Mouse Glomerular Endothelial Cells Research Articles

Notoginsenoside Fc, a novel renoprotective agent, ameliorates glomerular endothelial cells pyroptosis and mitochondrial dysfunction in diabetic nephropathy through regulating HMGCS2 pathway

BackgroundDiabetic nephropathy (DN) is the primary cause of end-stage renal disease (ESRD), and the therapeutic strategies for DN are limited. Notoginsenoside Fc (Fc), a novel saponin isolated from Panax Notoginseng (PNG), has been reported to alleviate vascular injury in diabetic rats. However, the protective effects of Fc on DN remain unclear. PurposeTo investigate the beneficial effects and mechanisms of Fc on DN. MethodsDb/db mice were treated with 2.5, 5 and 10 mg·kg−1·d−1 of Fc for 8 weeks. High glucose (HG) induced mouse glomerular endothelial cells (GECs) were treated with 2.5, 5 and 10 μM of Fc for 24 h. ResultsOur data found that Fc ameliorated urinary microalbumin level, kidney dysfunction and histopathological damage in diabetic mice. Moreover, Fc alleviated the accumulation of oxidative stress, the collapse of mitochondrial membrane potential and the expression of mitochondrial fission proteins, such as Drp-1 and Fis1, while increased the expression of mitochondrial fusion protein Mfn2. Fc also decreased pyroptosis-related proteins levels, such as TXNIP, NLRP3, cleaved caspase-1, and GSDMD-NT, indicating that Fc ameliorated GECs pyroptosis. In addition, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) expression was increased in diabetic group, which was partially abrogated by Fc. Our data further proved that knockdown of HMGCS2 could restrain HG-induced GECs mitochondrial dysfunction and pyroptosis. These results indicated that the inhibitory effects of Fc on mitochondrial damage and pyroptosis were associated with the suppression of HMGCS2. ConclusionTaken together, this study clearly demonstrated that Fc ameliorated GECs pyroptosis and mitochondrial dysfunction partly through regulating HMGCS2 pathway, which might provide a novel drug candidate for DN.

AR/RKIP pathway mediates the inhibitory effects of icariin on renal fibrosis and endothelial-to-mesenchymal transition in type 2 diabetic nephropathy

Ethnopharmacological relevanceHerba epimedium brevicornu maxim is traditionally known as a sexual enhancement, and has the effects of tonifying kidney yang. Icariin is a flavonoid extracted from epimedium brevicornu maxim, and has been shown to improve nephropathy disease. Aim of the studyThis study investigated the possible role of icariin in regulating renal EndMT in type 2 diabetic nephropathy (T2DN). Materials and methodsMale type 2 diabetic Sprague Dawley rats, Male D2.BKS(D)-Leprdb/J (db/db) mice, and mouse glomerular endothelial cells were utilized to evaluate the effect of icariin. Western blotting, Q-PCR, immunohistochemistry, H&E, Masson staining, immunofluorescence, and siRNA transfection, were performed in this study. ResultsThe inhibitory function of icariin in renal fibrosis and renal EndMT was verified in type 2 diabetic animals. Methyltestosterone suppressed renal fibrosis and EndMT in db/db mice. Androgen receptor (AR), the major receptor of testosterone, was upregulated by icariin. The AR antagonist MDV3100, blocked the inhibition by icariin in renal EndMT, revealing that icariin repressed renal EndMT by activating AR. In addition, icariin and methyltestosterone upregulated the Raf kinase inhibitor protein (RKIP) in db/db mice. Furthermore, siRNA-RKIP inhibited the effect of icariin on EndMT. The MEK/ERK pathway, as the downstream pathway of RKIP, was suppressed by icariin and methyltestosterone. Of note, the effect of icariin on the MEK/ERK pathway was abolished by MDV3100 or siRNA-RKIP. ConclusionsThese results supported that icariin targeted AR/RKIP/MEK/ERK pathway to suppress renal fibrosis and EndMT in T2DN.

Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3.

The main characteristics of diabetic nephropathy (DN) at the early stage are abnormal angiogenesis of glomerular endothelial cells (GECs) and macrophage infiltration. Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand, advanced glycation end products (AGEs). Catalpol, an iridoid glucoside extracted from Rehmannia glutinosa, has been found to ameliorate vascular inflammation, reduce endothelial permeability, and protect against endothelial damage in diabetic milieu. However, little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs. Mouse GECs (mGECs) and RAW 264.7 macrophages were treated with different concentrations of AGEs (0, 50, 100, 200 and 400 µg/mL) for different time (0, 6, 12, 24 and 48 h) to determine the optimal concentration of AGEs and treatment time. Cells were treated with catalpol (10 µmol/L), GB1107 (1 µmol/L, galectin-3 inhibitor), PX-478 (50 µmol/L, HIF-1α inhibitor), adenovirus-green fluorescent protein (Ad-GFP) [3×107 plaque-forming unit (PFU)/mL] or Ad-galectin-3-GFP (2×108 PFU/mL), which was followed by incubation with 50 µg/mL AGEs. The levels of galectin-3, vascular endothelial growth factor A (VEGFA) and pro-angiogenic factors angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tunica interna endothelial cell kinase-2 (Tie-2) were detected by enzymelinked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of these cells. The expression levels of galectin-3, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and hypoxia-inducible factor-1α (HIF-1α) in mGECs and those of galectin-3 and HIF-1α in RAW 264.7 macrophages were detected by Western blotting and immunofluorescence (IF) staining. The rat DN model was established. Catalpol (100 mg/kg) or GB1107 (10 mg/kg) was administered intragastrically once a day for 12 weeks. Ad-galectin-3-GFP (6×107 PFU/mL, 0.5 mL) or Ad-GFP (6×106 PFU/mL, 0.5 mL) was injected into the tail vein of rats 48 h before the sacrifice of the animals. The expression of galectin-3, VEGFR1, VEGFR2, and HIF-1α in renal cortices was analyzed by Western blotting. The expression of galectin-3, F4/80 (a macrophage biomarker), and CD34 (an endothelium biomarker) in renal cortices was detected by IF staining, and collagen accumulation by Masson staining. The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50 µg/mL AGEs for 48 h than those in untreated cells. Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages. Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells. Catalpol could significantly decrease the levels of Ang-1, Ang-2 and Tie-2 released by AGEs-treated mGECs, which could be reversed by over-expression of galectin-3. Catalpol could significantly inhibit AGEs-induced expression of galectin-3, HIF-1α, VEGFR1, and VEGFR2 in mGECs. The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478. Moreover, catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages, which was weakened by PX-478. Additionally, catalpol significantly inhibited the expression of galectin-3, macrophage infiltration, collagen accumulation, and angiogenesis in the kidney of diabetic rats. Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol. Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3. It could prevent the progression of diabetes-induced renal damage.

Positive association between circulating Caveolin-1 and microalbuminuria in overt diabetes mellitus in pregnancy.

Mounting evidence has shown that caveolin-1 plays a pathological role in the progression of albuminuria. Our study aimed to provide clinical evidence showing whether circulating caveolin-1 levels were associated with microalbuminuria (MAU) in women with overt diabetes mellitus in pregnancy (ODMIP). A total of 150 pregnant women were enrolled in different groups, including 40 women with ODMIP and MAU (ODMIP + MAU), 40 women with ODMIP, and 70 women without ODMIP (Non-ODMIP). Plasma caveolin-1 levels were determined by ELISA. The presence of caveolin-1 in the human umbilical vein vascular wall was evaluated by immunohistochemical and western blot analysis, respectively. Albumin transcytosis across endothelial cells was measured using an established nonradioactive in vitro approach. Significantly increased levels of plasma caveolin-1 were detected in ODMIP + MAU women. The Pearson's correlation analysis revealed a positive correlation between plasma caveolin-1 levels and Hemoglobin A1c (HbA1c %) as well as with MAU in the ODMIP + MAU group. Simultaneously, experimental knockdown or overexpression of caveolin-1 significantly decreased or increased the level of albumin transcytosis across both human and mouse glomerular endothelial cells (GECs), respectively. Our data showed a positive association between plasma caveolin-1 levels and microalbuminuria in ODMIP + MAU.

