Antiviral Properties of HIV-1 Capsid Inhibitor GSK878.

GSK878 is a newly described HIV-1 inhibitor that binds to the mature capsid (CA) hexamer in a pocket originally identified as the binding site of the well-studied CA inhibitor PF-74. Here, we show that GSK878 is highly potent, inhibiting an HIV-1 reporter virus in MT-2 cells with a mean 50% effective concentration (EC50) of 39 pM and inhibiting a panel of 48 chimeric viruses containing diverse CA sequences with a mean EC50 of 94 pM. CA mutations associated with reduced susceptibility to other inhibitors that bind to PF-74 binding site (L56I, M66I, Q67H, N74D, T107N, and Q67H/N74D) also reduced susceptibility to GSK878, with M66I, Q67H/N74D, and L56I having the greatest impact on antiviral activity. Amino acid substitutions in the CA cyclophilin A (CypA) binding loop (H87P and P90A), distal from the inhibitor binding site and associated with reduced CA-CypA binding, subtly, but reproducibly, also decreased GSK878 potency. Mechanism-of-action studies showed that GSK878 blocked both early (preintegration) and late (postintegration) steps in HIV-1 replication, with the early inhibition primarily determining the compound's antiviral activity. The early inhibition results from blocks to HIV-1 nuclear import and proviral integration and is associated with altered stability of the HIV-1 CA core.

Potent Long-Acting Inhibitors Targeting the HIV-1 Capsid Based on a Versatile Quinazolin-4-one Scaffold.

Long-acting (LA) human immunodeficiency virus-1 (HIV-1) antiretroviral therapy characterized by a ≥1 month dosing interval offers significant advantages over daily oral therapy. However, the criteria for compounds that enter clinical development are high. Exceptional potency and low plasma clearance are required to meet dose size requirements; excellent chemical stability and/or crystalline form stability is required to meet formulation requirements, and new antivirals in HIV-1 therapy need to be largely free of side effects and drug-drug interactions. In view of these challenges, the discovery that capsid inhibitors comprising a quinazolinone core tolerate a wide range of structural modifications while maintaining picomolar potency against HIV-1 infection in vitro, are assembled efficiently in a multi-component reaction, and can be isolated in a stereochemically pure form is reported herein. The detailed characterization of a prototypical compound, GSK878, is presented, including an X-ray co-crystal structure and subcutaneous and intramuscular pharmacokinetic data in rats and dogs.

Low-dose IL-2 expands CD4+ regulatory T cells with a suppressive function in vitro via the STAT5-dependent pathway in patients with chronic kidney diseases.

BACKGROUND: Patients with chronic kidney disease (CKD) often have CD4+ regulatory T cells (Tregs) dysfunction and chronic inflammation. We aim to investigate the effect, function, and related mechanism of low-dose IL-2 on CD4+ regulatory T cells expansion in vitro from patients with CKD. METHODS: A total of 148 newly diagnosed patients with CKD at Stage III and 35 healthy volunteer subjects were recruited into our studies. The number of peripheral Tregs in peripheral blood mononuclear cells isolated from CKD patients, which were characterized by FACS as CD4+CD25hi and CD4+CD25+FoxP3+. The effect of low-dose IL-2 on expansion of Tregs, and the suppressive function of expanded Tregs were also analyzed by FACS. The levels of FoxP3 mRNA were detected by qRT-PCR. The activation of IL-2 induced Stat5 and blocking experiments were assessed by Western Blotting and FACS. RESULTS: We found that the frequency of peripheral Tregs from CKD patients was significantly lower than that in healthy volunteer subjects. We also showed that IL-2 selectively expanded CD4+CD25hi and CD4+CD25+FoxP3+ regulatory T cells, and also upregulated the expression of FoxP3 mRNA. Our in vitro studies demonstrated that expanded CD4+ regulatory T cells from CKD patients suppressed proinflammatory Th1 and Th17 cell response. Furthermore, STAT5 activation is required for IL-2-induced expansion of regulatory T cells and expression of FoxP3 mRNA from CKD patients. CONCLUSIONS: Our findings support the clinical Treg defects in CKD patients with glomerular diseases, and the rationale of evaluating low-dose IL-2 treatment for selectively modulating CD4+ Tregs.

The Effects of Indoxyl Sulfate on Human Umbilical Cord-Derived Mesenchymal Stem Cells In Vitro.

