Dahuang Zhechong Pill Alleviates Liver Fibrosis Progression by Regulating p38 MAPK/NF-κ B/TGF-ß1 Pathway.

OBJECTIVE: To explore the effect and mechanism of Dahuang Zhechong Pill (DHZCP) on liver fibrosis. METHODS: Liver fibrosis cell model was induced by transforming growth factor-ß (TGF-ß) in hepatic stellate cells (HSC-T6). DHZCP medicated serum (DMS) was prepared in rats. HSC-T6 cells were divided into the control (15% normal blank serum culture), TGF-ß (15% normal blank serum + 5 ng/mL TGF-ß), DHZCP (15% DMS + 5 ng/mL TGF-ß), DHZCP+PDTC [15% DMS + 4 mmol/L ammonium pyrrolidine dithiocarbamate (PDTC)+ 5 ng/mL TGF-ß], and PDTC groups (4 mmol/L PDTC + 5 ng/mL TGF-ß). Cell activity was detected by cell counting kit 8 and levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the cell supernatant were determined by enzyme-linked immunosorbnent assay. Western blot was used to measure the expressions of p38 mitogen-activated protein kinase/nuclear factor kappa B/transforming growth factor-ß1 (p38 MAPK/NF-κ B/TGF-ß1) pathway related proteins, and the localization and expressions of these proteins were observed by immunofluorescence staining. RESULTS: DHZCP improves the viability of cells damaged by TGF-ß and reduces inflammatory cytokines and ALT and AST levels in the supernatant of HSC-T6 cells induced with TGF-ß (P<0.05 or P<0.01). Compared with the TGF-ß group, NF-κ B p65 levels in the DHZCP group were decreased (P<0.05). p38 MAPK and NF-κ B p65 levels in the DHZCP+PDTC were also reduced (P<0.01). Compared with the TGF-ß group, the protein expression of Smad2 showed a downward trend in the DHZCP, DHZCP+PDTC, and PDTC groups (all P<0.01), and the decreasing trend of Samd3 was statistically significant only in DHZCP+PDTC group (P<0.01), whereas Smad7 was increased (P<0.05 or P<0.01). CONCLUSION: DHZCP can inhibit the process of HSC-T6 cell fibrosis by down-regulating the expression of p38 MAPK/NF-κ B/TGF-ß1 pathway.

Bowman's capsule rupture and the Oxford MEST-C score in adult patients with Immunoglobulin a vasculitis nephritis: a single center experience.

OBJECTIVE: Bowman's capsule rupture (BCR) is a glomerular pathological change, but it is still not well recognized in immunoglobulin A vasculitis nephritis (IgAV-N). The Oxford MEST-C score is a classification for IgA nephropathy; however, its clinical correlation and prognostic value in adult patients with IgAV-N are unclear. METHODS: A retrospective study of 145 adult patients with IgAV-N diagnosed by renal biopsy was conducted. Clinical manifestations, pathological changes and the prognosis of IgAV-N patients were compared depending on the presence or absence of BCR, International Study of Kidney Disease in Children (ISKDC) classification and MEST-C score. The primary endpoint events were end-stage renal disease, renal replacement therapy and all-cause death. RESULTS: In total, 51 of 145 (35.17%) patients with IgAV-N presented with BCR. Patients with BCR had more proteinuria, lower serum albumin, and more crescents. Compared with IgAV-N patients with crescents only, 51/100 patients with crescents combined with BCR had a higher proportion of crescents in all glomeruli (15.79% vs. 9.09%; p = 0.003). Patients with higher ISKDC grades had more severe clinical presentation, but it did not reflect the prognosis. However, the MEST-C score not only reflected clinical manifestations but also predicted prognosis (p < 0.05). BCR contributed to the effectiveness of the MEST-C score in predicting the prognosis of IgAV-N (C-index: 0.845 to 0.855). CONCLUSIONS: BCR is associated with clinical manifestations and pathological changes in patients with IgAV-N. The ISKDC classification and MEST-C score are related to the patient's condition, but only the MEST-C score is correlated with the prognosis of patients with IgAV-N, while BCR can improve its predictive ability.

BCR was associated with clinical manifestations and pathological changes in patients with IgAV-N, particularly crescents.The ISKDC classification was related to clinical manifestations of patients with IgAV-N, but it wasn't associated with prognosis.The Oxford MEST-C score was correlated to clinical presentations and prognosis of patients with IgAV-N, while BCR can improve its predictive ability.

Epsin1-mediated exosomal sorting of Dll4 modulates the tubular-macrophage crosstalk in diabetic nephropathy.

