MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy.

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking. Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence. Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury. Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.

Risk factors for the mortality of hemodialysis patients with COVID-19: A multicenter study from the overall hemodialysis population in Wuhan.

INTRODUCTION: Maintenance hemodialysis (MHD) patients are highly threatened in the novel coronavirus disease 2019 (COVID-19) pandemic, but evidence of risk factors for mortality in this population is still lacking. METHODS: We followed outcomes of the overall MHD population of Wuhan, including 7154 MHD patients from 65 hemodialysis centers, from January 1 to May 4, 2020. Among them, 130 were diagnosed with COVID-19. The demographic and clinical data of them were collected and compared between survivors and nonsurvivors. RESULTS: Compared to the corresponding period of last year, the all-cause mortality rate of the Wuhan MHD population significantly rose in February, and dropped down in March 2020. Of the 130 COVID-19 cases, 51 (39.2%) were deceased. Advanced age, decreased oxygen saturation, low diastolic blood pressure (DBP) on admission, and complications including acute cardiac injury (HR 5.03 [95% CI 2.21-11.14], p < 0.001), cerebrovascular event (HR 2.80 [95% CI 1.14-6.86], p = 0.025) and acute respiratory distress syndrome (HR 3.50 [95% CI 1.63-7.51], p = 0.001) were identified as independent risk factors for the death of COVID-19. The median virus shedding period of survivors was 25 days, longer than the general population. CONCLUSIONS: Maintenance hemodialysis patients are a highly vulnerable population at increased risk of mortality and prolonged virus shedding period in the ongoing COVID-19 pandemic. Advanced age, decreased oxygen saturation, low DBP on admission, and complications like acute cardiac injury are parameters independently associated with poor prognosis.

MAD2B-mediated cell cycle reentry of podocytes is involved in the pathogenesis of FSGS.

Rationale: Focal segmental glomerulosclerosis (FSGS) is characterized by the dysfunction of "post-mitotic" podocytes. The reentry of podocytes in the cell cycle will ultimately result in cell death. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of anaphase-promoting complex (APC)/cyclosome, precisely controls the metaphase to anaphase transition and ordered cell cycle progression. However, the role of MAD2B in FSGS podocyte injury remains unknown. Methods: To explore MAD2B function in podocyte cell cycle reentry, we used conditional mutant mice lacking MAD2B selectively in podocytes in ADR-induced FSGS murine model. Additionally, KU-55933, a specific inhibitor of ataxia-telangiectasia mutated (ATM) was utilized in vivo and in vitro to explore the role of ATM in regulating MAD2B. Results: The expression of MAD2B in podocytes was dramatically increased in patients with FSGS and ADR-treated mice along with podocyte cell cycle reentry. Podocyte-specific knockout of MAD2B effectively attenuated proteinuria, podocyte injury, and prevented the aberrant cell cycle reentry. By bioinformatics analysis we revealed that ATM kinase is a key upstream regulator of MAD2B. Furthermore, inhibition of ATM kinase abolished MAD2B-driven cell cycle reentry and alleviated podocyte impairment in FSGS murine model. In vitro studies by site-directed mutagenesis and immunoprecipitation we revealed ATM phosphorylated MAD2B and consequently hampered the ubiquitination of MAD2B in a phosphorylation-dependent manner. Conclusions: ATM kinase-MAD2B axis importantly contributes to the cell cycle reentry of podocytes, which is a novel pathogenic mechanism of FSGS, and may shed light on the development of its therapeutic approaches.

MAD2B contributes to parietal epithelial cell activation and crescentic glomerulonephritis via Skp2.

