Mouse Mesangial Cells Research Articles

Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding

Event Abstract Back to Event Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding Valentina Magno1, Bella Roßbach2, Krithika Hariharan2, Jens Friedrichs1, Andreas Kurtz2 and Carsten Werner1, 3 1 Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Germany 2 Charité – Universitätsmedizin Berlin, Berlin-Brandenburg Center for Regenerative Therapies, Germany 3 Center for Regenerative Therapies Dresden, Technische Universität Dresden, Germany Introduction: Cell-derived extracellular matrices (ECMs) have shown in the latest years to have broad application in the field of tissue engineering and regenerative medicine. The recent introduction of macromolecular crowding (MMC) for the modulation of in vitro ECM production allowed, furthermore, to better mimic physiological microenvironments[1]. In this work, we aim to explore the effect of MMC on the secretion of ECM by primary mouse mesangial cells and to study the impact of decellularized ECMs produced under standard and MMC conditions on the growth and differentiation of human induced pluripotent stem cell (iPSC)-derived intermediate mesoderm (IM) progenitors[2]. Methods: Covalently attached mesangial cell-derived ECMs were produced on a maleic anhydride co-polymer platform[3]. The ECM secretion was carried out for 8 days either under standard (Std) or MMC culture conditions by supplementing the medium with the synthetic polysaccharide Ficoll. During the ECM deposition phase, cell viability was assessed using a PrestoBlue assay. Cell layers were decellularised using an hypotonic solution and DNA remnants were removed by DNase treatment. The impact of MMC on ECM composition and morphology was investigated by colorimetric assays, immunohistochemistry, atomic force microscopy and scanning electron microscopy. IM progenitors were seeded in parallel on the mesangial cell-derived ECMs as well as on Geltrex and collagen IV as control substrates, with or without the supplementation of the ROCK inhibitor Y-27632. Adhesion and proliferation of IM progenitors were analyzed respectively using a Crystal Violet and a PrestoBlue assay. Furthermore, the expression of relevant kidney markers was examined via real time PCR. Results and Discussion: PrestoBlue assay showed that MMC did not affect the viability of the mesangial cells during ECM deposition. MMC was found to influence the composition, the mechanical properties and the supramolecular assembly of the mesangial cell-derived ECM. IM progenitors showed significantly higher cell viability and adhesion on both types of ECM (Std and MMC) compared to Geltrex and collagen IV. Particularly, in presence of the ROCK inhibitor, a significantly higher adhesion was found on MMC-ECM compared to both Std-ECM and control substrates. Higher proliferation rates were observed on both types of ECM vs. the controls, with the MMC-ECM outperforming Std-ECM. A significant increase in the expression of some mesangial markers and other glomerular markers was found for IM progenitors cultivated on MMC-ECM vs. Std-ECM, with even less mesangial-like characteristics found on the control substrates. Conclusion: Our results indicate that matrix based signals support the differentiation of the IM progenitors toward the mesangial lineage without the addition of soluble cues. In the future, the combination of cell-derived ECM and MMC may be utilized to study kidney progenitor differentiation and to develop bioengineered kidney tissues. A systematic study on different crowder categories and concentrations may also allow to control progenitor differentiation to obtain a reliable cell source for regenerative approaches. This work was supported by EU Marie Curie NephroTools FP7 Initial Training Network (ITN)

Different Effects of EP2 and EP4 Receptors in TGF-β1 Induced Mesangial Cells Injury

Background: The prostaglandin E2 (PGE2) and its receptor subtypes 2 and 4 (EP2 and EP4) in mesangial cells (MCs) proliferation and glomerulosclerosis hasn’t been elaborated in detailed. Methods: We cultured EP2-/-, EP4flox/flox, and WT mice mesangial cells to detect the relevant indicators and downstream signalling. Additionally, 5/6 nephrectomy were introduced to confirm the in vitro outcomes. Results: We found that the expression of fibronectin (FN), COX-2 and the phosphorylation level of Smad3 in group EP2-/- obviously increased, with the falling of cyclic adenosine monophosphate (cAMP) level and no protein kinase (PKA) activity. While the relevant genes decreased in EP4 knock out (EP4flox/flox + AD-CRE) group, which represent no significant change in the level of cAMP, although with high level of PKA activity and extracellular signal transduction kinase (ERK) phosphorylation. In vivo study, 24-hour proteinuria, serum creatinine and urea nitrogen in EP4+/- group obviously declined, and the degree of glomerular sclerosis and the density of FN and COX-2 in glomerulus reduced significantly. EP2-/- show a totally opposite event to EP4+/- . Conclusion: EP2 receptor mainly depends on cAMP/PKA to inhibit effect of PGE2 induced by TGF-β1/Smad3 pathway and further alleviate the damage of MCs. While EP4 may coordinate the effect of MCs injury through ERK phosphorylation. Our finding suggests the pleiotropic functions of PGE2, and thus explores a potential therapy on the level of gene mediation.

