Glomeruli Of Diabetic Mice Research Articles

Long non-coding RNA lncMGC mediates the expression of TGF-β-induced genes in renal cells via nucleosome remodelers.

Background: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play key roles in diabetic kidney disease (DKD). The miR-379 megacluster of miRNAs and its host transcript lnc-megacluster (lncMGC) are regulated by transforming growth factor-β (TGF-β), increased in the glomeruli of diabetic mice, and promote features of early DKD. However, biochemical functions of lncMGC are unknown. Here, we identified lncMGC-interacting proteins by in vitro-transcribed lncMGC RNA pull down followed by mass spectrometry. We also created lncMGC-knockout (KO) mice by CRISPR-Cas9 editing and used primary mouse mesangial cells (MMCs) from the KO mice to examine the effects of lncMGC on the gene expression related to DKD, changes in promoter histone modifications, and chromatin remodeling. Methods: In vitro-transcribed lncMGC RNA was mixed with lysates from HK2 cells (human kidney cell line). lncMGC-interacting proteins were identified by mass spectrometry. Candidate proteins were confirmed by RNA immunoprecipitation followed by qPCR. Cas9 and guide RNAs were injected into mouse eggs to create lncMGC-KO mice. Wild-type (WT) and lncMGC-KO MMCs were treated with TGF-β, and RNA expression (by RNA-seq and qPCR) and histone modifications (by chromatin immunoprecipitation) and chromatin remodeling/open chromatin (by Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) were examined. Results: Several nucleosome remodeling factors including SMARCA5 and SMARCC2 were identified as lncMGC-interacting proteins by mass spectrometry, and confirmed by RNA immunoprecipitation-qPCR. MMCs from lncMGC-KO mice showed no basal or TGF-β-induced expression of lncMGC. Enrichment of histone H3K27 acetylation and SMARCA5 at the lncMGC promoter was increased in TGF-β-treated WT MMCs but significantly reduced in lncMGC-KO MMCs. ATAC peaks at the lncMGC promoter region and many other DKD-related loci including Col4a3 and Col4a4 were significantly lower in lncMGC-KO MMCs compared to WT MMCs in the TGF-β-treated condition. Zinc finger (ZF), ARID, and SMAD motifs were enriched in ATAC peaks. ZF and ARID sites were also found in the lncMGC gene. Conclusion: lncMGC RNA interacts with several nucleosome remodeling factors to promote chromatin relaxation and enhance the expression of lncMGC itself and other genes including pro-fibrotic genes. The lncMGC/nucleosome remodeler complex promotes site-specific chromatin accessibility to enhance DKD-related genes in target kidney cells.

LRH-1 activation alleviates diabetes-induced podocyte injury by promoting GLS2-mediated glutaminolysis.

Alteration of metabolic phenotype in podocytes directly contributes to the development of albuminuria and renal injury in conditions of diabetic kidney disease (DKD). This study aimed to identify and evaluate liver receptor homologue-1 (LRH-1) as a possible therapeutic target that alleviates glutamine (Gln) metabolism disorders and mitigates podocyte injury in DKD. Metabolomic and transcriptomic analyses were performed to characterize amino acid metabolism changes in the glomeruli of diabetic mice. Next, Western blotting, immunohistochemistry assays, and immunofluorescence staining were used to detect the expression of different genes in vitro and in vivo. Furthermore, Gln and glutamate (Glu) content as well as ATP generation were examined. A decrease in LRH-1 and glutaminase 2 (GLS2) expression was detected in diabetic podocytes. Conversely, the administration of LRH-1 agonist (DLPC) upregulated the expression of GLS2 and promoted glutaminolysis, with an improvement in mitochondrial dysfunction and less apoptosis in podocytes compared to those in vehicle-treated db/db mice. Our study indicates the essential role of LRH-1 in governing the Gln metabolism of podocytes, targeting LRH-1 could restore podocytes from diabetes-induced disturbed glutaminolysis in mitochondria.

TDAG51-Deficiency Podocytes are Protected from High-Glucose-Induced Damage Through Nrf2 Activation via the AKT-GSK-3β Pathway.