Tubular epithelial cells (TECs) play critical roles in the development of diabetic nephropathy (DN), and can activate macrophages through the secretion of exosomes. However, the mechanism(s) of TEC-exosomes in macrophage activation under DN remains unknown. By mass spectrometry, 1,644 differentially expressed proteins, especially Dll4, were detected in the urine exosomes of DN patients compared with controls, which was confirmed by western blot assay. Elevated Epsin1 and Dll4/N1ICD expression was observed in kidney tissues in both DN patients and db/db mice and was positively associated with tubulointerstitial damage. Exosomes from high glucose (HG)-treated tubular cells (HK-2) with Epsin1 knockdown (KD) ameliorated macrophage activation, TNF-α, and IL-6 expression, and tubulointerstitial damage in C57BL/6 mice in vivo. In an in vitro study, enriched Dll4 was confirmed in HK-2 cells stimulated with HG, which was captured by THP-1 cells and promoted M1 macrophage activation. In addition, Epsin1 modulated the content of Dll4 in TEC-exosomes stimulated with HG. TEC-exosomes with Epsin1-KD significantly inhibited N1ICD activation and iNOS expression in THP-1 cells compared with incubation with HG alone. These findings suggested that Epsin1 could modulate tubular-macrophage crosstalk in DN by mediating exosomal sorting of Dll4 and Notch1 activation.

The mechanisms underlying the actions of Xuefu Zhuyu decoction pretreatment against neurological deficits after ischemic stroke in mice: The mediation of glymphatic function by aquaporin-4 and its anchoring proteins.

Ischemic stroke (IS) has been associated with an impairment in glymphatic function. Xuefu Zhuyu Decoction (XFZYD) is widely used in the prevention and treatment of ischemic stroke. We hypothesized that Xuefu Zhuyu decoction pretreatment could attenuate early neurological deficits after ischemic stroke by enhancing the function of the glymphatic system. To prove our hypothesis, we carried out temporary middle cerebral artery occlusion and reperfusion surgery on C57BL/6 mice and then measured neurological score, infarct size and performed hematoxylin-eosin staining to assess stroke outcomes after 24 h of reperfusion. Subsequently, we injected fluorescent tracers in to the cisterna magna and evaluated tracer distribution in coronal brain sections. The polarization of aquaporin-4 (AQP4), colocalization of aquaporin-4, α-dystroglycan, ß-dystroglycan and agrin were determined by immunofluorescence. Our research showed that pretreatment with Xuefu Zhuyu decoction significantly alleviated neurological scores, neurological deficits and pathological abnormalities in a mouse model of ischemic stroke. Importantly, Xuefu Zhuyu decoction pretreatment enhanced cerebrospinal fluid influx, protected aquaporin-4 depolarization and promoted the colocalization of aquaporin-4 with its anchoring proteins in the brain. Our findings highlight novel mechanisms underlying the neuroprotective effect of Xuefu Zhuyu decoction pretreatment on ischemic stroke-induced brain damage through the glymphatic system. Xuefu Zhuyu decoction pretreatment may offer a promising approach to slow the onset and progression of ischemic stroke.

Clinic-pathological characteristics of rare tubulointerstitial diseases. / å°‘è§è‚¾å°ç®¡é—´è´¨æ€§ç–¾ç— çš„ä¸´åºŠåŠç— ç†ç‰¹å¾åˆ†æž.

