Objective    To describe and analyze the clinical characters of patients with FRG from 7 Chinese families. Then analyze and identify their mutations in SGLT2 gene, and explore the association of genotype and phenotype.    Methods    Quantitative test for 24-hour urine glucose and other laboratory tests were carried out among 7 probands (14 patients in all) and their family members from 7 pedigrees (totaling 23 subjects). All coding regions, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis.    Results    Five novel mutations in SLC5A2 gene were identified in this investigation, including four missense mutations (A Serine to Glycine at position 335 (c.1003A＞G, p.S335G), a Glutamine to Arginine at position 448 (c.1343A＞G, p.Q448R)，an alanine to proline at position 474 (p.A474P, c.1420G＞C) and a glycine to aspartic acid at position 580 (c.1739G＞A, p.G580D) and a deletion in intron 7 (c.886(-10_-31)del). By the minigene studies using the pSPL3 plasmids, we confirmed the deletion c.886(-10_-31)del as a splicing mutation. In this study, the mutation c.886(-10_-31)del accounted for about 43% of the total alleles (12/28). These patients with compound heterozygous or homozygous mutations manifested middle degree or severe glycosuria (Quantitative test for 24-hour urine glucose: 10.56-50.68 g/1.73 m2), however those with heterozygous variants presented with mild to moderate glycosuria (Quantitative test for 24-hour urine glucose ≤2.45 g/1.73 m2). This fits co-dominant inheritance pattern.    Conclusions    Five novel mutations which may be related to FRG are found in this study, and c.886(-10-31) del may be a high frequency mutation in Chinese patients. 

Objective    To evaluate whether three chronic kidney disease epidemiology collaboration (CKD-EPI) equations (CKD-EPI_2009Scr, CKD-EPI_2012SCysC and CKD-EPI_{2012Scr-SCysC}) are applicable in the prediction of glomerular filtration rate (GFR) in Chinese patients with diabetic nephropathy (DN).    Methods    One hundred and eight patients with DN who were hospitalized in the First Affiliated Hospital of Guangzhou Medical University with GFR being measured by dynamic renal imaging with ^99mTc-DTPA from June 2012 to April 2014 were enrolled in this study. GFR measured by dynamic renal imaging with ^99mTc-DTPA was used as the reference value (rGFR). GFR was estimated by the CKD-EPI_2009Scr equation, the CKD-EPI_2012SCysC equation, and the CKD-EPI_{2012Scr-SCysC} equation (labeled as eGFR1, eGFR2, eGFR3). The correlation, 30% accuracy, staging consistency, deviation and diagnostic accuracy were compared among the three CKD-EPI equations.    Results    The rGFR in 108 DN patients was (61.78±26.51) ml•min^-1•(1.73 m²)^-1. The correlation between three eGFRs and rGFR was significant (all P＜0.01), the correlation coefficients were 0.738, 0.708, 0.782. The 30% accuracy were 74.07%, 52.78%, 67.59%, The 30% accuracy of eGFR1 and eGFR3 were higher than eGFR2 (all P＜0.05), but there was no significant difference between eGFR3 and eGFR1 (χ²=0.874, P=0.436). The staging consistency was not ideal, Kappa values were 0.391, 0.180 and 0.422. For the deviations between three eGFRs and rGFR, there was no significant difference between eGFR3 and rGFR (P＞0.05), eGFR1 underestimated rGFR, eGFR2 overestimated rGFR (all P＜0.01). The results of the Bland-Altman chart showed that consistencies between three eGFRs and rGFR were poor, the degree of deviation of eGFR3 was the smallest. The area under the ROC curve (AUC) of three eGFRs was 0.878, 0.883 and 0.915. The AUC, sensitivity, specificity, overall compliance rate and Youden index of eGFR3 were the highest.    Conclusions    The eGFRs predicted by the three CKD-EPI equations showed good relevance, accuracy and diagnostic accuracy with the rGFR, but poor in consistencies. Comparatively, CKD-EPI_{2012Scr-SCysC} may be better than others, but its consistency limits exceeds the acceptable limits. Therefore, the applicability of using the three CKD-EPI equations to predict the GFR in Chinese DN patients requires a larger sample and multiple verifications as well as further improvement.

