Mouse Model Of Diabetic Nephropathy Research Articles

Automated Image Analyses of Glomerular Hypertrophy in a Mouse Model of Diabetic Nephropathy.

Glomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal diseases such as obesity-associated glomerulopathies and diabetic nephropathy (DN). Using light sheet fluorescent microscopy (LSFM) and 3D image analysis, we tested algorithms for automated and unbiased quantification of total glomerular numbers and individual glomerular volume in the uninephrectomized (UNx) db/db mouse model of DN. At 6 weeks after surgery, db/db and UNx db/db mice showed increased urine albumin-to-creatinine ratio (ACR) compared with db/+ control mice. Before euthanasia, glomeruli were labeled in vivo by injecting tomato lectin. Whole-kidney LSFM 3D image analysis revealed that mean glomerular volume was significantly increased in UNx db/db mice compared with db/+ mice. Moreover, analysis of individual glomerular volume showed a shift in volume distribution toward larger glomeruli and thereby demonstrated additive effects of diabetes and UNx on induction of glomerular hypertrophy. The automatized quantification showed no significant differences in glomerular numbers among db/+, db/db, and UNx db/db mice. These data correlated with glomerular numbers as quantified by subsequent stereologic quantification. Overall, LSFM coupled with automated 3D histomorphometric analysis was demonstrated to be advantageous for unbiased assessment of glomerular volume and numbers in mouse whole-kidney samples. Furthermore, we showed that injection of fluorescently labeled lectin and albumin can be used as markers of nephron segments in the mouse kidneys, thus enabling functional assessment of kidney physiology, pathology, and pharmacology in preclinical rodent models of kidney disease.

P0987MIR-181A IMPROVED RENAL INFLAMMATION VIA TARGETING TNF-Α IN DIABETIC NEPHROPATHY

Abstract Background and Aims Recent studies have revealed that miR-181a is involved in various inflammation associated diseases, however, its participation in pathogenesis of diabetic nephropathy (DN) remains unclear. Hence, we investigated the involvement of miR-181a in the pathogenesis of DN patients and in animal model. Method miR-181a level was measured in the kidney of DN patients and DN mice model (db/db mice) by RT-PCR. To investigate the interaction of miR-181 a with the 3’-UTR of potential target transcripts, Luciferase reporter assays were performed. miR-181a-GFP-AAV and miR-181a inhibitor was generated to over-express or downregulate miR-181a in the kidney of db/db mice respectively. The expression of target gene and inflammatory proteins were measured in the kidney of our animal model. Results In DN patients and DN mice, a significant decrease in miR-181a levels in kidney were revealed by Expression analyses performed. Overexpression of miR-181a reduced proinflammatory genes (TNF-ɑ, IL-1β, and IL-18 ) expression, whereas suppression of miR-181a worsened these changes. Furthermore, there was a negative linear correlation between miR-181a and TNF-ɑ, IL-1β, and IL-18 expression levels in the kidney from DN patients. Luciferase assays revealed TNF-ɑ as a direct target of miR-181a. Conclusion Our data emphasized on the anti-inflammatory actions of miR-181a via targeting TNF-ɑ in the context of renal inflammation in DN. Hence, miR-181a could be served as a potential therapeutic options for controlling renal inflammation in DN.

SO025DCR2 ACCELERATES RENAL FIBROSIS AND TUBULAR CELL SENESCENCE BY PHOSPHORYLATING TYR 194 OF PRDX1 IN DIABETIC NEPHROPATHY