Mitochondria-targeted peptide SS-31 attenuates renal injury via an antioxidant effect in diabetic nephropathy

Oxidative stress is implicated in the pathogenesis of diabetic kidney injury. SS-31 is a mitochondria-targeted tetrapeptide that can scavenge reactive oxygen species (ROS). Here, we investigated the effect and molecular mechanism of mitochondria-targeted antioxidant peptide SS-31 on injuries in diabetic kidneys and mouse mesangial cells (MMCs) exposed to high-glucose (HG) ambience. CD-1 mice underwent uninephrectomy and streptozotocin treatment prior to receiving daily intraperitoneal injection of SS-31 for 8 wk. The diabetic mice treated with SS-31 had alleviated proteinuria, urinary 8-hydroxy-2-deoxyguanosine level, glomerular hypertrophy, and accumulation of renal fibronectin and collagen IV. SS-31 attenuated renal cell apoptosis and expression of Bax and reversed the expression of Bcl-2 in diabetic mice kidneys. Furthermore, SS-31 inhibited expression of transforming-growth factor (TGF)-β1, Nox4, and thioredoxin-interacting protein (TXNIP), as well as activation of p38 MAPK and CREB and NADPH oxidase activity in diabetic kidneys. In vitro experiments using MMCs revealed that SS-31 inhibited HG-mediated ROS generation, apoptosis, expression of cleaved caspase-3, Bax/Bcl-2 ratio, and cytochrome c (cyt c) release from mitochondria. SS-31 normalized mitochondrial potential (ΔΨm) and ATP alterations, and inhibited the expression of TGF-β1, Nox4, and TXNIP, as well as activation of p38 MAPK and CREB and NADPH oxidase activity in MMCs under HG conditions. SS-31 treatment also could reverse the reduction of thioredoxin (TRX) biologic activity and upregulate expression of thioredoxin 2 (TRX2) in MMCs under HG conditions. In conclusion, this study demonstrates a protective effect of SS-31 against HG-induced renal injury via an antioxidant mechanism in diabetic nephropathy.

Cytosolic phospholipase A2α regulates G1 progression through modulating FOXO1 activity.

Group IVA phospholipase A2 [cytosolic phospholipase A2α (cPLA2α)] is a key mediator of inflammation and tumorigenesis. In this study, by using a combination of chemical inhibition and genetic approaches in zebrafish and murine cells, we identify a mechanism by which cPLA2α promotes cell proliferation. We identified 2 cpla2α genes in zebrafish, cpla2αa and cpla2αb, with conserved phospholipase activity. In zebrafish, loss of cpla2α expression or inhibition of cpla2α activity diminished G1 progression through the cell cycle. This phenotype was also seen in both mouse embryonic fibroblasts and mesangial cells. G1 progression was rescued by the addition of arachidonic acid or prostaglandin E2 (PGE2), indicating a phospholipase-dependent mechanism. We further show that PGE2, through PI3K/AKT activation, promoted Forkhead box protein O1 (FOXO1) phosphorylation and FOXO1 nuclear export. This led to up-regulation of cyclin D1 and down-regulation of p27(Kip1), thus promoting G1 progression. Finally, using pharmacologic inhibitors, we show that cPLA2α, rapidly accelerated fibrosarcoma (RAF)/MEK/ERK, and PI3K/AKT signaling pathways cooperatively regulate G1 progression in response to platelet-derived growth factor stimulation. In summary, these data indicate that cPLA2α, through its phospholipase activity, is a critical effector of G1 phase progression through the cell cycle and suggest that pharmacological targeting of this enzyme may have important therapeutic benefits in disease mechanisms that involve excessive cell proliferation, in particular, cancer and proliferative glomerulopathies.