Mitotic spindle assembly checkpoint protein 2 (MAD2B), a well-known anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase-ζ, is critical for mitotic control and DNA repair. Previously, we detected a strong increase of MAD2B in the glomeruli from patients with crescentic glomerulonephritis and anti-glomerular basement membrane (anti-GBM) rats, which predominantly originated from activated parietal epithelial cells (PECs). Consistently, in vitro MAD2B was increased in TNF-α-treated PECs, along with cell activation and proliferation, as well as extracellular matrix accumulation, which could be reversed by MAD2B genetic depletion. Furthermore, we found that expression of S phase kinase-associated protein 2 (Skp2), an APC/CCDH1 substrate, was increased in the glomeruli of anti-GBM rats, and TNF-α-stimulated PECs and could be suppressed by MAD2B depletion. Additionally, genetic deletion of Skp2 inhibited TNF-α-induced PEC activation and dysfunction. Finally, TNF-α blockade or glucocorticoid therapy administered to anti-GBM rats could ameliorate MAD2B and Skp2 accumulation as well as weaken PEC activation. Collectively, our data suggest that MAD2B has a pivotal role in the pathogenesis of glomerular PEC activation and crescent formation through induction of Skp2 expression.

Selenoprotein T protects against cisplatin-induced acute kidney injury through suppression of oxidative stress and apoptosis.

Previously, selenoprotein T (SelT) expression was shown to be induced in nervous, endocrine, and metabolic tissues during ontogenetic and regenerative processes. However, whether SelT plays a critical role in renal diseases remains unclear. Here, we explored the role of SelT in cisplatin-induced acute kidney injury (AKI). Results revealed that SelT was highly expressed in renal tubules, but its expression was significantly reduced in cisplatin-induced AKI. Importantly, knocking down of SelT expression in kidney cells in vitro resulted in cisplatin-induced cell apoptosis, as indicated by the elevation of cleaved-PARP and Bax expression, Caspase-3 activity, and number of TUNEL-positive cells. Moreover, SelT silencing-induced reactive oxygen species (ROS) production, accompanied by a decrease in intracellular superoxide dismutase (SOD) and catalase (CAT) activity and increase in malondialdehyde (MDA) content. Notably, the protein and mRNA levels of Nox4 were increased in response to SelT downregulation. Furthermore, suppression of Nox4 expression by GKT137831 partially alleviated SelT knockdown-induced ROS generation and cell apoptosis in cisplatin-treated kidney cells. Taken together, our findings provide the first evidence that SelT protects against cisplatin-induced AKI by suppression of oxidative stress and apoptosis.

Serologic Detection of SARS-CoV-2 Infections in Hemodialysis Centers: A Multicenter Retrospective Study in Wuhan, China.

RATIONALE & OBJECTIVE: Patients receiving maintenance hemodialysis (MHD) are highly vulnerable to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current study was designed to evaluate the prevalence of SARS-CoV-2 infection based on both nucleic acid testing (NAT) and antibody testing in Chinese patients receiving MHD. STUDY DESIGN: Cross-sectional study. SETTING & PARTICIPANTS: From December 1, 2019, to March 31, 2020, a total of 1,027 MHD patients in 5 large hemodialysis centers in Wuhan, China, were enrolled. Patients were screened for SARS-CoV-2 infection by symptoms and initial computed tomography (CT) of the chest. If patients developed symptoms after the initial screening was negative, repeat CT was performed. Patients suspected of being infected with SARS-CoV-2 were tested with 2 consecutive throat swabs for viral RNA. In mid-March 2020, antibody testing for SARS-CoV-2 was obtained for all MHD patients. EXPOSURE: NAT and antibody testing results for SARS-CoV-2. OUTCOMES: Morbidity, clinical features, and laboratory and radiologic findings. ANALYTICAL APPROACH: Differences between groups were examined using t test or Mann-Whitney U test, comparing those not infected with those infected and comparing those with infection detected using NAT with those with infection detected by positive serology test results. RESULTS: Among 1,027 patients receiving MHD, 99 were identified as having SARS-CoV-2 infection, for a prevalence of 9.6%. Among the 99 cases, 52 (53%) were initially diagnosed with SARS-CoV-2 infection by positive NAT; 47 (47%) were identified later by positive immunoglobulin G (IgG) or IgM antibodies against SARS-CoV-2. There was a spectrum of antibody profiles in these 47 patients: IgM antibodies in 5 (11%), IgG antibodies in 35 (74%), and both IgM and IgG antibodies in 7 (15%). Of the 99 cases, 51% were asymptomatic during the epidemic; 61% had ground-glass or patchy opacities on CT of the chest compared with 11.6% among uninfected patients (P<0.001). Patients with hypertensive kidney disease were more often found to have SARS-CoV-2 infection and were more likely to be symptomatic than patients with another primary cause of kidney failure. LIMITATIONS: Possible false-positive and false-negative results for both NAT and antibody testing; possible lack of generalizability to other dialysis populations. CONCLUSIONS: Half the SARS-CoV-2 infections in patients receiving MHD were subclinical and were not identified by universal CT of the chest and selective NAT. Serologic testing may help evaluate the overall prevalence and understand the diversity of clinical courses among patients receiving MHD who are infected with SARS-CoV-2.