T cell death-associated gene 51 (TDAG51) has been implicated in the development of various pathological conditions. However, whether TDAG51 plays a role in diabetic renal disease remains unknown. The current work investigated the possible function of TDAG51 in diabetic renal disease using high-glucose (HG)-stimulated podocytes in vitro. The elevation of TDAG51 was observed in podocytes in response to HG exposure and the glomeruli of diabetic mice. The siRNAs targeting TDAG51 were applied to deplete TDAG51 in HG-stimulated podocytes. Crucially, TDAG51 deficiency was sufficient to decrease the apoptosis, oxidative stress, and inflammation caused by HG. Mechanically, the inhibition of TDAG51 was capable of enhancing the activation of nuclear factor E2-related factor 2 (Nrf2) associated with the upregulation of AKT-glycogen synthase kinase-3β (GSK-3β) pathway. The reduction of AKT abolished the activation of Nrf2 elicited by TDAG51 deficiency. Additionally, the reduction of Nrf2 diminished the anti-HG injury effect elicited by TDAG51 deficiency. Overall, these data demonstrate that TDAG51 deficiency defends against HG-induced podocyte damage through Nrf2 activation by regulating AKT-GSK-3β pathway. This study suggests that TDAG1 may have a potential role in diabetic renal disease by affecting HG-induced podocyte damage.

POS-328 SUPPRESSION OF ER-ASSOCIATED DEGRADATION BY INTRAGLOMERULAR CROSS-COMMUNICATION BETWEEN MESANGIAL CELLS AND PODOCYTES CAUSES PODOCYTE INJURY IN DIABETIC KIDNEY DISEASE

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single cell analyses, the molecular mechanisms and its role in disease progression still remain elusive. Furthermore, our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. Thus, we hypothesized that the cross-communication between MCs and podocytes may affect the ER stress responses of these cells and thereby participate in the progression of DKD, especially in podocyte injury.

Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes.

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2−/− mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.

Regulation of Podocyte Injury by CircHIPK3/FUS Complex in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is a major microvascular complication of diabetes mellitus and is one of the leading causes of end-stage kidney disease. Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that play important roles in various diseases, yet their roles in DKD are poorly understood. CircRNA HIPK3 (circHIPK3), a highly conserved circRNA, is closely related to various cellular functions, including cell proliferation and apoptosis. The association between circHIPK3 and diabetic complications has been well demonstrated in multiple previous studies. However, the role of circHIPK3 in podocyte injury in DKD remains unclear. Herein, we discovered that circHIPK3 expression is markedly elevated in cultured podocytes under high-glucose (HG) conditions and glomeruli of diabetic mice, which is closely associated with podocyte injury in DKD. Functionally, lentivirus-mediated knockdown of circHIPK3 dramatically suppresses HG-induced podocyte apoptosis in vitro. Therapeutically, silencing circHIPK3 by adeno-associated virus-mediated RNA interference ameliorates podocyte injury and albuminuria in STZ-induced diabetic mice. Mechanistically, circHIPK3 facilitates the enrichment of fused in sarcoma (FUS) on the ectodysplasin A2 receptor (EDA2R) promoter, resulting in the upregulation of EDA2R expression and activation of apoptotic signaling. Taken together, these results indicate circHIPK3/FUS/EDA2R axis as a therapeutic target for podocyte injury and DKD progression.

MCC Regulator of WNT Signaling Pathway (MCC) Is a Podocyte Essential Gene.

