Objective To investigate the clinical and pathological characteristics of light chain proximal tubulopathy (LCPT). Methods Nine patients with LCPT diagnosed by renal biopsy in Peking University First Hospital from January 1, 2011 to September 30, 2016 were enrolled, and their clinical findings and pathological features were reviewed. Immunofluorescence (IF) of light chains (κ, λ) on paraffin sections after protease digestion and immunogold labeling of light chains (κ, λ) on ultrathin sections were performed in some cases. Results The main clinical manifestation of the nine patients was proteinuria of small molecules, with acute or chronic renal insufficiency, and six of them led to partial or complete Fanconi syndrome (FS). The hematologic diseases included 3 cases of multiple myeloma and 6 cases of monoclonal gammopathy of renal significance (MGRS). Pathological examination of renal biopsy showed two types: crystalline and noncrystalline LCPT. Seven cases of crystalline LCPT were stained for κ light chain, the proximal tubular epithelial cytoplasm exhibited fine granular vacuolation, with needle-shaped crystals and clear clefts by light microscopy, the intracytoplasmic inclusions of various shapes including rhomboidal, rectangular and rod-shaped crystals were identified by electron microscopy. Two cases of noncrystalline LCPT were stained for λ light chain, the prominent argyrophilic granules in cytoplasm of proximal tubular epithelia were observed by light microscopy, and intracytoplasmic large and irregular shaped phagolysosomes were found by electron microscopy, cast nephropathy were coexisted in these 2 cases, the additional light chain deposition disease were confirmed in one of them by electron microscopy and IF. All cases had monotypic staining of light chains in cytoplasm of proximal tubules by IF on frozen tissue and paraffin sections after protease digestion, with the latter method being more sensitive than the routine IF. The immunogold labeling showed specific monotypic labeling of κ and λ light chain on intracytoplasmic crystals and phagolysosomes respectively by immunoelectron microscopy. Conclusions LCPT is a rarely reported entity that manifested as acquired Fanconi syndrome and dysfunction of proximal tubules clinically. Pathologically it is divided into two types: crystalline and noncrystalline LCPT, with more prevalent of κ light chain related crystalline type, noncrystalline LCPT is mostly λ type, and is easily coexisted with cast nephropathy. The IF and immunoelectron microscopy of light chains(κ, λ) and ultrastructural examination by electron microscopy are important methods for the diagnosis of LCPT.

Objective To compare different equations for estimated glomerular filtration rate (eGFR) in patients with chronic kidney disease (CKD). Methods Hospitalized patients with CKD from the nephrology department of the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital) were recruited between December 2014 and May 2015. The calculations of eGFR and 24 h creatinine clearance rate (Ccr) were accomplished in three days after admission. The eGFRs were calculated separately using the 24 h creatinine clearance rate adjusted by the standard body surface area (Ccr_BSA), Cockcroft-Gault equation adjusted by the standard body surface area (eCcr_BSA), CKD-EPI creatinine equation (EPI_Cr), CKD-EPI cystatin C equation (EPI_CysC), CKD-EPI creatinine-cystatin C equation (EPI_Cr_CysC), simplified MDRD (MDRD) and China MDRD equations. The EPI_Cr_CysC equation was used as the standard and the precision and accuracy of the other six equations were compared and analyzed. Results A total of 403 CKD participants were enrolled in the study, with 228 male patients and a mean age of (54.9±18.4) years. The main primary diseases were chronic glomerulonephritis (43.7%) and diabetic nephropathy (13.2%). The median concentration of serum creatinine and cystatin C were 117.5 (69.7, 242.4) μmol/L and 1.80 (1.13, 3.31) mg/L, respectively. The median values of Ccr_BSA, eCcr_BSA, MDRD, China MDRD, EPI_Cr, EPI_CysC and EPI_Cr_CysC equations were 50.8 (21.1, 96.2), 51.9 (23.3, 93.2), 53.6 (23.0, 97.4), 52.2 (22.4, 94.1), 53.2 (22.1, 97.3), 35.1 (15.4, 67.0) and 49.1 (22.8, 82.3) ml?min-1?(1.73 m2)-1, respectively. There was well agreement among MDRD, China MDRD and EPI_Cr equations, while there were large differences between equations derived from CysC (EPI_Cr_CysC and EPI_CysC) and equations derived only from creatinine (EPI_Cr, MDRD, China MDRD, eCcr_BSA, Ccr_BSA equations). Compared with EPI_Cr_CysC equation (the reference equation), EPI_Cr equation showed the highest accuracy [percentage of other eGFR equation calculations that were ＞30% of the reference equation calculations (1-P30), 30.8%] while Ccr_BSA equation showed the lowest (1-P30, 42.4%). EPI_CysC equation showed the highest precision [inter-quartile range (IQR) of the difference, 11.7 ml?min-1?(1.73 m2)-1] while Ccr_BSA equation showed the lowest [IQR of the difference, 22.8 ml?min-1?(1.73 m2)-1]. Conclusions The agreement among equations derived only from creatinine is better; while it exhibits some differences between equations with cystatin C and equations derived only from creatinine. The accuracy of EPI_Cr equation is second only to EPI_Cr_CysC equation and it is currently the most suitable eGFR equation for clinical popularization of renal glomerular function assessment.

