Mesangial Cells In Response Research Articles

#1978 Proteomic analysis of exosomes derived from mesangial cells treated with galactose-deficient IgA1

Abstract Background and Aims IgA nephropathy (IgAN) is an autoimmune disease characterized by IgA deposits that build up in the kidneys, inflame and damage glomeruli causing hematuria and proteinuria. It is a leading cause of chronic kidney disease, which often results in end-stage kidney disease. There is no disease specific treatment for IgAN. The glomerulus, in which IgA1 lacking galactose residues (gdIgA1) are deposited, consists of mesangial cells, podocytes, and endothelial cells. Crosstalk between the cell types allows the glomerulus to act as a unit and react to its dynamic environment. There is very little evidence on mesangial cell derived exosomes and their cargo to understand the pathological crosstalk signaling in the glomerulus of IgAN. We isolated exosomes from mesangial cells that were treated with patient-derived gdIgA1 and IgA1 complexes. Proteomic analysis was performed to identify the exosome cargo and delineate the signaling pathways leading to maladaptive paracrine crosstalk. From our study, we aim to understand the molecular targets for therapeutic strategies in IgAN. Method Human mesangial cells were treated with/without IgA1, gdIgA1 or IgG for 24 h. Cell culture supernatant was collected for exosome isolations. Exosomes were analyzed by size, morphology and number using TEM and NanoFCM. Exosome protein extracts were processed for LC-Tandem mass spectrometry analysis using filter-aided sample preparation protocol. Proteomic analysis was performed using Qlucore omics explorer and pathway analysis using Ingenuity Pathway Analysis (IPA). Exosome marker expressions such as CD63, CD81 and FLOT1 and proteome validation was performed using Western blotting. Statistical analysis was performed using GraphPad Prism and values <0.05 were considered significant. Results We confirmed the release of exosomes by mesangial cells in response to IgA1/gdIgA1 complexes. Exosomes were fewer in unstimulated cells as compared to IgA1/gdIgA1 treated cells. They showed round morphology with a dense core and uniform size distribution. Exosomes from treated cells showed a mean size of about 75 nm and concentration in the range of 1-2 × 10^10 particles/ml. While untreated cells showed a mean size of about 100 nm and concentration of about 1 × 10^9 particles/ml. We confirmed the protein expression of exosome markers such as CD63, CD81 and FLOT1. For the first time, we demonstrated global proteomics on exosomes from mesangial cells followed by pathway analysis. We have identified 89 unique proteins and pathways in exosomes derived from gdIgA1 treated mesangial cells that could mediate pathological signaling in the glomerulus in comparison to exosomes derived from IgA1 treated mesangial cells. Conclusion Our study provides an understanding of the pathological crosstalk signaling through exosomes and key molecular targets for IgAN.

Dysregulation of miR-25-3p in Diabetic Nephropathy and Its Role in Inflammatory Response.

To investigate the expression level of miR-25-3p in patients with type 2diabetes mellitus (T2DM) and diabetic nephropathy (DN), and its effect on proliferation, apoptosis and inflammatory response of mesangial cells cultured with high glucose. Blood samples of all clinical subjects were collected for RT-qPCR analysis to detect serum miR-25-3p levels. Human mesangial cells (HMCs) cultured with high glucose were used to construct DN model in vitro. MTT assay, flow cytometry and ELISA were used to evaluate the effects of miR-25-3p on the proliferation, apoptosis, and inflammatory response of DN cell models. Serum miR-25-3p was decreased in both T2DM group and DN group, but more in DN group. Serum miR-25-3p was positively correlated with eGFR and negatively correlated with UAER. The expression of miR-25-3p was reduced in HMCs induced by high glucose. Transfection of miR-25-3p mimic could significantly up-regulate the miR-25-3p level in HMCs. Besides, high glucose culture resulted in abnormal proliferation of HMCs, reduced apoptotic cells, and increased inflammation. The addition of miR-25-3p mimic significantly inhibited cell proliferation and promoted cell apoptosis and reduced the production of inflammatory factors. The abnormal reduction of serum miR-25-3p in DN indicates that it may be a potential biomarker for clinical diagnosis of DN. In in vitro experiments, miR-25-3p was involved in the progression of DN by regulating cell proliferation, apoptosis, and inflammatory response.

Decreased DANCR contributes to high glucose-induced extracellular matrix accumulation in human renal mesangial cell via regulating the TGF-β/Smad signaling.