Clinical Characteristics of and Medical Interventions for COVID-19 in Hemodialysis Patients in Wuhan, China.

BACKGROUND: Reports indicate that those most vulnerable to developing severe coronavirus disease 2019 (COVID-19) are older adults and those with underlying illnesses, such as diabetes mellitus, hypertension, or cardiovascular disease, which are common comorbidities among patients undergoing maintenance hemodialysis. However, there is limited information about the clinical characteristics of hemodialysis patients with COVID-19 or about interventions to control COVID-19 in hemodialysis centers. METHODS: We collected data retrospectively through an online registration system that includes all patients receiving maintenance hemodialysis at 65 centers in Wuhan, China. We reviewed epidemiologic and clinical data of patients with laboratory-confirmed COVID-19 between January 1, 2020 and March 10, 2020. RESULTS: Of 7154 patients undergoing hemodialysis, 154 had laboratory-confirmed COVID-19. The mean age of the 131 patients in our analysis was 63.2 years; 57.3% were men. Many had underlying comorbidities, with cardiovascular disease (including hypertension) being the most common (68.7%). Only 51.9% of patients manifested fever; 21.4% of infected patients were asymptomatic. The most common finding on chest computed tomography (CT) was ground-grass or patchy opacity (82.1%). After initiating comprehensive interventions-including entrance screening of body temperature and symptoms, universal chest CT and blood tests, and other measures-new patients presenting with COVID-19 peaked at 10 per day on January 30, decreasing to 4 per day on February 11. No new cases occurred between February 26 and March 10, 2020. CONCLUSIONS: We found that patients receiving maintenance hemodialysis were susceptible to COVID-19 and that hemodialysis centers were high-risk settings during the epidemic. Increasing prevention efforts, instituting universal screening, and isolating patients with COVID-19 and directing them to designated hemodialysis centers were effective in preventing the spread of COVID-19 in hemodialysis centers.

Subcellular trafficking of tubular MDM2 implicates in acute kidney injury to chronic kidney disease transition during multiple low-dose cisplatin exposure.

Acute kidney injury (AKI) is the leading cause of renal failure, and quite a few patients will advance to chronic kidney disease (CKD) in the long term. Here, we explore the roles and mechanisms of tubular epithelial cells (TECs) during repeated cisplatin (CP) induced AKI to CKD transition (AKI-CKD). Previously, we reported that murine double minute 2 (MDM2), an E3-ubiquitin ligase, is involved in tubulointerstitial fibrosis. However, whether tubular MDM2 is implicated in AKI-CKD is undefined. Currently, we confirmed that during AKI-CKD, MDM2 shifts from nucleus to cell membrane in TECs both in vivo and in vitro. Whereas regulating MDM2 distribution chemically or genetically has a prominent impact on tubular disorders. And then we investigated the mechanisms of the above findings. First, in the nucleus, repeated CP administration leads to MDM2 reduction with escalated p53 and cell cycle G2/M arrest. On the other hand, multiple CP treatment increases the level of membranous MDM2 with ensuing integrin ß8 degradation and TGF-ß1 activation. More interestingly, anchoring MDM2 on cell membranes can mimic the reduction of integrin ß8 arousing by repeated CP exposure. Collectively, our findings provided the evidence that tubular MDM2 subcellular shuttling is involved in AKI-CKD through p53-G2/M arrest and integrin ß8 mediated TGF-ß1 activation.