Objective To explore the relationship between intermedin (IMD) and renal interstitial capillary loss in IgA nephropathy (IgAN) patients. Methods Renal biopsy specimens collected from primary IgAN patients in our hospital (n=80) were compared with normal renal tissues. Expressions of IMD, CD31 and VE-cadherin were examined by immunohistochemical method, and plasma concentrations of IMD and TGF-β1 in 37 cases from the 80 cases were compared. The relationship between IMD and renal interstitial capillary loss in IgAN patients was analyzed. Results IMD and VE-cadherin in renal tubule interstitium expressions increased compared to the control group at the early stage of IgAN (P＜0.05). CD31 expression remained unchanged at the early stage of pathological lesions of IgAN (P＞0.05), but decreased at the early stage of clinical stage of IgAN compared to the control (P＜0.05). Expressions of IMD, CD31 and VE-cadherin were reducing as the disease progressed, and the correlations of CD31 and VE-cadherin (r=0.517, P＜0.01), IMD and CD31 (r=0.655, P＜0.01) or IMD and VE-cadherin (r=0.576, P＜0.01) were positive. Plasma concentrations of IMD and TGF-β1 were higher than those of the control group at the early stage of IgAN (P＜0.05), and the changes of IMD and TGF-β1were correlated positively (r=0.582, P＜0.01). Conclusion Compared with the control group, expression of IMD in kidney tubules increases at the early stage of IgAN, and change of IMD correlates closely with the renal interstitial capillary loss. Plasma concentrations of IMD and TGF-β1 increase compared with the control group at the early stage of IgAN, and the changes of IMD and TGF-β1 are related closely.

Objective To explore the association of serum soluble Klotho (sKlotho) with nonfatal cardiovascular disease (CVD) and all-cause/CVD mortality in maintenance hemodialysis (MHD) patients. Methods A total of 132 MHD patients admitted during October 2011 were enrolled. Serum sKlotho was measured by ELISA. Demographic data, including age, gender and comorbid conditions, were obtained from their medical histories, and parameters including calcium, phosphorus and albumin were assessed. The occurrence time of nonfatal CVD and all-cause mortality were recorded during the 60 months follow-up. MHD patients were categorized into four groups according to the quartiles of sKlotho: group Ⅰ (sKlotho＜361.34 ng/L), group Ⅱ (361.34 ng/L≤sKlotho＜398.81 ng/L), group Ⅲ (398.81 ng/L≤sKlotho＜445.99 ng/L) and group Ⅳ (sKlotho≥445.99 ng/L). Spearman correlation analysis and binary Logistic regression analysis were used to test the association between sKlotho and nonfatal CVD events. The impacts of sKlotho on all-cause mortality and CVD mortality were assessed by Kaplan-Meier method with log-rank test. Cox regression model was applied to evaluate the effect of sKlotho on MHD patients outcomes. Results All 132 MHD patients had sKlotho ranging from 304.02 ng/L to 550.62 ng/L. And 87 patients suffered from nonfatal CVD, with 192 episodes of nonfatal CVD during the follow-up period. The sKlotho had negative correlations with coronary artery disease (r=-0.286, P=0.001), congestive heart failure (r=-0.190, P=0.029), cerebrovascular accident (r=-0.240, P=0.006) and peripheral arterial occlusion (r=-0.243, P=0.005). The sKlotho were risk factors of coronary artery disease (OR=0.989, P=0.023) and peripheral artery occlusion (OR=0.988, P=0.046). 35 patients died in the follow-up period, including 27 death from CVD. The all-cause mortality and CVD mortality rates were significantly different among four groups (P=0.036, P=0.047). Survival rates of all-cause death and CVD death varied among four groups (χ2=8.076, P=0.044; χ2=7.866, P=0.049). Patients in group Ⅳhad higher survival rates of all-cause death and CVD death than those in group Ⅰ and group Ⅱ (all P＜0.05). Multivariate Cox regression analyses revealed diabetes and age were independent risk factors for all-cause mortality and CVD mortality (all P＜0.05), but sKlotho was not associated with the poor prognosis (HR=0.996, P=0.256; HR=0.996, P=0.287). Conclusions Patients with lower sKlotho have worse nonfatal CVD ratio, especially coronary artery disease and peripheral arterial occlusion. Reduced serum sKlotho is associated with all-cause and CVD mortality, but sKlotho is still not a predictive indicator of prognosis of MHD patients.