Podocytes are an integral part of the glomerular filtration barrier. Many genes are already known to be essential for podocyte survival, structure and function, but there are more podocyte essential genes to be identified. By single-cell RNA-seq of mouse podocytes, we detected the expression of gene encoding MCC regulator of WNT signaling pathway (MCC) in majority of the podocytes and speculated that MCC is essential for podocytes. We confirmed MCC expression in mouse podocytes and further showed its expression in human podocytes. To experimentally prove the essentiality of MCC for podocytes, we knocked down MCC in cultured podocytes and found marked morphological change of cell shape, cytoskeletal F-actin stress fiber disruption, increased apoptosis, and downregulation of podocyte essential genes, CD2AP and WT1, demonstrating that MCC is essential for podocytes. Since MCC has been implicated in cell cycle and β-catenin signaling, we examined the expression of cell cycle related genes and activity of β-catenin in the MCC knockdown podocytes, but did not find significant changes. To further explore the mechanism underlying the role of MCC in podocytes, we performed RNA-sequencing and bioinformatics analysis of MCC knockdown podocytes and found a significant enrichment of the regulated genes in lamellipodia formation. Consistently, we found that MCC is present in lamellipodia and MCC knockdown resulted in loss of lamellipodia in the cells. Lastly, we found that MCC was downregulated in podocytes treated with puromycin aminonucleosides and in glomeruli of diabetic mice and FSGS patients, implicating MCC is involved in the development of podocytopathy and proteinuria. In conclusion, MCC is potentially essential for podocytes and its downregulation may be involved in podocytopathy.

TRIM32 Inhibition Attenuates Apoptosis, Oxidative Stress, and Inflammatory Injury in Podocytes Induced by High Glucose by Modulating the Akt/GSK-3β/Nrf2 Pathway.

Hyperglycemia-induced oxidative stress in podocytes exerts a major role in the pathological process of diabetic nephropathy. Tripartite motif-containing protein 32 (TRIM32) has been reported to be a key protein in the modulation of cellular apoptosis and oxidative stress under various pathological processes. However, whether TRIM32 participates in the regulation of high glucose (HG)-induced injury in podocytes has not been investigated. This work aimed to assess the possible role of TRIM32 in mediating HG-induced apoptosis, oxidative stress, and inflammatory response in podocytes in vitro. Our results showed a marked increase in TRIM32 expression in HG-exposed podocytes and the glomeruli of diabetic mice. Loss-of-function experiments showed that TRIM32 knockdown improves the viability of HG-stimulated podocytes and suppresses HG-induced apoptosis, oxidative stress, and inflammatory responses in podocytes. Further investigation revealed that TRIM32 inhibition enhances the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, which is associated with the modulation of the Akt/glycogen synthase kinase-3β (GSK-3β) axis in podocytes following HG exposure. However, Akt suppression abrogated the TRIM32 knockdown-mediated activation of Nrf2 in HG-exposed podocytes. Nrf2 knockdown also markedly abolished the protective effects induced by TRIM32 inhibition o in HG-exposed podocytes. In summary, this work demonstrated that TRIM32 inhibition protects podocytes from HG-induced injury by potentiating Nrf2 signaling through modulation of Akt/GSK-3β signaling. The findings reveal the potential role of TRIM32 in mediating podocyte injury during the progression of diabetic nephropathy.

POS-356 Suppression of ER-associated degradation by intraglomerular crosstalk between mesangial cells and podocytes causes podocyte injury in diabetic kidney disease

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single-nucleus RNA-sequencing analyses, the molecular mechanisms and role in disease progression remain elusive. Furthermore, our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology.

Tangeretin Ameliorates Glucose-Induced Podocyte Injury through Blocking Epithelial to Mesenchymal Transition Caused by Oxidative Stress and Hypoxia.

Podocyte injury inevitably results in leakage of proteins from the glomerular filter and is vital in the pathogenesis of diabetic nephropathy (DN). The underlying mechanisms of podocyte injury facilitate finding of new therapeutic targets for DN treatment and prevention. Tangeretin is an O-polymethoxylated flavone present in citrus peels with anti-inflammatory and antioxidant properties. This study investigated the renoprotective effects of tangeretin on epithelial-to-mesenchymal transition-mediated podocyte injury and fibrosis through oxidative stress and hypoxia caused by hyperglycemia. Mouse podocytes were incubated in media containing 33 mM glucose in the absence and presence of 1–20 μM tangeretin for up to 6 days. The in vivo animal model employed db/db mice orally administrated with 10 mg/kg tangeretin for 8 weeks. Non-toxic tangeretin inhibited glucose-induced expression of the mesenchymal markers of N-cadherin and α-smooth muscle actin in podocytes. However, the reduced induction of the epithelial markers of E-cadherin and P-cadherin was restored by tangeretin in diabetic podocytes. Further, tangeretin enhanced the expression of the podocyte slit diaphragm proteins of nephrin and podocin down-regulated by glucose stimulation. The transmission electron microscopic images revealed that foot process effacement and loss of podocytes occurred in diabetic mouse glomeruli. However, oral administration of 10 mg/kg tangeretin reduced urine albumin excretion and improved foot process effacement of diabetic podocytes through inhibiting loss of slit junction and adherenes junction proteins. Glucose enhanced ROS production and HIF-1α induction in podocytes, leading to induction of oxidative stress and hypoxia. Similarly, in diabetic glomeruli reactive oxygen species (ROS) production and HIF-1α induction were observed. Furthermore, hypoxia-evoking cobalt chloride induced epithelial-to-mesenchymal transition (EMT) process and loss of slit diaphragm proteins and junction proteins in podocytes, which was inhibited by treating submicromolar tangeretin. Collectively, these results demonstrate that tangeretin inhibited podocyte injury and fibrosis through blocking podocyte EMT caused by glucose-induced oxidative stress and hypoxia.