Glomerulosclerosis is one of the major histopathologic changes in diabetic kidney diseases (DKD), which is characterized by excessive deposition of extracellular matrix (ECM) in the glomerulus mainly produced by mesangial cells in response to transforming growth factor-β (TGF-β) stimuli under diabetic conditions. Despite TGF-β has been implicated as a major pathogenic factor in the development of diabetic glomerulosclerosis, clinical trials of monoclonal antibodies against TGF-β failed to demonstrate therapeutic benefits. Thus, developing alternative therapeutic strategies to effectively block the TGF-β/Smad signaling could be of paramount importance for DKD treatment. Emerging evidence indicates that dysregulation of certain lncRNAs can lead to aberrant activation of TGF-β/Smad signaling. Herein, we identified a novel lncRNA, named DANCR, which could efficiently function as a negative regulator of TGF-β/Smad signaling in mesangial cells. Ectopic expression of DANCR could specifically block the activation of TGF-β/Smad signaling induced by high-glucose or TGF-β in human renal mesangial cells (HRMCs). Mechanistically, DANCR functions to stabilize nemo-like kinase (NLK) mRNA through interaction with insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), resulting in enhanced phosphorylating on the linker region of activated Smad2/3 in the nucleus. Taken together, our data have uncovered an lncRNA-based regulatory modality of the TGF-β/Smad signaling and identified DANCR as an endogenous blocker of TGF-β/Smad signaling in HRMCs, which may represent a potential therapeutic target against the diabetic glomerulosclerosis.

Nanodisc delivery of liver X receptor agonist for the treatment of diabetic nephropathy.

Dyslipidemia is recognized to be an important contributor to the progression of diabetic nephropathy (DN), leading to lipoprotein dysregulation, excessive mesangium expansion as well as inflammation in the glomeruli. Thus, dual targeting of abnormal cholesterol metabolism and inflammatory responses of mesangial cells represents an alternative approach for DN treatment. Herein, we sought to develop a renal-targeting therapeutic strategy for diabetic nephropathy by modifying synthetic high-density lipoprotein (sHDL) nanodiscs with a kidney targeting ligand (KT peptide) and encapsulating a liver X receptor (LXR) agonist in the modified sHDL. LXR agonists delivered by sHDL can facilitate the removal of excessive lipids from mesangial cells, ameliorate inflammation and restore normal renal function. Overall, our data suggests that our optimized KT-targeted sHDL/TO nanodiscs (KT-sHDL/TO) generate potent therapeutic efficacy not only by more efficient cholesterol efflux, but also by suppressing mesangial cell proliferation. Most importantly, in a DN murine model, KT-sHDL/TO ameliorated dyslipidemia and inflammation superior to blank sHDL and non-targeting sHDL/TO formulations, showing promise for future clinical translation in DN treatment.

MO404: Autophagy and Fibrotic Response is Genetically Determined in Mouse Mesangial Cells

Abstract BACKGROUND AND AIMS C57Bl6/J (B6) mouse strain is resistant to several experimental models of renal fibrosis. Mesangial cells play an important role in the pathogenesis of glomerulosclerosis. Also, autophagy dysregulation was recently related to fibrosis. However, it is unclear whether autophagy dysregulation could be genetically determined in mice. Thus, we investigated autophagy and fibrotic response in primary mesangial cells isolated from fibrosis resistant (B6) and fibrosis prone (CBA) inbred mouse strains. METHOD Primary mesangial cells were isolated from 6-weeks old B6 and CBA male mice via magnetic bead separation technique. Glomerular outgrowths were maintained in RPMI medium for 21 days when cells were subcultured and characterized for mesangial markers. Mesangial cells with passage numbers P5 to P8 were seeded onto 24-well plates. Gene expressions were analyzed after 48-h of PBS (CTL) or TGF-β (10 ng/mL) treatment (n = 6/group). Data (mean ± SD) were analyzed using Kruskal–Wallis test. RESULTS Tgfb1 and Col1a1 expressions increased by 2.5-fold and 8-fold in TGF-β treated CBA cells as compared to B6 (P < 0.01), respectively. The expression of Egr1 (early growth response factor-1) that participates in both fibrosis and autophagy increased by 2-fold in TGF-β treated CBA cells but remained unchanged in B6 cells (P < 0.05). Similarly, the expression of core autophagy molecule Lc3b did not change in TGF-β treated B6 cells, but increased by 60% in CBA cells (P < 0.05), accompanied by 1.5-fold increase of Sqstm1/p62. On the other hand, the expression of Mtmr14 that negatively regulates autophagy decreased by 30% in TGF-β treated CBA cells but remained unchanged in B6. CONCLUSION We conclude that genetic background of mice highly influences the TGF-β induced fibrotic and autophagy response of mesangial cells. Our results corroborate previous reports on autophagy dysregulation associated to fibrosis. FUNDING: Hungarian Academy of Sciences BO/00 304/20/5, ÚNKP-21–5-SE-1, NTP-NFTÖ-21-B-0045