Transient Receptor Vanilloid Subtype 4-Mediated Ca2+ Influx Promotes Glomerular Endothelial Inflammation in Sepsis-Associated Acute Kidney Injury

Sepsis-associated acute kidney injury (S-AKI) is a frequent complication in patients who are critically ill, which is often initiated by glomerular endothelial cell dysfunction. Although transient receptor vanilloid subtype 4 (TRPV4) ion channels are known to be permeable to Ca2+ and are widely expressed in the kidneys, the role of TRPV4 on glomerular endothelial inflammation in sepsis remains elusive. In the present study, we found that TRPV4 expression in mouse glomerular endothelial cells (MGECs) increased after lipopolysaccharide (LPS) stimulation or cecal ligation and puncture challenge, which increased intracellular Ca2+ in MGECs. Furthermore, the inhibition or knockdown of TRPV4 suppressed LPS-induced phosphorylation and translocation of inflammatory transcription factors NF-κB and IRF-3 in MGECs. Clamping intracellular Ca2+ mimicked LPS-induced responses observed in the absence of TRPV4. In vivo experiments showed that the pharmacologic blockade or knockdown of TRPV4 reduced glomerular endothelial inflammatory responses, increased survival rate, and improved renal function in cecal ligation and puncture-induced sepsis without altering renal cortical blood perfusion. Taken together, our results suggest that TRPV4 promotes glomerular endothelial inflammation in S-AKI and that its inhibition or knockdown alleviates glomerular endothelial inflammation by reducing Ca2+ overload and NF-κB/IRF-3 activation. These findings provide insights that may aid in the development of novel pharmacologic strategies for the treatment of S-AKI.

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria's distribution, fusion, and fission events without staining the cells.

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Catalpol ameliorates endothelial dysfunction and inflammation in diabetic nephropathy via suppression of RAGE/RhoA/ROCK signaling pathway

Catalpol is an iridoid glycoside compound isolated from the root of Rehmannia glutinosa, which has been reported to be a promising candidate for the treatment of diabetic diseases. The present study aimed at investigating the effects and potential mechanism of catalpol on endothelial dysfunction and inflammation in diabetic nephropathy (DN). We constructed DN mice and advanced glycation end products (AGEs)-induced mouse glomerular endothelial cells (mGECs) injury model. The results demonstrated that catalpol effectively improved renal pathology and decreased levels of urine protein, serum creatinine, and blood urea nitrogen in DN mice. Catalpol significantly reduced endothelial dysfunction and inflammatory infiltration of macrophages in DN mice and AGEs-induced mGECs. To further study the protective mechanism of catalpol, we transfected DN mice with recombinant adeno-associated virus expressing receptor of AGEs (RAGE) and intervened AGEs-induced mGECs with inhibitors. Catalpol reversed endothelial dysfunction and inflammation aggravated by RAGE overexpression in DN mice. Meanwhile, catalpol significantly inhibited the RAGE/Ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) pathway in DN mice with RAGE overexpression. Moreover, the combination of catalpol with inhibitors of RAGE, RhoA and ROCK exerted stronger anti-endothelial dysfunction and anti-macrophage infiltration effects on AGEs-induced mGECs compared with catalpol alone. In short, this study indicated that catalpol could ameliorate endothelial dysfunction and inflammation via suppression of RAGE/RhoA/ROCK pathway, hereby delaying the progression of DN.