TGFβ acts through PDGFRβ to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression

Interaction of transforming growth factor-β (TGFβ)-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of the TGFβ receptor involved in the noncanonical signaling is unknown. Here, we show that TGFβ increased the catalytic loop phosphorylation of platelet-derived growth factor receptor β (PDGFRβ), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGFβ increased phosphorylation of the PI 3-kinase-interacting Tyr-751 residue of PDGFRβ, thus activating Akt. Inhibition of PDGFRβ using a pharmacological inhibitor and siRNAs blocked TGFβ-stimulated phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active myristoylated Akt reversed the siPDGFRβ-mediated inhibition of mTORC1 activity; however, co-expression of the phospho-deficient mutant of PRAS40 inhibited the effect of myristoylated Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFRβ in mesangial cells. Additionally, we demonstrate that PDGFRβ-initiated phosphorylation of PRAS40 is required for TGFβ-induced mesangial cell hypertrophy and fibronectin and collagen I (α2) production. Increased activating phosphorylation of PDGFRβ is also associated with enhanced TGFβ expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rats, respectively. Thus, pursuing TGFβ noncanonical signaling, we identified how TGFβ receptor I achieves mTORC1 activation through PDGFRβ-mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFRβ in TGFβ-driven renal fibrosis.

Suppressed ER-associated degradation by intraglomerular cross talk between mesangial cells and podocytes causes podocyte injury in diabetic kidney disease.

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single-nucleus RNA sequencing analyses, the molecular mechanisms and role in disease progression remain elusive. Our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. In vitro experiments revealed the suppression of the ER-associated degradation (ERAD) pathway and induction of apoptosis in podocytes that were stimulated with the supernatant of MCs cultured in high glucose conditions. In diabetic mice, ERAD inhibition resulted in exacerbated albuminuria, increased apoptosis in podocytes, and reduced nephrin expression associated with the downregulation of ERAD-related biomolecules. Flow cytometry analysis of podocytes isolated from MafB (a transcription factor known to be expressed in macrophages and podocytes)-GFP knock-in mice revealed that ERAD inhibition resulted in decreased nephrin phosphorylation. These findings suggest that an intraglomerular cross talk between MCs and podocytes can inhibit physiological ERAD processes and suppress the phosphorylation of nephrin in podocytes, which thereby lead to podocyte injury under diabetic conditions. Therapeutic intervention of the ERAD pathway through the cross talk between these cells is potentially a novel strategy for DKD.

Altered DNA methylation of TRIM13 in diabetic nephropathy suppresses mesangial collagen synthesis by promoting ubiquitination of CHOP.