BACKGROUND/AIMS: Indoxyl sulfate, an important protein-bound uremic toxin, can damage stem cells, thus hampering stem cell-based regenerative medicine approaches targeting chronic kidney diseases (CKD). Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are thought to have promising clinical application because of their high proliferative potential and ease of isolation than MSCs from other sources. In the present study, we aimed to determine the harmful effects of indoxyl sulfate on the phenotype and functional potential of hUC-MSCs in vitro. METHODS: The toxicity and cell viability was examined by Trypan blue exclusion and MTT assay. The cellular surface markers and the percentage of apoptotic cells by Annexin-V/PI staining were analyzed by flow cytometry. Proliferation was evaluated based on cell number counting and Ki-67 immunostaining. Cell senescence was measured using senescence-associated ß-Galactosidase activity. The ability to stimulate the development of CD4+CD25+FoxP3+ regulatory T cells was assessed by incubating hUC-MSCs with peripheral blood mononuclear cells from the healthy volunteers. RESULTS: Our results demonstrated that the immunophenotype of hUC-MSCs was not affected by indoxyl sulfate flow cytometry. However, a significant decrease in cell numbers and fraction of Ki-67 positive proliferating cells, along with a significant increase in cellular senescence were detected in hUC-MSCs after exposure to indoxyl sulfate. Additionally, their ability to stimulate CD4+CD25+FoxP3+ regulatory T cell production was compromised when hUC-MSCs were pretreated with indoxyl sulfate. CONCLUSION: Taken together, our study clearly demonstrated that the molecular alterations and functional incompetence in hUC-MSCs under the challenge of indoxyl sulfate in vitro.

BRG1-mediated immune tolerance: facilitation of Treg activation and partial independence of chromatin remodelling.

Treg activation in response to environmental cues is necessary for regulatory T cells (Tregs) to suppress inflammation, but little is known about the transcription mechanisms controlling Treg activation. We report that despite the known proinflammatory role of the chromatin-remodelling factor BRG1 in CD4 cells, deleting Brg1 in all αß T cell lineages led to fatal inflammation, which reflected essential roles of BRG1 in Tregs. Brg1 deletion impaired Treg activation, concomitant with the onset of the inflammation. Remarkably, as the inflammation progressed, Tregs became increasingly activated, but the activation levels could not catch up with the severity of inflammation. In vitro assays indicate that BRG1 regulates a subset of TCR target genes including multiple chemokine receptor genes. Finally, using a method that can create littermates bearing either a tissue-specific point mutation or deletion, we found the BRG1 ATPase activity partially dispensable for BRG1 function. Collectively, these data suggest that BRG1 acts in part via remodelling-independent functions to sensitize Tregs to inflammatory cues, thus allowing Tregs to promptly and effectively suppress autoimmunity.

Inducible mouse models illuminate parameters influencing epigenetic inheritance.

Environmental factors can stably perturb the epigenome of exposed individuals and even that of their offspring, but the pleiotropic effects of these factors have posed a challenge for understanding the determinants of mitotic or transgenerational inheritance of the epigenetic perturbation. To tackle this problem, we manipulated the epigenetic states of various target genes using a tetracycline-dependent transcription factor. Remarkably, transient manipulation at appropriate times during embryogenesis led to aberrant epigenetic modifications in the ensuing adults regardless of the modification patterns, target gene sequences or locations, and despite lineage-specific epigenetic programming that could reverse the epigenetic perturbation, thus revealing extraordinary malleability of the fetal epigenome, which has implications for 'metastable epialleles'. However, strong transgenerational inheritance of these perturbations was observed only at transgenes integrated at the Col1a1 locus, where both activating and repressive chromatin modifications were heritable for multiple generations; such a locus is unprecedented. Thus, in our inducible animal models, mitotic inheritance of epigenetic perturbation seems critically dependent on the timing of the perturbation, whereas transgenerational inheritance additionally depends on the location of the perturbation. In contrast, other parameters examined, particularly the chromatin modification pattern and DNA sequence, appear irrelevant.

A general approach for controlling transcription and probing epigenetic mechanisms: application to the CD4 locus.

