Objective To compare the effect of various antigen retrieval methods in paraffin sections of renal biopsy tissue, and explore the best antigen retrieval method. Methods Forty-five renal biopsy specimens were collected from the First Affiliated Hospital of Zhengzhou University, including lupus nephritis (n=10), membranous nephropathy (n=10), IgA nephropathy (n=10) and amyloidosis glomerulopathy (n=15). Five retrieval methods (including high pressure thermal retrieval combined with trypsin retrieval, microwave thermal retrieval combined with trypsin retrieval, high pressure thermal retrieval, microwave thermal retrieval and gastroprotease retrieval) were used for immunofluorescence staining of paraffin sections. Renal tissue specimens were divided into six groups according to different antigen retrieval methods, frozen section specimens used as a control group. The immunofluorescence semi-quantitative scores of paraffin sections of the five heat-repairing antigen methods and frozen sections were compared. Results Immunofluorescence staining of hyperbaric thermal retrieval combined with trypsin retrieval group and microwave thermal retrieval combined with trypsin retrieval group were similar with those of frozen sections. Compared with the control group, there were no significant difference in the semi-quantitative immunofluorescence scores between the two groups (all P>0.05). However, Immunofluorescence staining of hyperbaric thermal retrieval, microwave thermal retrieval, pepsin digestion had significantly higher false negative rate than those of frozen sections. Compared with the control group, the difference in semi-quantitative immunofluorescence score was statistically significant (all P<0.05). Conclusion High pressure heat retrieval combined with trypsin retrieval or microwave heat retrieval combined with trypsin retrieval is the first choice of antigen retrieval methods.

Objective To observe the changes of abdominal aortic calcification and biochemical indicators after parathyroidectomy (PTX) in the maintenance hemodialysis (MHD) patients with secondary hyperparathyroidism (SHPT). Methods The MHD patients with SHPT who were followed up for 2 years were analyzed retrospectively and divided into PTX surgery group (n=26) and non-surgery group (n=18) according to whether they underwent PTX，and then the abdominal aortic calcification score (AACS), intact parathyroid hormone (iPTH), blood calcium and phosphorus after 2 years were observed in the two groups. The PTX surgery group was divided into advanced group and non-advanced group according to whether abdominal aortic calcification had progressed or not 2 years after the operation. Indicators such as age, dialysis age, iPTH, blood calcium, blood phosphorus, calcium and phosphorus product were compared between the two groups to analyze the possible factors related to the development of abdominal aortic calcification. Results A total of 44 patients meeting the inclusion criteria were included, with 26 in the PTX surgery group and 18 in the non-surgery group. The baseline data of the PTX surgery group and the non-surgery group showed statistical difference in the age of dialysis (P<0.05), but no statistical differences in gender, age and history of hypertension. Compared with preoperative indicators, postoperative iPTH, blood calcium and phosphorus significantly reduced (all P<0.05), and there was no significant difference in AACS. There were 8 cases (30.77%) of accelerating progress of calcification, 8 cases (30.77%) of improvement in calcification, 10 cases (38.46%) of calcification stability. After 2 years, iPTH value of non-advanced group was significantly lower than advanced group [(20.62±6.44) ng/L vs (132.72±76.83) ng/L], while the preoperative AACS progress was higher in non-advanced group [(13.11±2.71) vs (2.00±1.41)] (all P<0.05). In non-surgery group, AACS was significantly higher after 2 years [(10.44±1.65) vs (8.05±1.26)], blood phosphorus and the product of blood calcium and phosphorus significantly decreased (all P<0.05), and the levels of iPTH and blood calcium did not significantly change. Pearson correlation analysis showed that the decreased value between preoperative AACS and 2-year postoperative AACS was positively correlated with the decreased value of iPTH (r=0.534, P=0.012), blood calcium (r=0.643, P=0.004), blood phosphorus (r=0.897, P<0.001) and calcium-phosphorus product (r=0.568, P=0.021）, and negatively correlated with preoperative AACS (r=-0.647，P=0.014). Conclusions Small sample data shows that PTX can correct parathyroid hormone, calcium and phosphorus for long term, and prevent abdominal aortic calcification progression, even reverse vascular calcification. Whether abdominal aortic calcification improves or not may be associated with the decrease of iPTH, calcium, phosphorus and the product of blood calcium and phosphorus.

