Animal Models Of Diabetic Nephropathy Research Articles

Protective effect of paeoniflorin in diabetic nephropathy: A preclinical systematic review revealing the mechanism of action.

Paeoniflorin (PF), the main active glucoside of Paeonia Lactiflora, has many pharmacological activities, such as inhibition of vasodilation, hypoglycemia, and immunomodulation. Although the current evidence has suggested the therapeutic effects of PF on diabetic nephropathy (DN), its potential mechanism of action is still unclear. A systematic review and meta-analysis of the existing literature on paeoniflorin treatment in DN animal models was performed to evaluate the efficacy and mechanism of PF in DN animal models. The risk of bias in each study was judged using the CAMARADES 10-item quality checklist with the number of criteria met varying from 4 / 10 to 7 / 10, with an average of 5.44. From inception to July 2022, We searched eight databases. We used the Cochrane Collaboration's 10-item checklist and RevMan 5.3 software to assess the risk of bias and analyze the data. Three-dimensional dose/time-effect analyses were conducted to examine the dosage/time-response relations between PF and DN. Nine animal studies were systematically reviewed to evaluate the effectiveness of PF in improving animal models of DN. Meta-analysis data and intergroup comparisons indicated that PF slowed the index of mesangial expansion and tubulointerstitial injury, 24-h urinary protein excretion rate, expression of anti-inflammatory mediators (mRNA of MCP-1, TNF-α, iNOS, and IL-1 β), and expression of immune downstream factors (P-IRAK1, TIRF, P-IRF3, MyD88, and NF-κBp-p65). Furthermore, modeling methods, animal species, treatment duration, thickness of tissue sections during the experiment, and experimental procedures were subjected to subgroup analyses. The present study demonstrated that the reno-protective effects of PF were associated with its inhibition on macrophage infiltration, reduction of inflammatory mediators, and immunomodulatory effects. In conclusion, PF can effectively slow down the progression of DN and hold promise as a protective drug for the treatment of DN. Due to the low bioavailability of PF, further studies on renal histology in animals are urgently needed. We therefore recommend an active exploration of the dose and therapeutic time frame of PF in the clinic and in animals. Moreover, it is suggested to actively explore methods to improve the bioavailability of PF to expand the application of PF in the clinic.

Protective effects and possible mechanisms of catalpol against diabetic nephropathy in animal models: a systematic review and meta-analysis.

Aim of the Study: Rehmannia glutinosa is a core Chinese herbal medicine for the treatment of diabetes and diabetic nephropathy (DN). It has been used for the treatment of diabetes for over 1,000years. Catalpol is the main active compound in Rehmannia roots. Current evidence suggests that catalpol exhibits significant anti-diabetic bioactivity, and thus it has attracted increasing research attention for its potential use in treating DN. However, no studies have systematically evaluated these effects, and its mechanism of action remains unclear. This study aimed to evaluate the effects of catalpol on DN, as well as to summarize its possible mechanisms of action, in DN animal models. Materials and Methods: We included all DN-related animal studies with catalpol intervention. These studies were retrieved by searching eight databases from their dates of inception to July 2022. In addition, we evaluated the methodological quality of the included studies using the Systematic Review Center for Laboratory animal Experimentation (SYRCLE) risk-of-bias tool. Furthermore, we calculated the weighted standard mean difference (SMD) with 95% confidence interval (CI) using the Review Manager 5.3 software and evaluated publication bias using the Stata (12.0) software. A total of 100 studies were retrieved, of which 12 that included 231 animals were finally included in this review. Results: As compared to the control treatment, treatment with catalpol significantly improved renal function in DN animal models by restoring serum creatinine (Scr) (p = 0.0009) and blood urea nitrogen (BUN) (p < 0.00001) levels, reducing proteinuria (p < 0.00001) and fasting blood glucose (FBG) (p < 0.0001), improving kidney indices (p < 0.0001), and alleviating renal pathological changes in the animal models. In addition, it may elicit its effects by reducing inflammation and oxidative stress, improving podocyte apoptosis, regulating lipid metabolism, delaying renal fibrosis, and enhancing autophagy. Conclusion: The preliminary findings of this preclinical systematic review suggest that catalpol elicits significant protective effects against hyperglycemia-induced kidney injury. However, more high-quality studies need to be carried out in the future to overcome the methodological shortcomings identified in this review.

Probiotic Formula Ameliorates Renal Dysfunction Indicators, Glycemic Levels, and Blood Pressure in a Diabetic Nephropathy Mouse Model.