Synthetic regulatory proteins such as tetracycline (tet)-controlled transcription factors are potentially useful for repression as well as ectopic activation of endogenous genes and also for probing their regulatory mechanisms, which would offer a versatile genetic tool advantageous over conventional gene targeting methods. In this study, we provide evidence supporting this concept using Cd4 as a model. CD4 is expressed in double-positive and CD4 cells but irreversibly silenced in CD8 cells. The silencing is mediated by heterochromatin established during CD8 lineage development via transient action of the Cd4 silencer; once established, the heterochromatin becomes self-perpetuating independently of the Cd4 silencer. Using a tet-sensitive Cd4 allele harboring a removable Cd4 silencer, we found that a tet-controlled repressor recapitulated the phenotype of Cd4-deficient mice, inhibited Cd4 expression in a reversible and dose-dependent manner, and could surprisingly replace the Cd4 silencer to induce irreversible Cd4 silencing in CD8 cells, thus suggesting the Cd4 silencer is not the (only) determinant of heterochromatin formation. In contrast, a tet-controlled activator reversibly disrupted Cd4 silencing in CD8 cells. The Cd4 silencer impeded this disruption but was not essential for its reversal, which revealed a continuous role of the silencer in mature CD8 cells while exposing a remarkable intrinsic self-regenerative ability of heterochromatin after forced disruption. These data demonstrate an effective approach for gene manipulation and provide insights into the epigenetic Cd4 regulatory mechanisms that are otherwise difficult to obtain.

Hepatitis C virus RNA elimination and development of resistance in replicon cells treated with BMS-790052.

BMS-790052, a first-in-class hepatitis C virus (HCV) replication complex inhibitor, targeting nonstructural protein 5A (NS5A), displays picomolar to nanomolar potency against genotypes 1 to 5. This exceptional potency translated into robust anti-HCV activity in clinical studies with HCV genotype 1-infected subjects. To date, all BMS-790052-associated resistance mutations have mapped to the N-terminal region of NS5A. To further characterize the antiviral activity of BMS-790052, HCV replicon elimination and colony formation assays were performed. Replicon was cleared from genotype 1a and 1b replicon cells in a time- and dose-dependent manner. Elimination of the genotype 1a replicon required longer treatment durations and higher concentrations of BMS-790052 than those for the genotype1b replicon. Single amino acid substitutions that conferred relatively low levels of resistance were observed at early time points and at low doses. Higher doses and longer treatment durations yielded mutations that conferred greater levels of resistance, including linked amino acid substitutions. Replicon cells that survived inhibitor treatment remained fully sensitivity to pegylated alpha interferon (pegIFN-α) and other HCV inhibitors. Moreover, genotype 1a replicon elimination was markedly enhanced when pegIFN-α and BMS-790052 were combined. Resistant variants observed in this study were very similar to those observed in a multiple ascending dose (MAD) monotherapy trial of BMS-790052, validating replicon elimination studies as a model to predict clinical resistance. Insights gained from the in vitro anti-HCV activity and resistance profiles of BMS-790052 will be used to help guide the clinical development of this novel HCV inhibitor.

In vitro activity of BMS-790052 on hepatitis C virus genotype 4 NS5A.

The antiviral profile of BMS-790052, a potent hepatitis C virus (HCV) replication complex inhibitor targeting nonstructural protein NS5A, is well characterized for HCV genotype-1. Here, we report that BMS-790052 inhibits hybrid replicons containing HCV genotype-4 NS5A genes with 50% effective concentrations (EC(50)s) ranging from 7 to 13 pM. NS5A residue 30 was an important site for BMS-790052-selected resistance in the hybrid replicons. Our results support the potential of BMS-790052 as a valuable component of combination therapy for HCV genotype-4 chronic infection.

[Sirolimus inhibits the expression of type I collagen and fibronectin in cultured renal cortical myofibroblasts].

