Hyperglycolysis in endothelial cells drives endothelial injury and microvascular alterations in peritoneal dialysis.

BACKGROUND: Endothelial cell (EC) dysfunction leading to microvascular alterations is a hallmark of technique failure in peritoneal dialysis (PD). However, the mechanisms underlying EC dysfunction in PD are poorly defined. METHODS: We combined RNA sequencing with metabolite set analysis to characterize the metabolic profile of peritoneal ECs from a mouse model of PD. This was combined with EC-selective blockade of glycolysis by genetic or pharmacological inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in vivo and in vitro. We also investigated the association between peritoneal EC glycolysis and microvascular alterations in human peritoneal samples from patients with end-stage kidney disease (ESKD). RESULTS: In a mouse model of PD, peritoneal ECs had a hyperglycolytic metabolism that shunts intermediates into nucleotide synthesis. Hyperglycolytic mouse peritoneal ECs displayed a unique active phenotype with increased proliferation, permeability and inflammation. The active phenotype of mouse peritoneal ECs can be recapitulated in human umbilical venous ECs and primary human peritoneal ECs by vascular endothelial growth factor that was released from high glucose-treated mesothelial cells. Importantly, reduction of peritoneal EC glycolysis, via endothelial deficiency of the glycolytic activator PFKFB3, inhibited PD fluid-induced increases in peritoneal capillary density, vascular permeability and monocyte extravasation, thereby protecting the peritoneum from the development of structural and functional damages. Mechanistically, endothelial PFKFB3 deficiency induced the protective effects in part by inhibiting cell proliferation, VE-cadherin endocytosis and monocyte-adhesion molecule expression. Pharmacological PFKFB3 blockade induced a similar therapeutic benefit in this PD model. Human peritoneal tissue from patients with ESKD also demonstrated evidence of increased EC PFKFB3 expression associated with microvascular alterations and peritoneal dysfunction. CONCLUSIONS: These findings reveal a critical role of glycolysis in ECs in mediating the deterioration of peritoneal function and suggest that strategies targeting glycolysis in peritoneal ECs may be of therapeutic benefit for patients undergoing PD.

Circular RNA circBNC2 inhibits epithelial cell G2-M arrest to prevent fibrotic maladaptive repair.

The mechanisms underlying fibrogenic responses after injury are not well understood. Epithelial cell cycle arrest in G2/M after injury is a key checkpoint for determining wound-healing leading to either normal cell proliferation or fibrosis. Here, we identify a kidney- and liver-enriched circular RNA, circBNC2, which is abundantly expressed in normal renal tubular cells and hepatocytes but significantly downregulated after acute ischemic or toxic insult. Loss of circBNC2 is at least partially mediated by upregulation of DHX9. Gain- and loss-of-function studies, both in vitro and in vivo, demonstrate that circBNC2 acts as a negative regulator of cell G2/M arrest by encoding a protein that promotes formation of CDK1/cyclin B1 complexes. Restoring circBNC2 in experimentally-induced male mouse models of fibrotic kidney and liver, decreases G2/M arrested cell numbers with secretion of fibrotic factors, thereby mitigating extracellular matrix deposition and fibrosis. Decreased expression of circBNC2 and increased G2/M arrest of epithelial cells are recapitulated in human ischemic reperfusion injury (IRI)-induced chronic kidney disease and inflammation-induced liver fibrosis, highlighting the clinical relevance. These findings suggest that restoring circBNC2 might represent a potential strategy for therapeutic intervention in epithelial organ fibrosis.

Magnetic Resonance Imaging Features on Deep Learning Algorithm for the Diagnosis of Nasopharyngeal Carcinoma.