Objective To investigate the effects of hyperglycemia on ubiquitination and endoplasmic reticulum stress in renal intrinsic cells (podocytes and proximal tubular epithelial cells) and its role in pathogenesis of diabetic nephropathy. Methods Diabetic mice were induced by streptozotocin injection. After 16 weeks of hyperglycemia, immunofluorescence was used to detect the expressions of ubiquitination and glucose-regulating protein 94 (GRP94) in renal cortex and medulla area of kidney sections. Primary mouse podocyte and proximal tubular epithelial cells were isolated by flow cytometry, and exposed to 30 mmol/L glucose for indicated time (1 d, 3 d and 7 d). Their ubiquitination and GRP94 expressions were evaluated by Western blotting. Results Diabetic mice presented microalbuminuria and slightly widened mesangium was found in glomerular area. Ubiquitinated proteins, mainly localized in podocytes and tubular epithelial cells, exhibited an apparently higher expression in diabetic mice than control mice (all P＜0.05). Hyperglycemia promoted the ubiquitination in a time-dependent manner. Compared with their normal cells, primary mouse podocyte and primary tubular epithilial cells treated with high glucose for 3 d and 7 d showed increased ubiquitinated protein (all P＜0.05). GRP94 was interspersed in podocytes and proximal tubular epithelial cells. Expression of GRP94 was significantly increased in glomerular area of diabetic mice and podocyte with 3 and 7 day-high glucose as compared with those in their control groups (all P＜0.05). GRP94 expression had no significant change in tubular area and tubular epithilial cells treated with high glucose. Conclusions Hyperglycemia may lead to accumulation of ubiquitinated proteins in intrinsic kidney cells. The imbalance of protein homeostasis in podocyte may contribute to podocyte injury during the onset of diabetic nephropathy.

Objective To observe the expression of microRNA-148b (miR-148b) induced by high glucose in rat mesangial cells, and to explore its effect on its target gene AMP-activated protein kinase α1 (AMPKα1) and extracellular matrix excretion. Methods Rat mesangial cells were divided into 3 groups: normal glucose (NG, 5.5 mmol/L glucose) group, hypertonic (MA, 5.5 mmol/L glucose+ 19.5 mmol/L mannitol) group and high-glucose (HG, 25.0 mmol/L glucose) group. MiR-148b expression was detected by real time PCR. Then miR-148b inhibitor was transfected to rat mesangial cells. Their protein expressions of AMPKα1, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), fibronectin (FN) and collagen Ⅳ were detected by Western blotting. The expression of AMPKα1 mRNA was detected by real time PCR. The expression of collagen Ⅳ was also detected by immunofluorescence. Results Compared with NG group, HG group showed up-regulated miR-148b expression, down-regulated AMPKα1 mRNA and protein expressions, and up-regulated CHOP, GRP78, collagen Ⅳ and FN expressions (all P＜0.05). HG-induced mesangial cells with miR-148b inhibitor had up-regulated AMPKα1 mRNA and protein expressions, and down-regulated CHOP, GRP78, collagen Ⅳ, FN expressions as compared with HG-induced cells without miR-148b inhibitor (all P＜0.05). Conclusions HG can up-regulate miR-148b expression and down-regulate AMPKα1 expression in rat mesangial cells, then activate endoplasmic reticulum stress to induce extracellular matrix excretion. MiR-148b inhibitor up-regulates AMPKα1 expression, inhibits endoplasmic reticulum stress and reduces extracellular matrix excretion.