Abstract Background and Aims Cell senescence of renal tubular epithelial cells (RTECs), which is involved in renal tubulointerstitial fibrosis (TIF), is a key event in the progression of diabetic nephropathy (DN). However, the underlying mechanism remains unclear. This study aims to investigate the role and mechanism of decoy receptor 2 (DcR2) in TIF and cell senescence of RTECs. Method Streptozotocin (STZ)-induced DN mice model and high glucose (HG)-induced cell senescent model were constructed. The expression of DcR2 were regulated by ultrasound-mediated gene transfer of DcR2-siRNA or DcR2-overexpression plasmid in vivo. The co-expression of DcR2 with senescent markers (p16, p21 and SA-β-gal) and fibrotic markers (FN, collagen I and α-SMA). The interaction of DcR2 and its ligand TRAIL and antagonistic receptor DR5 were detected by laser confocal and Co-IP. The DcR2-interaction proteins were screened in renal tissue of DN patients and HG-induced cells by Co-IP combining with LC-MS/MS. The interaction of DcR2 and PRDX1 was validated by Co-IP, pull down assay and immunofluorescence co-staining. Peroxidase activity of PRDX1 was assessed by the kits of reactive oxygen species (ROS) and specific 2-cys peroxidase activity. The level of PRDX1 phosphorylation was measured through WB and the phosphorylated site were studied by wild-type PRDX1 and mutant Tyr194 PRDX1. Results DcR2 was co-expressed with senescent markers and co-localization with fibrotic markers in DN patients and STZ-DN mice. Knockdown of DcR2 effectively decreased renal fibrosis and tubular cell senescence in STZ-induced DN mice. However, DcR2 overexpression showed the opposite effects. DcR2 didn’t interacted with TRAIL or DR5 in vivo and vitro. The following study indicated that PRDX1 is a novel DcR2-interacting protein using quantitative proteomics, which have peroxidase and antioxidant activity in cytoplasm. The interaction of DcR2-PRDX1 mediated RTEC senescence in vitro. Knockdown of DcR2 inhibited the level of ROS and promoted the peroxidase activity of PRDX1, resulting in oxidative stress and cell senescence, and DcR2 overexpression presented the contrary results. DcR2 regulated the level of PRDX1 phosphorylation by interacting Tyr194 of PRDX1, and the mutant Tyr194 of PRDX1 can’t be phosphorylated by DcR2. Conclusion DcR2 mediates renal fibrosis and RTEC senescence by phosphorylating with PRDX1, suggesting DcR2 could serve as a potential therapeutic target for the amelioration of DN progression.

Cyclo-RGD Truncated Polymeric Nanoconstruct with Dendrimeric Templates for Targeted HDAC4 Gene Silencing in a Diabetic Nephropathy Mouse Model.

Diabetic nephropathy (DN), a chronic progressive kidney disease, is a significant complication of diabetes mellitus. Dysregulation of the histone deacetylases (HDACs) gene has been implicated in the pathogenesis of DN. Hence, the HDAC-inhibitors have emerged as a critical class of therapeutic agents in DN; however, the currently available HDAC4-inhibitors are mostly nonselective in nature as well as inhibit multiple HDACs. RNA interference of HDAC4 (HDAC4 siRNA) has shown immense promise, but the clinical translation has been impeded due to lack of a targeted, specific, and in vivo applicable delivery modality. In the present investigation, we examined Cyclo(RGDfC) (cRGD) truncated polymeric nanoplex with dendrimeric templates for targeted HDAC4 Gene Silencing. The developed nanoplex exhibited enhanced encapsulation of siRNA and offered superior protection against serum RNase nucleases degradation. The nanoplex was tested on podocytes (in vitro), wherein it showed selective binding to the αvβ3 integrin receptor, active cellular uptake, and significant in vitro gene silencing. The in vivo experiments showed remarkable suppression of the HDAC4 and inhibition in the progression of renal fibrosis in the Streptozotocin (STZ) induced DN C57BL/6 mice model. Histopathological and toxicological studies revealed nonsignificant abnormality/toxicity with the nanoplex. Conclusively, nanoplex was found as a promising tactic for targeted therapy of podocytes and could be extended for other kidney-related ailments.

Ergosterol ameliorates renal inflammatory responses in mice model of diabetic nephropathy

Ergosterol (ERG) was reported to exhibit anti-inflammatory and anti-oxidative activities. Besides, ERG was found to attenuate kidney injury in the diabetic mouse. However, the protective effect of ERG in diabetic nephropathy-induced inflammation remains unclear. We aimed to study whether ERG could alleviate diabetic nephropathy-induced inflammation and explore the underlying mechanisms. The diabetic nephropathy mice model was induced by intraperitoneal injection of 30 mg/kg Streptozotocin (STZ). The inflammatory cytokines levels, and insulin concentration in the serum of both diabetic nephropathy patients and mouse model were determined by ELISA. mRNA and protein expression were analyzed by RT-PCR and Western blot, respectively. Fasting blood glucose levels were detected using a commercial kit. Blood biochemistry levels were determined by an automatic analyzer. Mesangium proliferation was detected by PAS staining. It was found that serum levels of IL-6, TNF-α, and MCP-1 dramatically increased in the diabetic nephropathy patients. In mice, ERG treatment greatly decreased fasting blood glucose levels, inflammatory cytokine levels, and renal injury, while it enhanced the insulin level. Mechanically, ERG treatment dramatically decreased NF-κB signaling pathway. Our findings highlight the potential of ERG as an effective agent to treat diabetic nephropathy.

