Objective To report four male COL4A5 mutation mosaicism patients with X-linked Alport syndrome, and to provide evidence for diagnosis, genetic counseling, and reproduction in the respective families and improve our knowledge of mosaicism in Alport syndrome. Methods Suspected male mosaic patients for COL4A5 who met the following criteria: clinical diagnosis of Alport syndrome, harbored COL4A5 mutations detected using next generation sequencing or Sanger sequencing, heterozygosity for the mutant and normal COL4A5 alleles in the DNA demonstrated by Sanger sequencing, registered in the on-line registry of hereditary kidney diseases, and admitted to Peking University First Hospital during the period of April 2018 to April 2019 were enrolled. Clinical data and karyotypes were retrospectively analyzed. Genetic DNA isolated from multiple tissues was analyzed for COL4A5 gene mutations by using PCR and Sanger sequencing. Related literatures published in PubMed, CNKI and Wanfang databases were reviewed. Results Four COL4A5 somatic and germline mosaic male patients with Alport syndrome were included in the study. Patient 1 was characterized by hematuria and proteinuria. His karyotype of peripheral blood was normal. COL4A5 c.3455-1G>A mosaicism was detected in multiple tissues (peripheral blood, saliva and urine). Patient 2 presented with hematuria and microalbuminuria. His karyotype of peripheral blood was normal. COL4A5 c.4994+1G>A mosaicism was detected in multiple tissues (peripheral blood, saliva and skin fibroblasts). Patients 3 showed hematuria without proteinuria. COL4A5 c.3535G>A mosaicism was found in genomic DNA of peripheral blood and hair. Laboratory and physical examinations of patient 4 showed hematuria and normal renal function, without proteinuria, megasoma or small testes. COL4A5 c.3106G>A mosaicism was detected in genomic DNA of skin fibroblasts. Although without karyotype analysis due to unavailable specimens, 47,XXY or 46,XY/47,XXY mosaicism was not considered according to the reproductive history and lack of clinical manifestations of megasoma and small testes in patients 3 and 4. Renal disease in 8 published male cases with mosaicism for COL4A5 was affected by mutant allelic fractions and genotype. Conclusions Compared with hemizygous males with X-linked Alport syndrome, the renal phenotype of mosaic males was milder, and associated with mutant allelic fractions and mutation type.

Objective To analyze the splicing mutation site of COL4A5 gene in a family with X-linked dominant Alport syndrome and explore the possibility of exon specific U1 small nuclear RNA (snRNA) gene therapy. Methods The clinical data of the proband and family members of Alport syndrome were collected, and the gene mutations in the whole exon of a series of nephropathy genes in the proband were detected by high-throughput sequencing. The splice site changes and pathogenicity caused by COL4A5 c.546+5G>A mutation were analyzed by online software. Minigene experiment was used to verify and analyze the effect of COL4A5 gene mutation site c.546+5G>A in the proband of Alport syndrome family, and transient transfection and introduction of modified U1 snRNA to correct splicing mutation. Results The results of gene sequencing showed that there was a hemizygous variation of COL4A5 gene in the proband and his half brother, and the variation site was c.546+5G>A. The results of online software for analyzing the pathogenicity of splice variation showed that the original donor splicing site could not be detected after mutation, suggesting that there was a great possibility of affecting splicing. The abnormal splicing mode of COL4A5 gene with c.546+5G>A mutation—deletion of exon 9 was verified by hybridized small gene detection. The abnormal splicing mutation could be partially corrected by the modified U1 snRNA. The correction ratios of ExSpeU1 (MT), ExSpeU1(E9+1), ExSpeU1(E9+9) and ExSpeU1(E9+11) to exon 9 deletion caused by c.546+5G>A were 0, 43.81%, 52.09% and 48.12%, respectively. Conclusions The pathogenicity of the new splicing mutation of COL4A5 is verified, and the modified U1 snRNA can partially correct the abnormal splicing.

Objective To investigate the incidence, risk factors and early prognosis of acute kidney injury (AKI) after heart valve surgery in adults, and to provide evidence for the early prevention and treatment of AKI after heart valve surgery. Methods Clinical data of patients undergoing heart valve surgery from January 2016 to March 2017 were collected retrospectively. Early postoperative AKI was diagnosed and staged according to Kidney Disease Improving Global Outcomes (KDIGO) clinical guidelines for AKI. Patients were divided into AKI group and non-AKI group according to whether AKI occurred within 48 hours after surgery, and the differences in clinical indexes between the two groups were compared and analyzed. Influencing factors for early postoperative AKI were screened by stepwise multivariate logistic regression. Results A total of 600 patients were enrolled, including 197 patients in the AKI group and 403 patients in the non-AKI group. The incidence of AKI was 32.83%. In the non-AKI and AKI groups, proportion of renal replacement therapy (RRT) during hospitalization, mechanical ventilation time, intensive care unit (ICU) time and proportion of hospital mortality were different (all P<0.001). In the AKI group, 160 patients (81.22%) were in stage I, 33 patients (16.75%) were in stageⅡ, and 4 patients (2.03%) were in stageⅢ. The proportion of patients receiving RRT, mechanical ventilation time, ICU time, hospital mortality were also different in patients with AKI of different stages (all P<0.05). Logistic regression analysis results showed that males, body mass index≥24.0 kg/m², previous history of cardiac surgery, baseline serum creatinine>115 mmol/L, albumin<35 g/L, aortic occlusion time (AOT)>90 min, blood glucose in ICU after operation>11.1 mmol/L, the difference between the highest blood glucose within 48 hours after the operation and the blood glucose at 0 h after the operation≥2 mmol/L, blood lactic acid in ICU after operation>4 mmol/L and maximum vasoactive drug score within 48 hours after surgery>9 were independent influencing factors for early AKI (all P<0.05). Conclusions The incidence of AKI within 48 hours after heart valve surgery is over 30%. The independent influencing factors include male, overweight/obesity, previous cardiac surgery, preoperative renal insufficiency, hypoproteinemia, long duration of AOT, post-operative stress hyperglycemia, fluctuating blood glucose levels wildly, hyperlactacidemia, and using more vasoactive drugs. AKI after cardiac surgery affects the early prognosis of patients, the later of the stage leads to the worse prognosis.