The objective of this research was to investigate the application values of magnetic resonance imaging (MRI) features of the deep learning-based image super-resolution reconstruction algorithm optimized convolutional neural network (OPCNN) algorithm in nasopharyngeal carcinoma (NPC) lesion diagnosis. A total of 54 patients with NPC were selected as research objects. Based on the traditional CNN structure, OPCNN was proposed. Besides, MRI processed by the traditional CNN model and the U-net network model was introduced to be analyzed and compared with its algorithm. The used assessment parameters included volume transfer constant (K trans), rate constant (K ep), volume fraction (V e), and apparent diffusion coefficient (ADC). The results showed that the values of Dice coefficient, peak signal-to-noise ratio (PSNR), and structural similarity (SSIM) of the OPCNN algorithm were significantly higher than those of the traditional CNN model and the U-net network model. Meanwhile, the difference was statistically significant (P < 0.05). K trans, K ep, and V e in tumor lesions were significantly higher than those in the healthy side, while the ADC was significantly lower than that in the healthy side (P < 0.05). The sensitivity, specificity, and accuracy of dynamic contrast-enhancement magnetic resonance imaging (DCE-MRI) in the diagnosis of nasopharyngeal carcinoma staging were slightly higher than those in T2-weighted imaging (T2WI) and diffusion-weighted imaging (DWI). The diagnostic sensitivity of DCE-MRI was more than 85%, its diagnostic specificity was more than 75%, and its diagnostic accuracy was more than 90%. The AUC area of NPC diagnosed by combination of the three was significantly different from that diagnosed by single T2WI, DWI, and DCE-MRI (P < 0.05). The diagnostic accuracy of MRI based on the OPCNN algorithm for nasopharyngeal carcinoma (93.2%) was significantly higher than that of single MRI (76.4%). In summary, the OPCNN algorithm proposed in this study could improve the quality of MRI images, and the effect was better than the traditional deep learning model, which had the value of clinical promotion. The application value of DCE-MRI in the diagnosis of pathogenic lesions of nasopharyngeal carcinoma was better than conventional MRI. The combined application of T2WI, DWI, and DCE-MRI in the screening of nasopharyngeal carcinoma lesions could greatly improve the diagnostic accuracy of nasopharyngeal carcinoma.

Clinicopathological Characteristics and Outcomes of PLA2R-Associated Membranous Nephropathy in Seropositive Patients Without PLA2R Staining on Kidney Biopsy.

RATIONALE & OBJECTIVE: Phospholipase A2 receptor (PLA2R)-associated membranous nephropathy (MN) with circulating serum autoantibodies to PLA2R (SAb+) but no deposits of PLA2R antigen in glomerular tissue by immunofluorescence (GAg-) has been reported. However, little is known about the clinicopathological characteristics or prognosis of this subtype of MN. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: 130 SAb+ patients in China with biopsy-proven MN who had follow-up data and received immunosuppressive therapy. The median follow-up was 16 (IQR, 9-25) months. PREDICTOR: PLA2R antigen detection by immunofluorescence staining of kidney biopsy specimens. OUTCOMES: Complete remission (CR) was defined as proteinuria levels <0.3 g/d and a >50% decrease compared with a previously established baseline. Partial remission (PR) was defined as proteinuria levels <3.5 g/d and a >50% decrease compared with a previously established baseline. The kidney function outcome was defined as a >40% decrease in estimated glomerular filtration rate (eGFR) at the end of the study compared with baseline. ANALYTICAL APPROACH: Kaplan-Meier analysis of PR and CR comparing SAb+/GAg+ and SAb+/GAg- patients. Cox proportional hazards models to examine these associations were adjusted for confounders. RESULTS: Among 130 SAb+ patients with PLA2R-associated MN, 18 were GAg-. Compared with SAb+/GAg+ patients, those who were SAb+/GAg- presented with more severe kidney injury as evidenced by higher SAb titer, greater proteinuria, lower serum albumin concentrations, lower eGFR (all P < 0.05), and more severe disease with higher chronicity scores (P < 0.001) on kidney biopsies. SAb+/GAg- patients exhibited a significantly lower probability of PR (P < 0.001) and CR (P = 0.03) and were more likely to experience a >40% decrease in eGFR (P = 0.008) than patients who were SAb+/GAg+. After adjusting for clinical and pathologic variables available at the time of biopsy, compared with SAb+/GAg+ patients, SAb+/GAg- patients had a lower rate of experiencing remission (hazard ratio, 0.32 [95% CI, 0.15-0.68]; P = 0.003) and a higher rate of the >40% eGFR decrease outcome (hazard ratio, 7.66 [95% CI, 1.54-38.08]; P = 0.01). LIMITATIONS: Retrospective study, small sample size, and lack of a uniform approach to treatment. CONCLUSIONS: Seropositive PLA2R-associated MN without PLA2R staining on kidney biopsy may represent a distinct clinical subtype with more severe disease and a worse prognosis. GAg- is independently associated with poor response to treatment and >40% eGFR decrease in seropositive PLA2R-associated MN.

