Immunoglobulin A nephropathy (IgAN) is the most common glomerulonephritis worldwide. The clinical spectrum of IgAN varies from minor urinary abnormalities to rapidly progressive renal failure. Evaluation of the disease by repeated renal biopsy is not practical due to its invasive procedure. Urinary sediment miRNAs promise to serve as non-invasive biomarkers to assess kidney injury of IgAN.

The miRNAs in urinary sediment are easy to obtain, which provides a new approach to searching for non-invasive biomarkers of IgA nephropathy (IgAN). Compared with normal controls (n = 3), 214 different miRNAs in the urinary sediment of IgAN (n = 9) were found by miRNA chip assay. By quantitative PCR analysis, miR-25-3p, miR-144-3p and miR-486-5p were confirmed to be significantly higher in IgAN (n = 93) than in the normal group (n = 82) or disease control (n = 40). These three miRNAs had good specificity and sensitivity for the diagnosis of IgAN by receiver operating characteristic curve analysis, in which the AUC value of miR-486-5p was the largest at 0.935. Urinary sediment miR-25-3p, miR-144-3p and miR-486-5p were demonstrated to be mainly derived from urinary erythrocytes, which were separated by CD235a magnetic beads. The increased expression of urinary erythrocyte miRNAs in IgAN patients was not associated with those in the blood erythrocytes. In addition, urinary supernatant microvesicles of miR-144-3p and miR-486-5p in the IgAN group were also significantly increased. This study showed that the miR-25-3p, miR-144-3p and miR-486-5p in urinary sediment were mainly derived from urinary erythrocytes, which could be non-invasive candidate biomarkers for IgA nephropathy.

Objective:Clarify the value of different standards hematuria in differential diagnosis of diabetic nephropathy and non-diabetic renal disease in type 2 diabetics. Analysis related factors of hematuria in diabetic nephropathy patients. Methods:Mi-croscopic hematuria was defined as ﹥3,﹥10 red blood cells per high-power field( RBCs/hpf). Glomerular hematuria was diag-nosed as dysmorphic erythrocytes ﹥80%. Analysis their respective value of differential diagnosis. Use multiple logistic regression an-alyses to screen the related factors of hematuria. Results:There were 100 patients in diabetic nephropathy group,200 patients in non-diabetic renal disease group. There were differences between both groups in three kinds of hematuria. Glomerular hematuria had the maximum value of specificity(94%),positive predictive value(90. 16%)and AUC. Nephrotic syndrome was an independent risk factor for hematuria(OR=2. 411,P=0. 04). Conclusion:Glomerular hematuria were superior to microscopic hematuria for predic-ting non-diabetic renal disease in type 2 diabetics. Patients with nephrotic syndrome were more likely to appear hematuria.

Aims/Introduction: There are sparse and limited studies on erythrocyte morphology in renal biopsy identifying nephropathic patients among type 2 diabetics. The present study sought to clarify the predictive value of dysmorphic erythrocytes in type 2 diabetics with non-diabetic renal disease and influences on hematuria.Materials and Methods: We examined 198 patients with type 2 diabetes who underwent kidney biopsies between 2012 and 2013. Hematuria was defined as >3 or >10 red blood cells per high-power field (RBCs/hpf) in urine sediment. If >80% of the erythrocytes were dysmorphic, glomerular hematuria was diagnosed. Clinical findings and predictive value of dysmorphic erythrocytes were compared between patients with hematuria (n = 19) and those without (n = 61). The potential risk factors for hematuria among diabetic nephropathy patients were also screened.Results: There was a statistically significant difference between the diabetic nephropathy group and the non-diabetic renal disease group (6.6 vs 16.8%; P = 0.04) when the demarcation point of hematuria was 10 RBCs/hpf. When the definition of hematuria was based on an examination of urinary erythrocyte morphology, a marked difference was seen (3.3 vs 24.8%; P < 0.001). Glomerular hematuria showed high specificity and a positive predictive value (0.97 and 0.94, respectively) in non-diabetic renal disease. A multivariate analysis showed that nephrotic syndrome was significantly associated with hematuria (odds ratio 3.636; P = 0.034).Conclusions: Dysmorphic erythrocytes were superior to hematuria for indicating non-diabetic renal disease in type 2 diabetics. Nephrotic syndrome was an independent risk factor for hematuria.