Ipragliflozin Ameliorates Diabetic Nephropathy Associated with Perirenal Adipose Expansion in Mice.

Sodium glucose cotransporter-2 (SGLT2) inhibitors inhibit the development of diabetic nephropathy (DN). We determined whether changes in perirenal fat (PRAT) by a SGLT2 inhibitor ipragliflozin (Ipra) contribute to the suppression of DN development. High-fat diet (HFD)-fed mice were used as a DN model and were treated with or without Ipra for 6 weeks. Ipra treatment reduced urinary albumin excretion (UAE) and glomerular hypertrophy in HFD-fed mice. In the PRAT of Ipra-treated mice, adipocyte size was increased, and inflammation, fibrosis, and adipocyte death were suppressed. In conditioned medium made from PRAT (PRAT-CM) of Ipra-treated mice, the concentration of leptin was significantly lower than PRAT-CM of mice without Ipra treatment. Serum leptin concentration in renal vein positively correlated with UAE. PRAT-CM from HFD-fed mice showed greater cell proliferation signaling in mouse glomerular endothelial cells (GECs) than PRAT-CM from standard diet-fed mice via p38MAPK and leptin-dependent pathways, whose effects were significantly attenuated in PRAT-CM from Ipra-treated mice. These findings suggest that Ipra-induced PRAT expansion may play an important role in the improvement of DN in HFD-fed mice. In vitro experiments suggest that reduced PRAT-derived leptin by Ipra could inhibit GECs proliferation, possibly contributing to the suppression of DN development.

Laminar flow substantially affects the morphology and functional phenotype of glomerular endothelial cells.

Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.

Exogenous hydrogen sulfide and miR-21 antagonism attenuates macrophage-mediated inflammation in ischemia reperfusion injury of the aged kidney.

Ischemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in the aging population. A reduction of hydrogen sulfide (H2S) production in the old kidney and renal IRI contribute to renal pathology and injury. Recent studies suggest that microRNAs (miRs) play an important role in the pathophysiology of AKI and a significant crosstalk exists between H2S and miRs. Among the miRs, miR-21 is highly expressed in AKI and is reported to have both pathological and protective role. In the present study, we sought to determine the effects of age-induced reduction in H2S and mir-21 antagonism in AKI. Wild type (WT, C57BL/6J) mice aged 12-14weeks and 75-78weeks underwent bilateral renal ischemia (27min) and reperfusion for 7days and were treated with H2S donor, GYY4137 (GYY, 0.25mg/kg/day, ip) or locked nucleic acid anti-miR-21 (20mg/kg b.w., ip) for 7days. Following IRI, old kidney showed increased macrophage polarization toward M1 inflammatory phenotype, cytokine upregulation, endothelial-mesenchymal transition, and fibrosis compared to young kidney. Treatment with GYY or anti-miR-21 reversed the changes and improved renal vascular density, blood flow, and renal function in the old kidney. Anti-miR-21 treatment in mouse glomerular endothelial cells showed upregulation of H2S-producing enzymes, cystathionine β-synthase (CBS), and cystathionineγ-lyase (CSE), and reduction of matrix metalloproteinase-9 and collagen IV expression. In conclusion, exogenous H2S and inhibition of miR-21 rescued the old kidney dysfunction due to IRI by increasing H2S levels, reduction of macrophage-mediated injury, and promoting reparative process suggesting a viable approach for aged patients sustaining AKI.

Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis.