Urinary Matrix Metalloproteinase 7 Activated by Oxidative Stress Predicts Kidney Prognosis in Myeloperoxidase-Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

Aims: Effective and applicable predictors of end-stage kidney disease (ESKD) are needed for patients with myeloperoxidase-antineutrophil cytoplasmic antibody-associated vasculitis (MPO-AAV) and kidney involvement. We investigated whether urinary matrix metalloproteinase-7 (uMMP7) was associated with kidney injury severity and incident ESKD in MPO-AAV. Results: A prospective two-stage study was conducted in 150 patients with newly diagnosed MPO-AAV in two independent cohorts. uMMP7 was measured on the days of initial and repeat kidney biopsies. In stage I, a higher initial uMMP7 level was associated with a lower estimated glomerular filtration rate (eGFR), higher level of proteinuria, and greater extent of kidney pathologic lesions. This elevated uMMP7 protein level is activated and potentially derived from the enhanced kidney production induced by oxidative stress. In stage II, uMMP7 at initial biopsy was independently associated with the incidence of ESKD over 6 years. The higher uMMP7 group (vs. lower) had an adjusted hazard ratio of 3.79 (95% confidence interval [CI], 1.49-6.09) for ESKD in the test cohort. Findings were similar in the validation cohort. A combination of data from the two cohorts revealed that adding uMMP7 into clinical or clinicopathologic models significantly improved risk discrimination for future ESKD. Innovation: An elevated uMMP7 level in MPO-AAV was independently associated with severe kidney injury and incident ESKD. Conclusions: uMMP7 in MPO-AAV improves identification of patients at risk of ESKD and may enable early and optimized therapy to improve outcomes. Antioxid. Redox Signal. 37, 246-256.

Trimethylamine-N-Oxide Aggravates Kidney Injury via Activation of p38/MAPK Signaling and Upregulation of HuR.

BACKGROUND: Trimethylamine-N-oxide (TMAO) is an intestinal metabolic toxin, which is produced by gut flora via metabolizing high-choline foods. TMAO is known to increase the risk of atherosclerosis and cardiovascular events in chronic kidney disease (CKD) patients. OBJECTIVES: The objective of this study was to explore the role and mechanism of TMAO aggravating kidney injury. METHOD: We used the five-sixths nephrectomy (5/6 Nx)-induced CKD rats to investigate whether TMAO could aggravate kidney damage and its possible mechanisms. Six weeks after the operation, the two groups of 5/6 Nx rats were subjected to intraperitoneal injection with 2.5% glucose peritoneal dialysis fluid (2.5% PDF) and 2.5% PDF plus TMAO 20 mg/kg/day. RESULTS: In this study, we provided evidence showing TMAO significantly aggravated renal failure as well as inflammatory cell infiltration and in five-sixths nephrectomy-induced CKD rats. We found that TMAO could upregulate inflammatory factors including MCP-1, TNF-α, IL-6, IL-1ß, and IL-18 by activating p38 phosphorylation and upregulation of human antigen R. TMAO could aggravate oxidative stress by upregulating NOX4 and downregulating SOD. The result also confirmed that TMAO promoted NLRP3 inflammasome formation as well as cleaved caspase-1 and IL-1ß activation in the kidney tissue. CONCLUSIONS: Taken together, the present study validates TMAO as a pro-inflammatory factor that causes renal inflammatory injury and renal function impairment. Inhibition of TMAO synthesis or promoting its clearance may be a potential therapeutic approach of CKD in the future.

Intensity of Macrophage Infiltration in Glomeruli Predicts Response to Immunosuppressive Therapy in Patients with IgA Nephropathy.

BACKGROUND: The lack of a tool for predicting the response to immunosuppressive therapy in IgA nephropathy (IgAN) limits patient-specific risk stratification and early treatment decision making. Models for predicting response to immunosuppression in IgAN that can be applied at the time of kidney biopsy are needed. METHODS: This prospective cohort study involved 621 Chinese patients with IgAN who were at high risk for disease progression and had persistent proteinuria ≥1 g/d, despite 3 months of optimized supportive care with renin-angiotensin system inhibitors. Participants received immunosuppressive therapy for a median of 18 months. We used immunochemistry to identify macrophage and lymphocyte infiltrates in biopsy specimens and digital image analysis to quantify them. The outcome was response to immunosuppression, defined as complete or partial remission within 12 months of immunosuppression. RESULTS: Kidney infiltration of CD68 + and CD206 + macrophages increased in patients with IgAN. Having higher levels of glomerular CD206 + macrophage infiltration was associated with a 40-fold increased probability of response to immunosuppression in adjusted analysis compared with having lower levels. Patients with a higher intensity of glomerular CD68 + infiltrates had a 13-fold increase in probability of responding to immunosuppression. Intensity of glomerular CD206 + and CD68 + macrophage infiltration predicted the response to immunosuppression (area under the curve [AUC], 0.84; 95% CI, 0.81 to 0.88). The AUC increased to 0.87 (95% CI, 0.84 to 0.91) in a model combining the infiltration score of CD206 + and CD68 + infiltrates with the MEST-C score and clinical data at biopsy. CONCLUSIONS: Intensity of glomerular macrophage infiltration predicted response to immunosuppressive therapy in patients with IgAN who were at high risk of progression, and may help physicians identify patients who will benefit from such treatment.

