Knockdown of GAS5 relieved renal tubular epithelial fibrosis. This effect was mediated by the downregulation and functional inactivation of miR-96-5p. Furthermore, miR-96-5p was downregulated in DKD mice, and this downregulation attenuated the repression of FN1(fibronectin, FN) and led to its upregulation. The decrease in miR-96-5p was partially attributed to the miRNA-sponge action of GAS5.Our research demonstrates that knockdown of lncRNA GAS5 leads to antifibrosis by competitively binding miR-96-5p, which inhibits the expression of FN1. These results indicate that targeting lncRNA GAS5 may be a promising therapeutic strategy for preventing DKD.

Krüppel‑like factor4 ameliorates diabetic kidney disease by activating autophagy via the mTOR pathway.

Diabetic kidney disease (DKD) is diagnosed increasingly frequently and represents a serious threat to human health. Krüppel-like factor 4 (KLF4) has aroused attention due to its potential effect on podocytes and in ameliorating proteinuria associated with glomerulopathy. The purpose of the present study was to investigate the potential role of KLF4 in DKD. It was hypothesized that KLF4 impacts diabetic nephropathy by mediating the podocyte autophagic process. A KLF4 plasmid vector was constructed, and podocytes were transfected and incubated with DKD mice serum for in vitro experiments. A db/db spontaneous DKD mouse model was also established for in vivo study. After treatment, the level of serum creatinine (Scr), blood urea nitrogen (BUN), and 24-h urinary protein was determined. Immunofluorescence and periodic acid-Schiff staining, western blotting, flow cytometry and a TUNEL assay were performed to observe changes in glomerular morphology and the level of apoptosis, cytoskeleton proteins, epithelial-mesenchymal transition (EMT) biomarkers, autophagic proteins and mTOR pathway proteins in each group. KLF4 overexpression significantly reduced the level of urinary albumin, Scr, BUN and attenuated mesangial matrix expansion, as well as mesangial cell proliferation in DKD mice. KLF4 overexpression also inhibited podocyte apoptosis and downregulated vimentin and α-smooth muscle actin, and upregulated E-cadherin and nephrin, both in vivo and in vitro. Moreover, the microtubule associated protein 1 light chain 3α (LC3)-II/LC3-I ratio and LC3-II fluorescence was significantly increased in the vector-KLF4 group compared to the negative control vector group both in vivo and in vitro. Finally, a decrease in the level of phosphorylated (p)-mTOR and p-S6K protein expression was observed following KLF4 overexpression in vitro. The present findings suggested that KLF4 plays a renoprotective role in DKD, which is associated with the activation of podocyte autophagy, and may be involved in the mTOR signaling pathway.

Podoplanin mediates the renoprotective effect of berberine on diabetic kidney disease in mice.

Hyperglycemia-caused podocyte injury plays a crucial role in the progress of diabetic kidney disease. Podoplanin, one of the podocyte-associated molecules, is closely related to the integrity of the glomerular filtration barrier. A number of studies demonstrate that berberine could ameliorate renal dysfunction in diabetic mice with nephropathy, but the molecular mechanisms have not been fully elucidated. In this study, we explored the relationship between the renoprotective effect of berberine and podoplanin expression in streptozotocin (STZ)-induced diabetic mice as well as mouse podocytes (MPC5 cells) cultured in high glucose (HG, 30 mM) medium. We found that the expression levels of podoplanin were significantly decreased both in the renal glomerulus of STZ-induced diabetic mice and HG-cultured MPC5 cells. We also demonstrated that NF-κB signaling pathway was activated in MPC5 cells under HG condition, which downregulated the expression level of podoplanin, thus leading to increased podocyte apoptosis. Administration of berberine (100, 200 mg/kg every day, ig, for 8 weeks) significantly improved hyperglycemia and the renal function of STZ-induced diabetic mice and restored the expression level of podoplanin in renal glomerulus. In high glucose-cultured MPC5 cells, treatment with berberine (30–120 μM) dose-dependently decreased the apoptosis rate, increased the expression of podoplanin, and inhibited the activation of NF-κB signaling pathway. When podoplanin expression was silenced with shRNA, berberine treatment still inhibited the NF-κB signaling pathway, but its antiapoptotic effect on podocytes almost disappeared. Our results suggest that berberine inhibits the activation of NF-κB signaling pathway, thus increasing the podoplanin expression to exert renoprotective effects.

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

520-P: Hyperglycemia-Induced Mitochondrial Dysfunction in Diabetic Kidney Disease May Be Mediated by Crabtree Effect in Kidney Proximal Tubule Epithelial Cells

Background: Recent studies indicate mitochondrial dysfunction is a dominant pathway in diabetic kidney disease (DKD), however underlying mechanisms are unclear. Here we examined if Crabtree effect by glucose is a cause of mitochondrial dysfunction in kidney. Methods: Lactate was measured by by YSI biochemistry analyzer. TCA cycle metabolites were measured by gas chromatography mass spectroscopy in urine samples of type 1 and type 2 DKD mouse models. Changes in oxygen consumption (OCR) and extracellular acidification i(ECAR) were analyzed on a Seahorse extracellular flux analyzer. Immunohistochemical analysis was performed on FFPE-embedded mouse kidney sections. Western blot analysis was performed on kidney cortical sections and HK2 cells. Results: Significantly increased urine and plasma lactate along with a decrease in several TCA cycle metabolites were observed in both type 1 and type 2 diabetic mice. Mitochondrial functional analysis in different kidney cells showed a profound respiratory inhibition and stimulation of lactate by glucose in proximal tubules, but not in mesangial cells and podocytes. Pyruvate at above physiological concentration reversed the glucose-induced inhibition of OCR. Western blotting of kidney cortical tissue sections showed a significant increase in phospho PDH in diabetic kidney tissues and in HK2 cells treated with high glucose. In normal conditions, phospho PDH was predominantly on distal tubules, however in diabetic conditions a significant increase in phospho PDH was noted in proximal tubule cells. Dichloroacetate, an inhibitor of PDK completely reversed the glucose induced respiratory inhibition in proximal tubule epithelial cells. Conclusions: Mitochondrial dysfunction in proximal tubule epithelial cells may potentially be due to high-glucose induced PDH phosphorylation resulting in limited pyruvate oxidation into mitochondria and enhanced lactate production. Disclosure M. Darshi: None. J.J. Kim: None. V. Drel: None. A.N. Murphy: Advisory Panel; Self; Cytokinetics Inc. Consultant; Self; Agilent Technologies. Funding JDRF; U.S. Department of Veterans Affairs