Role of FQQI motif in the internalization, trafficking, and signaling of guanylyl-cyclase/natriuretic peptide receptor-A in cultured murine mesangial cells

Binding of the cardiac hormone atrial natriuretic peptide (ANP) to transmembrane guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), produces the intracellular second messenger cGMP in target cells. To delineate the critical role of an endocytic signal in intracellular sorting of the receptor, we have identified a FQQI (Phe(790), Gln(791), Gln(792), and Ile(793)) motif in the carboxyl-terminal region of NPRA. Mouse mesangial cells (MMCs) were transiently transfected with the enhanced green fluorescence protein (eGFP)-tagged wild-type (WT) and mutant constructs of eGFP-NPRA. The mutation FQQI/AAAA, in the eGFP-NPRA cDNA sequence, markedly attenuated the internalization of mutant receptors by almost 49% compared with the WT receptor. Interestingly, we show that the μ1B subunit of adaptor protein-1 binds directly to a phenylalanine-based FQQI motif in the cytoplasmic tail of the receptor. However, subcellular trafficking indicated that immunofluorescence colocalization of the mutated receptor with early endosome antigen-1 (EEA-1), lysosome-associated membrane protein-1 (LAMP-1), and Rab 11 marker was decreased by 57% in early endosomes, 48% in lysosomes, and 42% in recycling endosomes, respectively, compared with the WT receptor in MMCs. The receptor containing the mutated motif (FQQI/AAAA) also produced a significantly decreased level of intracellular cGMP during subcellular trafficking than the WT receptor. The coimmunoprecipitation assay confirmed a decreased level of colocalization of the mutant receptor with subcellular compartments during endocytic processes. The results suggest that the FQQI motif is essential for the internalization and subcellular trafficking of NPRA during the hormone signaling process in intact MMCs.

Therapeutic potential of digitoflavone on diabetic nephropathy: nuclear factor erythroid 2-related factor 2-dependent anti-oxidant and anti-inflammatory effect.

Nuclear factor erythroid 2-related factor 2 (Nrf2) has emerged as a therapeutic target in many diseases, because it can induce antioxidant enzymes and other cytoprotective enzymes. Moreover, some Nrf2 activators have strong anti-inflammatory activities. Oxidative stress and inflammation are major components involved in the pathology of diabetic nephropathy. In the present study, we evaluated the Nrf2-dependent anti-oxidative and anti-inflammatory effects of digitoflavone in streptozotocin-induced diabetic nephropathy. The molecular mechanisms of digitoflavone were investigated in vitro using SV40-transformed mouse mesangial cells (SV40-Mes13). For the in vivo experiment, diabetes was induced in Nrf2+/+ and Nrf2−/− mice by STZ injection, and digitoflavone was administered 2 weeks after the STZ injection. Digitoflavone induced Nrf2 activation and decreased oxidative damage, inflammation, TGF-β1 expression, extracellular matrix protein expression, and mesangial cell hyperplasia in SV40-Mes13 cells. Digitoflavone-treated Nrf2+/+ mice, but not Nrf2−/− mice, showed attenuated common metabolic disorder symptoms, improved renal performance, minimized pathological alterations, and decreased oxidative damage, inflammatory gene expression, inflammatory cell infiltration, TGF-β1 expression, and extracellular matrix protein expression. Our results show that the anti-oxidative and anti-inflammatory effects of digitoflavone are mediated by Nrf2 activation and that digitoflavone can be used therapeutically to improve metabolic disorders and relieve renal damage induced by diabetes.

Steap4 attenuates high glucose and S100B-induced effects in mesangial cells.

Six-transmembrane epithelial antigen of prostate 4 (Steap4)-knockout mice develop hyperglycaemia and inflammation whereas Steap4 overexpression attenuates atherosclerosis in diabetic mice. Thus, we studied the roles of Steap4 in high glucose (HG, 27.5 mM) or S100B (1 μM, a ligand for the receptor for advanced glycation end-product or RAGE)-induced effects in mouse mesangial (MES13) cells. We found that HG-induced Steap4 protein expression was dependent on S100B. HG increased cell membrane, but not cytosolic, Steap4 protein expression. HG increased protein-protein interaction between Steap4 and S100B, which was confirmed by mass spectrometry of immunoprecipitated S100B. SP600125, LY294002 and AG490 attenuated S100B-induced Steap4 protein expression or gene transcriptional activity. A mutation in signal transducer and activator of transcription 3 (Stat3) site 2 of the Steap4 promoter constructs resulted in a marked decrease in HG or S100B-induced activation of Steap4 gene transcription. Overexpression of Steap4 attenuates HG or S100B-induced collagen IV, fibronectin and cyclooxygenase 2 protein expression. Overexpression of Steap4 attenuates HG or S100B-induced transforming growth factor-β (TGF-β). Moreover, overexpression of Steap4 attenuates S100B-induced signalling. Finally, overexpressing Steap4 attenuated renal expression of fibronectin, S100B, TGF-β, type IV collagen, p-Akt, p-extracellular signal regulated kinase 1/2 and p-Stat3 in streptozotocin-diabetic mice. Thus, overexpression of Steap4 attenuated HG or S100B-induced effects in MES13 cells and attenuated some of S100B-induced effects in diabetic mouse kidneys.

Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts.

Both of the sphingosine kinase (SK) subtypes SK-1 and SK-2 catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate (S1P). However, the subtype-specific cellular functions are largely unknown. In this study, we investigated the cellular function of SK-2 in primary mouse renal mesangial cells (mMC) and embryonic fibroblasts (MEF) from wild-type C57BL/6 or SK-2 knockout (SK2ko) mice. We found that SK2ko cells displayed a significantly higher proliferative and migratory activity when compared to wild-type cells, with concomitant increased cellular activities of the classical extracellular signal regulated kinase (ERK) and PI3K/Akt cascades, and of the small G protein RhoA. Furthermore, we detected an upregulation of SK-1 protein and S1P3 receptor mRNA expression in SK-2ko cells. The MEK inhibitor U0126 and the S1P1/3 receptor antagonist VPC23019 blocked the increased migration of SK-2ko cells. Additionally, S1P3ko mesangial cells showed a reduced proliferative behavior and reduced migration rate upon S1P stimulation, suggesting a crucial involvement of the S1P3 receptor. In summary, our data demonstrate that SK-2 exerts suppressive effects on cell growth and migration in renal mesangial cells and fibroblasts, and that therapeutic targeting of SKs for treating proliferative diseases requires subtype-selective inhibitors.

SiRNA delivery to the glomerular mesangium using polycationic cyclodextrin nanoparticles containing siRNA.

There is an urgent need for new therapies that can halt or reverse the course of chronic kidney disease with minimal side-effect burden on the patient. Small interfering RNA (siRNA) nanoparticles are new therapeutic entities in clinical development that could be useful for chronic kidney disease treatment because they combine the tissue-specific targeting properties of nanoparticles with the gene-specific silencing effects of siRNA. Recent reports have emerged demonstrating that the kidney, specifically the glomerulus, is a readily accessible site for nanoparticle targeting. Here, we explore the hypothesis that intravenously administered polycationic cyclodextrin nanoparticles containing siRNA (siRNA/CDP-NPs) can be used for delivery of siRNA to the glomerular mesangium. We demonstrate that siRNA/CDP-NPs localize to the glomerular mesangium with limited deposition in other areas of the kidney after intravenous injection. Additionally, we report that both mouse and human mesangial cells rapidly internalize siRNA/CDP-NPs in vitro and that nanoparticle uptake can be enhanced by attaching the targeting ligands mannose or transferrin to the nanoparticle surface. Lastly, we show knockdown of mesangial enhanced green fluorescent protein expression in a reporter mouse strain following iv treatment with siRNA/CDP-NPs. Altogether, these data demonstrate the feasibility of mesangial targeting using intravenously administered siRNA/CDP-NPs.

CXCL10 expression induced by Mxi1 inactivation induces mesangial cell apoptosis in mouse Habu nephritis

MAX interactor 1 (Mxi1) proteins are c-myc antagonists that primarily exert their biological functions by inhibiting Myc-dependent gene transcription. In this study, Mxi1−/− mice were used to generate a model of mesangial proliferative glomerulonephritis for the first time. In the present study, we demonstrated that Mxi1−/− mice exhibited a more typical and severe pathological phenotype, which was displayed primarily as a noticeable dissolution phenotype with a higher proportion of apoptotic cells and higher chemokine CXCL10 expression during the early days of modeling, compared with wild-type mice. Additionally, we determined that IRF3-mediated TLR4 signaling was likely involved in regulating CXCL10 expression, which might participate in the mesangial dissolution process. We also found increases in CXCL10 expression, caspase 3 activation, and the proportion of apoptotic cells when Mxi1 expression was inhibited in mouse mesangial cells. Furthermore, the proportion of apoptotic cells decreased after inhibiting CXCL10 expression. Therefore, we concluded that the mesangial cell apoptosis observed in this mesangial proliferative glomerulonephritis model was related to CXCL10 expression induced by Mxi1 inactivation. This finding provides a new theoretical basis for the mechanism of mesangial proliferative glomerulonephritis progression and reveals potential intervention targets for the early treatment of this disease.