OBJECTIVES: Tubulointerstitial diseases is one of the common causes of renal dysfunction. Some rare pathological types are easy to be misdiagnosed and missedly diagnosed because of their low prevalence and relatively insufficient understanding, which affects the treatment and prognosis of patients. This study aims to explore clinical manifestations and pathological characteristics of several rare tubulointerstitial diseases, and therefore to improve their diagnosis and treatment. METHODS: A total of 9 363 patients diagnosed by renal biopsy in the Department of Nephrology, Second Xiangya Hospital, Central South University from November 2011 to September 2021 were selected. Six cases of light chain cast nephropathy (LCCN), 2 cases of light chain proximal tubulopathy (LCPT), 1 case of LCCN with LCPT, 4 cases of genetic tubulointerstitial disease, and 6 cases of non-genetic related tubulointerstitial lesion were screened out, and their clinical manifestations and renal biopsy pathological results were collected, compared, and analyzed. RESULTS: Patients with LCCN presented with mild to moderate anemia, microscopic hematuria, and mild to moderate proteinuria. Compared with patients with LCPT, proteinuria and anemia were more prominent in patients with LCCN. Five patients with LCCN and 2 patients with LCPT had elevated serum free kappa light chain. Five patients with LCCN presented clinically with acute kidney injury (AKI). Two patients with LCPT and 1 patient with LCCN and LCPT showed CKD combined with AKI, and 1 LCPT patient presented with typical Fanconi syndrome (FS). Five patients with LCCN, 2 patients with LCPT, and 1 patient with LCCN and LCPT were diagnosed with multiple myeloma. Five patients with LCCN had kappa light chain restriction in tubules on immunofluorescence and a "fractured" protein casts with pale periodic acid-Schiff (PAS) staining on light microscopy. Immunohistochemical staining of 2 LCPT patients showed strongly positive kappa light chain staining in the proximal tubular epithelial cells. And monoclonal light chain crystals in crystalline LCPT and abnormal lysosomes and different morphological inclusion bodies in noncrystalline LCPT were observed under the electron microscope. Six patients with LCCN were mainly treated by chemotherapy. Renal function was deteriorated in 1 patient, was stable in 4 patients, and was improved in 1 patient. Two patients with LCPT improved their renal function after chemotherapy. Four patients with genetic tubulointerstitial disease were clinically presented as CKD, mostly mild proteinuria, with or without microscopic hematuria, and also presented with hyperuricemia, urine glucose under normal blood glucose, anemia, polycystic kidneys. Only 1 case had a clear family history, and the diagnosis was mainly based on renal pathological characteristics and genetic testing. Compared with patients with non-genetic related tubulointerstitial lesion, patients with genetic tubulointerstitial disease had an earlier age of onset, higher blood uric acid, lower Hb and estiated glomemlar fitration (eGFR), and less edema and hypertension. Renal pathology of genetic tubulointerstitial disease presented tubular atrophy and interstitial fibrosis, abnormal tubular dilation, glomerular capsuledilation, and glomerular capillary loop shrinkage. Glomerular dysplasia and varying degrees of glomerular sclerosis were observed. Genetic tubulointerstitial disease patients were mainly treated with enteral dialysis, hypouricemic and hypoglycemic treatment. Two genetic tubulointerstitial disease patients had significantly deteriorated renal function, and 2 patients had stable renal function. CONCLUSIONS: Patients with AKI or FS, who present serum immunofixation electrophoresis and/or serum free kappa light chain abnormalities, should be alert to LCCN or LCPT. Renal biopsy is a critical detection for diagnosis of LCCN and LCPT. Chemotherapy and stem cell transplantation could delay progression of renal function in patients with LCCN and LCPT. If the non-atrophic area of the renal interstitium presents glomerular capsule dilatation, glomerular capillary loop shrinkage, and abnormal tubular dilatation under the light microscopy, genetic tubulointerstitial disease might be considered, which should be traced to family history and can be diagnosed by genetic testing.

Digital Spatial Profiling of Individual Glomeruli From Patients With Anti-Neutrophil Cytoplasmic Autoantibody-Associated Glomerulonephritis.

We previously showed that the rupture of Bowman's capsule (BC) promotes the progression of crescentic glomerulonephritis by enhancing the entry of CD8+ T cells into the glomeruli. In the present study, we utilized digital spatial profiling to simultaneously profile the altered abundances of the messenger RNA (mRNA) transcripts and proteins in the glomerular and periglomerular areas of four biopsy samples of anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis (ANCA-GN) and two biopsy specimens of minimal change disease (MCD) controls. The paraffin-embedded biopsy samples were stained with collagen IV, CD45, and SYTO 13 to distinguish the glomeruli with periglomerular infiltration but intact BC, with focal BC rupture, and with extensive rupture of BC and glomeruli without crescent formation and leukocytic infiltration in ANCA-GN. By assessing multiple discrete glomerular areas, we found that the transcript expression levels of the secreted phosphoprotein-1 and its receptor CD44 were upregulated significantly in the glomeruli with more severe ruptures of BC, and their expression levels correlated positively with the fibrotic markers. We also found that both alternative and classic complement pathways were activated in the glomeruli from patients with ANCA-GN. Furthermore, M1 macrophages were involved mostly in the early stage of BC rupture, while M2 macrophages were involved in the late stage and may contribute to the fibrosis process of the crescents. Finally, loss of glomerular cells in ANCA-GN was likely mediated by apoptosis. Our results show that digital spatial profiling allows the comparative analysis of the mRNA and protein profiles in individual glomeruli affected differently by the disease process and the identification of potential novel mechanisms in ANCA-GN.

Epac activation ameliorates tubulointerstitial inflammation in diabetic nephropathy.