Background: Angiogenesis is a critical component of many pathological conditions, and microRNAs (miRNAs) are indispensable in angiogenesis. It is unclear whether miRNAs regulate angiogenesis in the presence of high concentrations of uric acid (HUA), and the underlying mechanisms remain unknown.Methods and Results: It was found that HUA inhibited the angiogenic ability of endothelial cells. miRNA expression profiling was conducted using microarray assays in HUA-stimulated endothelial cells. Eighteen differentially expressed miRNAs were subjected to bioinformatic analyses. The results indicated that miR-92a was negatively regulated and was closely related to angiogenesis. Furthermore, the effects of miR-92a on HUA-stimulated endothelial cell angiogenesis and the underlying mechanisms were investigated in dual-luciferase reporter assays, electrophoretic mobility shift assays, immunoblot assays, and tube formation assays. It was determined that Kruppell-like factor 2 (KLF2) is a target gene of miR-92a, and KLF2 binds the vascular endothelial growth factor-A (VEGFA) promoter to inhibit its expression. miR-92a and VEGFA overexpression or KLF2 downregulation alleviates the HUA-mediated inhibition of angiogenesis in endothelial cells in vitro.Conclusions: This study reported that there is a novel pathway regulating angiogenesis under HUA conditions. In the presence of HUA, miR-92a downregulation increased KLF2 expression, subsequently inhibiting VEGFA, which resulted in decreased angiogenesis. Thus, this study reports a possible mechanism for cardiovascular injury caused by hyperuricemia and suggests that the miR-92a-KLF2-VEGFA axis may be a target for hyperuricemia treatment.

The current study aimed to investigate the association between telomere length in peripheral blood leukocytes and kidney function in various age groups of a healthy population. A total of 139 healthy individuals were divided into five groups according to their age: 35‑44, 45‑54, 55‑64, 65‑74 and >75 years old. Peripheral blood leukocytes were obtained and the telomere restriction fragment (TRF) length was assayed using a digoxigenin‑labeled hybridization probe in Southern blot assays. Laboratory assays of kidney function were also performed. A correlation was observed between TRF length and age (r=‑0.314, P<0.001), with the telomere length of the individuals >75 years group being significantly shorter than the telomere length of the 35‑44, 45‑54 and 55‑64 years age groups (P<0.05). By contrast, the TRF length for males versus females did not differ for any of the age groups, while a correlation was observed between TRF length and serum levels of cystatin C (r=‑0.195, P<0.05). There was also a correlation between TRF length and glomerular filtration rate (r=‑0.184, P<0.05). The current study demonstrated that in this cohort, leukocyte telomere length reduced with age and was correlated with serum levels of cystatin C and glomerular filtration rate. Therefore, TRF length is associated with kidney function and may serve as a marker of aging.

The podocyte functions as a glomerular filtration barrier. Autophagy of postmitotic cells is an important protective mechanism that is essential for maintaining the homeostasis of podocytes. Exploring an in vivo rat model of passive Heymann nephritis and an in vitro model of puromycin amino nucleotide (PAN)-cultured podocytes, we examined the specific mechanisms underlying changing autophagy levels and podocyte injury. In the passive Heymann nephritis model rats, the mammalian target-of-rapamycin (mTOR) levels were upregulated in injured podocytes while autophagy was inhibited. In PAN-treated podocytes, mTOR lowered the level of autophagy through the mTOR-ULK1 pathway resulting in damaged podocytes. Rapamycin treatment of these cells reduced podocyte injury by raising the levels of autophagy. These in vivo and in vitro experiments demonstrate that podocyte injury is associated with changes in autophagy levels, and that rapamycin can reduce podocyte injury by increasing autophagy levels via inhibition of the mTOR-ULK1 pathway. These results provide an important theoretical basis for future treatment of diseases involving podocyte injury.