Trigonelline induces autophagy to protect mesangial cells in response to high glucose via activating the miR-5189-5p-AMPK pathway

BackgroundDiabetic nephropathy (DN) is a primary cause of end‐stage renal disease. Increasing evidence indicates that microRNAs (miRNAs) are involved in DN pathogenesis. Trigonelline (TRL) has been shown to lower blood sugar and cholesterol levels, promote nerve regeneration, and exert anti-cancer and sedative properties. MethodThe effect of TRL on human mesangial cell (HMC) growth was assessed using the MTT assay. Differentially expressed miRNAs were validated using real-time quantitative polymerase chain reaction (real-time PCR). Bioinformatics, cell transfection, and Western blot analyses were utilized to confirm the binding of miR-5189-5p to HIF1AN. The effects of miR-5189-5 expression on cell proliferation were also assessed. Western blot analysis was used to determine the activation of multiple signaling molecules including phosphorylated-(p)-AMPK, SIRT1, LC3B, p62, and Beclin-1 in the autophagy pathway. ResultsTRL improved proliferation, increased the expression of miR-5189-5p, reduced HIF1AN, and restored the inhibition of autophagy in HMCs induced by high glucose. MiR-5189-5p mimics inhibited HIF1AN expression, and the miR-5189-5p inhibitor increased HIF1AN expression. MiR-5189-5p mimics significantly improved the proliferation of HMCs induced by high glucose, reduced the relative protein expression of p-AMPK, SIRT1, LC3B, and Beclin-1, and significantly increased the relative protein expression of p62. ConclusionWe showed that TRL up-regulated miR-5189-5p expression, activated the AMPK pathway, and activated autophagy in HMCs. Our study demonstrates that TRL could be a new treatment strategy to protect mesangial cells in response to high glucose.

Krϋppel-like factor 15 suppresses renal glomerular mesangial cell proliferation via enhancing P53 SUMO1 conjugation.

Mesangial cell (MC) proliferation is a key pathological feature in a number of common human renal diseases, including mesangial proliferative nephritis and diabetic nephropathies. Knowledge of MC responses to pathological stimuli is crucial to the understanding of these disease processes. We previously determined that Krϋppel‐like factor 15 (KLF15), a kidney‐enriched zinc‐finger transcription factor, was required for inhibition of MC proliferation. In the present study, we investigated the direct target gene and the underlying mechanism by which KLF15 regulated mesangial proliferation. First, we screened small ubiquitin‐related modifier 1 (SUMO1) as the direct transcriptional target of KLF15 and validated this finding with ChIP‐PCR and luciferase assays. Furthermore, we demonstrated that overexpressing KLF15 or SUMO1 enhanced the stability of P53, which blocked the cell cycle of human renal MCs (HRMCs) and therefore abolished cell proliferation. Conversely, knockdown of SUMO1 in HRMCs, even those overexpressed with KLF15, could not inhibit HRMC proliferation rates and increase SUMOylation of P53. Finally, the results showed that the levels of SUMOylated P53 in the kidney cortices of anti‐Thy 1 model rats were decreased during proliferation periods. These findings reveal the critical mechanism by which KLF15 targets SUMO1 to mediate the proliferation of MCs.

HMGB1 regulates ferroptosis through Nrf2 pathway in mesangial cells in response to high glucose.