Tubulointerstitial inflammation plays an important role in the progression of diabetic nephropathy (DN), and tubular epithelial cells (TECs) are crucial promoters of the inflammatory cascade. Exchange protein activated by cAMP (Epac) has been shown to suppress the angiotensin II (Ang-II)-induced release of inflammatory cytokines in tubular cells. However, the role of Epac in TEC-mediated tubulointerstitial inflammation in DN remains unknown. We found that administering the Epac agonist 8-pCPT-2'-O-Me-cAMP (8-O-cAMP) to db/db mice inhibited tubulointerstitial inflammation characterized by macrophage infiltration and increased inflammatory cytokine release and consequently alleviated tubulointerstitial fibrosis in the kidney. Furthermore, 8-O-cAMP administration restored CCAAT/enhancer binding protein ß (C/EBP-ß) expression and further upregulated the expression of Suppressor of cytokine signaling 3 (SOCS3), while inhibiting p-STAT3, MCP-1, IL-6, and TNF-α expression in the kidney cortex in db/db mice. And in vitro study showed that macrophage migration and MCP-1 expression induced by high glucose (HG, 30 mM) were notably reduced by 8-O-cAMP in human renal proximal tubule epithelial (HK-2) cells. In addition, 8-O-cAMP treatment restored C/EBP-ß expression in HK-2 cells and promoted C/EBP-ß translocation to the nucleus, where it transcriptionally upregulated SOCS3 expression, subsequently inhibiting STAT3 phosphorylation. Under HG conditions, siRNA-mediated knockdown of C/EBP-ß or SOCS3 in HK-2 cells partially blocked the inhibitory effect of Epac activation on the release of MCP-1. In contrast, SOCS3 overexpression inhibited HG-induced activation of STAT3 and MCP-1 expression in HK-2 cells. These findings indicate that Epac activation via 8-O-cAMP ameliorates tubulointerstitial inflammation in DN through the C/EBP-ß/SOCS3/STAT3 pathway.

Relationships among the Dosage of Erythropoiesis-Stimulating Agents, Erythropoietin Resistance Index, and Mortality in Maintenance Hemodialysis Patients.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) constitute an important treatment option for anemia in hemodialysis (HD) patients. We investigated the relationships among the dosage of ESA, erythropoietin resistance index (ERI) scores, and mortality in Chinese MHD patients. METHODS: This multicenter observational retrospective study included MHD patients from 16 blood purification centers (n = 824) who underwent HD in 2011-2015 and were followed up until December 31, 2016. We collected demographic variables, HD parameters, laboratory values, and ESA dosages. Patients were grouped into quartiles according to ESA dosage to study the effect of ESA dosage on all-cause mortality. The ERI was calculated as follows: ESA (IU/week)/weight (kg)/hemoglobin levels (g/dL). We also compared outcomes among the patients stratified into quartiles according to ERI scores. We used the Cox proportional hazards model to measure the relationships between the ESA dosage, ERI scores, and all-cause mortality. Using propensity score matching, we compared mortality between groups according to ERI scores, classified as either > or ≤12.80. RESULTS: In total, 824 patients were enrolled in the study; 200 (24.3%) all-cause deaths occurred within the observation period. Kaplan-Meier analyses showed that patients administered high dosages of ESAs had significantly worse survival than those administered low dosages of ESAs. A multivariate Cox regression identified that high dosages of ESAs could significantly predict mortality (ESA dosage >10,000.0 IU/week, HR = 1.59, 95% confidence intervals (CIs) (1.04, 2.42), and p = 0.031). Our analysis also indicated a significant increase in the risk of mortality in patients with high ERI scores. Propensity score matching-analyses confirmed that ERI > 12.80 could significantly predict mortality (HR = 1.56, 95% CI [1.11, 2.18], and p = 0.010). CONCLUSIONS: Our data suggested that ESA dosages >10,000.0 IU/week in the first 3 months constitute an independent predictor of all-cause mortality among Chinese MHD patients. A higher degree of resistance to ESA was related to a higher risk of all-cause mortality.

Effects of HIF-1α on renal fibrosis in cisplatin-induced chronic kidney disease.