One-third of patients with end-stage chronic kidney disease (CKD) experience diabetic nephropathy (DN), which worsens the progression of renal dysfunction. However, preventive measures for DN are lacking. Lactobacillus acidophilus TYCA06, Bifidobacterium longum subsp. infantis BLI-02, and Bifidobacterium bifidum VDD088 probiotic strains have been demonstrated to delay CKD progression. This study evaluated their biological functions to stabilize blood-glucose fluctuations and delay the deterioration of renal function. The db/db mice were used to establish a DN animal model. This was supplemented with 5.125 × 109 CFU/kg/day (high dose) or 1.025 × 109 CFU/kg/day (low dose) mixed with probiotics containing TYCA06, BLI-02, and VDD088 for 8 weeks. Blood urea nitrogen (BUN), serum creatinine, blood glucose, and urine protein were analyzed. Possible mechanisms underlying the alleviation of DN symptoms by probiotic strains were evaluated through in vitro tests. Animal experiments revealed that BUN, serum creatinine, and blood glucose upon probiotic administration were significantly lower than in the control group. The rate of change of urine protein decreased significantly, and blood pressure, glucose tolerance, and renal fibrosis were improved. In vitro testing indicated that TYCA06 and BLI-02 significantly increased acetic acid concentration. TYCA06, BLI-02, and VDD088 were associated with better antioxidation, anti-inflammation, and glucose consumption activities relative to the control. A combination of the probiotics TYCA06, BLI-02, and VDD088 attenuated renal function deterioration and improved blood-glucose fluctuation in a diabetes-induced CKD mouse model.

Bone morphogenetic protein-7 attenuates pancreatic damage under diabetic conditions and prevents progression to diabetic nephropathy via inhibition of ferroptosis.

Approximately 30% of diabetic patients develop diabetic nephropathy, a representative microvascular complication. Although the etiological mechanism has not yet been fully elucidated, renal tubular damage by hyperglycemia-induced expression of transforming growth factor-β (TGF-β) is known to be involved. Recently, a new type of cell death by iron metabolism called ferroptosis was reported to be involved in kidney damage in animal models of diabetic nephropathy, which could be induced by TGF-β. Bone morphogenetic protein-7 (BMP7) is a well-known antagonist of TGF-β inhibiting TGF-β-induced fibrosis in many organs. Further, BMP7 has been reported to play a role in the regeneration of pancreatic beta cells in diabetic animal models. We used protein transduction domain (PTD)-fused BMP7 in micelles (mPTD-BMP7) for long-lasting in vivo effects and effective in vitro transduction and secretion. mPTD-BMP7 successfully accelerated the regeneration of diabetic pancreas and impeded progression to diabetic nephropathy. With the administration of mPTD-BMP7, clinical parameters and representative markers of pancreatic damage were alleviated in a mouse model of streptozotocin-induced diabetes. It not only inhibited the downstream genes of TGF-β but also attenuated ferroptosis in the kidney of the diabetic mouse and TGF-β-stimulated rat kidney tubular cells. BMP7 impedes the progression of diabetic nephropathy by inhibiting the canonical TGF-β pathway, attenuating ferroptosis, and helping regenerate diabetic pancreas.

The potential role of exosome-derived mesenchymal stem cells and Balanites aegyptiaca in diabetic nephropathy amelioration in rats.

The utilization of mesenchymal stem cell (MSC)-derived exosomes, which include numerous growth factors, cytokines, and microRNAs, is the primary aspect of the novel MSC activity models. The current research aims to: (i) identify the morphology of exosomes; (ii) determine exosomes secreted into MSCs conditioned cell culture medium; and (iii) perform a comprehensive characterization of isolated exosomes and elucidate their protective role in the diabetic nephropathy animal model. Ultracentrifugation was performed by utilizing the culture supernatant of MSCs. Transmission electron microscopy, nanoparticle tracking analysis, as well as Western blot, were utilized for isolated exosome characterization. The purified exosomes were used for in vivo implantation in a diabetic nephropathy animal model. The present research was carried out on 70 adult male albino rats weighing 180 to 200 grams. Rats were classified into seven groups: Group I: negative control group; Group II: diabetic nephropathy group; Group III: Balanites therapeutic group; Group IV: Balanites + MSCs therapeutic group; Group V: Balanites + exosome therapeutic group; Group VI: MSCs therapeutic group; and Group VII: exosome therapeutic group. By the end of the study period, total antioxidant capacity (TAC), malondialdehyde (MDA), and the histology of pancreatic tissue were assessed. Isolated exosomes with sizes ranging from 30 to 150 nm demonstrated the typical cup-shaped morphology. Additionally, exosome criteria were demonstrated by the exosome surface proteins CD81 and CD63, which were expressed by exosome marker proteins. Treatment with exosomes along with Balanites induced a significant reduction in pancreatic MDA with a substantial elevation in pancreatic TAC. Furthermore, treatment with exosomes and Balanites demonstrated normal pancreatic parenchyma and pancreatic lobules with normal pancreatic acini and acinar cells. These findings strongly suggest that ultracentrifugation is the most efficient tool for isolating exosomes. Also, these findings demonstrated that Balanites and exosomes had synergistic effects on one another, with more potent renoprotective activities in rats.