Kaempferol in ameliorating diabetes-induced fibrosis and renal damage: An in vitro and in vivo study in diabetic nephropathy mice model

BackgroundKaempferol is a natural polyflavonol that has gained considerable attention as antidiabetic therapeutics. Recent reports emphasize the role of hyperglycemia and RhoA/Rho Kinase activity in the pathogenesis of diabetic nephropathy (DN). This study aims to evaluate the GLP-1 and insulin release along with RhoA/Rho Kinase inhibition pertaining to the anti-fibrotic and reno-protective effects of Kaempferol in DN. MethodsThe effect of Kaempferol on GLP-1 and insulin release along with underlying mechanisms (Ca2+ and cAMP levels) in GLUTag and MIN6 cells as well as in their co-culture has been evaluated. Further, the effect of Kaempferol on GLP-1 and insulin release was evaluated under in-vivo circumstances in the DN C57BL/6 mouse model. Histology and fibrosis specific staining was performed to study the renal injuries and fibrosis, while the expression of mRNA and protein of interest was evaluated by RT-PCR and western blot analysis. ResultsKaempferol treatment promoted the GLP-1 and insulin release, which was accompanied by increased intracellular levels of cAMP and Ca2+ in GLUTag and MIN6 cells. In agreement with in vitro studies, Kaempferol also increased the release of GLP-1 and insulin in the DN mouse model. Notably, Kaempferol showed the potential to ameliorate the histological changes as well as renal fibrosis while decreasing the expression levels of DN markers including TGF-β1, CTGF, fibronectin, collagen IV, IL-1β, RhoA, ROCK2, and p-MYPT1 in DN kidney tissues. A rise in the expression of E-cadherin and nephrin was also noted in the same study. ConclusionThis study establishes that Kaempferol ameliorates renal injury and fibrosis by enhancing the release of GLP-1, insulin, and inhibition of RhoA/Rho Kinase. This study recommends Kaempferol for further clinical trials to be developed as novel therapeutics for improving the renal function in DN patients.

Mouse Umbilical Cord Mesenchymal Stem Cell Paracrine Alleviates Renal Fibrosis in Diabetic Nephropathy by Reducing Myofibroblast Transdifferentiation and Cell Proliferation and Upregulating MMPs in Mesangial Cells.

Transplantation of umbilical cord mesenchymal stem cells (UC-MSCs) is currently considered a novel therapeutic strategy for diabetic nephropathy (DN). However, the mechanisms by which UC-MSCs ameliorate renal fibrosis in DN are not well understood. Herein, we firstly investigated the therapeutic effects of mouse UC-MSC infusion on kidney structural and functional impairment in streptozotocin- (STZ-) induced diabetic mice. We found that the repeated injection with mUC-MSCs alleviates albuminuria, glomerulus injury, and fibrosis in DN mouse models. Next, mesangial cells were exposed to 5.6 mM glucose, 30 mM glucose, or mUC-MSC-conditioned medium, and then we performed western blotting, immunofluorescence, wound healing assay, and cell proliferation assay to measure extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs), myofibroblast transdifferentiation (MFT), and cell proliferation. We demonstrated that mUC-MSC paracrine decreased the deposition of fibronectin and collagen I by inhibiting TGF-β1-triggered MFT and cell proliferation mediated by PI3K/Akt and MAPK signaling pathways, and elevating the levels of MMP2 and MMP9. Importantly, we provided evidence that the antifibrosis role of mUC-MSC paracrine in DN might be determined by exosomes shed by MSCs. Together, these findings reveal the mechanisms underlying the therapeutic effects of UC-MSCs on renal fibrosis in DN and provide the evidence for DN cell-free therapy based on UC-MSCs in the future.

Low‐Dose Metformin Treatment Ameliorates Renal Dysfunction and Fibrosis in a Mouse Model of Diabetic Nephropathy