Inhibition of SIRT2 Alleviates Fibroblast Activation and Renal Tubulointerstitial Fibrosis via MDM2.

BACKGROUND/AIMS: Renal tubular epithelial cells and fibroblasts are the main sources of myofibroblasts, and these cells produce the extracellular matrix during tubulointerstitial fibrosis (TIF). Histone deacetylases (HDAC) inhibitors exert an antifibrogenic effect in the skin, liver and lung. Sirtuin 2 (SIRT2), which is a class III HDAC, is an important member of NAD+-dependent protein deacetylases. The current study evaluated the role of SIRT2 in renal TIF. METHODS: Immunohistochemical staining and Western blot were performed to evaluate SIRT2 expression in TIF patients and unilateral urethral obstruction (UUO) mice. Western blot was used to assess the protein levels of SIRT2, α-SMA, collagen III, fibronectin, and MDM2 in tubular epithelial cells and fibroblasts. The specific inhibitor AGK2 was used to inhibit SIRT2 activity, and targeted siRNA was used to suppress SIRT2 expression. RESULTS: SIRT2 expression increased in the tubulointerstitium of TIF patients and UUO mice. SIRT2 inhibition ameliorated TIF in UUO mice. SIRT2 expression in tubular cells was unchanged after exposure to TGF-ß1. The SIRT2-specifc inhibitor AGK2 did not attenuate TGF-ß1-induced tubular epithelial-mesenchymal transition. However, SIRT2 was upregulated in fibroblasts, and fibroblasts were activated after TGF-ß1 treatment. Genetic knockdown and chemical inhibition of SIRT2 attenuated TGF-ß1-induced fibroblast activation. We also explored the downstream signaling of SIRT2 during fibroblast activation. Genetic knockdown and chemical inhibition of SIRT2 suppressed TGF-ß1-induced increase in MDM2 expression, and inhibition of the MDM2-p53 interaction using Nutlin-3 did not suppress SIRT2 upregulation. CONCLUSION: Our results suggest that SIRT2 participates in the activation of fibroblasts and TIF, which is mediated via regulation of the MDM2 pathway, and the downregulation of SIRT2 may be a therapeutic strategy for renal fibrosis.

IL-6 increases podocyte motility via MLC-mediated focal adhesion impairment and cytoskeleton disassembly.

The disturbance of podocyte motility is an essential pathogenic mechanisms of foot process effacement during proteinuric diseases, and myosin light chain (MLC) is a pivotal component in regulating the motility of podocytes. Inflammatory cytokine interleukin-6 (IL-6) has been reported to induce podocyte abnormalities by various mechanisms, however, whether aberrant cell motility contributes to the IL-6-induced podocyte injury remains unknown. Here, by wound healing, transwell, and cell migration assays, we confirmed that IL-6 accelerates the motility of podocyte. Simultaneously, the phosphorylation of MLC is elevated along with perturbed focal adhesion (FAs) and cytoskeleton. Next, via genetic and pharmacologic interruption of MLC or its phosphorylation we revealed that the activation of MLC is implicated in IL-6-mediated podocyte hypermotility as well as the disassembly of FAs and F-actin. By using stattic, an inhibitor for STAT3 phosphorylation, we uncovered that STAT3 activation is the upstream event for MLC phosphorylation and the following aberrant motility of podocytes. Additionally, we found that calcitriol markedly attenuates podocyte hypermotility via blocking STAT3-MLC. In conclusion, our study demonstrated that IL-6 interrupts FAs dynamic, cytoskeleton organization, and eventually leads to podocyte hypermotility via STAT3/MLC, whereas calcitriol exerts its protective role by inhibiting this pathway. These findings enrich the mechanisms accounting for IL-6-mediated podocyte injury from the standpoint of cell motility and provide a novel therapeutic target for podocyte disorders.