BackgroundMesangial collagen synthesis in renal glomeruli contributes to the pathogenesis of diabetic nephropathy (DN) which is one of the most serious complications of diabetes mellitus. However, the underlying mechanism of mesangial collagen synthesis is largely unknown.MethodsThe differential expression of CHOP and TRIM13 which is a well-defined E3 ubiquitin ligase was compared in renal biopsy samples from DN/normal renal tissues, in isolated glomeruli of diabetic/control mice, as well as in high glucose (HG) or TGF-β1-stimulated renal mesangial cells. Then the relationship between TRIM13 and CHOP was explored using the ubiquitination assay.FindingsWe found that the expression of TRIM13 was downregulated in renal biopsies, isolated glomeruli of diabetic mice, and HG/TGF-β1-stimulated renal mesangial cells, while the expression of CHOP was upregulated. An increased level of TRIM13 promoter methylation contributed to the deregulation of TRIM13 in renal glomeruli of DN. The ubiquitination assay confirmed that TRIM13 promoted ubiquitination and degradation of CHOP. Meanwhile, overexpressing TRIM13 attenuated DN-induced collagen synthesis and restored renal function in vitro and in vivo via downregulating CHOP.InterpretationOur findings demonstrated that overexpressed TRIM13 suppresses mesangial collagen synthesis in DN by promoting ubiquitination of CHOP, suggesting TRIM13 as a potential therapeutic target in treating DN.

Arctigenin attenuates diabetic kidney disease through the activation of PP2A in podocytes

Arctigenin (ATG) is a major component of Fructus Arctii, a traditional herbal remedy that reduced proteinuria in diabetic patients. However, whether ATG specifically provides renoprotection in DKD is not known. Here we report that ATG administration is sufficient to attenuate proteinuria and podocyte injury in mouse models of diabetes. Transcriptomic analysis of diabetic mouse glomeruli showed that cell adhesion and inflammation are two key pathways affected by ATG treatment, and mass spectrometry analysis identified protein phosphatase 2 A (PP2A) as one of the top ATG-interacting proteins in renal cells. Enhanced PP2A activity by ATG reduces p65 NF-κB-mediated inflammatory response and high glucose-induced migration in cultured podocytes via interaction with Drebrin-1. Importantly, podocyte-specific Pp2a deletion in mice exacerbates DKD injury and abrogates the ATG-mediated renoprotection. Collectively, our results demonstrate a renoprotective mechanism of ATG via PP2A activation and establish PP2A as a potential target for DKD progression.

Tangeretin Ameliorates Podocyte Injury Through Blocking Epithelial to Mesenchymal Transition in High Glucose-exposed Podocytes and Diabetic Kidneys (P06-009-19)

ObjectivesEpithelial to mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties. This process contributes to the accumulation of matrix proteins in kidney and leads to renal glomeruli fibrosis. Tangeretin is an O-polymethoxylated flavone with anti-inflammatory and antioxidant properties that is found in citrus peels. This study investigated the renoprotective effects of tangeretin on EMT-mediated podocyte injury and fibrosis caused by hyperglycemia. MethodsMouse glomerular epithelial cells (podocyte) were incubated in media containing 5.5 mM glucose, 27.5 mM mannitol as an osmotic control or 33 mM glucose (high glucose) in the absence and presence of 1–20 μM tangeretin for up to 4 days. Antibodies of E-cadherin, N-cadherin, α-SMA, nephrin, podocin, P-cadherin and collagen1 were used for Western blotting. The in vivo animal model employed db/db mice orally administrated with 10 mg/kg tangeretin for 8 weeks. Kidney tissue extracts of were Western-blotted, and tissue sections cut down in 5 μM thickness were immunohistochemically stained. In addition, kidney tissue sections (ultrathin sections, 70 nm) were observed with transmission electron microscopy (TEM). ResultsNon-toxic tangeretin enhanced expression of the podocyte slit diaphragm proteins of nephrin, podocin and P-cadherin down-regulated by glucose stimulation. Also, tangeretin inhibited high glucose-induced expression of the mesenchymal markers of N-cadherin and α-smooth muscle actin, whereas the induction of the epithelial marker E-cadherin was enhanced. Furthermore tangeretin attenuated the fibronectin induction and collagen production elevated by the presence of high glucose. The TEM images revealed that podocyte foot process effacement occurred in diabetic mouse glomeruli. However, oral administration of 10 mg/kg tangeretin reduced urine albumin excretion and improved foot process effacement of diabetic podocytes through inhibiting loss of glomerular slit junction proteins. ConclusionsThese results demonstrated that tangeretin maintained the structures of podocyte slit diaphragm in a robust form, and inhibited podocyte injury through blocking epithelial to mesenchymal transition of podocytes. Therefore, tangeretin may be a potent renoprotective agent counteracting diabetes-associated loss of podocyte slit diaphragm and maintaining glomerular filtration barrier. Funding SourcesThis work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MEST) (NRF-2017R1A6A3A04011473).