Ferroptosis, a novel type of programmed cell death, is involved in inflammation and oxidation of various human diseases, including diabetic kidney disease. The present study explored the role of high-mobility group box-1 (HMGB1) on the regulation of ferroptosis in mesangial cells in response to high glucose. Compared with healthy control, levels of serum ferritin, lactate dehydrogenase (LDH), reactive oxygen species (ROS), malonaldehyde (MDA), and HMGB1 were significantly elevated in diabetic nephropathy (DN) patients, accompanied with deregulated ferroptosis-related molecules, including long-chain acyl-CoA synthetase 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2), NADPH oxidase 1 (NOX1), and glutathione peroxidase 4 (GPX4). In vitro assay revealed that erastin and high glucose both induced ferroptosis in mesangial cells. Suppression of HMGB1 restored cellular proliferation, prevented ROS and LDH generation, decreased ACSL4, PTGS2, and NOX1, and increased GPX4 levels in mesangial cells. Furthermore, nuclear factor E2-related factor 2 (Nrf2) was decreased in DN patients and high glucose-mediated translocation of HMGB1 in mesangial cells. Knockdown of HMGB1 suppressed high glucose-induced activation of TLR4/NF-κB axis and promoted Nrf2 expression as well as its downstream targets including HO-1, NQO-1, GCLC, and GCLM. Collectively, these findings suggest that HMGB1 regulates glucose-induced ferroptosis via Nrf2 pathway in mesangial cells.

Abstract P2028: Paracrine And Intracellular Angiotensin II Activate Protein Kinase C, Map Kinase Erk1/2, And Nuclear Factor-кB Signaling Responses In Murine Glomerular Mesangial Cells.

The glomerular mesangial cells (MC) are a key target for the endocrine, paracrine, autocrine, and intracrine angiotensin II (ANG II). Increased local paracrine and intracrine ANG II formation in MCs may cause glomerular injury by promoting MC cell growth, hypertrophy and fibrosis. In the present study, we investigated the phospho-proteomic signaling and mitochondrial responses to paracrine or intracellular ANG II in murine MCs. MCs were grown to ~80% confluence in 6-well plates in DMEM and Ham’s F-12 medium, containing 5% fetal bovine serum and 1% penicillin-streptomycin, at 37°C under 95% O 2 /5% CO 2 . MCs were fasted in a serum-free medium for 24 h before being treated with ANG II (0.1 to 100 nM) for 5 to 60 min, or transfected with a mitochondria-targeting intracellular ANG II for 48 h (mito-ECFP/ANG II, 4 μg/well), with or without pretreatment with the AT 1 receptor blocker losartan (1 μM), AT 2 receptor blocker PD123319 (1 μM), or expression of mito-AT 2 receptors. Proteins were extracted for Western blot analysis of p-PKCα, p-ERK1/2, p38 MAPK, p65 and p50 subunits of NF-кB, or mitochondria protein complexes I to V, respectively. ANG II significantly increased p-ERK1/2 (Control: 0.20 ±0.07 vs. ANG II: 0.04 ± 0.07, p&lt0.05), p-PKCα (Control: 0.50 ± 0.10 vs. ANG II: 1.30 ±0.20, p&lt0.01), p38 MAPK (Control: 0.30 ±0.04 vs. ANG II: 0.60 ±0.06, p&lt0.01), and NF-кB responses (Control: 0.30 ±0.02 vs. ANG II: 0.55 ± 0.08, p&lt0.01) in dose- and time-dependent manners, with peak responses to ANG II occurred between 0.1 and 1.0 nM, 5-10 min ( p &lt0.05). These phopho-proteomic signaling responses were attenuated by losartan (p&lt0.01) or PD123319 (p&lt0.01), respectively. mito-ECFP/ANG II increased mitochondrial protein complex I &gt38%, Complex III &gt56%, Complex IV &gt89%, and Complex V &gt63%, respectively (p&lt0.01). The effects of mito-ECFP/ANG II on mitochondrial protein complexes were attenuated by losartan or the expression of mito-AT 2 receptors (p&lt0.01). These results suggest that paracrine and intracellular ANG II may activate AT 1 and AT 2 receptors to induce important MAP kinases ERK1/2, p-PKCα, p38 MAPK, NF-кB signaling, or mitochondria responses in MCs, which may contribute to ANG II-induced glomerular injury in hypertension and kidney diseases.