Many patients with idiopathic focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation (TX). Although several circulating permeability factors (CPFs) responsible for recurrence have been suggested, there is no consensus. To facilitate CPF identification and predict recurrence after TX, there is a need for robust methods that demonstrate the presence of CPFs. Cultured human podocytes (hPods) and human and mouse glomerular endothelial cells (ciGEnC, mGEnC) were exposed to plasmas of FSGS patients with presumed CPFs, and of (disease) controls. A visual scoring assay and flow cytometry analysis of side scatter were used to measured changes in cellular granularity after exposure to plasma. Nine out of 13 active disease plasmas of 10 FSGS patients with presumed CPFs induced granularity in hPod in a dose- and time-dependent manner. Corresponding remission plasmas induced no or less granularity in hPod. Similar results were obtained with ciGEnC and mGEnC, although induced granularity was less compared with hPod. Notably, foetal calf serum, healthy plasma and a remission plasma partially blocked FSGS plasma-induced hPod granularity. We developed a novel assay in which active disease, presumably CPF-containing, FSGS plasmas induced granularity in cultured hPod. Our results may indicate the presence of CPF inhibitor(s) in healthy and remission plasma. We suggest the presence of a delicate balance between CPF and a CPF inhibitory factor, which is disturbed in patients with active disease. Our novel assays can be applied in future research to identify CPF and CPF inhibitors, and possibly to predict recurrence after TX.

Collagen receptor- and metalloproteinase-dependent hypertensive stress response in mesangial and glomerular endothelial cells.

Progressive alteration of the extracellular matrix (ECM) is the characteristic of hypertensive nephropathy (HN). Both mesangial and endothelial cells have the ability to synthesize and degrade ECM components, including collagens through the activation of matrix metalloproteinases (MMPs) in stress conditions, such as in hypertension. On the other hand, hydrogen sulfide (H2S) has been shown to mitigate hypertensive renal matrix remodeling. Surprisingly, whether H2S ameliorates receptor-mediated (urokinase plasminogen activator receptor-associated protein, uPARAP/Endo180) collagen dysregulation in Ang-II hypertension is not clear. The purpose of this study was to determine whether Ang-II alters the expression of Endo180, tissue plasminogen activator (tPA), MMPs, and their tissue inhibitors (TIMPs) leading to the dysregulation of cellular collagen homeostasis and whether H2S mitigates the collagen turnover. Mouse mesangial cells (MCs) and glomerular endothelial cells (MGECs) were treated without or with Ang-II and H2S donor GYY (GYY4137) for 48h. Cell lysates were analyzed by Western blot and RT-PCR, and cells were analyzed by immunocytochemistry. The results indicated that, while Ang-II differentially expressed MMP-13 and TIMP-1 in MCs and in MGECs, it predominantly decreased tPA, Endo 180, and increased plasminogen activator inhibitor-1 (PAI-1), MMP-14, and collagen IIIA and IV in both the cell types. Interestingly, H2S donor GYY treatment normalized the above changes in both the cell types. We conclude that Ang-II treatment causes ECM remodeling in MCs and MGECs through PAI-1/tPA/Endo180 and MMP/TIMP-dependent collagen remodeling, and H2S treatment mitigates remodeling, in part, by modulating these pathways.

Isolation, purification, and conditional immortalization of murine glomerular endothelial cells of microvascular phenotype

Glomerular endothelial cells (GEnC) are a specialized microvascular subset of endothelial cells that, when injured, result in many types of diseases within the kidney. Thus, techniques to study GEnC in a cell culture system are important to investigate mechanisms of GEnC injury. Studies of endothelial cell function in culture have predominately relied on using macrovascular endothelial cells from vascular areas other than the glomerulus. Over the last 15 years, glomerular endothelial cells lines have been created but were isolated by targeting cells expressing CD31. Some studies identified endothelial cells isolated from the microvasculature do not express CD31 and some suggest that CD31+ cells are phenotypically different than endothelial cells found in capillaries. Here we detail our method of isolation, purification, and conditional immortalization of mouse glomerular endothelial cells targeting endothelial cells that do not express CD31.•This method allows for isolation, purification, and conditional immortalization of glomerular endothelial cells for continued passage of GEnCs beyond that of primary cell culture.•This method can be used in genetically modified mice to investigate how a modification of a specific gene or protein affects the glomerular endothelium at the cellular level.