LRG1 Promotes Diabetic Kidney Disease Progression by Enhancing TGF-β-Induced Angiogenesis.

Glomerular endothelial dysfunction and neoangiogenesis have long been implicated in the pathogenesis of diabetic kidney disease (DKD). However, the specific molecular pathways contributing to these processes in the early stages of DKD are not well understood. Our recent transcriptomic profiling of glomerular endothelial cells identified a number of proangiogenic genes that were upregulated in diabetic mice, including leucine-rich α-2-glycoprotein 1 (LRG1). LRG1 was previously shown to promote neovascularization in mouse models of ocular disease by potentiating endothelial TGF-β/activin receptor-like kinase 1 (ALK1) signaling. However, LRG1's role in the kidney, particularly in the setting of DKD, has been unclear. We analyzed expression of LRG1 mRNA in glomeruli of diabetic kidneys and assessed its localization by RNA in situ hybridization. We examined the effects of genetic ablation of Lrg1 on DKD progression in unilaterally nephrectomized, streptozotocin-induced diabetic mice at 12 and 20 weeks after diabetes induction. We also assessed whether plasma LRG1 was associated with renal outcome in patients with type 2 diabetes. LRG1 localized predominantly to glomerular endothelial cells, and its expression was elevated in the diabetic kidneys. LRG1 ablation markedly attenuated diabetes-induced glomerular angiogenesis, podocyte loss, and the development of diabetic glomerulopathy. These improvements were associated with reduced ALK1-Smad1/5/8 activation in glomeruli of diabetic mice. Moreover, increased plasma LRG1 was associated with worse renal outcome in patients with type 2 diabetes. These findings identify LRG1 as a potential novel pathogenic mediator of diabetic glomerular neoangiogenesis and a risk factor in DKD progression.

Apelin inhibited epithelial\u2212mesenchymal transition of podocytes in diabetic mice through downregulating immunoproteasome subunits \u03b25i

The epithelial−mesenchymal transition (EMT) of podocytes had been reported to be involved in the glomerular fibrosis in diabetic kidney diseases, which was regulated by TGFβ and NFκB pathways. And apelin, an adipokine which is upregulated in diabetic kidney diseases, was reported to be negatively correlated to TGFβ in polycystic kidney disease and attenuate EMT in renal tubular cells. Therefore, it is hypothesized that apelin might inhibit the EMT of podocytes through downregulating the expression and activation of TGFβ/Smad pathway in diabetic kidney diseases. The results showed that apelin in glomeruli of diabetic mice were increased and exogenous apelin inhibited the EMT of podocytes in diabetic mice, which were accompanied with the decreased expression of proteasome subunits β5i. The results from β5iKO mice confirmed that the inhibiting effects of apelin on EMT of podocytes in diabetic mice were dependent on β5i. The results from culture podocytes showed that apelin decreased the degradation of pIκB and promoted the translocation of IκB into nucleus through decreasing the expression of β5i, which would inhibit the promoting effects of NFκB on expression of TGFβ and followed by decreased activation of Smad pathway and EMT in podocytes. In conclusion, apelin might act as an EMT suppressor for podocytes to decrease the process of glomerular fibrosis in diabetic mice.

Hordenine protects against hyperglycemia-associated renal complications in streptozotocin-induced diabetic mice