Cisplatin (Cis) can cause chronic kidney disease (CKD) and promote renal fibrosis, but the underlying mechanism is not fully understood. Hypoxia inducible factor-1α (HIF-1α) can promote renal fibrosis in some kidney diseases, but its role in Cis-induced CKD is still unknown. Notch-1 is a recognized molecule that promotes renal fibrosis under pathological circumstances, and evidence shows that HIF-1α and Notch-1 are closely related to each other. In the present study, mice with HIF-1α gene knockout in proximal tubular cells (PTCs) (PT-HIF-1α-KO) were generated and treated with Cis to induce CKD. A human proximal tubular cell line (HK-2) and primary mouse PTCs were used for in vitro studies. The results showed that HIF-1α was increased in the kidneys of Cis-treated wild-type mice, accompanied by elevated Notch-1, Notch-1 intracellular domain (N1ICD), Hes-1 and renal fibrosis. However, these alterations were partially reversed in PT-HIF-1α-KO mice. Similar results were observed in HK-2 cells and primary mouse PTCs. In addition, treating the cells with Cis induced a marked interaction of HIF-1α and N1ICD. Further inhibiting Notch-1 significantly reduced cellular fibrogenesis but did not affect HIF-1α expression. The data suggested that HIF-1α could promote renal fibrosis in Cis-induced CKD by activating Notch-1 both transcriptionally and post-transcriptionally and that HIF-1α may serve as a potential therapeutic target for Cis-induced CKD.

Predictive value of sub classification of focal segmental glomerular sclerosis in Oxford classification of IgA nephropathy.

BACKGROUND: The Oxford classification of IgA nephropathy (IgAN) was revised in 2016 which lacked sufficient evidence for prognostic value of subclassification of focal segmental glomerular sclerosis (S lesion), and the proper proportion of S lesion for subclassification remains undetermined. AIM: This study aimed to explore the predictive value of the new subclassification of S score on renal outcomes of IgAN patients. METHODS: 348 patients with IgAN-associated S lesion were enrolled. According to the optimal cut-off of 25% established by receiver operating characteristic (ROC) curves, we divided S1 patients into two groups: S1a group (S lesion < 25%) and S1b group (S lesion ≥ 25%). IgAN patients with mild lesion (M0E0S0T0C0) were set as the control group. The clinical features at renal biopsy, pathological findings, and follow-up parameters (follow-up time ranged from 1 to 5 years) were collected. We used univariate and multivariate analyses to assess whether the subclassification of S score could refine risk prediction and clinical utility. RESULTS: We demonstrated that S lesion ≥ 25% was associated with a more rapid GFR loss and a lower rate of complete remission of proteinuria even adjusted for multiple clinic pathological variables, compared to S1a group (All p values <.05). And the ratio of glomeruli with T lesion and crescents were higher in patients with S lesion ≥ 25%. Data showed that IgAN patients with S lesion ≥ 25% were at an increased risk of poor renal outcomes even with immunosuppression. CONCLUSION: This study might recommend new subclassification of S scores (S0 (no S lesion), S1 (S lesion <25% of glomeruli), and S2 (S lesion ≥ 25% of glomeruli)) for the Oxford classification. This model may also help to evaluate pros and cons of immunosuppressive therapy in IgAN patients with different level of S lesion.KEY MESSAGESS lesion ≥ 25% is an independent risk factor for poor renal outcome in IgAN patients.This new subclassification of S scores may help to evaluate pros and cons of immunotherapy in IgAN patients with different level of S lesion.

Nocardiosis in glomerular disease patients with immunosuppressive therapy.

BACKGROUND: Glomerular disease patients have a high risk of infection, which contributes to the progression of disease per se and mortality, especially in those with long-term use of glucocorticoids and (or) immunosuppressive agents. Cases of sporadic nocardiosis have been reported in glomerular disease patients, and this observation was conducted to comprehensively understand the manifestations of and treatments for nocardiosis, which is commonly misdiagnosed as pneumonia or tuberculosis or even as lung cancer or metastatic tumors in glomerular disease patients. METHODS: We reviewed the demographic characteristics, laboratory abnormalities, radiological features, and treatments of 7 patients with nocardiosis and glomerular disease receiving steroids and immunosuppression therapy at the nephrology department of the Second Xiangya Hospital between 2012 and 2019. RESULTS: It was found that all 7 patients had been receiving methylprednisolone for renal disease at a median dose of 20 mg per day and a median duration of 4 months before developing nocardiosis. There were 4 males and 3 females, and the median age was 52.14 years. All 7 patients had hypoalbuminemia at the time of admission. In addition, various cystic abscesses in the subcutaneous tissue, with or without lung and brain involvement, were observed in these patients. Encouragingly, body temperatures returned to normal, and subcutaneous abscesses diminished or disappeared with compound sulfamethoxazole treatment alone or in combination with linezolid, imipenem and mezlocillin/sulbactam. CONCLUSIONS: It was shown that multisite abscesses, including subcutaneous, pulmonary and cerebral abscesses, were the common manifestations of nocardiosis in glomerular disease patients. Sulfonamide was the first-line antibiotic therapy for nocardiosis, and combinations of other antibiotics were also needed in some serious cases.