OBJECTIVE To investigate high-concentration uric acid-induced endothelial dysfunction mediated by miR-155. METHODS Human umbilical vein endothelial cells (HUVECs) were incubated with 600 µmol/L uric acid for 24 and 48 h, and eNOS expression and NO content in the cell culture were measured. The target genes of miR-155 were predicted using on-line analysis software and validated by dual-luciferase system. Real-time PCR was used to detect the expression of miR-155 in endothelial cells incubated with high-concentration uric acid. The effect of miR-155 on endothelial dysfunction was assessed by transfection of its inhibitor into HUVECs. RESULTS The expression of eNOS and NO secretion decreased obviously in HUVECs incubated with 600 µmol/L uric acid. MicroRNA on-line analysis software and dual luciferase reporter experiments suggested that the level of eNOS translation was directly regulated by miR-155. The expression of miR-155 in endothelial cells was upregulated after stimulation with high-concentration uric acid, and was inhibited by transfection of miR-155 inhibitor. Expression of eNOS and secretion of NO were elevated in endothelial cells transfected with miR-155 inhibitor after incubation with high-concentration uric acid. CONCLUSION High-concentration uric acid can down-regulate eNOS expression via miR-155 to induce endothelial dysfunction.

Membranous nephropathy is a common cause of nephrotic syndrome in adults. Although many mechanisms have been proposed, whole proteomic research is still lacking. We analyzed the passive Heymann nephritis animal model using label-free quantitative proteome technology. Results showed 160 differential proteins between control and PHN model groups at days 14 and 21. The expression level of endoplasmic reticulum stress (ERS)-associated protein GRP78 and GRP94 protein was up-regulated on day 14 or 21, which was confirmed by Western blotting. The results also showed that the autophagy marker LC3 was up-regulated in the models. Furthermore, we used tunicamycin to induce ERS of podocytes in vitro to investigate the mechanism. Results of Western blotting revealed that the expression of GRP78, GRP94, and LC3 was up-regulated, while that of the cytoskeletal protein tubulin-β was down-regulated, and immunofluorescence displayed disordered distribution of tubulin-β. These suggest that ERS plays an important role in podocyte damage. Autophagy can repair the cytoskeleton damage caused by ERS as a protective mechanism. This provides an important basis for a thorough understanding of the mechanism of podocyte damage and the pathogenesis of membranous nephropathy.

Objective:To clone a full-length cDNA gene of rat high-affinity sodium-dependent dicarboxylate transporter (SDCT2) and to determine intracellular localization of SDCT2 protein in renal tubular epithelial cells. Methods:The total RNA was extracted from kidney tissue of rat. Then full-length cDNA gene of SDCT2 was amplified by RT-PCR technique using 8-peptide FLAG labeled primers,sequenced and inserted into eukaryotic expression vector to construct SDCT2 protein expression vector. The expression vector was then transfected into swine renal tubular epithelial cell LLC-PK1 and the subcellular localization of this protein was observed using laser confocal microscope. Results:The 2 kb of full-length cDNA gene of SDCT2 was successfully amplified. Western blot analysis revealed that SDCT2 protein has been correctly expressed in LLC-PK1 cells. Confocal analysis showed that the normal SDCT2 protein was mainly localized at the cellular plasma membrane,consistent with the predicting result obtained by bioinformatics. To explore the effect of different ligation temperature on the ligation efficiency of recombinat DNA,the transformation efficiency in 3 different ligation temperatures was observed. The results showed that the ligation efficiency of temperature circulation method was obviously higher than those of two regular ligation temperature methods. Conclusion: Rat full-length SDCT2 gene was successfully cloned and SDCT2 protein is mainly expressed at the cellular membrane of renal tubular epithelial cells.

AIM: To compare the cDNA sequences of ASBT (apical Na+/bile acid cotransporter) gene cloned from human kidney and intestine tissues,and to determine subcellular localization of ASBT in renal tubular epithelial cells and expression site of ASBT in human kidney tissue.METHODS: The total RNA was extracted from human kidney and intestine tissues.The full-length cDNA gene of ASBT was amplified by RT-PCR technique using primers with 8-peptide FLAG tag,sequenced and inserted into eukaryotic expression vector to construct ASBT protein expression vector,which was then transfected into swine renal tubular epithelial cell line LLC-PK1.The subcellular localization of ASBT protein was determined by immunofluorescence staining and laser confocal microscope.Immunohistochemical analysis was used to observe expression position of ASBT in kidney tissue.RESULTS: The results revealed that the sequence of kidney ASBT cDNA gene was identical to that of human intestine ASBT gene.Western blotting analysis indicated that ASBT protein was correctly expressed in LLC-PK1 cells.Confocal scanning analysis showed that ASBT protein was mainly localized at the cellular plasma membrane,consistent with the predicting result obtained by bioinformatics.The results of immunohistochemistry revealed that ASBT was expressed at brush border membrane of proximal renal tubular cells,but not expressed in distal tubule and renal interstitium.CONCLUSION: The gene sequence of kidney ASBT is the same as that of intestine ASBT and ASBT protein is expressed at the lumen(apical) membrane of proximal renal tubular epithelial cells.