Lipid Deposition in Kidney Diseases: Interplay Among Redox, Lipid Mediators, and Renal Impairment.

Significance: The relationship between lipid disturbances and renal diseases has been studied for several decades, and it is well recognized that when the balance of renal lipid uptake, synthesis, oxidation, and outflow is disrupted, lipids will undergo oxidation, be sequestrated as lipid droplets, generate toxic metabolites, and cause nephrotoxicity in diverse renal diseases. Recent Advances: During renal disorders, redox signaling is a pivotal event promoting or resulting from lipid disorders. Accordingly, a vicious cycle of lipid redox dysregulation could be developed, accelerating the renal damage. Critical Issues: The aim of this concise review is to introduce the connection among redox, lipid abnormalities and kidney damage in various conditions. And we summarized current understanding of the lipid redox loop implicated in acute kidney injury, chronic kidney disease, metabolic abnormalities, aging, and genetic pitfalls. Future Directions: Despite recent advances, further investigations are required to clarify the complicated molecular and regulatory mechanisms among redox, lipid mediators and renal disorders. Moreover, exploring an ideal target for potential therapies should be discussed and studied in future. Antioxid. Redox Signal. 28, 1027-1043.

The classic signalling and trans-signalling of interleukin-6 are both injurious in podocyte under high glucose exposure.

Interleukin-6 (IL-6) is a multifunctional cytokine that employs IL-6 classic and trans-signalling pathways, and these two signal channels execute different or even opposite effects in certain diseases. As a cardinal event of diabetic kidney disease (DKD), whether the podocyte abnormalities are associated with IL-6 signalling, especially classic or trans-signalling respectively, remains unclear. In this study, we identified that the circulatory IL-6, soluble IL-6R (sIL-6R) and soluble glycoprotein 130 (sgp130) levels are elevated in patients with DKD. The expressions of membrane-bound IL-6R (mIL-6R), sIL-6R and gp130 are enhanced in kidney cortex of diabetic mice accompanying with activated STAT3 by tyrosine 705 residue phosphorylation, while not serine 727. Above data infer both classic signalling and trans-signalling of IL-6 are activated during DKD. In cultured podocyte, high glucose (HG) up-regulates the expression of mIL-6R and gp130, as well as STAT3 tyrosine 705 phosphorylation, in a time-dependent manner. Entirely blocking IL-6 signalling by gp130 shRNA, gp130 or IL-6 neutralizing antibodies attenuates HG-induced podocyte injury. Interestingly, either inhibiting IL-6 classic signalling by mIL-6R shRNA or suppressing its trans-signalling using sgp130 protein dramatically alleviates HG-induced podocyte injury, suggesting both IL-6 classic signalling and trans-signalling play a detrimental role in HG-induced podocyte injury. Additionally, activation of IL-6 classic or trans-signalling aggravates podocyte damage in vitro. In summary, our observations demonstrate that the activation of either IL-6 classic or trans-signalling advances podocyte harming under hyperglycaemia. Thus, suppressing IL-6 classic and trans-signalling simultaneously may be more beneficial in podocyte protection and presents a novel therapeutic target for DKD.

MDM2 Contributes to High Glucose-Induced Glomerular Mesangial Cell Proliferation and Extracellular Matrix Accumulation via Notch1.

Murine double minute 2 (MDM2) is an E3-ubiquitin ligase critical for various biological functions. Previous data have revealed an indispensable role of MDM2 in kidney homeostasis. However, its role in glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation during hyperglycemia condition remains unclear. In our present study, we found that MDM2 protein level was significantly upregulated in high glucose-treated GMCs, while knocking down MDM2 by siRNA could attenuate high glucose-induced ECM accumulation and GMCs proliferation. Unexpectedly, Nutlin-3a, a MDM2-p53 interaction blocker, had no benefit in protecting diabetic mice from renal impairment in vivo and in alleviating high glucose-induced ECM accumulation in vitro. Intriguingly, we found that Notch1 signaling activation was obviously attenuated by MDM2 depletion in GMCs with high glucose exposure. However, Numb, a substrate of MDM2 which suppresses Notch1 signaling, was found not to be involved in the MDM2 and Notch1 association. Moreover, our findings demonstrated that MDM2 interacted with Notch1 intracellular domain (NICD1) independent of Numb and regulated the ubiquitination status of NICD1. Collectively, our data propose a pivotal role of MDM2 in high glucose-induced GMC proliferation and ECM accumulation, via modulating the activation of Notch1 signaling pathway in an ubiquitination-dependent way.