Objective    To evaluate the potential association of serum sclerostin with the development of coronary artery calcifications(CAC)in maintenance hemodialysis (MHD) patients. Methods    Ninety-two patients who were on MHD between Jan 2014 and Jan 2015 in the dialysis center were enrolled prospectively. Serum sclerostin was tested. CAC was measured by multi-slice computed tomography (MSCT) scanning, and the CAC score (CACs) was calculated. Logistic regression analysis was used to determine the risk factor of CAC in MHD patients. The diagnostic value of serum sclerostin for CAC was assessed using receiver operator characteristic curve (ROC).    Results    CAC (Agatston score>100) was present in 65.2% (60/92) patients, the median CAC score was 446 (26, 1 000). The median of serum sclerostin levels was 37.05 (29.99, 49.04) ng/L. The serum sclerostin levels were significantly elevated in the group of CACs＞400 compared to that in the group of CACs＜100 [40.71(36.69, 74.21) ng/L vs 28.16 (25.27, 33.64) ng/L, P＜0.05]. Multivariate logistic regression analysis showed that serum sclerostin level was independent risk factor for CAC (OR=1.292, 95%CI 1.017-1.641, P＜0.05). The area under the ROC curve (AUC) of serum sclerostin for CAC was 0.846 (95%CI 0.717-0.975, P=0.001), sensitivity was 0.826, and specificity was 0.769 for a cutoff value of 35.165 ng/L.    Conclusions    Serum sclerostin level is associated with CAC. Serum sclerostin level may have a diagnostic value for CAC in MHD patients.

Objective    To observe the effect of JLP deficiency on the progression of  renal interstitial fibrosis in mice model of unilateral ureteral obstruction (UUO), and to investigate the role and underlying mechanism of JLP in the development of renal fibrosis in obstructive nephropathy. Methods    jlp Wild type (jlp^+/+) and jlp deficient (jlp^-/-) mice were divided into four groups: jlp^+/+- and jlp^-/--sham-operated groups(jlp^-/--sham group and jlp^+/+-sham group), jlp^+/+- and jlp^-/--unilateral ureteral obstruction (UUO)-operated groups (jlp^-/--UUO group and jlp^+/+-UUO group). Mice were sacrificed at 7 days and 14 days after the operation respectively to evaluate the fibrosis by Masson staining. The expression of JLP in jlp^+/+renal tissue was assayed by immunohistochemistry staining, immunofluorescence and Western blotting. Immunohistochemical staining was used to detect the expression of α-smooth muscle actin (α-SMA), collagen Ⅰ(COL-Ⅰ), collagen Ⅲ(COL-Ⅲ) and transforming growth factor-β1 (TGF-β1) in sham and UUO groups. Besides, the α-SMA, COL-Ⅰ, COL-Ⅲ, TGF-β1, p-Smad2 and p-Smad3 protein levels were also analyzed by Western blotting in four groups.    Results  The expression of JLP was mainly demonstrated in the renal tubules of mice. A large amount of collagen deposition was observed in the renal interstitial area in jlp^-/--UUO group compared to jlp^+/+-UUO group. Similarly, the expression of α-SMA, COL-Ⅰ, COL-Ⅲ and TGF-β1 was significantly increased in the kidney cortices in jlp^-/-- UUO-operated groups. Meanwhile, Western blotting showed that the expression of α-SMA, COL-Ⅰ, COL-Ⅲ, and TGF-β1 protein was obviously higher in jlp^-/--UUO group. Moreover, the expression of p-Smad2 and p-Smad3 protein was markedly higher in jlp^-/--UUO group.    Conclusion    Scaffolding protein JLP is critical in preventing renal fibrosis through the inhibition of TGF-β1 expression and myo-fibroblast production.

Objective    To investigate the role of transforming growth factor-β activated kinase-1 (TAK1) signaling pathway in the activation of bone marrow derived macrophages (BMDM) induced by high glucose.    Methods    Purity of mouse BMDM was detected by flow cytometry. The mice macrophages cultured in vitro were stimulated by high glucose and treated with TAK1 specific inhibitor 5Z-7-oxozeaenol. Cells were divided into normal control group (RPMI 1640), osmolality control group (25 mmol/L mannitol), high glucose group (33 mmol/L D-glucose) and inhibitor group (33 mmol/L         D-glucose+300 nmol/L 5Z-7-oxozeaenol). Immunocytochemistry and flow cytometry were used to detect macrophage subtype. The expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis Factor-α (TNF-α) mRNA were determined by real time PCR. Expressions of p-TAK1, TAK1 binding protein (TAB1), p-JNK, p-p38 MAPK and NF-κB p65 proteins were analyzed by Western blotting.    Results    The purity of BMDM was about 99.36%. Compared with normal control group, high glucose group had increased percentage of M1 macrophages, increased expression of MCP-1 and TNF-α mRNA (all P＜0.05). Moreover, p-TAK1, TAB1, p-JNK, p-p38 MAPK and NF-κB p65 proteins expression also increased significantly in high glucose group (all P＜0.05). After treatment with inhibitor 5Z-7-oxozeaenol, the effects induced by high glucose were inhibited (P＜0.05).    Conclusions    High glucose can induce M1 macrophage activation and expression of inflammatory cytokine of BMDM, which can be inhibited 5Z-7-oxozeaenol through inhibiting TAK1/MAPK and TAK1/NF-κB pathway.