Objective:To study the clinical and pathological characteristics of IgA nephropathy patients with nephrotic syndrome and the clinical risk factors associated with the renal pathologic lesions.Method:118 patients with nephrotic syndrome were collected from the inpatients who were diagnosed as IgA nephropathy by renal biopsy.The clinical and pathological features of patients were analyzed.And the clinical index of different severity of renal pathologic lesions was compared;the relationship between renal pathologic lesions and clinical factors were studied;the predicted factors were analyzed by step-wise multiple variance regression analysis.Results:The incidence of hypertension,renal failure,full field of microscopic hematuria was lower in group A than those in group B(11.8% vs 63.1%,15% vs 41.7%,14.7% vs 50%,respectively.P0.01).But the incidence of urinary protein(UP)≥6 g/24 h was higher in group A than that in group B(58.8% vs 32.1%,P0.01).Mean arterial pressure(MAP),serum creatine(Scr)were lower in group A than those in group B(P0.01),urinary protein excretion,hemoglobin were higher in group A than those in group B(P0.01).MAP,UP6 g/24 h,microscopic hematuria 5.0×107/L were independent predictors of the severity of renal pathologic lesion in IgA nephropathy patients with nephrotic syndrome(OR=1.048,3.227,6.578,P=0.034,0.047,0.002.respectively.)Hemoglobin was a favorable factor for renal pathologic lesion.Meanwhile,with the decreasing of hemoglobin,the increasing of hypertension and number of microscopic hematuria the renal pathologic lesion are more severe.Conclusion:IgA nephropathy patients with nephrotic syndrome have different clinical manifestations and pathologic lesions;patients with decreased hemoglobin,accompanied by hypertension and mass microscopic hematuria have more severe renal lesions.

High-affinity, sodium-dependent dicarboxylate transporter (NaDC3) is responsible for transport of Krebs cycle intermediates and may involve in regulation of aging and life span. Hydropathy analysis predicts that NaDC3 contains 11 or 12 hydrophobic transmembrane (TM)domains. However, the actual membrane topological structure of NaDC3 remains unknown. In this study, confocal immunofluorescence microscopy and membrane biotinylation of epitope-tagged N and C termini of NaDC3 provide evidence of an extracellular C terminus and an intracellular N terminus, indicating an odd number of transmembrane regions. The position of hydrophilic loops within NaDC3 was identified with antibodies against the loops domains combined with cysteine accessibility methods. A confocal image of membrane localization and transport activity assay of the cysteine insertion mutants show behavior similar to that of wild-type NaDC3 in transfected HEK293 cells, suggesting that these mutants retain a native protein configuration. We find that NaDC3 contains 11 transmembrane helices. The loops 1, 3, 5, 7, and 9 face the extracellular side, and loops 2, 4, 6, and 10 face the cytoplasmic side. A re-entrant loop-like structure between TM8 and TM9 may protrude into the membrane. Our results support the topography of 11 transmembrane domains with an extracellular C terminus and an intracellular N terminus of NaDC3, and for the first time provide experimental evidence for a novel topological model for NaDC3

为了探讨细胞周期抑制因子p19ARF对人二倍体细胞复制性衰老的影响,构建了重组p19ARF真核表达载体,并通过脂质体的介导将p19ARF基因转染到人二倍体成纤维细胞WI-38中过表达,观察其对WI-38细胞衰老的影响.结果发现与对照细胞相比,在p19ARF基因导入后,细胞中p53和p21的表达水平明显上调,细胞传代数减少10～12代,生长速率降低,细胞周期阻滞于G1期,衰老标志物SA-β-gal染色阳性率上升,线粒体膜电位下降,细胞形态呈衰老细胞样变化,这些结果表明p19ARF高表达可促进人二倍体细胞的衰老进程.

To investigate the effect of cell cycle inhibitor p14ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results revealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10–12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.