The 5-hydroxytryptamine receptor 1F stimulates mitochondrial biogenesis and angiogenesis in endothelial cells

A hallmark of acute kidney injury (AKI) is vascular rarefication and mitochondrial dysfunction. Promoting vascular recovery following AKI could facilitate kidney repair as the vasculature is responsible for oxygen and nutrient delivery to extravascular tissues. Little is known about mitochondrial biogenesis (MB) in endothelial cells, and the role of 5-HT1F receptor signaling in MB has only been studied in epithelial cells. Our laboratory has shown that stimulating MB through the 5-HT1F receptor promotes recovery from AKI and that 5-HT1F receptor knockout mice have decreased MB and poor renal recovery. We hypothesized that the 5-HT1F receptor plays a role in vascular homeostasis and mediates MB in renal endothelial cells. 5-HT1F receptor knockout mice had decreased renal vascular content, as evidenced by decreased CD31+ endothelial cells and αSMA+ vessels. Human glomerular endothelial cells (HEC) and mouse glomerular endothelial cells (MEC) expressed the 5-HT1F receptor. Treatment of HEC and MEC with 5-HT1F receptor agonists LY344864 or lasmiditan (0–500 nM) induced MB as evidenced by maximal mitochondrial respiration, a marker of MB. HEC and MEC treated with lasmiditan or LY344864 also had increased nuclear- and mitochondrial-encoded proteins (PGC1α, COX-1, and VDAC), and mitochondrial number, confirming MB. Treatment of HEC with LY344864 or lasmiditan enhanced endothelial branching morphogenesis and migration, indicating a role for 5-HT1F receptor stimulation in angiogenic pathways. We propose that stimulation of 5-HT1F receptor is involved in MB in endothelial cells and that treatment with 5-HT1F receptor agonists could restore stimulate repair and recovery following kidney injury.

Hydrogen sulfide inhibits Ca2+-induced mitochondrial permeability transition pore opening in type-1 diabetes.

Hydrogen sulfide (H2S) attenuates N-methyl-d-aspartate receptor-R1 (NMDA-R1) and mitigates diabetic renal damage; however, the molecular mechanism is not well known. Whereas NMDA-R1 facilitates Ca2+ permeability, H2S is known to inhibit L-type Ca2+ channel. High Ca2+ activates cyclophilin D (CypD), a gatekeeper protein of mitochondrial permeability transition pore (MPTP), thus facilitating molecular exchange between matrix and cytoplasm causing oxidative outburst and cell death. We tested the hypothesis of whether NMDA-R1 mediates Ca2+ influx causing CypD activation and MPTP opening leading to oxidative stress and renal injury in diabetes. We also tested whether H2S treatment blocks Ca2+ channel and thus inhibits CypD and MPTP opening to prevent renal damage. C57BL/6J and Akita (C57BL/6J-Ins2Akita) mice were treated without or with H2S donor GYY4137 (0.25 mg·kg-1·day-1 ip) for 8 wk. In vitro studies were performed using mouse glomerular endothelial cells. Results indicated that low levels of H2S and increased expression of NMDA-R1 in diabetes induced Ca2+ permeability, which was ameliorated by H2S treatment. We observed cytosolic Ca2+ influx in hyperglycemic (HG) condition along with mitochondrial-CypD activation, increased MPTP opening, and oxidative outburst, which were mitigated with H2S treatment. Renal injury biomarker KIM-1 was upregulated in HG conditions and normalized following H2S treatment. Inhibition of NMDA-R1 by pharmacological blocker MK-801 revealed similar results. We conclude that NMDA-R1-mediated Ca2+ influx in diabetes induces MPTP opening via CypD activation leading to increased oxidative stress and renal injury, and H2S protects diabetic kidney from injury by blocking mitochondrial Ca2+ permeability through NMDA-R1 pathway.