BackgroundDiabetes mellitus is the leading cause of chronic kidney disease and end‐stage renal disease in the United States. About one‐third of patients with diabetes develop renal dysfunction, commonly referred to as diabetic nephropathy. Metformin is a widely prescribed antidiabetic medication for patients with type 2 diabetes; however, its direct effects on the renal function besides normalizing glucose levels in patients with diabetic nephropathy are poorly understood. Thus, the objective of our study was to investigate the effects of a non‐glucose lowering, low‐dose metformin treatment on the diabetic kidney using a genetic mouse model of type 2 diabetes.MethodsOur study utilized db/db mice (Leprdb), which harbor a mutation in their leptin receptors, and hence, show the signs and symptoms similar to those experienced in patients with type 2 diabetes. To accelerate the progression of nephropathy, each mouse had their left kidney removed at ten weeks of age. Heterozygous mice were used as a control group for the study. Our study had four treatment groups (n=8 per group): 1) Vehicle‐treated control (non‐diabetic), 2) Metformin‐treated control (non‐diabetic), 3) Diabetic, vehicle‐treated, and 4) Diabetic, metformin‐treated. Mice were treated with either vehicle or metformin (100 mg/kg/day) for four weeks. Fasting blood glucose and urine samples were collected at the beginning and end of the study. Plasma and kidney tissue samples were collected at the end of the study. Markers of renal dysfunction such as elevations in urine albumin‐creatinine ratio (UACR) and plasma creatinine were measured in the samples collected from the study. Alpha‐smooth muscle (α‐SMA), a marker of renal fibrosis, were measured in kidney samples via Western blotting.ResultsOur results showed that the fasting blood glucose (FBG) levels were elevated in the diabetic mice (470.29 ±73.35 mg/dL) at the beginning of the study and were gradually increased with disease progression (672.57 ±107.88 mg/dL). Our results also demonstrated that low‐dose metformin treatment did not lower FBG levels in diabetic mice (583.38 ±195.77 mg/dL). However, metformin treatment was able to prevent the increase in UACR in diabetic mice (205.3 ±112.5 vs. 117.53 ±61.56 μg albumin/mg creatinine). Results from the plasma creatinine levels show that diabetic mice had increased plasma creatinine levels (0.72 ±0.07 mg/dL) compared to the control mice (0.51 ±0.06 mg/dL). Intriguingly, low‐dose metformin treatment did not prevent the diabetes‐induced elevations in plasma creatinine levels. Western blot analysis for α‐SMA indicated that only the diabetic mice expressed α‐SMA in the kidney and low‐dose metformin treatment diminished renal expression of α‐SMA in diabetic mice.ConclusionOur findings suggest that low‐dose metformin treatment improves renal function, and ameliorates renal fibrosis observed with diabetic nephropathy. Further studies are required to investigate the mechanisms that underlie the nephroprotective effects of low‐dose metformin therapy in diabetic mice.Support or Funding InformationFundingThis study was supported by intramural funds received from Drake University through Harris Research Endowment, Kresge Endowment, and Provost fund.

The potential role of the gut microbiota in modulating renal function in experimental diabetic nephropathy murine models established in same environment

Recent studies have shown that laboratory murine autoimmunity models under the same environment display different outcomes. We established diabetic nephropathy model mice under the same environment using the classic streptozotocin method. Renal dysfunction was different among the mice. Proteinuria was more significant in the severe proteinuria group (SP) than in the mild proteinuria group (MP). We hypothesized a role for the gut microbiota in the outcome and reproducibility of induced DN models. 16S rDNA gene sequencing technology was used to analyze the differences in the gut microbiota between the two groups. Here, through fecal microbiota transplantation (FMT) and gas chromatography mass spectrometry (GC–MS), we verified the role of the gut microbiota and its short-chain fatty acid (SCFA) generation in DN mouse renal dysfunction. In the SP group, there was a reduced abundance of Firmicutes (P < 0.0001), and the dominant genus Allobaculum [linear discriminant analysis (LDA) >3, P < 0.05] was positively correlated with body weight (Rho = 0.767, P < 0.01) and blood glucose content (Rho = 0.648, P < 0.05), while the dominant genus Anaerosporobacter (LDA > 3, P < 0.05) was positively correlated with 24-hour urinary protein content (Rho = 0.773, P < 0.01). In the MP group, the dominant genus Blautia (LDA > 3, P < 0.05) was negatively correlated with 24-hour urinary protein content (Rho = −0.829, P < 0.05). The results indicated that Allobaculum and Anaerosporobacter may worsen renal function, while Blautia may be a protective factor in DN. These findings suggested that the gut microbiota may contribute to the heterogeneity of the induced response since we observed potential disease-associated microbial taxonomies and correlations with DN.

Smad3 promotes AKI sensitivity in diabetic mice via interaction with p53 and induction of NOX4-dependent ROS production

The incidence and severity of acute kidney injury (AKI) is increased yearly in diabetic patients. Although the mechanisms for this remain unclear, the prevention of AKI in diabetic nephropathy is feasible and of value. As we detected highly activation of TGF-β/Smad3 signaling in both human biopsy and mouse model of diabetic nephropathy, we hypothesized that Smad3 activation in diabetic kidneys may increase AKI sensitivity. We tested our hypothesis in vitro using TGF-β type II receptor (TGF-βRII) disrupted tubular epithelial cells (TECs) and in vivo in mice with streptozotocin (STZ)-induced diabetic nephropathy before the induction of ischemia/reperfusion (I/R) injury. We found that high glucose (HG)-cultured TECs showed increased inflammation, apoptosis and oxidative stress following hypoxia/reoxygenation (H/R) injury. Disruption of TGF-βRII attenuated cell injury induced by H/R in HG-treated TECs. Consistently, Smad3 knockdown in diabetic kidney attenuated I/R-induced AKI. Mechanistically, Smad3 binds to p53 and enhances p53 activity in cells treated with HG and H/R, which may lead to TECs apoptosis. Additionally, ChIP assay showed that Smad3 bound with the promoter region of NOX4 and induced ROS production and inflammation. In conclusion, our results demonstrate that Smad3 promotes AKI susceptibility in diabetic mice by interacting with p53 and NOX4.