Aristolochic acid induces renal fibrosis by arresting proximal tubular cells in G2/M phase mediated by HIF-1α.

Renal tubulointerstitial fibrosis (TIF) is a common pathological feature of aristolochic acid (AA) nephropathy (AAN). G2/M arrest of proximal tubular cells (PTCs) is implicated in renal fibrosis of AAN, but the upstream regulatory molecule remains unknown. Hypoxia inducible factor-1α (HIF-1α) promotes renal fibrosis in kidney disease, but the role of HIF-1α in AAN is unclear. Evidence shows that HIF-1α and p21, a known inducer of cellular G2/M arrest, are closely related to each other. To investigate the role of HIF-1α in renal fibrosis of AAN and its effects on p21 expression and PTCs G2/M arrest, mice with HIF-1α gene knockout PTCs (PT-HIF-1α-KO) were generated, and AAN was induced by AA. In vitro tests were conducted on the human PTCs line HK-2 and primary mouse PTCs. HIF-1α and p21 expression, fibrogenesis, and G2/M arrest of PTCs were determined. Results showed that HIF-1α was upregulated in the kidneys of wild-type (WT) AAN mice, accompanied by p21 upregulation, PTCs G2/M arrest and renal fibrosis, and these alterations were reversed in PT-HIF-1α-KO AAN mice. Similar results were observed in HK-2 cells and were further confirmed in primary PTCs from PT-HIF-1α-KO and WT mice. Inhibiting p21 in HK-2 cells and primary PTCs did not change the expression of HIF-1α, but G2/M arrest and fibrogenesis were reduced. These data indicate that HIF-1α plays a key role in renal fibrosis in AAN by inducing PTCs G2/M arrest modulated through p21. HIF-1α may serve as a potential therapeutic target for AAN.

Design and validation of a scoring model for differential diagnosis of diabetic nephropathy and nondiabetic renal diseases in type 2 diabetic patients.

BACKGROUND: We aim to design a scoring model for differential diagnosis between diabetic nephropathy (DN) and nondiabetic renal disease (NDRD) in type 2 diabetic patients through a combination of clinical variables. METHODS: A total of 170 patients with type 2 diabetes who underwent kidney biopsies were included and divided into three groups according to pathological findings: DN group (n = 46), MIX group (DN + NDRD, n = 54), NDRD group (n = 70). Clinical characteristics and laboratory data were collected and compared among groups. Variables with a significant statistical difference between DN and NDRD patients were analyzed by logistic regression to predict the presence of NDRD; then a scoring model was established based on the regression coefficient and further validated in an independent cohort of 67 patients prospectively. RESULTS: On biopsy, 72.9% of patients had NDRD, and the most common pathological type was membranous nephropathy. The established scoring model for predicting NDRD included five predictors: age, systolic blood pressure, hemoglobin, duration of diabetes, and absence of diabetic retinopathy. The model demonstrated good discrimination and calibration (area under curve [AUC] 0.863, 95% CI, 0.800-0.925; Hosmer-Lemeshow [H-L] P = .062). Furthermore, high prediction accuracy (AUC = 0.900; 95% CI, 0.815-0.985) in the validation cohort proved the stability of the model. CONCLUSIONS: We present a simple, robust scoring model for predicting the presence of NDRD with high accuracy (0.85) for the first time. This decision support tool provides a noninvasive method for differential diagnosis of DN and NDRD, which may help clinicians assess the risk-benefit ratio of kidney biopsy for type 2 diabetic patients with renal impairment.

The deacetylase sirtuin 6 protects against kidney fibrosis by epigenetically blocking ß-catenin target gene expression.

Fibrosis is a common pathologic pathway of progressive kidney disease involving complex signaling networks. The deacetylase sirtuin 6 (sirt6) was recently implicated in kidney injury. However, it remains elusive whether and how sirt6 contributes to the regulation of kidney fibrosis. Here, we demonstrate that sirt6 protects against kidney interstitial fibrosis through epigenetic regulation of ß-catenin signaling. Sirt6 is markedly upregulated during fibrogenesis following obstructed nephropathy and kidney ischemia-reperfusion injury. Pharmacological inhibition of sirt6 deacetylase activity aggravates kidney fibrosis in obstructed nephropathy. Consistently, knockdown of sirt6 in mouse kidney proximal tubular epithelial cells aggravates transforming growth factor-ß-induced fibrosis in vitro. Mechanistically, sirt6 deficiency results in augmented expression of the downstream target proteins of ß-catenin signaling. We further show that sirt6 interacts with ß-catenin during transforming growth factor-ß treatment and binds to the promoters of ß-catenin target genes, resulting in the deacetylation of histone H3K56 to prevent the transcription of fibrosis-related genes. Thus, our data reveal the anti-fibrotic function of sirt6 by epigenetically attenuating ß-catenin target gene expression.