Exercise Induced Irisin Alleviates Type 1 Diabetic Nephropathy by Promoting Mitochondria Biogenesis and Function

Mitochondrial dysfunction is known to play an important role in the development and progression of chronic kidney disease. Improving mitochondrial homeostasis therefore has the potential to restore renal function in diabetic kidney disease. Exercise training was shown to be beneficial in improving skeletal muscle and cardiovascular system pathology by improving mitochondrial function, reduction of oxidative stress and metabolic reprogramming. Recent studies have shown that Irisin, a molecule secreted by the skeletal muscles during exercise protects the mitochondria from ischemia reperfusion injury in the lung and heart. In clinical studies, involving type 1 diabetes, high and low levels of Irisin has been reported however, its functional significance remains unclear. Exercise induces the expression of PGC‐1α, leading to the expression of the fibronectin type III domain containing 5 (FNDC5). Irisin derives from the proteolytic cleavage of the peptide FNDC5 by yet unknown enzymes. The purpose of our study was to investigate whether exercise induced Irisin improves mitochondrial biogenesis and function in type 1 diabetic nephropathy. C57BL/6‐Ins2Akita/J (Type 1 diabetes, Akita) and C57BL/6J (Wild type, WT) mice were subjected to 16‐week exercise regimen of 280m run on treadmill at speed of 7m/min for 5 days/week for 2 weeks and increased to 12m/min for the remaining 14 weeks. In vitro experiments were done in mouse glomerular endothelial cells (MGECs) treated with recombinant Irisin. In Akita and WT mice, exercise training improved renal vascularity, cortical blood flow and glomerular filtration rate compared to sedentary groups. Exercise upregulated Irisin expression in Akita and WT mice compared to sedentary mice but to a greater extent in WT mice. The protein and mRNA expression of PGC1α, PPARα and PPARγ were decreased in sedentary Akita mice and increased following exercise training suggesting induction of mitochondrial biogenesis. Sedentary Akita mice showed decreased expression of regulatory markers of mitochondrial DNA transcription, Nrf1, Nrf2, and Tfam and exercise normalized their expression. The expression of MnSOD and catalase were decreased in Akita mice suggesting impaired antioxidant defense and exercise restored their levels to normal. In conclusion, our results suggest that exercise induced Irisin protects the kidney in type 1 diabetes by restoring mitochondrial homeostasis.Support or Funding InformationThis study was supported, in part, by NIH grant HL‐104103 and DK 104653 to US and AHA grant: 15SDG25840013 to SP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Catalpol ameliorates advanced glycation end product-induced dysfunction of glomerular endothelial cells via regulating nitric oxide synthesis by inducible nitric oxide synthase and endothelial nitric oxide synthase.

Catalpol (Cat.) is an iridoid glucoside extracted from the root of Rehmannia glutinosa Libosch. In this study, we investigated whether Cat. could protect the mouse glomerular endothelial cells against the deleterious effect induced by advanced glycation end products (AGEs) and explored potential mechanisms. We found that 10 μM Cat. showed a protective effect on dead cells stimulated by AGEs. Cat. significantly decreased the expression of p-NF-κBp65 and inducible nitric oxide synthase (iNOS) and increased the expression of phosphorylated-endothelial nitric oxide synthase (p-eNOS; Ser1177), PI3K, p-Akt (Thr308), and total-Akt. Moreover, Cat. restored the integrity of glomerular endothelial barrier by increasing endothelial tight gap junction protein and ameliorated the endothelial hyperpermeability induced by AGEs via modulating the nitric oxide (NO) production. Additionally, Cat. attenuated the massive release of NO induced by AGEs, inhibiting the macrophage infiltration by modulating the NO production, accompanied by the decrease in the release of monocyte chemoattractant protein-1 and intercellular cell adhesion molecule-1 in vitro. Therefore, Cat. ameliorated AGEs-induced endothelial dysfunction via inhibiting the NF-κB/iNOS pathway and activating the PI3K/Akt/eNOS pathway. © 2019 IUBMB Life, 71(9):1268-1283, 2019.