Comparison of Local Metabolic Changes in Diabetic Rodent Kidneys Using Mass Spectrometry Imaging.

Understanding the renal region-specific metabolic alteration in different animal models of diabetic nephropathy (DN) is critical for uncovering the underlying mechanisms and for developing effective treatments. In the present study, spatially resolved metabolomics based on air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was used to compare the local metabolic changes in the kidneys of HFD/STZ-induced diabetic rats and db/db mice. As a result, a total of 67 and 59 discriminating metabolites were identified and visualized in the kidneys of the HFD/STZ-induced diabetic rats and db/db mice, respectively. The result showed that there were significant region-specific changes in the glycolysis, TCA cycle, lipid metabolism, carnitine metabolism, choline metabolism, and purine metabolism in both DN models. However, the regional levels of the ten metabolites, including glucose, AMP, eicosenoic acid, eicosapentaenoic acid, Phosphatidylserine (36:1), Phosphatidylserine (36:4), Phosphatidylethanolamine (34:1), Phosphatidylethanolamine (36:4), Phosphatidylcholine (34:2), Phosphatidylinositol (38:5) were changed in reversed directions, indicating significant differences in the local metabolic phenotypes of these two commonly used DN animal models. This study provides comprehensive and in-depth analysis of the differences in the tissue and molecular pathological features in diabetic kidney injury in HFD/STZ-induced diabetic rats and db/db mice.

A Review of Medicinal Plants with Renoprotective Activity in Diabetic Nephropathy Animal Models

Diabetic nephropathy (DN), also recognized as diabetic kidney disease, is a kidney malfunction caused by diabetes mellitus. A possible contributing factor to the onset of DN is hyperglycemia. Poorly regulated hyperglycemia can damage blood vessel clusters in the kidneys, leading to kidney damage. Its treatment is difficult and expensive because its causes are extremely complex and poorly understood. Extracts from medicinal plants can be an alternative treatment for DN. The bioactive content in medicinal plants inhibits the progression of DN. This work explores the renoprotective activity and possible mechanisms of various medicinal plant extracts administered to diabetic animal models. Research articles published from 2011 to 2022 were gathered from several databases including PubMed, Scopus, ProQuest, and ScienceDirect to ensure up-to-date findings. Results showed that medicinal plant extracts ameliorated the progression of DN via the reduction in oxidative stress and suppression of inflammation, advanced glycation end-product formation, cell apoptosis, and tissue injury-related protein expression.

Sestrin2 Signaling Pathway Regulates Podocyte Biology and Protects against Diabetic Nephropathy.

Sestrin2 regulates cell homeostasis and is an upstream signaling molecule for several signaling pathways. Sestrin2 leads to AMP-activated protein kinase- (AMPK-) and GTPase-activating protein activity toward Rags (GATOR) 1-mediated inhibition of mammalian target of rapamycin complex 1 (mTORC1), thereby enhancing autophagy. Sestrin2 also improves mitochondrial biogenesis via AMPK/Sirt1/peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) signaling pathway. Blockade of ribosomal protein synthesis and augmentation of autophagy by Sestrin2 can prevent misfolded protein accumulation and attenuate endoplasmic reticulum (ER) stress. In addition, Sestrin2 enhances P62-mediated autophagic degradation of Keap1 to release nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 release by Sestrin2 vigorously potentiates antioxidant defense in diabetic nephropathy. Impaired autophagy and mitochondrial biogenesis, severe oxidative stress, and ER stress are all deeply involved in the development and progression of diabetic nephropathy. It has been shown that Sestrin2 expression is lower in the kidney of animals and patients with diabetic nephropathy. Sestrin2 knockdown aggravated diabetic nephropathy in animal models. In contrast, upregulation of Sestrin2 enhanced autophagy, mitophagy, and mitochondrial biogenesis and suppressed oxidative stress, ER stress, and apoptosis in diabetic nephropathy. Consistently, overexpression of Sestrin2 ameliorated podocyte injury, mesangial proliferation, proteinuria, and renal fibrosis in animal models of diabetic nephropathy. By suppressing transforming growth factor beta (TGF-β)/Smad and Yes-associated protein (YAP)/transcription enhancer factor 1 (TEF1) signaling pathways in experimental models, Sestrin2 hindered epithelial-mesenchymal transition and extracellular matrix accumulation in diabetic kidneys. Moreover, modulation of the downstream molecules of Sestrin2, for instance, augmentation of AMPK or Nrf2 signaling and inhibition of mTORC1, has been protective in diabetic nephropathy. Regarding the beneficial effects of Sestrin2 on diabetic nephropathy and its interaction with several signaling molecules, it is worth targeting Sestrin2 in diabetic nephropathy.