Tubular Numb promotes renal interstitial fibrosis via modulating HIF-1α protein stability.

Tubulointerstitial fibrosis is the ultimate common pathway of all manners of chronic kidney disease. We previously demonstrated that specific deletion of Numb in proximal tubular cells (PTCs) prevented G2/M arrest and attenuated renal fibrosis. However, how Numb modulates cell cycle arrest remains unclear. Here, we showed that Numb overexpression significantly increased the protein level of hypoxia-inducible factor-1α (HIF-1α). Numb overexpression-induced G2/M arrest was blocked by silencing endogenous HIF-1α, subsequently downregulated the expression of cyclin G1 which is an atypical cyclin to promote G2/M arrest of PTCs. Further analysis revealed that Numb-augmented HIF-1α protein was blocked by simultaneously overexpressing MDM2. Moreover, silencing Numb decreased TGF-ß1-induceded HIF-1α protein expression. While endogenous MDM2 was knocked down this reduction was reversed, indicating that Numb stabilized HIF-1α protein via interfering MDM2-mediated HIF-1α protein degradation. Interestingly, HIF-1α overexpression significantly upregulated the expression of Numb and silencing endogenous HIF-1α blocked CoCl2 or TGF-ß1-induced Numb expression. Chromatin immunoprecipitation (ChIP) assays demonstrated that HIF-1α binded to the promoter region of Numb. This binding was significantly increased by TGF-ß1. Collectively, these data indicate that Numb and HIF-1α cooperates to promote G2/M arrest of PTCs, and thus aggravates tubulointerstitial fibrosis. Numb might be a potential target for the therapy of tubulointerstitial fibrosis.

Association of Urinary Matrix Metalloproteinase 7 Levels With Incident Renal Flare in Lupus Nephritis.

OBJECTIVE: Flares of lupus nephritis (LN) are frequent and associated with impaired renal prognosis. One major management obstacle in LN flare is the lack of effective methods to identify at-risk patients earlier in their disease course. This study was undertaken to test the utility of measurement of urinary matrix metalloproteinase 7 (MMP-7) for the dynamic surveillance of renal disease activity and prediction of renal flares in LN. METHODS: A prospective, 2-stage cohort study was performed in patients with LN. Urinary MMP-7 levels at the time of biopsy were evaluated in 154 patients with newly diagnosed LN in 2 independent cohorts. Urinary MMP-7 levels were assessed for correlation with renal histologic activity. Furthermore, after a minimum period of 12 months of renal disease remission, urinary MMP-7 levels were monitored bimonthly for 2 years in 65 patients with LN. The association between urinary MMP-7 levels and development of LN flare was analyzed. RESULTS: Urinary MMP-7 levels were elevated in patients with LN. A higher urinary MMP-7 level in LN was associated with greater renal histologic activity. As a marker for identifying LN patients with more severe renal histologic activity (i.e., a histologic activity index of ≥7), the level of urinary MMP-7 outperformed other clinical markers and improved their predictive performance, thus linking urinary MMP-7 levels to renal disease activity. Furthermore, among patients who had follow-up measurements of urinary MMP-7 after achievement of long-term remission of renal disease activity, an elevated urinary MMP-7 level during follow-up was independently associated with an increased risk of LN flare. This elevation in the urinary MMP-7 level hinted at the risk of an LN flare at an earlier time point prior to indications using conventional laboratory measures. Thus, use of the urinary MMP-7 level in conjunction with other clinical measures improved the prognostic value for prediction of an LN flare. CONCLUSION: Urinary MMP-7 levels in LN are correlated with renal histologic activity. An elevated urinary MMP-7 level detected after achievement of long-term renal disease remission is associated with a higher risk of incident renal flare in patients with LN.

Seropositive PLA2R-associated membranous nephropathy but biopsy-negative PLA2R staining.