Urine-derived stem cells for the therapy of diabetic nephropathy mouse model.

Diabetic nephropathy (DN) is one of the most representative diabetic microangiopathy complications. So far, there have been no satisfactory therapeutic strategies, and the injection of stem cells provides a target for DN therapy. Urine-derived stem cells (USCs) were obtained from 9 healthy men. 24 mice were randomly and equally divided into control group, DN model group, DN+hUSC group (treated with USCs for 3 times). Hematoxylin-eosin (HE) and Masson staining were used to detect histological changes of kidney injury. Creatinine and blood urea nitrogen (BUN) were measured to assess renal function. Besides, myofibroblast accumulation, macrophage infiltration, cell proliferation, and oxidative stress were detected by immunohistochemical analysis. Compared with DN model group, DN+hUSC group showed lower function loss, cell infiltration, and oxidative stress, as well as less renal fibrosis, histological damage, and cell proliferation. USC can alleviate inflammation and oxidative stress, reduce renal interstitial fibrosis, improve renal tissue structure and protect renal function through paracrine effect.

Luteolin Attenuates Diabetic Nephropathy through Suppressing Inflammatory Response and Oxidative Stress by Inhibiting STAT3 Pathway.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). DN has many pathological changes, but tubular injury is considered to be a crucial pathological feature and plays a key role in the progression of DN. Accumulating studies have confirmed that Luteolin (3,4,5,7-tetrahydroxyflavone, Lut) possesses anti-inflammatory and antioxidant activities, which may play a role in kidney protection in DN. This paper described the effects of Lut on appropriated tubular injury in the kidneys of db/db mice and searched the possible mechanisms underlying the kidney protection effect in DN. Twelve-week-old male C57BL/6 J db/db and C57BL/6 J db/m mice were used for the animal experiments. They were organized into the following five groups for the animal experiments: a db/m group (control, n=6); a db/db group(n=8) ; a db/db group receiving Lut (10 mg/kg/day, n=8)treatment by oral gavage; a db/db group receiving stattic (a selective STAT3 inhibitor,50 mg/Kg/day, n=8) treatment by oral gavage and a db/db group receiving both stattic and Lut treatment by oral gavage. In this study, we found that Lut might ameliorate glomerular sclerosis and interstitial fibrosis in DN mouse models through inhibiting the inflammatory response and oxidative stress. And it might play its biological function mainly through repressing the STAT3 activation. Lut attenuates DN mainly via suppression of inflammatory response and oxidative response. STAT3 pathway is the potential target, which ultimately reduces renal fibrosis and delays the progress of DN.

Fibroblast Growth Factor 21 Attenuates Diabetes-Induced Renal Fibrosis by Negatively Regulating TGF-β-p53-Smad2/3-Mediated Epithelial-to-Mesenchymal Transition via Activation of AKT.

BackgroundEpithelial-to-mesenchymal transition (EMT) is required for renal fibrosis, which is a characteristic of diabetic nephropathy (DN). Our previous study demonstrated that fibroblast growth factor 21 (FGF21) prevented DN associated with the suppressing renal connective tissue growth factor expression, a key marker of renal fibrosis. Therefore, the effects of FGF21 on renal fibrosis in a DN mouse model and the underlying mechanisms were investigated in this study.MethodsType 1 diabetes mellitus was induced in C57BL/6J mice by intraperitoneal injections of multiple low doses of streptozotocin. Then, diabetic and non-diabetic mice were treated with or without FGF21 in the presence of pifithrin-α (p53 inhibitor) or 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride (10-DEBC) hydrochloride (Akt inhibitor) for 4 months.ResultsDN was diagnosed by renal dysfunction, hypertrophy, tubulointerstitial lesions, and glomerulosclerosis associated with severe fibrosis, all of which were prevented by FGF21. FGF21 also suppressed the diabetes-induced renal EMT in DN mice by negatively regulating transforming growth factor beta (TGF-β)-induced nuclear translocation of Smad2/3, which is required for the transcription of multiple fibrotic genes. The mechanistic studies showed that FGF21 attenuated nuclear translocation of Smad2/3 by inhibiting renal activity of its conjugated protein p53, which carries Smad2/3 into the nucleus. Moreover pifithrin-α inhibited the FGF21-induced preventive effects on the renal EMT and subsequent renal fibrosis in DN mice. In addition, 10-DEBC also blocked FGF21-induced inhibition of renal p53 activity by phosphorylation of mouse double minute-2 homolog (MDM2).ConclusionFGF21 prevents renal fibrosis via negative regulation of the TGF-β/Smad2/3-mediated EMT process by activation of the Akt/MDM2/p53 signaling pathway.