Knockdown of ANGPTL-4 inhibits inflammatory response and extracellular matrix accumulation in glomerular mesangial cells cultured under high glucose condition

Diabetic nephropathy (DN) is one of the major diabetic complications that lead to end-stage renal failure. Angiopoietin-like protein-4 (ANGPTL-4) has been reported to be dysregulated in diabetes mellitus and diabetic complications. However, the role of ANGPTL-4 in glomerular mesangial cells (MCs) during DN remains unclear. In the present study, we evaluated the role of ANGPTL-4 in MCs in response to high glucose (HG) condition and the potential mechanism. The results proved that ANGPTL-4 expression is significantly increased in HG-stimulated MCs. Knockdown of ANGPTL-4 suppressed HG-induced cell proliferation of MCs. The production of pro-inflammatory cytokines including TNF-α, IL-1β, IL-6 were decreased in ANGPTL-4 knocked down MCs. Inhibition of ANGPTL-4 markedly suppressed the expressions of extracellular matrix (ECM) proteins, collagen IV (Col IV) and fibronectin (FN), in HG-stimulated MCs. Furthermore, ANGPTL-4 knockdown inhibited the HG-induced activation of NF-κB signaling pathway in MCs. Collectively, knockdown of ANGPTL-4 suppressed HG-induced cell proliferation, inflammatory response, and ECM accumulation inhibiting NF-κB signaling pathway in MCs. These findings suggested that ANGPTL-4 might be a therapeutic target for the prevention and treatment of DN.

Effect of mycophenolate and rapamycin on renal fibrosis in lupus nephritis.

Lupus nephritis (LN) leads to chronic kidney disease (CKD) through progressive fibrosis. Mycophenolate inhibits inosine monophosphate dehydrogenase and is a standard treatment for LN. The mammalian or mechanistic target of rapamycin (mTOR) pathway is activated in LN. Rapamycin inhibits mTOR and is effective in preventing kidney transplant rejection, with the additional merits of reduced incidence of malignancies and viral infections. The effect of mycophenolate or rapamycin on kidney fibrosis in LN has not been investigated. We investigated the effects of mycophenolate and rapamycin in New Zealand Black and White first generation (NZB/W F1) murine LN and human mesangial cells (HMCs), focusing on mechanisms leading to kidney fibrosis. Treatment of mice with mycophenolate or rapamycin improved nephritis manifestations, decreased anti-double stranded (ds) DNA antibody titer and reduced immunoglobulin G (IgG) deposition in the kidney. Both mycophenolate and rapamycin, especially the latter, decreased glomerular mTOR Ser2448 phosphorylation. Renal histology in untreated mice showed mesangial proliferation and progressive glomerulosclerosis with tubular atrophy, and increased expression of transforming growth factor β1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle actin (α-SMA), fibronectin (FN) and collagen. Both mycophenolate and rapamycin ameliorated the histopathological changes. Results from in vitro experiments showed that both mycophenolate and rapamycin decreased mesangial cell proliferation and their binding with anti-dsDNA antibodies. Mycophenolate and rapamycin also down-regulated mTOR and extracellular signal-regulated kinase (ERK) phosphorylation and inhibited fibrotic responses in mesangial cells that were induced by anti-dsDNA antibodies or TGF-β1. Our findings suggest that, in addition to immunosuppression, mycophenolate and rapamycin may reduce fibrosis in LN, which has important implications in preventing CKD in patients with LN.

Hyperglycemia induces NF-κB activation and MCP-1 expression via downregulating GLP-1R expression in rat mesangial cells: inhibition by metformin.

Hyperglycemia impairs glucagon-like peptide-1 receptor (GLP-1R) signaling in multiple cell types and thereby potentially attenuates the therapeutic effects of GLP-1R agonists. We hypothesized that the downregulation of GLP-1R by hyperglycemia might reduce the renal-protective effects of GLP-1R agonists in diabetic nephropathy (DN). In this study, we examined the effects of high glucose on the expression of GLP-1R and its signaling pathways in the HBZY-1 rat mesangial cell line. We found that high glucose reduced GLP-1R messenger RNA (mRNA) levels in HBZY-1 cells and in the renal cortex in db/db mice comparing with control groups. In consistence, GLP-1R agonist exendin-4 induced CREB phosphorylation was attenuated by high glucose but not low glucose treatment, which is paralleled with abrogated anti-inflammatory functions in HBZY-1 cells linked with nuclear factor-κB (NF-κB) activation. In consistence, GLP-1R inhibition aggravated the high glucose-induced activation of NF-κB and MCP-1 protein levels in cultured HBZY-1 cells while overexpression of GLP-1R opposite effects. We further proved that metformin restored high glucose-inhibited GLP-1R mRNA expression and decreased high glucose evoked inflammation in HBZY-1 cells. On the basis of these findings, we conclude that high glucose lowers GLP-1R expression and leads to inflammatory responses in mesangial cells, which can be reversed by metformin. These data support the rationale of combinative therapy of metformin with GLP-1R agonists in DN.