BACKGROUND: Serum phospholipase A2 receptor (PLA2R) antibody (SAb) and glomerular deposits of PLA2R antigen (GAg) have been tested widely in idiopathic membranous nephropathy (MN). Recently, we noticed a special form of PLA2R-associated MN with positive circulating PLA2R antibody but negative PLA2R deposits in the glomeruli by immunofluorescence on frozen tissue (IF-F). The significance of this form of PLA2R-associated MN is yet to be elucidated. This study aimed to explore the clinicopathological features of these PLA2R-associated MN patients. METHODS: This study enrolled 229 biopsy-proven PLA2R-associated MN patients with SAb+. SAb was measured by enzyme-linked immunosorbent assay, and GAg was detected by IF-F. These patients were divided into SAb+/GAg+ and SAb+/GAg- groups. Clinicopathological characteristics of SAb+/GAg+ and SAb+/GAg- PLA2R-associated MN patients were compared. PLA2R antigens of 19 SAb+/GAg- PLA2R-associated MN patients were verified by immunohistochemistry on paraffin tissue (IHC-P). RESULTS: Among 229 SAb+ PLA2R-associated MN patients, 210 (91.70%) were GAg+ and 19 (8.3%) were GAg-. These 19 SAb+/GAg- PLA2R-associated MN patients presented positive PLA2R deposits by IHC-P. Compared with SAb+/GAg+ PLA2R-associated MN patients, SAb+/GAg- PLA2R-associated MN patients had higher levels of serum PLA2R antibody (P = 0.004), increased proteinuria (P = 0.008), lower serum albumin (P = 0.019), more prominent chronic pathological lesions in terms of glomerulosclerosis score (P = 0.025), interstitial fibrosis score (P = 0.016), tubular atrophy score (P = 0.010) and total renal chronicity score (P = 0.010), and were more likely to be accompanied by focal segmental glomerulosclerosis (P = 0.014). Higher SAb level was associated with the total renal chronicity score (odds ratio per 100 RU/mL, 1.16; 95% confidence interval 1.01-1.33; P = 0.033). CONCLUSIONS: PLA2R-associated MN patients with seropositive PLA2R antibody but negative PLA2R deposits in the glomeruli by IF-F have higher levels of SAb and worse clinicopathological manifestations compared with their double-positive counterparts. IHC-P can be an alternative technique to reveal PLA2R glomerular deposits.

Urinary angiotensinogen: an indicator of active antineutrophil cytoplasmic antibody-associated glomerulonephritis.

BACKGROUND: One of the major challenges in improving the management of antineutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN) is the lack of a disease-specific indicator for histological lesions and disease activity. Here we tested the utility of urinary angiotensinogen (UAGT) as a biomarker of renal disease activity in ANCA-GN. METHODS: A prospective, two-stage cohort study was performed in ANCA-GN patients. In Stage I, UAGT was measured at the time of renal biopsy in 69 patients from two centers (test set) and 25 patients from two other centers (validation set). In Stage II, UAGT was monitored in 50 subjects in the test set for 24 months. RESULTS: In Stage I, UAGT significantly increased in ANCA-GN patients, correlating well with cellular crescents formation and active interstitial inflammation. Patients with crescentic ANCA-GN exhibited the highest UAGT compared with other histopathological classes of ANCA-GN. After multivariable adjustment, the highest quartile of UAGT, compared with the lowest quartile, associated with a 6-fold increased risk of crescentic ANCA-GN. For predicting crescentic ANCA-GN, UAGT [area under the receiver operating characteristics curve (AUC) = 0.88] outperformed albuminuria (AUC = 0.73) and estimated glomerular filtration rate (AUC = 0.69). UAGT improved the performance of those clinical markers in diagnosing crescentic ANCA-GN (P < 0.034), suggesting a role of UAGT in identifying active crescentic ANCA-GN. In Stage II, UAGT decreased after immunotherapy and increased at the time of renal relapse during the 2-year follow-up, suggesting the usefulness of UAGT to monitor disease activity over time. CONCLUSIONS: These results suggest the potential use of UAGT for assessing disease activity and renal relapse in ANCA-GN.

Hyperhomocysteinemia Accelerates Acute Kidney Injury to Chronic Kidney Disease Progression by Downregulating Heme Oxygenase-1 Expression.

AIMS: The risk factors promoting acute kidney injury (AKI) to chronic kidney disease (CKD) progression remain largely unknown. The aim of the present study was to investigate whether hyperhomocysteinemia (Hhcy) accelerates the development of renal fibrosis after AKI. RESULTS: Hhcy aggravated ischemia-reperfusion-induced AKI and the subsequent development of renal fibrotic lesions characterized by excessive extracellular matrix deposition. Mechanistically, the RNA binding protein human antigen R (HuR) bound to the 3'-untranslated region (3'-UTR) of heme oxygenase-1 (HO-1) messenger RNA (mRNA). Homocysteine (Hcy) downregulated HuR expression, reduced the binding of HuR to the 3'-UTR of HO-1, and thereafter decreased HO-1 expression. Administration of the HO-1 inducer cobalt protoporphyrin-IX significantly hindered Hhcy-augmented reactive oxygen species production and renal fibrotic lesions. Innovation and Conclusion: These data indicate that Hhcy might be a novel risk factor that promotes AKI to CKD progression. Lowering Hcy level or HO-1 induction might be a potential therapeutic strategy to improve the outcome of AKI.