Silencing of long noncoding RNA XIST protects against renal interstitial fibrosis in diabetic nephropathy via microRNA-93-5p-mediated inhibition of CDKN1A.

Long noncoding RNAs (lncRNAs) have been reported to play an important role in diabetic nephropathy (DN). However, the molecular mechanism involved in this process remains poorly understood. Thus, the present study aimed to explore the function and molecular mechanism of dysregulated lncRNA X-inactive specific transcript (XIST) in DN. DN mouse models were established by streptozotocin treatment, and human renal tubular epithelial HK-2 cells were exposed to high glucose to produce an in vitro model. XIST was highly expressed in renal tissues of patients with DN, mice with DN, and high glucose-exposed HK-2 cells. To identify the interaction among XIST, miR-93-5p, and cyclin-dependent kinase inhibitor 1A (CDKN1A) and to analyze the functional significance of their interaction in renal interstitial fibrosis, we altered endogenous expression of XIST and miR-93-5p and CDKN1A. Dual-luciferase reporter assay results suggested that XIST was highly expressed in the kidney tissue of DN mice and high glucose-exposed HK-2 cells. XIST was identified to be a lncRNA that could bind to miR-93-5p, and CDKN1A was a target of miR-93-5p. Downregulated expression of XIST led to an increase in miR-93-5p expression, thereby decreasing CDKN1A and suppressing renal interstitial fibrosis in DN. Consistently, XIST knockdown reduced the expression of fibrosis markers (fibronectin, collagen type IV, and transforming growth factor-β1). Restoration of CDKN1A or decreasing miR-93-5p yielded a reversed effect on renal interstitial fibrosis. In conclusion, our study demonstrated that silenced XIST inducing miR-93-5p-dependent CDKN1A inhibition was beneficial for preventing renal interstitial fibrosis in DN, which may provide a future strategy to prevent the progression of DN.

Cyclo-RGD Truncated Polymeric Nanoconstruct with Dendrimeric Templates for Targeted HDAC4 Gene Silencing in Diabetic Nephropathy Mouse Model

Diabetic Nephropathy (DN), a chronic progressive kidney disease, is a major complication of diabetes mellitus. Dysregulation of histone deacetylases (HDACs) gene has been implicated in the pathogenesis of DN. Hence, the HDAC-inhibitors have emerged as a novel class of therapeutic agents in DN, however, the currently available HDAC4-inhibitors are mostly nonselective in nature as well as inhibit multiple HDACs. RNA interference of HDAC4 (HDAC4 siRNA) has shown immense promise, but the clinical translation has been impeded due to lack of a targeted, specific and in vivo applicable delivery modality. In the present investigation, we examined Cyclo(RGDfC) (cRGD) truncated polymeric nanoplex with dendrimeric templates for targeted HDAC4 Gene Silencing. The developed nanoplex exhibited enhance encapsulation of siRNA and offered superior protection against serum RNase nucleases degradation. The nanoplex was tested on podocytes (in vitro) wherein it showed selective binding to the αvβ3 integrin receptor, effective cellular uptake and significant in vitro gene silencing. The in vivo experiments showed remarkable suppression of the HDAC4 and inhibition in the progression of renal fibrosis in STZ induced DN C57BL/6 mice model. Histopathological and toxicological studies revealed non-significant abnormality/toxicity with the nanoplex. Conclusively, nanoplex was found as a promising tactic for targeted therapy of podocytes and could be extended for other kidney related ailments.

Bergenin impedes the generation of extracellular matrix in glomerular mesangial cells and ameliorates diabetic nephropathy in mice by inhibiting oxidative stress via the mTOR/β-TrcP/Nrf2 pathway

Bergenin, a plant polyphenol, has been reported to lower the blood glucose level and ameliorate kidney function in streptozotocin (STZ)-induced diabetic rats. Herein, its protective effect on diabetic nephropathy (DN) was explored in view of extracellular matrix (ECM) generation in glomerular mesangial cells. Glomerular mesangial cells were treated with high glucose, and Q-PCR as well as western blot were used to determine the expression of ECM. To establish the participation and role of mammalian target of rapamycin (mTOR) and nuclear factor erythroid-derived 2-related factor 2 (Nrf2) in ECM generation, a combination of l-leucine (activator of mTOR) and Nrf2 shRNA transfection were performed, respectively. Moreover, a DN model was established in mice using high-glucose/high-fat diet and STZ. Bergenin impeded the generation of TGF-β1 and ECM, decreased the levels of intracellular superoxide anion and hydrogen peroxide, and increased the activity of antioxidant enzymes in the glomerular mesangial cells (HBZY-1 and HRMC cells) treated with high glucose. The inhibition of ECM generation by bergenin was dependent on the down-regulation of oxidative stress as confirmed via a superoxide overexpression system. The activation of Nrf2 was required for bergenin to inhibit the oxidative stress and ECM generation. Moreover, bergenin was found to inhibit the phosphorylation of mTOR, which is located at the upstream of Nrf2. Bergenin did not interfere with the expression of Nrf2 mRNA and Keap1 (the classic degradation control factor of Nrf2), but markedly inhibited the protein expression of the β-TrcP, an effect which could be abolished by l-leucine. In DN model mice, l-leucine diminished the ability of bergenin to reduce the levels of superoxide anion, hydrogen peroxide and ECM, which contributed to the eradication of the ameliorative effect of bergenin on nephropathy. Bergenin can inhibit glucose-induced ECM production in glomerular mesangial cells through the down-regulation of oxidative stress via the mTOR/β-TrcP/Nrf2 pathway, and it might be a candidate drug for the prevention and treatment of DN.