The worldwide prevalence of diabetes and associated metabolic diseases has dramatically increased. Pharmacological treatment of diabetes is still limited. Hordenine (HOR), a phenethylamine alkaloid, is a natural constituent in many plants. The present study was designed to explore the possible anti-diabetic effect of HOR in streptozotocin (STZ)-induced diabetic mice. Combined treatment of HOR and insulin significantly reduced fasting and postprandial blood glucose level in diabetic mice. HOR and insulin did not show evident protective effect against structural and functional injuries of pancreas. Renal histological and functional injuries were significantly improved by HOR or insulin treatment. Moreover, combined treatment of HOR and insulin resulted in a more significant amelioration of renal histological and functional injuries in diabetic mice. HOR induced a decrease of renal IL-1α/β and IL-6 expression, and a reduction of Col1α1 and MMP9 expression and PAS-stained mesangial expansion in glomeruli of diabetic mice. In diabetic mice, HOR significantly decreased Nrf2 expression and increased hnRNPF and hnRNPK expression in kidney. Moreover, HOR showed a synergistic effect with insulin on the expression of these regulators. Renal ROS level and TBARS content in diabetic mice were decreased by HOR. The reduction of renal expression of antioxidant enzymes in diabetic mice was inhibited by HOR and insulin. Furthermore, HOR and insulin function synergistically to play an antioxidant role against oxidative injury in diabetic nephropathy. In conclusion, to the best of our knowledge, we, for the first time, found the anti-diabetic, anti-inflammatory, and anti-fibrotic role of HOR in combination with insulin. HOR functions synergistically with insulin and prevents diabetic nephropathy. However, the molecular mechanism of the synergistic effect of HOR and insulin needs to be elucidated.

Hemicentin 1 influences podocyte dynamic changes in glomerular diseases.

Different complex mechanisms control the morphology of podocyte foot processes and their interactions with the underlying basement membrane. Injuries to this system often cause glomerular dysfunction and albuminuria. The present study aimed at identifying early markers of glomerular damage in diabetic nephropathy. For this purpose, we performed a microarray analysis on kidneys of 3-wk-old peroxisome proliferator-activated receptor-γ (PPARγ)-null and AZIP/F1 mice, which are two models of diabetic nephropathy due to lipodystrophy. This was followed by functional annotation of the enriched clusters of genes. One of the significant changes in the early stages of glomerular damage was the increase of hemicentin 1 (HMCN1). Its expression and distribution were then studied by real-time PCR and immunofluorescence in various models of glomerular damage and on podocyte cell cultures. HMCN1 progressively increased in the glomeruli of diabetic mice, according to disease severity, as well as in puromycin aminonucleoside (PA)-treated rats. Studies on murine and human podocytes showed an increased HMCN1 deposition upon different pathological stimuli, such as hyperglycemia, transforming growth factor-β (TGF-β), and PA. In vitro silencing studies showed that HMCN1 mediated the rearrangements of podocyte cytoskeleton induced by TGF-β. Finally, we demonstrated an increased expression of HMCN1 in the kidneys of patients with proteinuric nephropathies. In summary, our studies identified HMCN1 as a new molecule involved in the dynamic changes of podocyte foot processes. Its increased expression associated with podocyte dysfunction points to HMCN1 as a possible marker for the early glomerular damage occurring in different proteinuric nephropathies.

Modulation of glutathione peroxidase activity by age-dependent carbonylation in glomeruli of diabetic mice

AimsLow levels of reactive oxygen species and resulting oxidative protein modifications may play a beneficial role in cellular function under stress conditions. Here we studied the influence of age-dependent protein carbonylation on expression and activity of the anti-oxidative selenoenzyme glutathione peroxidase (GPx) in insulin-deficient Ins2Akita mice and type 2 diabetic obese db/db mice in context of diabetic nephropathy. MethodsProtein carbonylation, GPx expression and activity were examined in kidney tissue and lysates by common histological and protein biochemical methods. ResultsIn kidneys of Ins2Akita mice, carbonylated proteins, GPx-1 and GPx-4 expression were mainly detected in podocytes and mesangial cells. GPx activity was increased in kidney cortex homogenates of these mice. Remarkably, young animals did not show a concomitant increase in GPx expression but enhanced GPx carbonylation. No carbonylation-dependent modification of GPx activity was detected in db/db mice. In cultured podocytes hyperglycemia induced an increase in GPx expression but had no effect on activity or carbonylation. In kidney tissue sections of type 1 or type 2 diabetes patients, GPx-1 and GPx-4 expression but not overall protein carbonylation was significantly decreased. ConclusionsThese results indicate the existence of a threshold for beneficial carbonylation-dependent redox signaling during the progression of diabetic nephropathy.