The caveolin-1 regulated protein follistatin protects against diabetic kidney disease

Glomerular matrix protein accumulation, mediated largely by mesangial cells, is central to the pathogenesis of diabetic kidney disease. Our previous studies showed that the membrane microdomains caveolae and their marker protein caveolin-1 regulate matrix protein synthesis in mesangial cells in response to diabetogenic stimuli, and that caveolin-1 knockout mice are protected against diabetic kidney disease. In a screen to identify the molecular mechanism underlying this protection, we also established that secreted antifibrotic glycoprotein follistatin is significantly upregulated by caveolin-1 deletion. Follistatin potently neutralizes activins, members of the transforming growth factor-β superfamily. A role for activins in diabetic kidney disease has not yet been established. Therefore, invitro, we confirmed the regulation of follistatin by caveolin-1 in primary mesangial cells and showed that follistatin controls both basal and glucose-induced matrix production through activin inhibition. Invivo, we found activin A upregulation by immunohistochemistry in both mouse and human diabetic kidney disease. Importantly, administration of follistatin to type 1 diabetic Akita mice attenuated early diabetic kidney disease, characterized by albuminuria, hyperfiltration, basement membrane thickening, loss of endothelial glycocalyx and podocyte nephrin, and glomerular matrix accumulation. Thus, activin A is an important mediator of high glucose-induced profibrotic responses in mesangial cells, and follistatin may be a potential novel therapy for the prevention of diabetic kidney disease.

Cell surface expression of 78-kDa glucose-regulated protein (GRP78) mediates diabetic nephropathy

The 78-kDa glucose-regulated protein (GRP78) is a well-established endoplasmic reticulum (ER)-resident chaperone that maintains protein homeostasis and regulates the unfolded protein response. Under conditions of ER stress, GRP78 is also expressed at the cell surface and implicated in tumorigenesis, immunity, and cellular signaling events. The role of cell surface-associated GRP78 (csGRP78) in the pathogenesis of diabetic nephropathy has not yet been defined. Here we explored the role of csGRP78 in regulating high glucose (HG)-induced profibrotic AKT Ser/Thr kinase (AKT) signaling and up-regulation of extracellular matrix proteins. Using primary kidney mesangial cells, we show that HG treatment, but not the osmotic control mannitol, induces csGRP78 expression through an ER stress-dependent mechanism. We found that csGRP78, known to be located on the outer membrane leaflet, interacts with the transmembrane protein integrin β1 and activates focal adhesion kinase and downstream PI3K/AKT signaling. Localization of GRP78 at the cell surface and its interaction with integrin β1 were also required for extracellular matrix protein synthesis in response to HG. Surprisingly, both the N and C termini of csGRP78 were necessary for this profibrotic response. Increased localization of GRP78 at the plasma membrane was also found in the glomerular mesangial area of type 1 diabetic mice in two different models (streptozotocin-induced and Akita). In freshly isolated glomeruli from Akita mice, csGRP78 co-localized with the mesangial cell surface marker α8-integrin. In conclusion, our work reveals a role for csGRP78 in HG-induced profibrotic responses in mesangial cells, informing a potential approach to treating diabetic nephropathy.

Parathyroid hormone-related protein induces fibronectin up-regulation in rat mesangial cells through reactive oxygen species/Src/EGFR signaling.

Parathyroid hormone-related protein (PTHrP) is known to be up-regulated in both glomeruli and tubules in patients with diabetic kidney disease (DKD), but its role remains unclear. Previous studies show that PTHrP-induced hypertrophic response in mesangial cells (MCs) and epithelial-mesenchymal transition (EMT) in tubuloepithelial cells can be mediated by TGF-β1. In the present study, although long-term PHTrP (1–34) treatment increased the mRNA and protein level of TGF-β1 in primary rat MCs, fibronectin up-regulation occurred earlier, suggesting that fibronectin induction is independent of TGF-β1/Smad signaling. We thus evaluated the involvement of epidermal growth factor receptor (EGFR) signaling and found that nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species mediates PTHrP (1–34)-induced Src kinase activation. Src phosphorylates EGFR at tyrosine 845 and then transactive EGFR. Subsequent PI3K activation mediates Akt and ERK1/2 activation. Akt and ERK1/2 discretely lead to excessive protein synthesis of fibronectin. Our study thus demonstrates the new role of PTHrP in fibronectin up-regulation for the first time in glomerular MCs. These data also provided new insights to guide development of therapy for glomerular sclerosis.