MDM2 is implicated in high-glucose-induced podocyte mitotic catastrophe via Notch1 signalling.

Podocyte injury and depletion are essential events involved in the pathogenesis of diabetic nephropathy (DN). As a terminally differentiated cell, podocyte is restricted in 'post-mitosis' state and unable to regenerate. Re-entering mitotic phase will cause podocyte disastrous death which is defined as mitotic catastrophe (MC). Murine double minute 2 (MDM2), a cell cycle regulator, is widely expressed in renal resident cells including podocytes. Here, we explore whether MDM2 is involved in podocyte MC during hyperglycaemia. We found aberrant mitotic podocytes with multi-nucleation in DN patients. In vitro, cultured podocytes treated by high glucose (HG) also showed an up-regulation of mitotic markers and abnormal mitotic status, accompanied by elevated expression of MDM2. HG exposure forced podocytes to enter into S phase and bypass G2/M checkpoint with enhanced expression of Ki67, cyclin B1, Aurora B and p-H3. Genetic deletion of MDM2 partly reversed HG-induced mitotic phase re-entering of podocytes. Moreover, HG-induced podocyte injury was alleviated by MDM2 knocking down but not by nutlin-3a, an inhibitor of MDM2-p53 interaction. Interestingly, knocking down MDM2 or MDM2 overexpression showed inhibition or activation of Notch1 signalling, respectively. In addition, genetic silencing of Notch1 prevented HG-mediated podocyte MC. In conclusion, high glucose up-regulates MDM2 expression and leads to podocyte MC. Notch1 signalling is an essential downstream pathway of MDM2 in mediating HG-induced MC in podocytes.

Interleukin-6 Signaling Pathway and Its Role in Kidney Disease: An Update.

Interleukin-6 (IL-6) is a pleiotropic cytokine that not only regulates the immune and inflammatory response but also affects hematopoiesis, metabolism, and organ development. IL-6 can simultaneously elicit distinct or even contradictory physiopathological processes, which is likely discriminated by the cascades of signaling pathway, termed classic and trans-signaling. Besides playing several important physiological roles, dysregulated IL-6 has been demonstrated to underlie a number of autoimmune and inflammatory diseases, metabolic abnormalities, and malignancies. This review provides an overview of basic concept of IL-6 signaling pathway as well as the interplay between IL-6 and renal-resident cells, including podocytes, mesangial cells, endothelial cells, and tubular epithelial cells. Additionally, we summarize the roles of IL-6 in several renal diseases, such as IgA nephropathy, lupus nephritis, diabetic nephropathy, acute kidney injury, and chronic kidney disease.

PKC-α Triggers EGFR Ubiquitination, Endocytosis and ERK Activation in Podocytes Stimulated with High Glucose.

BACKGROUND: Protein Kinase C-α (PKC-α) and epidermal growth factor receptor (EGFR) are both involved in diabetic kidney disease; however, the connection between these two proteins during high glucose-induced podocyte injury remains uncertain. METHODS: Diabetes was induced in SD rats by streptozotocin (STZ). Fourteen days later, the kidney cortex was removed and subjected to plasma membrane isolation and lipid raft fractionation. In vitro study human podocyte cell line was differentiated and subjected to various treatments. The levels of membranous protein and endocytosis were assessed by biotinylation and sodium 2-mercaptoethane sulfonate (MesNa) treatment. Gö6976 and PYR-41 were used as inhibitors of PKC-α and ubiquitin activating E1 enzyme, respectively. RESULTS: In diabetic rats, the abundance of PKC-α in the membranous fraction and the lipid raft domain is elevated, whereas the EGFR level is reduced. Consistently, in vitro high glucose treated podocytes, membranous EGFR is downregulated with increased PKC-α. Furthermore, the ubiquitination and endocytosis of EGFR are enhanced accompanied by extracellular signal-regulated kinase (ERK) signaling activation and podocyte damage during hyperglycemia. However, these processes can be ameliorated by inhibition of either PKC-α or ubiquitin activating E1 enzyme. CONCLUSION: During hyperglycemia, PKC-α mediates podocytic EGFR ubiquitination, endocytosis from cell surface and the subsequent ERK activation, which contributes to podocyte injury.