Objective To investigate the effect of cyclosporine A (CsA) on autophagy-lysosomal pathway in tubular epithelial cells. Methods Human renal tubular epithelial cell line (HK-2 cell) was treated with different concentrations (3, 5 and 10 μmol/L) of CsA for 24 h. Then the viability and apoptosis of cells were measured by MTT assay or AnnexinV-PI staining followed by flow cytometry analysis, respectively. Autophagy-related protein LC3-Ⅱ and p62 were detected by immunofluorescence assay. Autophagic flux was analyzed in HK-2 cells transfected with a tandem mRFP-GFP fluorescent-tagged LC3 (tfLC3) plasmid by laser confocal microscope. The lysosomal degradation was evaluated by DQ-ovalbumin staining followed by flow cytometry analysis. Results The viability of HK-2 cells was significantly decreased with CsA stimulation when compared with control group (P＜0.01), but the number of apoptotic cells was markedly increased by CsA treatment (P＜0.05). Compared with the control group, different doses of CsA dramatically increased the expressions of LC3-Ⅱ(P＜0.01) and p62 (P＜0.05) in HK-2 cells. Moreover, HK-2 cells treated with CsA displayed a significant increase in autophagosomes but a marked decrease in autolysosomes. In HK-2 cells, exposured to CsA caused a decrease in lysosomal degradation by DQ-ovalbumin staining when compared with control group (P＜0.01). Conclusion Blockade of autophagy via disrupting lysosome degradation may represent a novel mechanism of CsA-induced tubular epithelial cells injury.

Objective To explore the role and mechanism of microRNA-15b in the regulation of epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs). Methods PCR assay was used to determine the expression of microRNA-15b in the HMrSV5 induced by 138 mmol/L high glucose for 24 h. MicroRNA-15b mimic or inhibitor was transfected into human peritoneal mesothelial cells (HMrSV5) to over-express or down-regulate microRNA-15b. The cells were then incubated with 138 mmol/L high glucose for 24 h, and the expressions of E-cadherin(E-Cad), Vimentin(VIM), Fibronectin(FN) and Smad7 were detected by real-time PCR and Western blotting respectively. Results microRNA-15b in the HMrSV5 cells was over-expressed and down-regulated. Increased level of microRNA-15b was obtained in HMrSV5 cells treated with high glucose. In vitro, high glucose led to the up-regulation of vimentin as well as fibronectin and the down-regulation of E-cadherin in HMrSV5 cells (all P＜0.05), which indicated EMT and fibrosis. Suppression of microRNA-15b by transfection with microRNA-15b inhibitor partially reversed the EMT and fibrosis changes (P＜0.05), while over-expression of microRNA-15b by transfection with microRNA-15b mimic obviously enhanced the EMT and fibrosis changes (P＜0.05). Conclusions MicroRNA-15b mediates high glucose induced EMT in human peritoneal mesothelial cells by the inhibition of Smad7 possibly. MicroRNA-15b maybe a new target for the prevention and treatment of peritoneal fibrosis during peritoneal dialysis (PD).

Objective To investigate the role of OMA1 in acute kidney injury (AKI) induced by lipopolysaccharide (LPS). Methods OMA1 wild-type and knocked out mice (8 week old) were injected with 10 mg/kg body weight of LPS. The model was confirmed by testing mouse serum creatinine and blood urea nitrogen. The apoptosis in mouse kidney cortex was examined by TUNEL staining and cleaved caspase 3. In vitro, in humam kidney proximal tubular cells (HK2) were knocked down OMA1 by transfecting OMA1 shRNA, with the scramble shRNA being used as negative control of transfection. HK2 cells were cultured with 5 μg/ml of LPS for 24 hours to induce apoptosis. DAPI staining of cells and caspase-3 activity were applied to test apoptosis. The images of mitochondria in cells were obtained by transfection of mito-green plasmid and OMA1 shRNA. Western blotting was used to exam the OMA1 and Cytochrome C expressions. Results Compared with OMA1 KO mice, LPS induced more severe AKI of WT mice with higher Scr [(97.2±26.5) μmol/L vs (53.0±17.7) μmol/L, P＜0.05] and BUN [(43.3±13.7) mmol/L vs (29.7±7.7) mmol/L, P＜0.05]. Moreover, there were more apoptosis cells in kidney cortex in WT mice than in OMA1 KO mice [(75.4± 26.1)/mm2 vs (38.3± 14.4)/mm2, P＜0.05]. About 46% of OMA1 expressions in HK2 cells were inhibited by OMA1 shRNA transfection (P＜0.05). Further, OMA1 shRNA cells with LPS stimulation had decreased mitochondria fragmentation [(29.8±10.9)% vs (43.2±6.8)% , P＜0.05], Cytochrome C release [(37.0±12.3)% vs (76.0±26.2)%, P＜0.05], and cell apoptosis [(13.2±3.9)% vs (25.0±7.1)%, P＜0.05] as compared with control cells. Conclusion Knockdown of OMA1 alleviated septic AKI through inhibition of cell apoptosis, mitochondria fragmentation, and Cytochrome C release.