OBJECTIVE: To investigate the anti-fibrotic effect of sirolimus (rapamycin) at the cell level. METHODS: The primary cultured rat renal cortical myofibroblasts were divided into two groups, control group and sirolimus 40 mg/L group at each time point. The protein levels of alpha-SMA, Col-I, fibronectin(FN) were analyzed by Western blot in both the whole cell lysates and supernatant culture media 12 h , 24 h and 48 h after incubation, respectively. Real-time quantitative PCR was carried out to measure the levels of procollagen-I mRNA 1 h, 2 h, 4 h, and 6 h after cell incubation. The activities of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell media were assayed by gelatin zymography. RESULTS: (1) Sirolimus had no effect on the expression of alpha-SMA of myofibroblasts at different time points. (2) The expression of Col-I in the whole cell lysates both reduced at the end of 24 h and 48 h in sirolimus group significantly [(0.58+/-0.05) and (0.63+/-0.18), P < 0.05] compared with control group at each time point, respectively. (3) The levels of procollagen-I mRNA reduced significantly at the end of 1 h and 2 h compared with control group at each time point [(0.38+/-0.05) and (0.55+/-0.16), P < 0.05], but increased to basic level at the end of 4 h. (4) The myofibroblasts had basic expression of Col-I early at the end of 12 h, its expression in supernatant culture medium reduced obviously both at 24 h and 48 h in sirolimus group compared with control group of each time point [(0.59+/-0.25) and (0.52+/-0.21), P < 0.05]. (5) The expression of FN in the whole cell lysates had the same trend as that in supernatant culture medium, which reduced obviously at the end of 24 h in sirolimus group compared with control group at each time point [(0.44+/-0.09) and (0.40+/-0.15), P < 0.05], but the inhibitive effect of sirolimus on FN disappeared at the end of 48 h. (6) The activities of MMP-2 and MMP-9 in the supernatant culture media were not significantly changed along with the experimental time points. CONCLUSION: Sirolimus may exert its anti-fibrotic effect through the inhibition of the expression of Col-I and/or FN in cultured renal cortical myofibroblasts.

Connective tissue growth factor stimulates renal cortical myofibroblast-like cell proliferation and matrix protein production.

Myofibroblasts primarily contribute to the pathogenesis of renal interstitial fibrosis by unregulated cell proliferation and synthesis of excessive amounts of extracellular matrix (ECM) proteins. We used cultured myofibroblast-like cells obtained by outgrowth from explants of rat kidney cortex to study the effects and relevant signaling pathway of connective tissue growth factor (CTGF) on cell proliferation and ECM production. Exogenous CTGF stimulated proliferation of myofibroblast-like cells in a dose- and time-dependent manner. CTGF also increased the secretion of fibronectin and collagen I protein in the supernatant medium. Nevertheless, CTGF did not affect matrix-degrading metalloproteinases-2 and -9 activities in supernatant medium measured by gelatin zymography. CTGF induced activation of extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinase pathway as early as 5 minutes. Inhibition of ERK1/2 activation with PD98059 completely blocked CTGF-induced cell proliferation as well as secretion of fibronectin and collagen I protein. The above results indicate that CTGF triggers cell proliferation and production of ECM proteins in cultured myofibroblast-like cells through the ERK1/2 mitogen-activated protein kinase pathway.

Connective tissue growth factor increases matrix metalloproteinase-2 and suppresses tissue inhibitor of matrix metalloproteinase-2 production by cultured renal interstitial fibroblasts.

The involvement of gelatinase (matrix metalloproteinase-2 [MMP-2] and MMP-9) in the matrix remodeling and development of tubulointerstitial fibrosis has been studied recently, but relatively little is known about the regulators and the mechanisms controlling the activation and expression of gelatinase in renal fibroblasts. In these studies, the production and underlying signaling pathway for gelatinase by exogenous connective tissue growth factor (CTGF) treatment were investigated. Here, we show that CTGF acts as a potent promoter of the activation and expression of MMP-2, but not MMP-9 in normal rat kidney fibroblasts cell line (NRK-49F). We found that CTGF significantly increased the activity of MMP-2, as well as MMP-2 protein in conditioned medium and MMP-2 mRNA levels in cells. In studies to address the mechanisms involved in the regulation of MMP-2 activity, we found that the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), the inhibitor of MMP-2, decreased significantly when cells were treated with CTGF. Further studies showed that extracellular signal-regulated kinase (ERK) signaling is responsible for most of the CTGF-induced MMP-2 expression and TIMP-2 suppression. When NRK-49F fibroblasts were incubated with CTGF, activation of ERK1/2 signaling was observed. Suppression of ERK1/2 activation with nontoxic concentrations of PD98059, a specific inhibitor of ERK activation, was associated with a reduction of CTGF-stimulated MMP-2 activity and protein expression. In addition, the CTGF-mediated reduction of TIMP-2 activity and protein expression was prevented when ERK1/2 activation was inhibited by PD98059. These results provide evidence that CTGF augments activation of MMP-2 through an effect on MMP-2 protein expression and TIMP-2 suppression, and that these effects are dependent on the activation of the ERK1/2 pathway.