Long noncoding RNA lnc-TSI inhibits renal fibrogenesis by negatively regulating the TGF-ß/Smad3 pathway.

Transforming growth factor-ß (TGF-ß) is a well-established central mediator of renal fibrosis, a common outcome of almost all progressive chronic kidney diseases. Here, we identified a poorly conserved and kidney-enriched long noncoding RNA in TGF-ß1-stimulated human tubular epithelial cells and fibrotic kidneys, which we termed TGF-ß/Smad3-interacting long noncoding RNA (lnc-TSI). Lnc-TSI was transcriptionally regulated by Smad3 and specifically inhibited TGF-ß-induced Smad3 phosphorylation and downstream profibrotic gene expression. Lnc-TSI acted by binding with the MH2 domain of Smad3, blocking the interaction of Smad3 with TGF-ß receptor I independent of Smad7. Delivery of human lnc-TSI into unilateral ureteral obstruction (UUO) mice, a well-established model of renal fibrosis, inhibited phosphorylation of Smad3 in the kidney and attenuated renal fibrosis. In a cohort of 58 patients with biopsy-confirmed IgA nephropathy (IgAN), lnc-TSI renal expression negatively correlated with the renal fibrosis index (r = -0.56, P < 0.001) after adjusting for cofounders. In a longitudinal study, 32 IgAN patients with low expression of renal lnc-TSI at initial biopsy had more pronounced increases in their renal fibrosis index and experienced stronger declines in renal function at repeat biopsy at a mean of 48 months of follow-up. These data suggest that lnc-TSI reduced renal fibrogenesis through negative regulation of the TGF-ß/Smad pathway.

Urinary angiotensinogen predicts progressive chronic kidney disease after an episode of experimental acute kidney injury.

One of the major obstacles to prevent AKI-CKD transition is the lack of effective methods to follow and predict the ongoing kidney injury after an AKI episode. In the present study, we test the utility of urinary angiotensinogen (UAGT) for dynamically evaluating renal structural changes and predicting AKI-CKD progression by using both mild and severe bilateral renal ischemia/reperfusion injury mice. UAGT returns to pre-ischemic levels 14 days after mild AKI followed by kidney architecture restoration, whereas sustained increase in UAGT accompanies by ongoing renal fibrosis after severe AKI. UAGT at day 14-42 correlates with renal fibrosis 84 days after AKI. For predicting fibrosis at day 84, the area under receiver operating characteristics curve of UAGT at day 14 is 0.81. Persistent elevation in UAGT correlates with sustained activation of intrarenal renin-angiotensin system (RAS) during AKI-CKD transition. Abrogating RAS activation post AKI markedly reduced renal fibrosis, with early RAS intervention (from 14 days after IRI) more beneficial than late intervention (from 42 days after IRI) in alleviating fibrosis. Importantly, UAGT decreases after RAS intervention, and its level at day 14-28 correlates with the extent of renal fibrosis at day 42 post RAS blockade. A pilot study conducted in patients with acute tubular necrosis finds that compared with those recovered, patients with AKI-CKD progression exhibits elevated UAGT during the 3-month follow-up after biopsy. Our study suggests that UAGT enables the dynamical monitoring of renal structural recovery after an AKI episode and may serve as an early predictor for AKI-CKD progression and treatment response.

Wnt9a Promotes Renal Fibrosis by Accelerating Cellular Senescence in Tubular Epithelial Cells.