Locally transplanted human urine-induced nephron progenitor cells contribute to renal repair in mice kidney with diabetic nephropathy

Chronic Kidney Disease (CKD) is increasingly recognized as a global public health issue. Diabetic nephropathy (DN), also known as diabetic kidney disease, is a leading cause of CKD. Regenerative medicine strategy employing nephron progenitor cells (NPCs) is worthy of consideration as an alternative to shortage of donor organs for kidney transplantation. In previous study, we successfully generated induced NPCs (iNPCs) from human urine-derived cells that resembled human embryonic stem cell-derived NPCs. Here, we aimed to investigate the therapeutic potential of iNPCs in DN animal model. The results revealed the therapeutic effect of iNPCs as follows: (1) diminished glomerular hypertrophy, (2) reduced tubulointerstitial fibrosis, (3) low blood urea nitrogen, serum creatinine and albuminuria value, (4) decreased inflammation/fibrosis, (5) enhanced renal regeneration and (6) confirmed safety. This study demonstrates that human iNPCs have a therapeutic potential as a cell source for transplantation in patients with kidney diseases.

MiR-181a Improved Renal Inflammation by Targeting TNF-α in a Diabetic Nephropathy Animal Model

Introduction: Recent studies have elucidated that miR-181a plays a vital role in various inflammation-associated diseases; however, its role in the pathogenesis of diabetic nephropathy (DN) has not been elucidated clearly. Hence, we investigated the involvement of miR-181a in the pathogenesis of DN in patients and animal models. Methods: miR-181a levels were measured in the kidneys of DN patients and a DN mouse model (db/db mice) by RT-PCR. The interaction of miR-181a with the 3′-UTR of potential target transcripts was investigated using luciferase reporter assays. miR-181a-GFP-AAV and miR-181a inhibitor were used to overexpress or downregulate miR-181a in the kidney of db/db mice, respectively. The expression of inflammatory proteins and target genes was measured in the kidneys of the animals. Results: In DN patients and DN mouse models, expression analyses showed a significant decrease in miR-181a levels in the kidney. In addition, a negative linear correlation between miR-181a and proinflammatory gene (IL-1β and TNF-α) levels was observed in the kidneys of DN patients. Overexpression of miR-181a reduced IL-1β and TNF-α expression, whereas suppression of miR-181a worsened these changes, which was attenuated by combined treatment with TNF-α neutralizing antibody in db/db mice. Moreover, luciferase assays revealed that TNF-α is a direct target of miR-181a. Conclusion: Our data emphasized the anti-inflammatory actions of miR-181a by targeting TNF-α in the context of renal inflammation in DN. Hence, we concluded that miR-181a could be a potential therapeutic option for minimizing renal inflammation in DN.

Combination of mulberry leaf active components possessed synergetic effect on SD rats with diabetic nephropathy by mediating metabolism, Wnt/β-catenin and TGF-β/Smads signaling pathway

Ethnopharmacological relevanceMulberry leaf has attracted much attention due to its excellent curative effect on diabetes and its complications, whether the combination of its effective components have protective and synergistic effect on diabetic nephropathy (DN) in vivo remain unclear. Aim of the studyThe aim of this study was to investigate the protective and synergistic effect of the combination (MAF1:1 and MAF1:5) of mulberry leaf alkaloids (MA) and flavonoids extract (MF) on DN. Materials and methodsA step by step method consisted of network pharmacological prediction, animal in vivo validation and metabolic mechanism research was used to construct the multi-component-target-pathway network of mulberry leaf against DN. Firstly, the potential components and mechanism of mulberry leaf against DN was explored by network pharmacology analysis. Secondly, DN animal model was established to validate the anti-DN activity of these potential compounds. Thirdly, the metabolomics of serum and urine samples from animal experiments was analyzed to explore the anti-DN mechanism of these potential compounds. ResultsThe results of network pharmacology demonstrated that a total of 7 compounds detected in MA and MF exhibited anti-DN activity, their mechanism were strongly in connection with metabolic pathways, arachidonic acid metabolism, sphingolipid signaling pathway, etc. The results of animal experiment indicated that MAF1:1 and MAF1:5 significantly relieved metabolic disorders through regulating Wnt/β-catenin and TGF-β/Smads signaling pathway, just like MF or MA alone. Metabolomics suggested they could regulate 16 serum and 7 urine endogenous metabolites through arachidonic acid metabolism, phenylalanine metabolism and sphingolipid metabolism, thus alleviated DN. Significantly, MAF1:1 and MAF1:5 might possess synergistic effect considering their therapeutic effects on DN rats were superior to the single use of MA or MF. ConclusionsMAF1:1 and MAF1:5 possessed protective and synergistic effect on DN rats through multi-target and multi-pathways. These findings were of great scientific significance and application value to reveal the advantage of mulberry leaf in preventing and treating DN.