Role of HMGN1 in epithelial-mesenchymal transition of kidney tubules in diabetic nephropathy mice

Objective This study aimed to investigate the renal expression change of high mobility of nucleosome binding protein 1 (HMGN1) in epithelia-mesenchymal transition (EMT) process, and to study the effect of HMGN1 on renal fibrosis in the diabetic nephropathy mice model. Methods 20 C57BL/6 mice were randomly divided into control group, model group, benazepril group, and insulin group. After 8 weeks of drug intervention, blood, urine and kidney tissue samples were taken from mice. The routine physiological and biochemical indexes were detected. Renal structure and fibrosis were detected by HE and Sirius red staining, respectively. Immunohistochemistry and in situ hybridization were used to investigate the protein and mRNA expression levels of HMGN1, CD68, F4/80, α-smooth muscle actin (α-SMA), and E-cadherin in renal tissue. Results Blood glucose, renal index, and urinary albumin to creatinine ratio (UACR) were significantly higher in the model group than those in the normal group. In the model group, HE staining showed glomerular hypertrophy and interstitial inflammatory cell infiltration, and Sirius red showed collagen deposition in the renal tissue. Compared with normal group, HMGN1, CD68, F4/80 positive cell counts and α-SMA protein expression were all increased, while E-cadherin protein expression was downregulated in the model group (all P<0.05). The above indexes were not improved significantly in the benazepril group. And after intervention of insulin, the expression levels of CD68 positive cell count and α-SMA protein were decreased and the expression levels of E-cadherin protein were increased compared with the model group (all P<0.05). The correlation analysis showed that the level of HMGN1 was correlated with CD68, F4/80, α-SMA, E-cadherin and collagen protein, while CD68 and f4/80 were correlated with α-SMA, collagen protein and blood glucose, respectively (all P<0.05). Conclusion HMGN1 is involved in the progression of diabetic nephropathy fibrosis, and its underlying mechanism might be related to the macrophage-mediated EMT process. Key words: High-mobility group nucleosome-binding protein 1; Epithelial-mesenchymal transition; Diabetic nephropathies; Renal fibrosis

SUN-299 TUBULAR ANGIOTENSIN II TYPE 1 RECEPTOR-ASSOCIATED PROTEIN AMELIORATES DIABETIC GLOMERULAR INJURIES IN STREPTOZOTOCIN-INDUCED MICE

Activated tissue renin-angiotensin system and enhanced infiltration of immune cells, especially macrophages, are critical factors in the development and progression of diabetic nephropathy. We previously identified that angiotensin II type 1 (AT1) receptor-associated protein (ATRAP) specifically binds to the AT1 receptor and suppresses the over-activation of AT1 receptor signaling. ATRAP is abundantly expressed in renal tubules, but rarely in glomeruli. ATRAP regulates blood pressure abnormality through the alteration of renal sodium handling in animal models of activated renin-angiotensin system. However, the role of ATRAP in the pathophysiology of progressive kidney diseases, such as diabetic nephropathy, is poorly elucidated. Our aim was to determine the protective role of ATRAP in a mouse model of diabetic nephropathy. To examine the effect of ATRAP deficiency on systemic and renal phenotypes under diabetic conditions, diabetes was induced in wild-type mice (WT) and systemic ATRAP knock-out mice (KO) by intraperitoneal injection of streptozotocin 55 mg/kg for 5 consecutive days. To examine the causal relationships between diabetic glomerular injury and numbers of alternatively-activated macrophages (M2Mφ) in the tubulointerstitium, which reduce inflammation and aid tissue repair, we performed adoptive transfer of ex vivo polarized M2Mφ into diabetic WT and KO at 8 and 16 weeks after streptozotocin injection. To examine whether tubular ATRAP regulates tubulointerstitial M2Mφ expression in diabetic nephropathy, diabetes was also induced in proximal tubule-specific ATRAP knock-out mice (PKO) and their littermate wild-type mice (CTL). All mice were sacrificed at 24 weeks after streptozotocin injection. The glycemic and blood pressure status of diabetic WT and KO mice were comparable throughout the study period. We confirmed increased urinary albumin excretion, increased glomerular area, decreased podocyte number (estimated by WT1 immunohistochemistry) and increased foot process width in diabetic KO compared with diabetic WT mice (Figure). The severity of tubulointerstitial injury was comparable in diabetic WT and KO mice. The number of CD206 positive-cells, a marker of M2Mφ, in the tubulointerstitium was increased in diabetic WT mice, but suppressed in diabetic KO. Adoptive transfer of M2Mφ almost completely diminished the glomerular injuries present in diabetic KO, which was equivalent to those in M2Mφ-treated diabetic WT. Moreover, the number of tubulointerstitial CD206 positive-cells in diabetic KO was restored by the adoptive transfer of M2Mφ. The number of tubulointerstitial CD206 positive-cells in diabetic PKO was significantly lower than in diabetic CTL mice. These results suggest that mainly renal tubular ATRAP plays a protective role in the progression of diabetic glomerular injury via enhancing the numbers of tubulointerstitial M2Mφ, indicating that ATRAP may be a novel therapeutic target for diabetic nephropathy.