Cellular senescence is associated with renal disease progression, and accelerated tubular cell senescence promotes the pathogenesis of renal fibrosis. However, the underlying mechanism is unknown. We assessed the potential role of Wnt9a in tubular cell senescence and renal fibrosis. Compared with tubular cells of normal subjects, tubular cells of humans with a variety of nephropathies and those of several mouse models of CKD expressed high levels of Wnt9a that colocalized with the senescence-related protein p16INK4A Wnt9a expression level correlated with the extent of renal fibrosis, decline of eGFR, and expression of p16INK4A Furthermore, ectopic expression of Wnt9a after ischemia-reperfusion injury (IRI) induced activation of ß-catenin and exacerbated renal fibrosis. Overexpression of Wnt9a exacerbated tubular senescence, evidenced by increased detection of p16INK4A expression and senescence-associated ß-galactosidase activity. Conversely, shRNA-mediated knockdown of Wnt9a repressed IRI-induced renal fibrosis in vivo and impeded the growth of senescent tubular epithelial cells in culture. Notably, Wnt9a-induced renal fibrosis was inhibited by shRNA-mediated silencing of p16INK4A in the IRI mouse model. In a human proximal tubular epithelial cell line and primary renal tubular cells, Wnt9a remarkably upregulated levels of senescence-related p16INK4A, p19ARF, p53, and p21 and decreased the phosphorylation of retinoblastoma protein. Wnt9a also induced senescent tubular cells to produce TGF-ß1, which promoted proliferation and activation in normal rat kidney fibroblasts. Thus, Wnt9a drives tubular senescence and fibroblast activation. Furthermore, the Wnt9a-TGF-ß pathway appears to create a reciprocal activation loop between senescent tubular cells and activated fibroblasts that promotes and accelerates the pathogenesis of renal fibrosis.

Long Noncoding RNA LINC01619 Regulates MicroRNA-27a/Forkhead Box Protein O1 and Endoplasmic Reticulum Stress-Mediated Podocyte Injury in Diabetic Nephropathy.

AIMS: Altered activities of long noncoding RNAs (lncRNAs) have been implicated in the regulation of microRNAs. microRNA-27a (miR-27a) upregulation has been shown to induce endoplasmic reticulum (ER) stress podocyte injury in diabetic nephropathy (DN). Herein, we aim to interrogate the mutually regulated network of miR-27a with long intergenic noncoding RNA 1619 (LINC01619) and the target gene. RESULTS: LINC01619 downregulation was found in human DN renal biopsy tissues and contributed to proteinuria and diminished renal function. LINC01619 was expressed in podocyte cytoplasm and involved in ER stress signaling pathway. LINC01619 exerted biological function by serving as a "sponge" for miR-27a, which negatively targeted forkhead box protein O1 (FOXO1) and activated ER stress. In diabetic rats and high-glucose cultured podocytes, LINC01619 triggered oxidative stress and podocyte injuries as demonstrated by increased apoptosis, diffuse podocyte foot process effacement, and decreased renal function. Innovation and Conclusion: This study demonstrates that LINC01619 functions as a competing endogenous RNA and regulates miR-27a/FOXO1-mediated ER stress and podocyte injury in DN. Antioxid. Redox Signal. 29, 355-376.

WT1 ameliorates podocyte injury via repression of EZH2/ß-catenin pathway in diabetic nephropathy.

Epigenetic modulation of podocyte injury plays a pivotal role in diabetic nephropathy (DN). Wilm's tumor 1 (WT1) has been found to have opposing roles with ß-catenin in podocyte biology. Herein, we asked whether the histone methyltransferase enzyme enhancer of zeste homolog 2 (EZH2) promotes WT1-induced podocyte injury via ß-catenin activation and the underlying mechanisms. We found that WT1 antagonized EZH2 and ameliorated ß-catenin-mediated podocyte injury as demonstrated by attenuated podocyte mesenchymal transition, maintenance of podocyte architectural integrity, decreased podocyte apoptosis and oxidative stress. Further, we provided mechanistical evidence that EZH2 was required in WT1-mediated ß-catenin inactivation via repression of secreted frizzled-related protein 1 (SFRP-1), a Wnt antagonist. Moreover, EZH2-mediated silencing of SFRP-1 was due to increased histone 3 lysine 27 trimethylation (H3K27me3) on its promoter region. WT1 favored renal function and decreased podocyte injury in diabetic rats and DN patients. Notably, WT1 exhibited clinical and biological relevance as it was linked to dropped serum creatinine, decreased proteinuria and elevated estimated glomerular filtration rate (eGFR). We propose an epigenetic process via the WT1/EZH2/ß-catenin axis in attenuating podocyte injury in DN. Targeting WT1 and EZH2 could be potential therapeutic approaches for DN.

MicroRNA-27a promotes podocyte injury via PPARγ-mediated ß-catenin activation in diabetic nephropathy.