Effects of Paracrine and Intracellular Angiotensin II on Protein Kinase C, MAP kinase ERK1/2, and Nuclear Factor‐κB Signaling Responses in Mouse Glomerular Mesangial cells

Angiotensin II (ANG II) acts not only as a vasoconstrictor, but also as a growth‐promoting cytokine and chemokine, proinflammatory and profibrotic factor in the kidney. Glomerular mesangial cells (MC) are a key target for endocrine, paracrine, autocrine, and intracrine ANG II. Augmentation of local paracrine and intracrine ANG II generation in MCs may induce glomerular injury by promoting MC cell growth, hypertrophy and fibrosis. In the present study, we performed phosphoproteomic characterization of ANG II‐induced protein kinase C (p‐PKCα), MAP kinase ERK1/2 (p‐ERK1/2), p38 MAPK, and nuclear factor‐κB (p65 and p50 subunits) signaling responses in murine glomerular MCs. Murine mesangial cells (MES‐13, ATCC) were grown to ~80% confluence in 6‐well plates in DMEM and Ham's F‐12 medium, containing 5% fetal bovine serum, and 1% penicillin‐streptomycin, maintained at 37°C and 95% O2/5% CO2. After starved at a serum‐free medium for 24 h, MCs were then treated with low to high levels of ANG II (0.1 to 100 nM) for 5 to 20 min, or transfected with a mitochondria‐targeting intracellular ANG II for 48 h (mito‐ECFP/ANG II, 4 μg/well), with or without pretreatment with the AT1 receptor blocker losartan (1 μM), AT2 receptor blocker PD123319 (1 μM) or both blockers. Proteins were extracted for Western blot analysis of p‐PKCα, p‐ERK1/2, p38 MAPK, and p65 and p50 subunits of NF‐κB, respectively. ANG II significantly increased p‐ERK1/2 and p‐PKCα in both dose‐ and time‐dependent manners, with peak responses to ANG II occurred at 0.1 and 1.0 nM, 5–10 min (p<0.05), respectively. ANG II also increased p38 MAPK response, but it peaked at 1 nM, 20 min (p<0.05). By contrast, ANG II appeared to decrease the NF‐κB responses at 1 nM, 10 min, which were reversed by losartan and PD123319. The expression of mito‐ECFP/ANG II selectively in the MC mitochondria significantly increased mitochondrial complex proteins (CI‐CV), which were blocked by losartan (p<0.01). Our results suggest that both paracrine and intracellular ANG II may activate AT1 receptors to induce important intracellular PKC, MAP kinase ERK1/2, p38 MAPK, and NF‐κB signaling responses in MCs, which may contribute to ANG II‐induced glomerular injury in hypertension and kidney diseases.Support or Funding InformationSupported by National Institute of Health grants, 2R01DK067299‐10A1 and 2R01DK102924‐03A1 to Zhuo, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ CAPES to Leite.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Estrogen-related receptor-α mediates puromycin aminonucleoside-induced mesangial cell apoptosis and inflammatory injury.

Glomerular diseases are the leading cause of chronic kidney disease, and mesangial cells (MCs) have been demonstrated to be involved in the pathogenesis. Puromycin aminonucleoside (PAN) is a nephrotoxic drug that induces glomerular injury with elusive mechanisms. The present study was undertaken to investigate the role of PAN in MC apoptosis, as well as the underlying mechanism. Here we found that PAN induced MC apoptosis accompanied by declined cell viability and enhanced inflammatory response. The apoptosis was further evidenced by increments of apoptosis regulator BAX (BAX) and caspase-3 expression. In line with the apoptotic response in MCs following PAN treatment, we also found a remarkable induction of estrogen-related receptor-α (ERRα), an orphan nuclear receptor, at both mRNA and protein levels. Interestingly, ERRα silencing by an siRNA approach resulted in an attenuation of the apoptosis and inflammatory response caused by PAN. More importantly, overexpression of ERRα in MCs significantly triggered MC apoptosis in line with increased BAX and caspase-3 expression. In PAN-treated MCs, ERRα overexpression further aggravated PAN-induced apoptosis. In agreement with the in vitro study, we also observed increased ERRα expression in line with enhanced apoptotic response in renal cortex from PAN-treated rats. These data suggest a detrimental effect of ERRα on PAN-induced MC apoptosis and inflammatory response, which could help us to better understand the pathogenic mechanism of MC injury in PAN nephropathy.