496-P: A New Method to Image Sweet Taste Receptors in the Pathogenesis of Diabetic Nephropathy: Detection of 18F Lactisole with PET/CT

Previous studies showed that the expression of sweet taste receptors (STRs) was obviously downregulated in the pathogenesis of diabetic nephropathy (DN), suggesting that STRs may act as new therapeutic targets of DN. However, STRs are weakly expressed in kidneys, it is necessary to find a non-invasive method for detection of STRs distribution in the whole body and kidney. Lactisole, a broad-acting sweet antagonist, is commercially available and may be useful to assess the function and physiological role of the STRs. Whether the detection of isotope 18F labeled lactisole (18F-lactisole) with PET/CT can be used as a probe for imaging STRs expression differences is unclear. In this study, DN mouse model was induced by streptozotocin (STZ) in vivo, mouse glomerular mesangial cells (GMCs) and human proximal tubular cells (HK-2) were stimulated by high glucose in vitro, exogenous lactisole was used as an intervention reagent, 18F-lactisole was used as probe agent to evaluate the expression and distribution of STRs in the pathogenesis of DN. Our results showed that both the expression and distribution of STRs in mice were clearly imaged by PET/CT via intravenous injection of 18F-lactisole, 18F-lactisole signal in renal tissue was attenuated under the condition of diabetes, this trend persisted 2 hours after furosemide injection. Furthermore, exogenous lactisole significantly mitigated the effect of high glucose on STRs in vitro and vivo, which were detected by PET/CT or radioactive dosimeter via 18F-lactisole. These combined results support the hypothesis that detection of 18F-lactisole with PET/CT can be used as a new probe to image STRs and distinguish its expression level, suggesting a new method for non-invasive diagnosis of DN. Disclosure L. Zhou: None. W. Huang: None. N. Liu: None. X.M. Ma: None. M. Guo: None. Q. Chen: None. C. Gao: None. Y. Xu: None.

Whole transcriptome analysis of diabetic nephropathy in the db/db mouse model of type 2 diabetes.

Whole-transcriptome analysis using RNA sequencing (RNA-seq) affords broader insights about gene expression regulatory networks in diabetic nephropathy (DN). To better explore the molecular basis of DN, kidney tissue from db/db DN model mice and control mice weresubmitted to RNA-seq analysis. Thousands of long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) werefound to be significantly differentially expressed in the DN group relative to the control group. To research the regulatory mechanism of these lncRNAs and mRNAs, the integrated co-expression networks wereconstructed for 322 mRNAs and 27 lncRNAs that revealed significantly correlated expression patterns in DN. The potential roles of these co-expressed mRNAs wereclassified by Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses. The co-expression networks involved 27 lncRNAs interacting with 38 key mRNAs related to metabolic processes, including ND4/4L, Ndufa2/5, Ndufb4/7, Ndufs3, Uqcrc1, Aco2, Alad, Alas1, Alpl, Atp5j2, Coq5, Coq6, Cth, and CytB, all of which are highly related to encoding subunits of the mitochondrial complexes. Thus, mitochondrial dysfunction could result in renal function decline in DN. Seven dysregulated lncRNAs and nine dysregulated mRNAs in the DN model were confirmed by quantitative real-time polymerase chain reaction. The lncRNA-mRNA co-expression network provides novel evidence to support the contention that metabolic changes are associated with metabolic reprogramming in the kidneys, and that these changes playa critical role during the progression of DN.