Podocyte injury has a pivotal role in the pathogenesis of diabetic nephropathy (DN). MicroRNA-27a (miR-27a), peroxisome proliferator-activated receptor γ (PPARγ) and ß-catenin pathways have been involved in the pathogenesis of DN. Herein, we asked whether miR-27a mediates podocyte injury through PPARγ/ß-catenin signaling in DN. The functional relevance of miR-27a, PPARγ and ß-catenin were investigated in cultured podocytes and glomeruli of diabetic rats and patients using in vitro and in vivo approaches. Podocyte injury was assessed by migration, invasion and apoptosis assay. Biological parameters were analyzed using enzyme-linked immunosorbent assay. We found that high glucose stimulated miR-27a expression, which, by negatively targeting PPARγ, activated ß-catenin signaling as evidenced by upregulation of ß-catenin target genes, snail1 and α-smooth muscle actin (α-SMA) and downregulation of podocyte-specific markers podocin and synaptopodin. These changes caused podocyte injury as demonstrated by increased podocyte mesenchymal transition, disrupted podocyte architectural integrity and increased podocyte apoptosis. Furthermore, we provide evidence that miR-27a contributed to unfavorable renal function and increased podocyte injury in diabetic rats. Notably, miR-27a exhibited clinical and biological relevance as it was linked to elevated serum creatinine, proteinuria and reduced creatinine clearance rate. In addition, miR-27a upregulation and activation of PPARγ/ß-catenin signaling were verified in renal biopsy samples from DN patients. We propose a novel role of the miR-27a/PPARγ/ß-catenin axis in fostering the progression toward more deteriorated podocyte injury in DN. Targeting miR-27a could be a potential therapeutic approach for DN.

Overexpression of Intrarenal Renin-Angiotensin System in Human Acute Tubular Necrosis.

BACKGROUND/AIMS: Acute tubular necrosis (ATN), a leading cause of acute kidney injury (AKI), is associated with decreased survival and increased progression of chronic kidney disease. A barrier to improving the clinical outcomes is the incomplete understanding of the pathogenesis of AKI. Our objective is to test the hypothesis that intrarenal renin-angiotensin system (RAS) is overexpressed in patients with ATN and could be an indicator of ATN severity. METHODS: A transversal study was conducted in patients with biopsy-proven ATN. Intrarenal expression of angiotensinogen and angiotensin II, and urinary angiotensinogen were measured. RESULTS: Patients with ATN demonstrated upregulation of intrarenal RAS, evidenced by upregulation of intrarenal angiotensinogen and angiotensin II. Patients with ATN also have elevated urinary angiotensinogen level that correlated with the overexpressed intrarenal RAS. Moreover, this increase in intrarenal RAS expression and urinary angiotensinogen was associated with the extent of acute tubular injury and urinary albumin excretion, respectively. CONCLUSIONS: We demonstrate that the intrarenal RAS is upregulated in patients with ATN and is associated with the severity of ATN. Urinary angiotensinogen reflects intrarenal RAS status, and is of value to assess the severity of ATN.

Anti-PLA2R Antibodies in Chinese Patients with Membranous Nephropathy.

BACKROUND ~This study used two standardized methods to evaluate anti-PLA2R antibody in serum of primary membranous nephropathy (PMN) among Chinese patients to determine  Anti-PLA2R antibody distribution and whether immunological reactivity reflected by antibody titer correlates with kidney function parameters. MATERIAL AND METHOD ~Overall, 82 subjects with biopsy-proven primary membranous nephropathy (PMN) , 22 cases with secondary membranous nephropathy (SMN), 40 non-MN patients with established glomerulonephritis, 20 healthy volunteers were recruited from the Division of Nephrology, Nanfang Hospital, China. Anti-PLA2R antibody in the serum of each patient was evaluated by both recombinant cell-based indirect immunofluorescence assay (RC-IFA) and enzyme linked immunosorbent assay (ELISA). Kidney function was assessed by proteinuria for 24 hours, serum albumin, blood urea nitrogen (BUN), serum creatine, serum cystatin C. We assessed the correlation between anti-PLA2R antibody levels and clinical parameter in the PMN patients. RESULTS ~ Fifty-three patients with PMN (64.6%) were positive for anti-PLA2R antibody. The level of antibody determined by RC-IFA ranged from 1:10 to 1:1000 and 0 to 1423 RU/ml by ELISA. The two anti-PLA2R test systems correlated very well with each other and reached an agreement of 95.7% for PMN patients. The level of antibody detected by ELISA in patients with PMN also significantly correlated with proteinuria and nephritic-range proteinuria (> 3.5g/day) . CONCLUSIONS ~Anti-PLA2R antibody is sensitive and extremely specific for diagnosis of Chinese patients with primary membranous nephropathy. Concentration of autoantibody against PLA2R is an ideal marker for monitoring the activity of immunological disease.