ONO-1301, a sustained-release prostacyclin analog, ameliorates the renal alterations in a mouse type 2 diabetes model possibly through its protective effects on mesangial cells.

Diabetic nephropathy is the most common pathological disorder predisposing patients to end-stage renal disease. Considering the increasing prevalence of type 2 diabetes mellitus worldwide, novel therapeutic approaches are urgently needed. ONO-1301 is a novel sustained-release prostacyclin analog that inhibits thromboxane A2 synthase. Here we examined the therapeutic effects of the intermittent administration of slow-release ONO-1301 (SR-ONO) on diabetic nephropathy in obese type 2 diabetes mice, as well as its direct effects on mesangial cells. The subcutaneous injection of SR-ONO (3mg/kg) every 3 wks did not affect the obesity or hyperglycemia in the db/db obese mice used as a model of type 2 diabetes, but it significantly ameliorated their albuminuria, glomerular hypertrophy, glomerular accumulation of type IV collagen, and monocyte/macrophage infiltration, and also the increase of TGF-β1, α-smooth muscle actin (α-SMA) and MCP-1 compared to vehicle treatment. In cultured mouse mesangial cells, ONO-1301 concentration-dependently suppressed the increases in TGF-β, type IV collagen, α-SMA, MCP-1 and fibronectin induced by high ambient glucose, at least partly through prostacyclin (PGI2) receptor-mediated signaling. Taken together, these results suggest the potential therapeutic efficacy of the intermittent administration of SR-ONO against type 2 diabetic nephropathy, possibly through protective effects on mesangial cells.

Hypoxia Inducible Factor-1 Alpha Promotes Mesangial Cell Proliferation in Lupus Nephritis

Background: Evidence has accumulated that hypoxia plays a significant role in the pathogenesis and progression of both acute renal injury and chronic renal disease. However, little was known about the effects of hypoxia on lupus nephritis (LN). In the current study, we investigated the expression of hypoxia inducible factor-1 alpha (HIF-1α) in LN. Methods: Renal biopsies from 22 LN patients and 20 patients with renal carcinoma were obtained. In situ HIF-1α expression was examined by immunohistochemical staining, and the relationship between HIF-1α and clinical/pathological features was analyzed. HIF-1α expression in kidney from both MRL/lpr and C57BL/6 mice was detected by immunohistochemical technology. Dimethyloxaloylglycine (DMOG), an inhibitor of HIF-degrading prolylhydroxylases, was utilized to prevent HIF-1α degradation in mouse mesangial cells (MCs). After DMOG treatment, the proliferation and apoptosis rates of mouse MCs were determined. Results: LN patients showed larger amounts of HIF-1α in both glomerular and tubulointerstitial areas. The levels of intraglomerular HIF-1α were closely associated with renal pathology activity index and clinical manifestations in LN patients. In MRL/lpr mice, intraglomerular HIF-1α-positive cells were also significantly increased. Interestingly, the levels of HIF-1α positively correlated with cell density in glomerulus in both LN patients and MRL/lpr mice. Upon treatment with DMOG, the proliferation of MCs was upregulated, and apoptosis was downregulated. Conclusion: HIF-1α is highly expressed in both glomerular and tubulointerstitial tissues in LN, especially in proliferative LN. HIF-1α may promote MCs growth through the induction of proliferation and inhibition of apoptosis, and hence plays an important role in the pathogenesis of LN. © 2014 S. Karger AG, Basel

S100B is required for high glucose-induced pro-fibrotic gene expression and hypertrophy in mesangial cells.

The advanced glycation end‑product (AGE)‑receptor for AGE (RAGE) axis induces transforming growth factor‑β (TGF‑β) expression, cell hypertrophy and increases extracellular matrices that are indicated in the pathogenesis of diabetic nephropathy (DN). RAGE binds to numerous ligands besides AGE, including S100B. In the present study, the roles of S100B in high glucose‑induced p21WAF1, extracellular matrices, TGF‑βl and cell hypertrophy in mouse mesangial (MES13) cells were investigated. High glucose (30 mM) time‑dependently (24‑72 h) induced S100B expression. High glucose and exogenous S100B (1 µM) time‑dependently increased p21WAF1 gene transcription and protein expression, increased type IV collagen and fibronectin protein expression, and TGF‑β gene transcription and bioactivity. Exogenous S100B also time‑dependently activated the extracellular regulated kinases (ERK1/2), p38 kinase and c‑Jun N‑terminal kinase (JNK) signaling pathways. Exogenous S100B‑induced TGF‑β gene transcription and bioactivity were attenuated by SB203580 (p38 kinase inhibitor) and PD98059 (ERK1/2 inhibitor). Finally, the knockdown of S100B by small interfering RNA (siRNA) attenuated high glucose‑induced TGF‑β gene transcription and bioactivity, type IV collagen and fibronectin protein expression and p21WAF1 protein expression. Thus, S100B induced TGF‑β via the ERK1/2 and p38 kinase pathways in mesangial cells. Additionally, high glucose‑induced pro‑fibrotic genes (TGF‑β, type IV collagen and fibronectin) and cell hypertrophy‑related p21WAF1 are dependent on S100B.