Type 1 diabetes results from autoimmune destruction of the insulin-producing β-cells of pancreatic islets, of which the capacity for self-replication in the adult is too limited to restore following extensive tissue injury. Tissue inhibitor of metalloproteinase (TIMP)-1 inhibits matrix metalloproteinase activity and regulates proliferation and apoptosis of a variety of cells types, depending on the context. Here, we show that overexpression of human TIMP-1 in pancreatic β-cells of transgenic mice counteracts the cytotoxicity and insulitis induced by multiple low-dose streptozotocin (MLDS). Nontransgenic mice developed severe hyperglycemia, hypoinsulinemia, and insulitis 2 weeks after streptozotocin administration and died within 17 weeks. However, MLDS-treated transgenic mice gradually normalized the metabolic parameters and survived. β-Cell mass increased in parallel as a result of enhancement of β-cell replication. Thus, our results have demonstrated for the first time that overexpression of TIMP-1 in β-cells enhances the replication of pancreatic islets β-cells and counteracts type 1 diabetes, indicating that the TIMP-1 gene may be a potential target to prevent, or even reverse, type 1 diabetes.

目的探讨大鼠低亲和力钠依赖二羧酸转运蛋白(SDCT1)对枸橼酸及草酸的转运特点.方法克隆出大鼠SDCT1全长cDNA基因.应用爪蟾卵母细胞异源性表达SDCT1,并使用双电极电压钳法记录通道电流.通过改变灌流液中枸橼酸、草酸的浓度,以及两种底物转运时钠离子的浓度及pH值,对其特性进行了研究.结果SDCT1对枸橼酸及草酸的转运均为底物浓度及钠离子依赖.pH值变化显著影响2者的转运.但低pH对草酸的转运影响要远大于枸橼酸.结论在临床实践中,虽然升高尿液pH值可以抑制枸橼酸的重吸收,发挥其抑制钙盐沉积的功能.但过度地碱化尿液也会使草酸的重吸收受到抑制,使草酸大量存留在尿中而导致结石.保持适当的尿酸碱度是必要的.

OBJECTIVE:To obtain full-length human urate transporter (hUAT) gene.METHODS:Primers was designed according to the sequence of hUAT reported in Genbank. The target fragments were obtained by reverse transcriptional (RT) PCR from the mRNA extracted from human renal tubular epithelial cell lines (HK-2), followed by cloning into pEGFP-C1 plasmid. The cloned fragment was subjected to restriction mapping and sequence analysis for confirmation.RESULTS AND CONCLUSION:hUAT gene with correct sequence is successfully cloned from HK-2 cells. The restriction maps and sequence analysis of the selected clones were consistent with the sequence reported in Genbank.

Specific anti-hURAT1 rabbit polyclonal antibody was obtained, and both Western-blot and immunohistochemistry showed that hURAT1 was expressed in the human kidney brush border, localized in the apical membrane of the LLC-PK1 cell. hURAT1 protein was a membrane protein located in renal proximal tubule, which could be detected in the apical membrane. The anti-hURAT1 polyclonal antibody could be used for studying the physiological function of hURAT1 and its pathology.

Extracellular matrix (ECM) expansion and mesangial cell (MC) proliferation are prominent features of most types of glomerulosclerosis. A delicate balance between the ECM and MC regulates cell survival. Increasing evidence shows that a loss of ECM components can cause mitochondrial dysfunction and induce cell apoptosis. It is proposed that directly blocking the synthesis of ECM components could lighten ECM accumulation and suppress cell overproliferation status. Fibronectin, one of the predominant adhesive glycoproteins of the mesangial ECM, provides the survival signal for cells. Its accumulation can be observed in most types of glomerulosclerosis. In this study, angiotensin II-induced fibronectin was suppressed by an RNA interference technique. It is interesting that MC slowly underwent apoptosis after infection with a retrovirus that continuously suppressed fibronectin synthesis. It was found that MC apoptosis occurred in a mitochondria-dependent manner mainly as a result of cytochrome c release and downstream caspase-3 and -9 activation. Furthermore, it was demonstrated that fibronectin knockdown affected mitochondrial handling of Ca2+ release from the endoplasmic reticulum. Importantly, blocking the inositol 1,4,5-triphosphate receptor with, 3,4,5-trimethoxybenzoate or decreasing Ca2+ in the ECM with EGTA partially saved the cells from apoptosis. These studies, which explored a new method for simultaneously inhibiting MC proliferation and ECM accumulation, may represent a novel therapeutic approach to glomerulosclerosis.