Objective    To investigate the effects of angiotensinⅡ(AngⅡ) or high glucose on the toll-like receptor 4 (TLR4) expression, inflammatory cytokines and fibrotic factors in human tubular epithelial cells (HK-2), revealing the innate immune-related pathogenesis of diabetic nephropathy (DN) which may have clinical implications.    Methods    Three TLR4 siRNA sequences were designed and synthetized. After transfection, the most effective siRNA was selected to use for further expriments. The experiment consisted of 2 parts. Part 1: Cells were divided into three groups:  normal-glucose group (NG, 5.5mmol/L glucose), mannose group (M, 5.5 mmol/L glucose+19.5 mmol/L mannose), High-glucose group (HG, 25 mmol/L glucose), preliminary validated the effects of high glucose and high osmotic pressure. Part 2: Cells were divided into seven groups: NG group, HG group, AngⅡgroup, AngⅡ+ negative group, HG+ negative group, AngⅡ+ siRNA group and HG+ siRNA group. Real time PCR was used to analyze the mRNA expression of TLR4, myeloid differentiation factor 88 (MyD88), heat shock protein 47 (HSP47). Western blotting was used to observe the protein expression of TLR4, MyD88, HSP47, NF-κB, type Ⅳ collagen (ColIV). ELISA was used to detect the expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6).    Results    Compared with NG group, TLR4, MyD88, HSP47 mRNA and TLR4, MyD88, NF-κB, ColⅣ, HSP47 protein were highly expressed under high glucose or AngⅡconditions (P＜0.01), and the expression levels of MCP-1 and IL-6 also increased significantly (P＜0.01). Compared with HG or AngⅡ group, the above indicators were obviously inhibited in the TLR4 siRNA groups (P＜0.01). Comparison between blank vector transfected groups and HG group as well as AngⅡ group indicated no statistic significance (P＞0.05).  Conclusions    Both AngⅡ and high glucose stimulate TLR4 expression, which result in the up-regulation of inflammatory and fibrotic factors in HK-2. Specific target of TLR4 gene silencing can block the TLR4 pathway that is activated by high glucose and AngⅡ, and thus reduce the inflammatory and fibtogenic factors' release. TLR4 signal is the common innate immune response pathway which induces the release of inflammatory and fibrotic factors in HK-2 under high glucose or high angiotension conditions.

Objective    To explore the effect and the possible pathway of different concentrations of QLT0267, which was the inhibitor of the integrin-linked kinase (ILK), on the process of high glucose-induced tubularepithelial-myofibroblast transdifferentiation (TEMT) in human renal tubular epithelial cells (HK-2).    Methods    HK-2 cells were exposed to 30 mmol/L GS, and TEMT model was established. After excluding the effect of high osmotic in TEMT, HK-2 cells were divided into 6 groups by different concentrations of GS and QLT0267 for 48 hours. The rate of the cell proliferation was calculated by MTT. The expression of ILK and α-smooth muscle actin (α-SMA) were determined by immunofluorescence and Western blot, and the expression of protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), and E-cadherin were  determined by Western blot.    Results (1) The expression of ILK, p-AKT, and α-SMA in HK-2 cells were unregulated and the expression of  E-cadherin was downregulated for 48 hours with glucose treating vs control (P＜0.05);  (2) The proliferation rate in high glucose group was higher than the group which concentration of QLT0267 was greater than 5 μmol/L (P＜0.05); (3)   With the concentrations of QLT0267 increased, the expression of p-AKT, α-SMA was gradually decreased (all P＜0.05), and the expression of E-cadherin was  gradually increased (all P＜0.05).    Conclusions    30 μmol/L of GS can lead to TEMT in HK-2 cell. The QLT0267 with concentration greater than 5 μmol/L may prevent the activation of ILK downstream proteins, then partially inhibits cell proliferation and TEMT in HK-2 cell.