RhoA required for acid-induced stress fiber formation and trafficking and activation of NHE3.

Exposure to an acid load increases apical membrane Na(+)/H(+) antiporter (NHE3) activity, a process that involves exocytic trafficking of the transporter to the apical membrane. We have previously shown that an intact microfilament structure is required for this exocytic process (Yang X, Amemiya M, Peng Y, Moe OW, Preisig PA, Alpern RJ. Am J Physiol Cell Physiol 279: C410-C419, 2000). The present studies demonstrate that acid-induced stress fiber formation is required for stimulation of NHE3 activity. Formation of stress fibers is associated with acid-induced tyrosine phosphorylation and increases in protein abundance of two focal adhesion proteins, p125(FAK) and paxillin. The Rho kinase inhibitor Y27632 completely blocks acid-induced stress fiber formation and the increases in apical membrane NHE3 abundance and activity, but it has no effect on acid-induced tyrosine phosphorylation of p125(FAK) or paxillin. Herbimycin A completely blocks acid-induced tyrosine phosphorylation of p125(FAK) and paxillin but only partially blocks stress fiber formation and NHE3 activation. These studies demonstrate that Rho kinase mediates acid-induced stress fiber formation, which is required for NHE3 exocytosis, and increases in NHE3 activity. Acid-induced tyrosine phosphorylation of the focal adhesion proteins p125(FAK) and paxillin is not Rho kinase dependent. Thus these two acid-mediated effects are associated, yet independent processes.

[The significance of urinary podocytes in patients with active lupus nephritis].

OBJECTIVE: To assess the significance of urinary podocyte and its possible implication as a marker of activity of lupus nephritis. METHODS: The presence of podocytes in urinary sediment was detected with immunochemical staining using anti-podocalyxin antibody. The correlation of the number of urinary podocytes with activity index of renal pathological lesions, hematuria, and proteinuria was analyzed respectively. The proliferating podocytes in renal biopsy tissue and urine from patients with class IV lupus nephritis were examined with double immunohistochemical staining. RESULTS: Thirty-one patients with lupus nephritis undergoing renal biopsy were enrolled into the study. Renal pathological findings of the patients could be classified into WHO class III (25.8%), class IV (64.5%) and class V (9.7%). 90% of the patients had positive urinary podocytes. The number of urinary podocytes was strongly and positively correlated with the severity of hematuria (r=0.639, P=0.000) and glomerular pathological activity index (r=0.487, P=0.014) in patients of class III and class IV. The amount of proteinuria was not correlated with pathological activity index, even though all the patients had proteinuria. Furthermore, the number of urinary podocytes, the severity of hematuria and the amount of proteinuria were all decreased after treatment with methyl prednisone, cyclophosphamide or mycophenolate mofetil. Interestingly, the urinary podocytes could disappear even before the remission of hematuria and proteinuria after treatment. Proliferative podocytes were observed both in biopsied kidney tissue and urinary sediments in patients of class IV. CONCLUSION: The presence and the number of urinary podocytes can be used as a valuable marker to grade the activity of lupus nephritis and to evaluate the efficacy of steroid therapy.

[High glucose downregulates the expression of podocalyxin protein in glomerular podocytes of mice].

OBJECTIVE: To examine the expression of podocalyxin protein in glomerular podocytes by long-term high glucose exposure in vitro and in vivo. METHODS: Immunohistochemical staining and computer image analysis were applied to detect the expression of podocalyxin protein in glomeruli from db/db mice and Wt mice. The effects of high glucose on the expression of podocalyxin protein were analyzed by Western blotting. The activation of MAPKS signaling pathway (ERK, p38 and JNK) by high glucose was also examined. RESULTS: The expressions of podocalyxin protein in db/db mice were obviously less than that in Wt mice [(0.18+/-0.07) vs (0.25+/-0.05),P<0.05] assessed by immunostaining and semiquantitative analysis. Basal levels of podocalyxin protein were observed in cultured mouse podocytes. The level of podocalyxin protein declined at each time point by high glucose incubation, reached the lowest level on the 6th day (5.5% of control group, P<0.01), but no significant changes were observed in normal glucose and mannitol glucose incubation groups. High glucose medium induced phosphorylation of ERK1/2 as early as 30 minutes, reached the peak at hour 6; maintained the activation from hour 12 to 24, and declined to the basal level at hour 48. However, activation of ERK1/2 was not detected in normal glucose and mannitol glucose groups. Blockade of activation of ERK1/2 with PD98059, a specific ERK1/2 activation inhibitor, attenuated the high glucose-induced expression of podocalyxin protein on the 6th day. CONCLUSION: High ambient glucose decreases the protein level of podocalyxin by podocyte in vitro and in vivo, and the decrease in podocalyxin protein is ERK1/2jdependent in cultured podocytes.