Role of the prostaglandin E2/E-prostanoid 2 receptor signalling pathway in TGFβ-induced mice mesangial cell damage.

The prostaglandin E2 receptor, EP2 (E-prostanoid 2), plays an important role in mice glomerular MCs (mesangial cells) damage induced by TGFβ1 (transforming growth factor-β1); however, the molecular mechanisms for this remain unknown. The present study examined the role of the EP2 signalling pathway in TGFβ1-induced MCs proliferation, ECM (extracellular matrix) accumulation and expression of PGES (prostaglandin E2 synthase). We generated primary mice MCs. Results showed MCs proliferation promoted by TGFβ1 were increased; however, the production of cAMP and PGE2 (prostaglandin E2) was decreased. EP2 deficiency in these MCs augmented FN (fibronectin), Col I (collagen type I), COX2 (cyclooxygenase-2), mPGES-1 (membrane-associated prostaglandin E1), CTGF (connective tissue growth factor) and CyclinD1 expression stimulated by TGFβ1. Silencing of EP2 also strengthened TGFβ1-induced p38MAPK (mitogen-activated protein kinase), ERK1/2 (extracellular-signal-regulated kinase 1/2) and CREB1 (cAMP responsive element-binding protein 1) phosphorylation. In contrast, Adenovirus-mediated EP2 overexpression reversed the effects of EP2-siRNA (small interfering RNA). Collectively, the investigation indicates that EP2 may block p38MAPK, ERK1/2 and CREB1 phosphorylation via activation of cAMP production and stimulation of PGE2 through EP2 receptors which prevent TGFβ1-induced MCs damage. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the damage induced by TGFβ1.

Repression of let-7 by transforming growth factor-β1-induced Lin28 upregulates collagen expression in glomerular mesangial cells under diabetic conditions.

Accumulation of mesangial extracellular matrix (ECM) proteins such as collagen type 1-α2 (Col1a2) and collagen type 4-α1 (Col4a1) is a key feature of diabetic nephropathy (DN). Transforming growth factor (TGF)-β1 plays important roles in ECM accumulation in DN, and evidence shows a mediatory role for microRNAs. In the present study, we found that microRNA let-7 family members (let-7b/c/d/g/i) were downregulated in TGF-β-treated mouse mesangial cells (MMCs) along with upregulation of Col1a2 and Col4a1. Ectopic expression of let-7b in TGF-β-treated MMCs attenuated Col1a2 and Col4a1 upregulation. Conversely, let-7b inhibitors increased Col1a2 and Col4a1 levels. Cotransfection of MMCs with mouse Col1a2 or Col4a1 3'-untranslated region luciferase constructs and let-7b inhibitors increased luciferase activity. However, constructs with let-7 target site mutations were unresponsive to TGF-β. TGF-β-induced 3'-untranslated region activity was attenuated by let-7b mimics, suggesting that Col1a2 and Col4a1 are direct targets of let-7b. In addition, Lin28b, a negative regulator of let-7 biogenesis, was upregulated in TGF-β-treated MMCs. Luciferase assays showed that the Lin28b promoter containing the Smad-binding element (SBE) responded to TGF-β, which was abolished in constructs without SBE. Chromatin immunoprecipitation assays showed TGF-β-induced enrichment of Smad2/3 at the Lin28b promoter, together suggesting that Lin28b is transcriptionally induced by TGF-β through SBE. Furthermore, let-7b levels were decreased, whereas Lin28b, Col1a2, and Col4a1 levels were increased, in glomeruli of diabetic mice compared with nondiabetic control mice, demonstrating the in vivo relevance of this Lin28/let-7/collagen axis. These results identify Lin28 as a new TGF-β target gene and suggest a novel role for the Lin28/let-7 pathway in controlling TGF-β-induced collagen accumulation in DN.

Prostaglandin E2 EP1 receptor enhances TGF-β1-induced mesangial cell injury.