MDM2 mediates fibroblast activation and renal tubulointerstitial fibrosis via a p53-independent pathway.

It is well recognized that murine double minute gene 2 (MDM2) plays a critical role in cell proliferation and inflammatory processes during tumorigenesis. It is also reported that MDM2 is expressed in glomeruli and involved in podocyte injury. However, whether MDM2 is implicated in renal fibrosis remains unclear. Here we investigated the role of MDM2 in tubulointerstitial fibrosis (TIF). By immunohistochemical staining and Western blotting we confirmed that MDM2 is upregulated in the tubulointerstitial compartment in patients with TIF and unilateral urethral obstruction (UUO) mice, which mainly originates from myofibroblasts. Consistently, in vitro MDM2 is increased in TGF-ß1-treated fibroblasts, one of the major sources of collagen-producing myofibroblasts during TIF, along with fibroblast activation. Importantly, genetic deletion of MDM2 significantly attenuates fibroblast activation. We then analyzed the possible downstream signaling of MDM2 during fibroblast activation. p53-dependent pathway is the classic downstream signaling of MDM2, and Nutlin-3 is a small molecular inhibitor of MDM2-p53 interaction. To our surprise, Nutlin-3 could not ameliorate fibroblast activation in vitro and TIF in UUO mice. However, we found that Notch1 signaling is attenuated during fibroblast activation, which could be markedly rescued by MDM2 knockdown. Overexpression of intracellular domain of Notch1 (NICD) by plasmid could obviously minimize fibroblast activation induced by TGF-ß1. In addition, the degradation of NICD is strikingly suppressed by PYR-41, an inhibitor of ubiquitin-activating enzyme E1, and proteasome inhibitor MG132. Taken together, our findings provide the first evidence that MDM2 is involved in fibroblast activation and TIF, which associates with Notch1 ubiquitination and proteasome degradation.

Effects of dexamethasone and HA1077 on actin cytoskeleton and ß-catenin in cultured human trabecular meshwork cells.

AIM: To investigate the effects of dexamethasone (DEX) and 1-(5-isoquinolinesulfonyl)-homopiperazine (HA1077) on actin cytoskeleton and ß-catenin in cultured human trabecular meshwork (HTM) cells. METHODS: The HTM cells were separated from human eyeball and cultured in vitro. They were divided into control group, DEX (1×10-6 mol/L) group, HA1077 (3×10-5 mol/L) group, and DEX (1×10-6 mol/L) and HA1077 (3×10-5 mol/L) group. Actin cytoskeleton and ß-catenin in HTM cells of the four groups were examined by immunofluorescence and Western blot analyses. RESULTS: In DEX group, there were reorganization of actin cytoskeleton and formation of cross linked actin networks (CLANs), which were partially reversed in DEX and HA1077 group. DEX treatment also induced an increased expression of ß-catenin, which was obviously reduced in DEX and HA1077 group. Meanwhile, the cultured HTM cells in HA1077 group had lower expression of ß-catenin than that in the control group. CONCLUSION: Our results show that HA1077 can reverse the changes of actin organization and expression of ß-catenin induced by DEX in cultured HTM cells, suggesting that HA1077 may play an important role in increasing outflow and reducing intraocular pressure.

Role and Association of Inflammatory and Apoptotic Caspases in Renal Tubulointerstitial Fibrosis.