Objective To explore the correlation between hemoglobin (Hb) and progression of renal function in patients with proliferative lupus nephritis (PLN). Methods Data of biopsy-proven PLN patients from January 2010 to February 2019 in Department of Nephrology, the First Affiliated Hospital of Shenzhen University were retrospectively analyzed. The patients were divided into stable renal function group and renal function progression group according to changes of renal function including serum creatinine doubling/end-stage renal disease (ESRD) and the demographic, clinical, and pathological characteristics were compared between the two groups. Cox regression and smooth curve fitting of generalized additive model analysis were used to explore the correlation between Hb and progression of renal function. Results A total of 87 patients were included in this study. The age was (34.97±11.95) years old and 79 cases (90.80%) were females. During follow-up of 19.0(6.5, 43.5) months, renal function of 15 patients (17.24%) progressed. Compared with stable renal function group, Hb level of patients in renal function progression group were lower (t=3.887, P<0.001), and serum creatinine (Z=-2.466, P=0.003) and uric acid (t=-2.154, P=0.034) were higher. As to the pathological characteristics, the proportion of class lupus nephritis-IV, renal tubular atrophy and interstitial fibrosis in renal function progression group were higher than those in stable renal function group, but there was no statistical difference between the two groups (all P>0.05). Multivariate Cox regression analysis indicated that high Hb was an independent protective factor of renal function progression in PLN patients (HR=0.893, 95%CI 0.836-0.954, P=0.001). The risk of progression to serum creatinine doubling/ESRD would decrease by 10.7% when Hb increased by 1 unit (g/L). Smooth curve fitting of generalized additive model analysis showed that Hb was linearly correlated with the risk of renal function progression (P=0.100). Receiver operating characteristic curve indicated that the risk of doubling serum creatinine/ESRD in PLN patients would be relatively low when Hb level was above 77 g/L (area under the curve 0.788, best threshold 77 g/L, sensitivity 0.600, specificity 0.903). Conclusions Hb is closely related to progression of renal function in patients with PLN. More attention and management of Hb levels in patients with PLN can play an important role in improving renal prognosis.

Objective To investigate the association between cognitive impairment and all-cause mortality in middle and elderly adult patients undergoing maintenance hemodialysis (HD). Methods A prospective cohort study was conducted. Patients from 11 HD centers in Beijing between April and June 2017 were enrolled. Baseline data were collected, and a series of neuropsychological batteries covered 5 domains of cognitive function were applied for the assessment of cognitive function. The patients were then classified as normal and cognitive impairment groups according to the fifth version of the Diagnostic and Statistical Manual of Mental Disorders criteria (DSM-V) and followed-up until June 2018. The clinical characteristics of the two groups of patients were compared. Kaplan-Meier survival analysis was used to compare the difference in the cumulative survival rate between the two groups. Multivariate Cox regression model was used to analyze the independent influencing factors of all-cause mortality, to determine the relationship between cognitive impairment and different cognitive domain impairments and all-cause death. Results A total of 613 patients were enrolled, of which 496(80.91%) patients had cognitive impairment. Compared with the normal cognitive function group, the patients in the cognitive impairment group tended to be older, longer dialysis vintage, a higher proportion of diabetes, hypertension, and stroke, increased serum iPTH level, and lower education level and urea clearance index (Kt/V) (all P<0.05). After (49.53±8.42) weeks of follow-up, Kaplan-Meier survival analysis showed that the cumulative survival rate of cognitive impairment group was significantly lower than that of cognitive normal group (Log-rank χ²=8.610, P=0.003). Multivariate Cox regression analysis showed that history of diabetes (HR=2.742, 95%CI 1.598-4.723, P<0.001), coronary heart disease (HR=1.906, 95%CI 1.169-3.108, P=0.010), dialysis vintage (every increase of 1 month, HR=1.007, 95%CI 1.003-1.011, P=0.001), serum level of albumin (every increase of 1 g/L, HR=0.859, 95%CI 0.809-0.912, P<0.001), cognitive impairment (HR=2.719, 95%CI 1.088-6.194, P=0.032) were independently associated with all-cause mortality. Multivariate Cox regression analysis on different cognitive domains also indicated that memory impairment (HR=2.571, 95%CI 1.442-4.584, P<0.001), executive function impairment (HR=3.311, 95%CI 1.843-5.949, P=0.001) and three, four, five domains combined impairment (HR=5.746, 95%CI 1.880-17.565, P=0.002; HR=12.420, 95%CI 3.690-41.802, P<0.001; HR=13.478, 95%CI 3.381-53.728, P<0.001) were independently related to all-cause mortality. Conclusions Cognitive impairment is an independent risk factor of all-cause mortality in middle and elderly adult patients undergoing maintenance hemodialysis, and the risk is significantly increased in patients with the impairment of the domains of memory, executive function, or in the combination of three to five cognitive domains.