[Hypoxia induces the expression and secretion of connective tissue growth factor and fibronectin by cultured renal cortical myofibroblasts].

OBJECTIVE: To investigate whether hypoxia can affect the expression and secretion of connective tissue growth factor(CTGF) and fibronectin(FN) in primary cultured rat renal cortical myofibroblasts . METHODS: The primary cultured rat renal cortical myofibroblasts were subjected to hypoxic (1%O(2)) or normoxic (21% O(2)) conditions for a variety of times. The protein levels of HIF-1alpha, CTGF and FN protein were analyzed by Western blotting in both the whole cell lysates and supernatant culture medium 6 h, 12 h and 24 h after incubation, respectively. RT-PCR was carried out to measure the levels of FN mRNA at different time points (2 h,3 h,6 h and 12 h). The activity of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell medium was assayed by gelatin zymography. RESULTS: The expression of HIF-1alpha was induced at h6 in cells under hypoxia incubation. The levels of cellular CTGF protein were increased in hypoxia treated myofibroblasts at h6 (175%+/-52%),significantly elevated at h12 (347%+/-67%, P<0.05 ) , and sustained the high levels by 24 h (143%+/-27%). The protein level of CTGF in supernatant culture medium reached 3.48 times higher than that in normoxic group of cells at h24 (348%+/-99% , P<0.05 ). The levels of secreted FN by myofibroblasts were elevated under hypoxia at h6 (187%+/-42%), h12 (199%+/-51%) and reached the peak level at h24 (210%+/-29%, P<0.05), whereas the levels of cellular FN was declined at the same time points. Furthermore, we found the expression of FN mRNA was increased in cells under hypoxia condition at h3, reached the peak level at h6(135%+/-13%, P<0.05), and then decreased to the comparable level of cells in normoxic group at h12. The activities of MMP-2 and MMP-9 in the supernatant cultured medium were not significantly changed along with the experimental time points. CONCLUSION: Hypoxia could potentiate renal interstitial fibrosis through stimulating the expression and secretion of CTGF and FN in cultured cortical myofibroblasts.

[High glucose regulates the expression of connective tissue growth factor and its receptor (low density lipoprotein receptor-related protein) in cultured podocytes].

OBJECTIVE: To observe the expression of connective tissue growth factor (CTGF) and its receptor-low density lipoprotein receptor-related protein (LRP), and the relevant signaling pathway for the regulation by long-term high glucose exposure in cultured podocytes. METHODS: The effects of high glucose on the expression of CTGF and its receptor LRP were analyzed by western blotting. The activation of mitogen activated protein kinase (MAPKS) signaling pathway by high glucose was also examined. RESULTS: Basal levels of CTGF were observed in cultured mouse podocytes, the levels of CTGF protein were increased by high glucose medium groups on the 2nd day, reached the peak on the 4th day (P< 0.05), began to decline on the 6th day, returned to the basal level on the 8th day (P>0.05). The levels of CTGF expression in normal glucose and mannitol glucose groups did not change markly. High glucose medium induced phosphorylation of ERK1/2 at as early as minute 30, reached the peak at hour 6; maintained the activity at hours 12 and 24, and declined to the basal level at hour 48. However, phosphorylation of ERK1/2 was not detected in normal glucose and mannitol glucose groups. Blockade of phosphorylation of ERK1/2 with PD98059, a specific ERK1/2 activation inhibitor, did decrease the high glucose-triggered expression of CTGF protein in 4 days. High glucose had no effect on the expression of LRP protein at each time point. CONCLUSION: Acute high glucose (2-4 days) stimulated the expression of CTGF protein via ERK1/2-dependent signaling pathway in cultured podocytes, while cultured in high glucose for 6-8 days, the podocytes did not increase its CTGF level. Long-term high glucose had no effect on the expression of LRP in podocytes.