A8324 Suppression of Endothelial to Mesenchymal Transition by SIRT3 Alleviated the Development of Hypertensive Renal Injury

Objectives: Endothelial to mesenchymal transition (EndoMT) has recently emerged as a potentially important contributor in promoting fibrosis in chronic kidney disease. However, little is known about the role and molecular basis of its involvement in hypertensive renal injury. Here, we aim to determine the role of SIRT3 on EndoMT in hypertensive renal injury and to explore its underlying mechanisms. Methods: Adult wild-type, SIRT3 knockout (SIRT3−/−), non transgenic and SIRT3 endothelial cell-specific transgenic (SIRT3-TgEC) mice were infused with Ang II for 4 weeks. Results: We found that SIRT3 expression was significantly reduced in Ang II-induced hypertensive model, accompanied with induction of EndoMT and increased reactive oxygen species (ROS) and renal fibrosis. In SIRT3−/− mice subjected to Ang II infusion, renal dysfunction was aggravated with an increased EndoMT and ROS level, while in SIRT3-TgEC mice, the Ang II-induced renal fibrosis and EndoMT and oxidative stress were ameliorated. With primary mouse glomerular endothelial cells we confirmed that Ang II treatment initiated EndoMT and decreased catalase expression, which were suppressed by SIRT3 overexpression. Using immunoprecipitation, luciferase and chromatin immunoprecipitation assay we demonstrated that SIRT3-mediated deacetylation and nuclear localization of forkhead box O3a (Foxo3a) resulted in activated Foxo3a-dependent catalase expression. Moreover, Foxo3a knockdown abolished SIRT3-mediated suppression of EndoMT. Conclusion: In conclusion, these results established the SIRT3-Foxo3a-catalase pathway as a critical factor in the maintenance of endothelial homeostasis and point to an important role of EndoMT in the vascular pathology of renal fibrosis, which may provide a new therapeutic target to impede the progression of hypertensive renal injury.

CD8+iTregs attenuate glomerular endothelial cell injury in lupus-prone mice through blocking the activation of p38 MAPK and NF-κB

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease. Endothelial cell injury plays an important role in the inflammatory processes associated with SLE. CD4+Foxp3+regulatory T cells (Tregs) reduce the injury to endothelial cells induced by inflammatory factors. As a newly identified regulatory T cell, we previously reported that CD8+CD103+iTregs had similar effects to those of CD4+iTregs in the process of immunoregulation. In this paper, we further explored the effect and mechanism of CD8+iTregs on endothelial cell injury. The expressions of vascular cellular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in MRL/lpr mouse glomerular endothelial cells (lupus-MGECs) were estimated by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blotting. The lupus-MGEC apoptosis rate was detected by flow cytometry and the adhesion of monocyte-like cells to lupus-MGECs exposed to lipopolysaccharide (LPS) was determined by the adhesion assay. Additionally, the expressions of P-p38, P-NF-κB and P-IκBα were detected by Western blotting. The results showed that LPS increased the expressions of VCAM-1, ICAM-1, IFN-γ, TNF-α, IL-6 and MCP-1 in lupus-MGECs, while CD8+iTregs significantly decreased the levels of these adhesion molecules and inflammatory mediators. Furthermore, CD8+iTregs alleviated lupus-MGEC apoptosis and inhibited the adhesion of monocyte-like cells to lupus-MGECs. Both nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK), activated by LPS, were suppressed by CD8+iTregs. These findings suggest that CD8+iTregs attenuate LPS-induced glomerular endothelial cell injury through blocking the activation of p38 MAPK and NF-κB in lupus-MGECs. The protective effect of CD8+iTregs indicates their possible therapeutic application in Lupus nephritis.