Increasing evidence indicates that transforming growth factor-β1 (TGF-β1) is a pivotal mediator in the pathogenesis of renal fibrosis. Mesangial cells (MCs) are important for glomerular function under both physiological and pathological conditions. Studies have found that the expression level of prostaglandin E2 (PGE2) in MCs increases under high glucose conditions, that PGE2 affects the proliferation and hypertrophy of MCs mainly through the EP1 pathway, and that the proliferation of MCs and the accumulation of extracellular matrix are the main events leading to glomerular fibrosis. In this study, we investigated the effects and mechanisms of action of the EP1 receptor, which is induced by transforming growth factor (TGF)-β1, on the proliferation of mouse MCs, the accumulation of extracellular matrix and the expression of PGE2 synthase. Primary mouse glomerular MCs were isolated from EP1 receptor-deficient mice (EP1-/- mice, in which the EP1 receptor was knocked down) and wild-type (WT) mice (WT MCs). In our preliminary experiments, we found that cell proliferation, as well as the mRNA and protein expression of cyclin D1, proliferating cell nuclear antigen (PCNA), fibronectin (FN), collagen I (ColI), membrane-associated PGE2 synthase-1 (mPGES-1) and cyclooxygenase-2 (COX-2) in the WT MCs were significantly increased following treatment with 10 ng/ml TGF-β1 for 24 h. Compared with the WT MCs, following the knockdown of the EP1 gene, the TGF-β1-induced MC injury was markedly suppressed. The aforementioned changes were notably enhanced following treatment with the EP1 agonist, 17-phenyl trinor PGE2 ethyl amide. Additionally, TGF-β1 induced extracellular signal-regulated kinase (ERK) phosphorylation. We found that the TGF-β1-induced ERK phosphorylation was alleviated by EP1 knockdown and promoted by EP1 expression. These results suggest that the EP1 receptor plays a role in the proliferation of mouse MCs, in the accumulation of extracellular matrix and in the expression of mPGES-1 induced by TGF-β1. Its mechanisms of action are possibly related to the reinforcement of ERK phosphorylation.

Transforming Growth Factor β1 (TGF-β1) Enhances Expression of Profibrotic Genes through a Novel Signaling Cascade and MicroRNAs in Renal Mesangial Cells

Increased expression of transforming growth factor-β1 (TGF-β1) in glomerular mesangial cells (MC) augments extracellular matrix accumulation and hypertrophy during the progression of diabetic nephropathy (DN), a debilitating renal complication of diabetes. MicroRNAs (miRNAs) play key roles in the pathogenesis of DN by modulating the actions of TGF-β1 to enhance the expression of profibrotic genes like collagen. In this study, we found a significant decrease in the expression of miR-130b in mouse MC treated with TGF-β1. In parallel, there was a down-regulation in miR-130b host gene 2610318N02RIK (RIK), suggesting host gene-dependent expression of this miRNA. TGF-β receptor 1 (TGF-βR1) was identified as a target of miR-130b. Interestingly, the RIK promoter contains three NF-Y binding sites and was regulated by NF-YC. Furthermore, NF-YC expression was inhibited by TGF-β1, suggesting that a signaling cascade, involving TGF-β1-induced decreases in NF-YC, RIK, and miR-130b, may up-regulate TGF-βR1 to augment expression of TGF-β1 target fibrotic genes. miR-130b was down-regulated, whereas TGF-βR1, as well as the profibrotic genes collagen type IV α 1 (Col4a1), Col12a1, CTGF, and PAI-1 were up-regulated not only in mouse MC treated with TGF-β1 but also in the glomeruli of streptozotocin-injected diabetic mice, supporting in vivo relevance. Together, these results demonstrate a novel miRNA- and host gene-mediated amplifying cascade initiated by TGF-β1 that results in the up-regulation of profibrotic factors, such as TGF-βR1 and collagens associated with the progression of DN.