Protective effects and mechanisms of omega-3 polyunsaturated fatty acid on intestinal injury and macrophage polarization in peritoneal dialysis rats.

AIM: This study was conducted to investigate the chronic injury of peritoneal glucose injection on the peritoneum and intestine and the protective effects of omega-3 polyunsaturated fatty acid (ω-3PUFA) during peritoneal dialysis (PD). METHODS: Peritoneal dialysis animal models were established by intraperitoneal injection of 4.25% glucose for 28 days. Protein expression in ileum and peritoneum was measured by immunofloresence and immunohistochemistry. Protein expression in macrophages was measured by Western blot. Fibrosis was analyzed by Masson staining. RESULTS: Peritoneal dialysis significantly increased the structural injury and decreased junction-related protein ZO-1 and occludin expression in ileum, the expression of proteins relating to the activation of M2 (Erg2, IRF4), but not M1 (CD38, IRF5) macrophages. PD significantly increased the expression of TGF-ß1, VEGF and ALK5 protein in peritoneal tissues. PD significantly increased fibrosis (Masson staining) and the expression of fibroblast marker α-SMA in peritoneal tissues. Injection of macrophage clean reagent and ω-3PUFA significantly inhibited M2 activation, and decreased Masson staining, α-SMA, TGF-ß1, VEGF and ALK5 protein expression in peritoneal tissues in PD treated rats. ω-3PUFA injection significantly decreased PD-induced injury in ileum and normalized the expression of ZO-1 and occludin in the ileum of PD rats. CONCLUSION: Omega-3 fatty acids can provide a protective role on PD-induced peritoneal fibrosis and injury of the intestine.

Disulfide-bond A oxidoreductase-like protein protects against ectopic fat deposition and lipid-related kidney damage in diabetic nephropathy.

Ectopic fat deposition (EFD) in the kidney has been shown to play a causal role in diabetic nephropathy; however, the mechanism underlying EFD remains elusive. By transcriptome analysis, we found decreased expression levels of disulfide-bond A oxidoreductase-like protein (DsbA-L) in the kidneys of diabetic mice (induced by high-fat diet plus Streptozotocin) compared with control mice. Increased expression of adipocyte differentiation-related protein and abnormal levels of collagen I, fibronectin, and phosphorylated 5'AMP-activated kinase (p-AMPK), adipose triglyceride lipase (p-ATGL), and HMG-CoA reductase (p-HMGCR) were also observed in diabetic mice. These alterations were accompanied by deposition of lipid droplets in the kidney, and were more pronounced in diabetic DsbA-L knockout mice. In vitro, overexpression of DsbA-L ameliorated high glucose-induced intracellular lipid droplet deposition in a human proximal tubular cell line, and DsbA-L siRNA aggravated lipid droplet deposition and reduced the levels of p-AMPK, p-ATGL, and p-HMGCR. High glucose and palmitic acid treatment enhanced the expression of interleukin-1ß and interleukin-18; these enhancements were further increased after treatment with DsbA-L siRNA but alleviated by co-treatment with an AMPK activator. In kidney biopsy tissue from patients with diabetic nephropathy, DsbA-L expression was negatively correlated with EFD and tubular damage. Collectively, these results suggest that DsbA-L has a protective role against EFD and lipid-related kidney damage in diabetic nephropathy. Activation of the AMPK pathway is a potential mechanism underlying DsbA-L action in the kidney.

MicroRNA-302c modulates peritoneal dialysis-associated fibrosis by targeting connective tissue growth factor.