Objective To investigate the injury effect of hyperoxali acid on human arterial endothelial cells (HAECs) and its mechanism. Methods HAECs were divided into intervention group and control group according to whether oxalic acid was used for intervention. The cells in the intervention group were stimulated with 30, 100, 200 and 300 μmol/L oxalic for different time. The effect of oxalic acid on the proliferation of HAECs was detected by MTT colorimetry. The change of cell cycle was analyzed by flow cytometry. The content of intracellular calcium was detected by fluorescence detection technology. The protein and mRNA expressions of cell cycle and anion transporter-related proteins were detected by Western blotting and fluorescence quantitative PCR. Besides, JAK2/STAT3 signaling pathway-related proteins were measured by Western blotting. Results MTT colorimetry results showed that the intervention groups with high concentration of oxalic acid (100, 200, 300 μmol/L) could significantly inhibit the proliferation of HAECs, which was significantly different from the control group (all P<0.05). Fluorescence detection showed that the contents of intracellular calcium of HAECs in the intervention groups with high concentration of oxalic acid (100, 200, 300 μmol/L) were significantly higher than those in the control group after 48 hours (P<0.05, P<0.001, P<0.001, respectively). Flow cytometry showed that the proportion of S phase of cells in the 200 μmol/L oxalic acid intervention group was significantly higher than that in the control group (P<0.05). The results of Western blotting and PCR showed that the relative protein and mRNA expressions of anion transporter-related proteins slc26a1, slc26a5, slc26a11 in the intervention groups were higher than those in the control group (all P<0.05). Western blotting showed that the expression of p-JAK2 and p-STAT3 in the intervention groups after 24 hours were significantly higher than those in control group (all P<0.05). Conclusions Hyperoxalic acid may enter HAECs through transporters slc26a1, slc26a5 and slc26a11 to inhibit cell proliferation and increase the intracellular calcium concentration. The mechanism may be through the activation of JAK2/STAT3 signaling pathway. Therefore, oxalic acid may be one of the uremic toxins leading to atherosclerosis.

Objective To investigate the role and mechanism of (histone deacetylase 6, HDAC6) in the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells and the activation of renal interstitial fibroblasts. Methods Human renal tubular epithelial cells (HK-2) and rat renal interstitial fibroblast (NRK-49F) were cultured in vitro, and divided into 4 groups: control group, Tubastatin A (TA) group (treated with 10 μmol/L HDAC6 inhibitor TA for 36 h), transforming growth factor-β1 (TGF-β1) group (10 ng/ml TGF-β1 for 36 h), and TGF-β1+TA group (treated with 10 ng/ml TGF-β1 and 10 μmol/L TA for 36 h). The expression levels of fibronectin, α-smooth muscle actin (α-SMA), collagen I, E-cadherin, HDAC6, acetyl histone H3, histone H3, acetyl α-tubulin, α-tubulin, TGF-β receptor (TGF-βR) 1, p-Smad3, Smad3, connective tissue growth factor (CTGF), epidermal growth factor receptor (EGFR) and p-EGFR in HK-2 and NRK-49F cell samples were detected by Western blotting, and quantitative analysis was performed according to gray level. Results (1) In HK-2 cells stimulated by TGF-β1, TA decreased the expression of fibronectin, α-SMA, collagen I, and increased the expression of epithelial cell marker E-cadherin. Meanwhile, TA decreased the expression of HDAC6 and increased the expression levels of acetyl histone H3 and acetyl α-tubulin (all P<0.05). (2) Compared with the TGF-β1 group, the expressions of TGF-βR1, p-Smad3, CTGF and p-EGFR in TGF-β1+TA group were decreased (all P<0.05), while the total protein levels of Smad3 and EGFR were not significantly different (both P>0.05). (3) In NRK-49F cells stimulated by TGF-β1, TA decreased the expressions of fibronectin, α-SMA, collagen I, TGF-βR1 and p-Smad3 (all P<0.05). Conclusions Blockade of HDAC6 by TA may inhibit the EMT of renal tubular epithelial cells and the activation of renal interstitial fibroblasts via regulating multiple signaling pathways including TGF-β/Smad3, CTGF and EGFR.