MiR-92d-3p suppresses the progression of diabetic nephropathy renal fibrosis by inhibiting the C3/HMGB1/TGF-β1 pathway.

The pathogenesis of diabetic nephropathy (DN) has not been fully elucidated. MicroRNAs (miRNAs) play an important role in the onset and development of DN renal fibrosis. Thus, the present study aimed to investigate the effect of miR-92d-3p on the progression of DN renal fibrosis. We used qRT-PCR to detect the expression levels of miR-92d-3p in the kidneys of patients with DN. Then, after transfecting lentiviruses containing miR-92d-3p into the kidneys of a DN mouse model and HK-2 cell line, we used qRT-PCR to detect the expression levels of miR-92d-3p, C3, HMGB1, TGF-β1, α-SMA, E-cadherin, and Col I. The expression levels of interleukin (IL) 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the HK-2 cells were detected through enzyme-linked immunosorbent assay (ELISA), and Western blotting and immunofluorescence were used in detecting the expression levels of fibronectin, α-SMA, E-cadherin, and vimentin. Results showed that the expression levels of miR-92d-3p in the kidney tissues of patients with DN and DN animal model mice decreased, and C3 stimulated HK-2 cells to produce inflammatory cytokines. The C3/HMGB1/TGF-β1 pathway was activated, and epithelial-to-interstitial transition (EMT) was induced in the HK-2 cells after human recombinant C3 and TGF-β1 protein were added. miR-92d-3p inhibited inflammatory factor production by C3 in the HK-2 cells and the activation of the C3/HMGB1/TGF-β1 pathway and EMT by C3 and TGF-β1. miR-92d-3p suppressed the progression of DN renal fibrosis by inhibiting the activation of the C3/HMGB1/TGF-β1 pathway and EMT.

Integrated metabolomics coupled with pattern recognition and pathway analysis to reveal molecular mechanism of cadmium-induced diabetic nephropathy.

Diabetic nephropathy (DN) is becoming a worldwide public health problem and its pathophysiological mechanism is not well understood. Emerging evidences indicated that cadmium (Cd), an industrial material but also an environmental toxin, may be involved in the development and progression of diabetes and diabetes-related kidney disease. However, the underlying mechanism is still unclear. Herein, a DN animal model was constructed by exposing to Cd, the metabolomic profiling of DN mice were obtained by using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), pattern recognition and pathway analysis were performed to screen potential biomarker. Moreover, western blotting was employed to verify the possible mechanism involved in the occurrence of Cd-induced DN. A total of 66 metabolites in serum have been screened out and identified as biomarkers, including free fatty acids, phospholipids, sphingomyelins, glycerides, and others. Significant differences were demonstrated between the metabolic profiles, including decreased levels of phospholipid and increased content of triglyceride, diacylglycerols, ceramide, lysophosphatidylcholine in Cd-induced DN mice compared with control. Protein expression level of p38 MAPK and Wnt/β-catenin were significantly increased. UPLC-Q-TOF/MS-based serum metabolomics coupled with pattern recognition methods and pathway analysis provide a powerful approach to identify potential biomarkers and is a new strategy to predict the underlying mechanism of disease caused by environmental toxicant.

MiR-30b-5p modulate renal epithelial-mesenchymal transition in diabetic nephropathy by directly targeting SNAI1