Long-term peritoneal dialysis (PD) can lead to the induction of mesothelial/epithelial-mesenchymal transition (MMT/EMT) and fibrosis; these effects eventually result in ultrafiltration failure and the discontinuation of PD. MicroRNA-302c (miR-302c) is believed to be involved in regulating tumour cell growth and metastasis by suppressing MMT, but the effect of miR-302c on MMT in the context of PD is unknown. MiR-302c levels were measured in mesothelial cells isolated from the PD effluents of continuous ambulatory peritoneal dialysis patients. After miR-302c overexpression using lentivirus, human peritoneal mesothelial cell line (HMrSV5) and PD mouse peritoneum were treated with TGF-ß1 or high glucose peritoneal dialysate respectively. MiR-302c expression level and MMT-related factors alteration were observed. In addition, fibrosis of PD mouse peritoneum was alleviated by miR-302c overexpression. Furthermore, the expression of connective tissue growth factor (CTGF) was negatively related by miR-302c, and LV-miR-302c reversed the up-regulation of CTGF induced by TGF-ß1. These data suggest that there is a novel TGF-ß1/miR-302c/CTGF pathway that plays a significant role in the process of MMT and fibrosis during PD. MiR-302c might be a potential biomarker for peritoneal fibrosis and a novel therapeutic target for protection against peritoneal fibrosis in PD patients.

Ectopic lipid accumulation: potential role in tubular injury and inflammation in diabetic kidney disease.

Emerging studies suggest that lipid accumulates in the kidneys during diabetic kidney disease (DKD). However, the correlation between ectopic lipid accumulation with tubular damage has not been thoroughly elucidated to date. Using Oil Red staining, lipid accumulation was observed in the kidneys of type 2 DKD patients (classes II-III) and db/db mice compared with the control and was predominantly located in the proximal tubular compartment. Immunohistochemistry (IHC) staining showed that the intensity of adipose differentiation related protein (ADRP) and sterol regulatory element binding protein-1 (SREBP-1) was clearly up-regulated, which was positively correlated with the tubulointerstitial damage score and inflammation. Furthermore, the urine ADRP content significantly increased in DKD patients compared with the control, which positively correlated with abnormal lipid metabolism, serum creatinine, urine N-acetyl-ß-glucosaminidase (NAG), albumin excretion (albumin-to-creatinine ratio (ACR)), and tumor necrosis factor-α (TNF-α) expression. However, there was no significant difference observed in plasma ADRP levels. In addition, the expression of SREBP-1 protein was dramatically increased in peripheral blood mononuclear cells (PBMCs) isolated from DKD patients, which was also tightly correlated with urine NAG, ACR, and TNF-α levels. In vitro studies demonstrated increased ADRP and SREBP-1 expression accompanied by lipid accumulation in HK-2 cells cultured in high glucose (HG). HG induced high levels of TNF-α expression, which was partially blocked by transfection of ADRP siRNA or SREBP-1 siRNA. These data indicated that ADRP and SREBP-1 are crucial factors that mediate lipid accumulation with tubular damage and inflammation in DKD, and ectopic lipid accumulation may serve as a novel therapeutic target for amelioration of tubular injury in DKD.

The Role of TLR4 on PGC-1α-Mediated Oxidative Stress in Tubular Cell in Diabetic Kidney Disease.

The role and precise mechanism of TLR4 in mitochondria-related oxidative damage and apoptosis of renal tubules in diabetic kidney disease (DKD) remain unclear. We examined the expression of TLR4 in renal biopsy tissues. Db/db diabetic mice and HK-2 cells cultured under high glucose (HG) were used as in vivo and vitro models. Real-time RT-PCR, Western blot, and immunohistochemistry were performed to examine the mRNA and protein levels of TLR4, NF-κΒ, PGC-1α, cytochrome C, and cleaved caspase-3. ATP level, activity of electron transport chain complex III, and antioxidant enzymes were investigated for mitochondrial function. Electron microscopy (EM) and MitoTracker Red CMXRos were used for mitochondrial morphology alteration. DHE staining and TUNEL assay were detected for ROS accumulation and apoptosis. PGC-1α plasmids were used for the overexpression of PGC-1α in HK-2. TAK242 and parthenolide were used as TLR4 and NF-κB blockers, respectively. Results showed that TLR4 was extensively expressed in the renal tubules of DKD patients and db/db diabetic mice, which was positively related to the tubular interstitial damage score and urinary ß-NAG levels. In diabetic mice, inhibition of TLR4 could reverse the decreased expression of PGC-1α, increased expression of cytochrome C and cleaved caspase-3, mitochondrial dysfunction and deformation, increased accumulation of ROS, and activation of tubular cell apoptosis. In vitro, inhibition of TLR4 or NF-κB showed consistent results. PGC-1α overexpression could reverse the mitochondrial dysfunction, increased cleaved caspase-3, and apoptosis in HK-2 cells treated with HG. Data indicated that the TLR4/NF-κB signaling pathway might be the upstream pathway of PGC-1α and promote the tubular damage of DKD by modulating the mitochondria-related oxidative damage and apoptosis.