A Maladaptive Role for EP4 Receptors in Mouse Mesangial Cells

Roles of the prostaglandin E2 E-prostanoid 4 receptor (EP4) on extracellular matrix (ECM) accumulation induced by TGF-β1 in mouse glomerular mesangial cells (GMCs) remain unknown. Previously, we have identified that TGF-β1 stimulates the expression of FN and Col I in mouse GMCs. Here we asked whether stimulation of EP4 receptors would exacerbate renal fibrosis associated with enhanced glomerular ECM accumulation. We generated EP4Flox/Flox and EP4+/− mice, cultured primary WT, EP4Flox/Flox and EP4+/− GMCs, AD-EP4 transfected WT GMCs (EP4 overexpression) and AD-Cre transfected EP4Flox/Flox GMCs (EP4 deleted). We found that TGF-β1-induced cAMP and PGE2 synthesis decreased in EP4 deleted GMCs and increased in EP4 overexpressed GMCs. Elevated EP4 expression in GMCs augmented the coupling of TGF-β1 to FN, Col I expression and COX2/PGE2 signaling, while TGF-β1 induced FN, Col I expression and COX2/PGE2 signaling were down-regulated in EP4 deficiency GMCs. 8 weeks after 5/6 nephrectomy (Nx), WT and EP4+/− mice exhibited markedly increased accumulation of ECM compared with sham-operated controls. Albuminuria, blood urea nitrogen and creatinine (BUN and Cr) concentrations were significantly increased in WT mice as compared to those of EP4+/− mice. Urine osmotic pressure was dramatically decreased after 5/6 Nx surgery in WT mice as compared to EP4+/− mice. The pathological changes in kidney of EP4+/− mice was markedly alleviated compared with WT mice. Immunohistochemical analysis showed significant reductions of Col I and FN in the kidney of EP4+/− mice compared with WT mice. Collectively, this investigation established EP4 as a potent mediator of the pro-TGF-β1 activities elicited by COX2/PGE2 in mice GMCs. Our findings suggested that prostaglandin E2, acting via EP4 receptors contributed to accumulation of ECM in GMCs and promoted renal fibrosis.

Suppression of mesangial cell proliferation and extracellular matrix production in streptozotocin-induced diabetic mice by adiponectin in vitro and in vivo.

Renal growth, particularly hypertrophy, is a feature of diabetic nephropathy (DN). Adiponectin, an adipocyte-derived hormone, is an important regulator of cell proliferation. Recent studies have suggested that adiponectin has a protective effect in the kidney. The purpose of this study was to investigate the therapeutic effects and the underlying mechanisms of adiponectin in early DN. Mouse mesangial cells (MMCs) were cultured in media containing different concentrations of platelet-derived growth factor-BB (PDGF-BB) with or without adiponectin. MMC proliferation and expression of type IV collagen, laminin, and fibronectin were investigated. Streptozotocin-induced diabetic mice were injected intravenously with recombinant lentivirus encoding the mouse adiponectin gene (Lenti-Acdc-IRES-EGFP). Urinary microalbumin, serum adiponectin level, and expression of proliferating cell nuclear antigen, type IV collagen, laminin, and fibronectin were determined. Adiponectin inhibited the increases in MMC proliferation and expression of type IV collagen, laminin, and fibronectin induced by PDGF-BB. Adiponectin also effectively reduced renal cell proliferation and expression of type IV collagen, laminin, and fibronectin when it was introduced in vivo by lentivirus-mediated gene transfer. These findings suggest that adiponectin exerts renoprotective effects by inhibiting renal cell proliferation and reducing synthesis of extracellular matrix proteins, thus suppressing the development and progression of DN.

The PTEN/PI3K/Akt signaling pathway mediates HMGB1-induced cell proliferation by regulating the NF-κB/cyclin D1 pathway in mouse mesangial cells.

Our previous experiment confirmed that high-mobility group box chromosomal protein 1 (HMGB1) was involved in the pathogenesis of Lupus nephritis (LN) by upregulating the proliferation of the mouse mesangial cell line (MMC) through the cyclin D1/CDK4/p16 system, but the precise mechanism is still unknown. Therefore, in the present study, we demonstrated that HMGB1 induced the proliferation of MMC cells in a time- and concentration-dependent manner, downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression, increased the level of Akt serine 473 phosphorylation, and induced p65 subunit nuclear translocation. The overexpression of PTEN prevented the upregulation of HMGB1-induced proliferation by blocking the activation of Akt. The knockdown of Akt by siRNA technology and blocking the nuclear factor-κB (NF-κB) pathway using pyrrolidine dithiocarbamate (PDTC) and SN50, inhibitors of NF-κB, both attenuated the HMGB1-induced proliferation by counteracting the activation of the cyclin D1. In addition, while sh-Akt partly blocked the nuclear translocation of the p65 subunit, PDTC did not affect the activation of the Akt induced by HMGB1 in MMC cells. These findings indicate that HMGB1 induced the proliferation of MMC cells by activating the PTEN/phosphoinositide-3-kinase (PI3K)/Akt/NF-κB signaling pathway.