ObjectRenal tubulointerstitial fibrosis plays a significant role in the development of diabetic nephropathy (DN). SNAI1 is a main activator of epithelial-to-mesenchymal transition (EMT) in the process of fibrosis. This study aimed to investigate the effect of miR-30b-5p targeting SNAI1 on the EMT in DN. MethodsBioinformatics and miRNAs microarray analyses were used to predict the candidate miRNA targeting SNAI1, that is miR-30b-5p. The db/db mice was as DN animal model and renal tissues of mice were stained with PAS. The miR-30b-5p expression in mouse and human renal tissue were examined by quantitative RT-PCR (qRT-PCR) and fluorescence in situ hybridization (FISH), while SNAI1 expression was determined by qRT-PCR and immunohistochemistry. Luciferase reporter gene assay was used to confirm miR-30b-5p directly target 3′-UTR of the SNAI1 mRNA. In vitro, HK-2 cells were treated with high glucose to establish hyperglycemia cell model and transfected with miR-30b-5p mimics to overexpress miR-30b-5p. Expression of miR-30b-5p, SNAI1 and EMT related indicators (E-cadherin, a-SMA and Vimentin) in HK-2 cells under different treatments were determined by qRT-PCR and/or western-blot. In addition, immunofluorescence was performed to evaluate a-SMA expression in HK-2 cells under different treatments. ResultsBioinformatics analyses revealed miR-30b-5p had complementary sequences with SNAI1 mRNA and the seed region of miR-30b-5p was conserved in human and a variety of animals, including mice. Microarray analysis showed miR-30b expression decreased in DN mice, which was further verified in db/db mice by qRT-PCR and in human DN by FISH. Contrary to miR-30b-5p, SNAI1 expression level was upregulated in db/db mice. Correlation analysis suggested SNAI1 mRNA level was negatively with miR-30b-5p level in renal tissue of db/db mice. Luciferase reporter gene assay confirmed miR-30b-5p directly targeted SNAI1 mRNA. In high glucose induced HK-2 cells, expression levels of miR-30b-5p and E-cadherin were decreased, while SNAI1, a-SMA and Vimentin were increased. Overexpression miR-30b-5p in high glucose induced HK-2 cells could reverse that phenomenon to some extent. ConclusionThese findings suggest that miR-30b-5p play a protective role by targeting SNAI1 in renal EMT in DN.

Inhibition of notch signalling and mesangial expansion by combined glucagon like peptide-1 agonist and crocin therapy in animal model of diabetic nephropathy

Diabetic nephropathy (DN) is one of the devastating complications in diabetes mellitus (DM). Glucagon-like peptide-1 (GLP-1) is one of the incretins secreted from L cells in the intestine. Crocin (a carotenoid component of saffron) has antioxidants properties. We investigated the renal effects of Exendin-4 as a GLP-1 agonist and Crocin in DN. Thirty male rats were divided into five groups: control, type II DM, type II DM + Exendin-4, type II DM + Crocin ‏ and type II DM + Exendine-4 + Crocin. At the end of the experimental period, systolic and diastolic blood pressures were measured, and GFR was calculated. Blood and urine samples were collected for biochemical analysis. Tissue samples were collected from the kidney for histological examination and biochemical measurements of protein expression. Treatment with GLP-1 agonist or Crocin caused a significant improvement in renal function. Better results were achieved with simultaneous administration of both drugs with inhibition of notch signalling pathway and the related proteins.

Metformin attenuates renal interstitial fibrosis through upregulation of Deptor in unilateral ureteral obstruction in rats

Renal interstitial fibrosis (RIF) is a common pathological process that accompanies chronic kidney disease (CKD) and that progresses to end-stage renal failure (ESRD). Accumulating evidence has revealed that persistent mammalian target of rapamycin (mTOR) activation in kidneys is closely associated with the occurrence and progression of CKD. The DEP domain-containing mTOR interacting protein (Deptor) is an endogenous negative regulator of mTOR. Metformin can attenuate renal fibrosis in an animal model of diabetic nephropathy. Previous studies demonstrated that metformin can attenuate renal fibrosis in several models of CKD. However, the precise mechanisms of this effect are not well understood. The present study aimed to examine the mechanism of action of metformin on unilateral ureteral obstruction (UUO)-induced RIF in rats in vivo. Sprague-Dawley rats were randomly divided into a sham-operated group, three UUO groups examined at different time points (3, 7 and 14 days after UUO surgery), and three metformin-treated groups, treated with three different concentrations of metformin. The metformin-treated groups were administered metformin orally every day for 14 consecutive days following surgery. The protein expression levels of Deptor, α-smooth muscle actin (α-SMA), phosphorylated (p-)mTOR, p-ribosomal protein S6 kinase (p-p70S6K) and CD68 were assessed. The present results suggested that, following UUO, there was a significant reduction of Deptor expression, and an increase in collagen deposition in the extracellular matrix over time, accompanied by an increased expression of several proteins including CD68, α-SMA, p-mTOR and p-p70S6K. Notably, metformin treatment reversed these effects. In conclusion, the present results suggested that metformin attenuated RIF of UUO rats, and the mechanism of action was found to be associated with the increase in Deptor expression and inhibition of the mTOR/p70S6K pathway in the kidneys of UUO rats.

Circ-AKT3 inhibits the accumulation of extracellular matrix of mesangial cells in diabetic nephropathy via modulating miR-296-3p/E-cadherin signals.