BACKGROUND/AIMS: Caspases, an evolutionary conserved family of aspartate-specific cystein proteases, play pivotal roles in apoptotic and inflammatory signaling. Thus far, 14 mammalian caspases are identified and categorized into 3 distinct sub-types: inflammatory caspases, apoptotic initiator and apoptotic executioner. Caspase-1 is an inflammatory caspase, while caspase-7 belongs to apoptotic executioner. The roles and association of these two distinct types of caspases in renal tubulointerstitial fibrosis (TIF) have not been well recognized. METHODS: Caspase-1 inhibitor Z-YVAD-FMK and caspase-7 siRNA were used in tubular epithelial cell line NRK-52E (TECs) to test their effects on transforming growth factor-beta1 (TGF-ß1) stimulation. In vivo, Unilateral ureteral obstruction (UUO) animal model was employed in wild-type (WT) and caspase-1 knock out (KO) (caspase-1-/-) mice. RESULTS: In current study, we found that caspase-7 was obviously activated in cultured TECs stimulated by TGF-ß1 and in UUO model of WT mice. While in UUO model of caspase-1 KO mice, the increased caspase-7 activation was suppressed significantly along with reduced trans-differentiation and minimized extracellular matrix (ECM) accumulation, as demonstrated by western blot, Masson trichrome staining and immunohistochemistry. In addition, pharmacological inhibition of caspase-1 dampened caspase-7 activation and TECs' transdifferentiation induced by TGF-ß1 exposure, which was consistent with in vivo study. Notably, caspase-7 gene knock down by specific siRNA abrogated TGF-ß1 driven TECs' trans-differentiation and reduced ECM accumulation. CONCLUSIONS: Our study associated inflammatory and apoptotic caspases in TIF for the first time and we further confirmed that caspase-1 activation is an upstream event of apoptotic caspase-7 induction in TIF triggered by UUO and in TECs mediated by TGF-ß1 induced transdifferentiation.

MAD2B promotes tubular epithelial-to-mesenchymal transition and renal tubulointerstitial fibrosis via Skp2.

The mitotic arrest deficient protein MAD2B is a well-defined anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase zeta. It is critical for mitotic control and DNA repair. However, the pathological role of MAD2B in kidney diseases has not been fully elucidated. In the present study, we aim to explore the role of MAD2B in the pathogenesis of renal tubulointerstitial fibrosis (TIF) and the underlying mechanism. By immunofluorescence and immunohistochemistry, we found an obvious MAD2B enhancement in tubular area of TIF patients and unilateral ureteral obstruction (UUO) mice. In vitro, transforming growth factor-ß1 (TGF-ß1) induced a time-dependent MAD2B accumulation prior to tubular epithelial-to-mesenchymal transition (EMT) in a rat proximal tubular epithelial cell line, NRK-52E. Knocking down MAD2B using siRNA dramatically inhibited TGF-ß1-induced tubular EMT process and subsequent extracellular matrix (ECM) production. We also found that Skp2, a confirmed APC/C-CDH1 substrate and E-cadherin destroyer, was increased in TGF-ß1-treated proximal tubular epithelial cells, which could be blocked by MAD2B depletion. In addition, Skp2 expression was also found to be increased in the renal tubular area of UUO mice. Locally knocking down MAD2B expression in the renal cortex using lentiviral transfection inhibited Skp2 expression, tubular EMT, and subsequent ECM accumulation. Taken together, our data suggests a pro-fibrotic role of MAD2B in the pathogenesis of tubular EMT and TIF by inducing Skp2 expression. MAD2B might be a potential target of promising interventions for renal TIF. KEY MESSAGES: Renal fibrosis activates MAD2B expression in renal tubules of human and mouse. TGF-ß1 contributes to MAD2B enhancement in rat tubular epithelial cells. MAD2B depletion alleviates renal tubulointerstitial fibrosis in vivo and in vitro. MAD2B promotes EMT transition in rat tubular epithelial cells by inducing Skp2.