The Use of Targeted Next Generation Sequencing to Explore Candidate Regulators of TGF-β1's Impact on Kidney Cells.

Aims/Hypothesis: Transforming growth factor-beta (TGF-β1) plays an important regulatory role in the progression of chronic kidney failure. Further, damage to kidney glomerular mesangial cells is central to the progression of diabetic nephropathy. The aim of this study was to explore the genetic associations between mRNA, microRNA, and epigenetics in mesangial cells in response to TGF-β1.Methods: The regulatory effects of TGF-β1 on mesangial cells were investigated at different molecular levels by treating mesangial cells with TGF-β1 for 3 days followed by genome-wide miRNA, RNA, DNA methylation, and H3K27me3 expression profiling using next generation sequencing (NGS).Results: Our results provide the first comprehensive, computationally integrated report of RNA-Seq, miRNA-Seq, and epigenomic analyses across all genetic variations, confirming the occurrence of DNA methylation and H3K27me3 in response to TGF-β1. Our findings show that the expression of KLF7 and Gja4 are involved in TGF-β1 regulated DNA methylation. Our data also provide evidence of the association between epigenetic changes and the expression of genes closely related to TGF-β1 regulation.Conclusion: This study has advanced our current knowledge of mechanisms that contribute to the expression of TGF-β1-regulated genes involved in the pathogenesis of kidney disease. The molecular underpinnings of TGF-β1 stimulation of kidney cells was determined, thereby providing a robust platform for further target exploration.

Carbohydrate response element binding protein (ChREBP) modulates the inflammatory response of mesangial cells in response to glucose.

Diabetic nephropathy (DN) is one of the most devastating complications of diabetes mellitus. Carbohydrate response element binding protein (ChREBP) is a basic helix–loop–helix leucine zipper transcription factor that primarily mediates glucose homeostasis in the body. The present study investigated the role of ChREBP in the pathogenesis of DN. The expression of ChREBP was detected in patients with type 2 diabetes mellitus (T2DM), diabetic mice, and mesangial cells. ELISA was used to measure cytokine production in mesangial cells. Flow cytometry analysis was performed to detect the apoptosis of mesangial cells in the presence of high glucose. The expression levels of ChREBP and several cytokines (TNF-α, IL-1β, and IL-6) were up-regulated in T2DM patients. The mRNA and protein levels of ChREBP were also significantly elevated in the kidneys of diabetic mice. Moreover, glucose treatment promoted mRNA levels of TNF-α, IL-1β, and IL-6 in mesangial cells. Glucose stimulation induced significant apoptosis of SV40 MES 13 cells. In addition, transfection with ChREBP siRNA significantly inhibited ChREBP expression. Consequently, the inflammatory responses and apoptosis were inhibited in SV40 MES 13 cells. These results demonstrated that ChREBP could mediate the inflammatory response and apoptosis of mesangial cells, suggesting that ChREBP may be involved in the pathogenesis of DN.

Daphnetin inhibits high glucose-induced extracellular matrix accumulation, oxidative stress and inflammation in human glomerular mesangial cells

Diabetic nephropathy (DN) is one of the most common causes of end-stage renal disease (ESRD). Oxidative stress and inflammation have been documented to play important roles in the pathogenesis of DN. Daphnetin, a natural coumarin compound, possesses antioxidant and anti-inflammatory activities. However, the role of daphnetin in DN has not yet been investigated. The aim of the present study was to explore the function of daphnetin in DN and the underlying mechanism in vitro. Our results demonstrated that daphnetin alleviated cell proliferation induced by high glucose (HG) in human mesangial cells (MCs). Daphnetin strikingly reduced reactive oxygen species (ROS) and malonaldehyde (MDA) levels, and induced the superoxide dismutase (SOD) activity in HG-stimulated MCs. Besides, the production of TNF-α, IL-1β, IL-6, fibronectin (FN) and collagen IV (Col IV) was also inhibited by daphnetin in HG-stimulated MCs. In addition, daphnetin enhanced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and inhibited the levels of p-Akt and p-p65 in HG-stimulated MCs. The results indicated that daphnetin inhibited HG-induced oxidative stress, inflammatory response, and ECM accumulation in human MCs. The effect is partially mediated by Nrf2/keap1 and Akt/NF-κB pathways. The findings suggested that daphnetin might be a therapeutic or preventive agent for DN.