Diabetic nephropathy is a leading cause of end‐stage renal disease globally. The vital role of circular RNAs (circRNAs) has been reported in diabetic nephropathy progression, but the molecular mechanism linking diabetic nephropathy to circRNAs remains elusive. In this study, we investigated the significant function of circ‐AKT3/miR‐296‐3p/E‐cadherin regulatory network on the extracellular matrix accumulation in mesangial cells in diabetic nephropathy. The expression of circ‐AKT3 and fibrosis‐associated proteins, including fibronectin, collagen type I and collagen type IV, was assessed via RT‐PCR and Western blot analysis in diabetic nephropathy animal model and mouse mesangial SV40‐MES13 cells. Luciferase reporter assays were used to investigate interactions among E‐cadherin, circ‐AKT3 and miR‐296‐3p in mouse mesangial SV40‐MES13 cells. Cell apoptosis was evaluated via flow cytometry. The level of circ‐AKT3 was significantly lower in diabetic nephropathy mice model group and mouse mesangial SV40‐MES13 cells treated with high‐concentration (25 mmol/L) glucose. In addition, circ‐AKT3 overexpression inhibited the level of fibrosis‐associated protein, such as fibronectin, collagen type I and collagen type IV. Circ‐AKT3 overexpression also inhibited the apoptosis of mouse mesangial SV40‐MES13 cells treated with high glucose. Luciferase reporter assay and bioinformatics tools identified that circ‐AKT3 could act as a sponge of miR‐296‐3p and E‐cadherin was the miR‐296‐3p direct target. Moreover, circ‐AKT3/miR‐296‐3p/E‐cadherin modulated the extracellular matrix of mouse mesangial cells in high‐concentration (25 mmol/L) glucose, inhibiting the synthesis of related extracellular matrix protein. In conclusion, circ‐AKT3 inhibited the extracellular matrix accumulation in diabetic nephropathy mesangial cells through modulating miR‐296‐3p/E‐cadherin signals, which might offer novel potential opportunities for clinical diagnosis targets and therapeutic biomarkers for diabetic nephropathy.

P0336INHIBITION C-C CHEMOKINE RECEPTOR TYPE 2 (CCR2) ON ACTIVATED KIDNEY PARIETAL EPITHELIAL CELLS REVEALS A NEW THERAPEUTIC APPROACH FOR CHRONIC KIDNEY DISEASES

Abstract Background and Aims We have recently demonstrated that small molecule inhibitors of CCR2 rapidly reduced proteinuria and preserved renal structure in animal models of diabetic nephropathy (DN) and focal segmental glomerular sclerosis (FSGS). Similar beneficial effects of CCR2 inhibition were also observed in DN patients in a large diabetic nephropathy Phase 2 clinical trial. Despite its rapid and robust effects in these kidney diseases, the mechanism by which CCR2 inhibition affords such renal protection remains unclear. CCR2 is well-known to be expressed on cells of the immune system, where it mediates inflammation and immunological functions by controlling the trafficking of monocytes and T cells. However, immune cell expression of CCR2 may not adequately explain the rapid pharmacologic benefits of CCR2 inhibitors in DN and FSGS because these diseases do not exhibit appreciable kidney inflammation. To better understand the protective mechanism(s) of CCR2 inhibition in renal diseases, we have used a variety of in vivo nephropathy models to determine if CCR2 is expressed on kidney parenchymal cells. Our results suggest that a subset of CD45 negative, renal parietal epithelial cells express CCR2. Method To characterize CCR2 expression in the kidney we used CCR2 RFP heterozygous mice (CCR2RFP/+) in which one copy of CCR2 was replaced by red fluorescent protein (RFP), whose expression is under the control of the endogenous CCR2 promoter. This approach provided a highly sensitive method for CCR2 detection by both flow cytometry and immunofluorescence, and compensated for the lack of validated, commercially available anti-CCR2 antibodies. Enriched glomerular cells were obtained by a non-enzymatic disruption of kidneys, followed by centrifugation at low speed (80gs), and sieving through 300µm and 150µm meshes. The Adriamycin (ADR)-nephropathy model with CCR2 RFP heterozygous mice was used to determine CCR2 expression on renal cells. 5/6 nephrectomy models of FSGS, as well as the db/db diabetic nephropathy model were used to access the change of activated parietal epithelial cells and proteinuria, both in the presence and absence of a CCR2 inhibitor in the setting of kidney injury. Results We observed dramatic increases in the number of activated parietal epithelial cells (PECs, identified as CD133+CD24+CD44hi cells by flow cytometry) in all three renal disease models. In the ADR nephropathy model, we found that in 30-40% of activated PEC cells within an enriched population of glomerular cells expressed CCR2/RFP. CD44hi RFP+ increased much more dramatically than CD44hi RFP- cells in response to ADR, and this was highly correlated with proteinuria, suggesting that CCR2 plays an important role in PEC cell activation and kidney pathology in ADR induced kidney injury. Furthermore, we found that blocking CCR2 with the CCR2-specific antagonist CCX872 significantly reduced both proteinuria and the number of activated PECs in the 5/6 nephropathy and db/db diabetic nephropathy models. Conclusion This is the first report that CCR2 is present on a subset of activated renal PECs. This population is markedly upregulated during kidney injury in a variety of murine models of nephropathy. Changes in the abundance CCR2+ activated PECs in the glomerular enriched preparations in response to CCR2 inhibition suggests that CCR2 antagonism may represent a novel approach for the treatment of chronic kidney disease.