[Regulation of heat shock protein 27/activating transcription factor-5 complex and its implication in podocyte apoptosis induced by high glucose].

OBJECTIVE: To investigate the regulation of heat shock protein (HSP)27/activating transcription factor (ATF)-5 complex in podocytes induced by high glucose and relevant mechanisms. METHODS: Mice kidney podocytes were cultured in culture fluid with D-glucose at normal concentration (5.5 mol/L) (Group NG) or with D-glucose at high concentration (30 mmol/L) (Group HG) cells of these 2 groups were collected at different time points after glucose stimulation to detect the cell apoptosis by Hoechst 33342 staining and fluorescence microscopy and flow cytometry. Western blotting was used to analyze the activation of extracellular signal-regulated kinase (ERK = MAPK) and p38 signaling pathway. The HSP27/ATF5 complex was assessed by co-immunoprecipitation. ERK pathway blocker PD98059 and p38 signal pathway blocker SB203580 were added into the culture fluid of Group HG and Group NG respectively, and then the podocytes were collected at different time points to detect the high glucose-induced HSP27/ATF5 complex and cell apoptosis. RESULTS: The apoptotic rate of the podocytes of Group HG 24 hours after high glucose incubation was 14.3% +/- 6.2%, and that 48 h after was 27.2% +/- 8.9%, significantly higher than that of Group NG (10.6% +/- 2.7%, P < 0.05). HSP27/ATF5 complex could detected in the cells of Group NG too, however, the level of HSP27/ATF5 complex in Group HG 12 hours after incubation was 195% +/- 36% that of Group NG (P < 0.05). Both the ERK signal pathway and p38 signal pathway of Group HG began to be activated 10 min after incubation, peaked 30 min after, remained at the highest level till 1 hour after, and returned almost to the baseline level 2 hours after. No activation of these 2 pathways was observed in Group NG. The HSP27/ATF5 complex level of the PD98059 + high glucose group was 109% +/- 19% that of Group NG, significantly lower than that of Group HG (211% +/- 46% that of Group NG, P < 0.05). The apoptotic rate of the PD98059 + high glucose group was 51% +/- 4%, significantly higher than that of PD98059 + normal glucose group (16% +/- 3%, P < 0.05) and that of Group HG (27% +/- 9%, P < 0.05). The apoptotic rate of the SB203580 + HG group was 16% +/- 6%, significantly lower than that of Group HG (27% +/- 9%, P < 0.05). The HSP27/ATF5 complex level of the SB230580 + HG group was 290% +/- 43% that of Group NG, not significantly different from that of Group HG (231% +/- 20% that of Group NG, P > 0.05). CONCLUSION: High glucose stimulates the formation of HSP27/ATF5 complex in podocytes through ERK signaling pathway but not P38 signaling pathway, and the HSP27/ATF5 complex may have a regulatory effect in podocyte apoptosis induced by high glucose.

High ambient glucose levels modulates the production of MMP-9 and alpha5(IV) collagen by cultured podocytes.

Recent evidences have demonstrated an important role for glomerular visceral epithelial cell (podocyte) in the development and progression of diabetic nephropathy. We investigated the high-glucose (HG)-triggered signaling pathway and its role in matrix metalloproteinase (MMP) production in murine podocytes. The activity of 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2), in an HG medium significantly increased during incubation of 2 to 3 days and decreased during incubation of more than 5 days revealed by Gelatin zymography. Opposite to the increases in MMP-9 activity, HG medium produced significant decreases in the protein levels of alpha5(IV) collagen. Changes in MMP-9 activity were associated with the same pattern as MMP-9 mRNA levels in podocytes exposed to HG media. HG medium rapidly activated ERK1/2 MAPK in podocytes. Moreover, ERK1/2 activation was required for HG-induced enhancement of MMP-9 activity and a decrease in the level of alpha5(IV) collagen. HG incubation rapidly induced an increase in the nuclear accumulation of Ets-1 protein. Blocking the ERK pathway suppressed HG-induced expression and nuclear accumulation of transcriptional factor Ets-1, and MMP-9 mRNA expression. We suggest that short- or long-term exposure to HG concentrations increases or decreases MMP-9 production and alpha5(IV) collagen expression in podocytes, this may contribute to the GBM abnormality caused by an imbalance in extracellular matrix (ECM) synthesis and degradation, and may play a critical role in the pathogenesis of proteinuria in diabetic nephropathy.