Connexin 43‑autophagy loop in the podocyte injury of diabetic nephropathy.

The reduction of podocyte injury is a key strategy in controlling proteinuria, which is the main early clinical manifestation of diabetic nephropathy (DN). Impaired autophagic flux is the primary mechanism responsible for podocyte injury in DN. The aim of the present study was to elucidate the effect of connexin 43 (Cx43) on impaired autophagic flux in podo-cyte injury and to explore its molecular mechanism of action in DN. Sprague-Dawley rats were administered streptozocin (STZ) to construct a DN animal model. Podocytes were incubated in media containing either buffer or high glucose (HG; 30 mM) for variable time periods. The podocytes were then examined and the mechanism of injury was investigated using an Annexin V/PI assay, immunofluorescence staining, western blotting, and RNA interference. In vivo, STZ-induced DN rats with or without Cx43 knockdown were established to observe the role of Cx43 in autophagic flux and podocyte injury. We observed that HG induced podocyte injury, accompanied by increases in Cx43 expression and impaired autophagic flux, as evidenced by the accumulation of LC3II/LC3I and p62. Interestingly, the silencing of Cx43 expression ameliorated autophagic flux impairment and reduced podocyte injury via suppression of the mammalian target of rapamycin pathway. Furthermore, impaired autophagic flux also blocked the degradation of Cx43. In vitro studies indicated that higher numbers of Annexin V/PI-positive podocytes, impaired autophagic flux and increased Cx43 expression were observed in HG-induced podocyte injury relative to the control group. The pathogenic effect of Cx43 on impaired autophagic flux and podocyte injury was also confirmed by Cx43 knockdown. The present study provided preliminary evidence indicating that the interdependence of Cx43 and impaired autophagic flux represents a novel mechanism of podocyte injury in DN. Hence, the Cx43-autophagy loop is a potentially relevant therapeutic target for the treatment of DN.

Fourteen-Day Study of Praliciguat, a Soluble Guanylate Cyclase Stimulator, in Patients with Diabetes and Hypertension

Background: Praliciguat (IW-1973), a soluble guanylate cyclase stimulator, has been shown to augment nitric oxide (NO) signaling and reduce fasting plasma glucose and proteinuria in an animal model of diabetic nephropathy. In healthy adults, repeated oral doses of 15-40 mg were tolerated and lowered blood pressure (BP). Methods: This Phase 2a, double-blind, placebo (PBO)-controlled study assessed the effects of oral praliciguat in 26 patients with type 2 diabetes mellitus (T2DM) and hypertension on stable glycemic and BP-lowering therapy. Most (69%) were also on statins. Two praliciguat regimens were tested: (1) 40 mg QD for days 1-14 (N=10), and (2) 20 mg BID for days 1-7 then 40 mg QD for days 8-14 (N=10). Assessments included serum chemistry, fasting plasma glucose, BP and heart rate, platelet function, reactive hyperemia index (RHI), plasma biomarkers, and adverse events (AEs). Results: Results were similar for both praliciguat regimens and are here combined. After 14 days of treatment, praliciguat-treated patients had notable differences in least squares mean change from baseline (95% CI) relative to PBO in 24-hour mean arterial pressure [-4.7 mmHg (-10, 0.8)], heart rate [2.9 bpm (-0.7, 6.5)], fasting glucose [-13 mg/dL (-32, 7)], HOMA-IR [-26 (-58, 7)], total serum cholesterol [-26 mg/dL (-44, -7)], and LDL cholesterol [-20 mg/dL (-37, -3)]. Praliciguat had no effect on platelet function or RHI. Among the exploratory biomarkers tested, levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine were lower in praliciguat vs. PBO recipients. Both praliciguat dose regimens were generally well tolerated. Except for one serious AE of upper gastrointestinal hemorrhage, AEs in praliciguat-treated patients were mild. Conclusion: In this initial trial, praliciguat had beneficial effects on BP and metabolic parameters in T2DM patients with hypertension on standard therapies. Further clinical investigation of praliciguat in T2DM patients is warranted. Disclosure J.P. Hanrahan: Employee; Self; Ironwood. J.D. Wakefield: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. P.J. Wilson: None. P. Miller: None. J. Chickering: None. L. Morrow: Stock/Shareholder; Self; ProSciento. Employee; Spouse/Partner; Eli Lilly and Company. M.L. Hall: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G. Milne: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. Employee; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. A.T. Profy: Other Relationship; Self; Ironwood Pharmaceuticals.