Objective To explore the association of abdominal aortic calcification score (AACS) with cardiovascular disease (CVD) outcomes in peritoneal dialysis (PD) patients. Methods The patients who underwent regular PD at Renji Hospital between July 2011 and July 2014 were recruited and prospectively followed up until the end of the study (August 31, 2018), death, or dropout PD. Abdomen lateral X-ray was used to determine AACS for each patient at enrollment. Patients were divided into three groups based on the tertiles of AACS: non-calcified group, AACS group (AACS=0), mild-moderate calcification group AACS group (0<AACS≤4) and severe calcification group (4<AACS≤24). Cumulative incidences of cardiovascular outcomes among three groups were estimated using competing risk model and compared through Gray test. Competing risk regression model was used to evaluate the association of AACS and cardiovascular events as well as CVD mortality. Results Two hundred and ninety-two PD patients were enrolled in this study. The cohort consisted of 160 males (54.8%) with the age (57.1±15.2) years and median PD vintage 28.4 (IQR 12.0, 57.8) months, and their average AACS was 2.0 (0.0, 6.0). Order logistic regression analysis showed that older age (OR=1.081, 95%CI 1.057-1.106, P<0.001) and longer PD vintage (OR=1.012, 95%CI 1.004-1.019, P=0.003), CVD history (OR=1.919, 95%CI 1.108-3.325, P=0.020) and diabetes (OR=2.554, 95%CI 1.415-4.609, P=0.002) were independent risk factors of escalating AACS in PD patients. During the follow-up, 65 cases CVD events and 50 cases CVD-related deaths developed. Patients in the upper AACS tertile had significantly higher estimated cumulative incidences of CVD occurrence (Gray=27.81, P<0.001) and CVD mortality (Gray=20.91, P<0.001). AACS was an independent predictor of both CVD occurrence (medium AACS group vs low AACS group: SHR=2.823, 95%CI 1.333-5.970, P=0.007; high AACS group vs medium AACS group: SHR=3.063, 95%CI 1.460-6.430, P=0.003) and CVD mortality (SHR=2.590, 95%CI 1.132-5.920, P=0.024) in competing risk regression models. Conclusions Age, PD vintage, diabetes and preexisting CVD are associated with higher AACS in the present cohort. AACS can predict CVD morbidity and mortality in PD population and therefore may help with the early identification of PD patients with adverse cardiovascular outcomes.

Objective To investigate the relationship between peritoneal thickness and baseline solute transport function in peritoneal dialysis (PD) patients, and analyze the factors affecting the function of peritoneal transport. Methods Non-diabetic end-stage renal disease (ESRD) patients admitted to the Second Hospital of Longyan City from January 2017 to June 2019 were enrolled in this study. The thickness of the peritoneal membrane was measured by color ultrasound instrument before the peritoneal catheterization. Standard peritoneal equilibration test (PET) was performed after one month of peritoneal dialysis. The ratio of corrected creatine in 4 h dialysate to 2 h serum creatine (D/Pcr) was used as a solute baseline transport index, and according to the D/Pcr evaluation results, the patients were divided into high/high average transfer (H) group (D/Pcr≥0.65) and low/low average transfer (L) group (D/Pcr<0.65). The clinical data, peritoneal thickness and peritoneal dialysis related indicators between the two groups of patients were compared. Binary logistic regression was used to analyze the factors affecting the function of peritoneal transport. Results The amount of peritoneal ultrafiltration in H group was significantly lower than that in L group, intraperitoneal creatinine clearance (Ccr) and peritoneal thickness were significantly higher than those in L group (both P<0.05). Pearson and Spearman correlation results showed that the thickness of peritoneal membrane positively correlated with D/Pcr (r=0.673, P<0.05), peritoneal Ccr (r=0.261, P<0.05), and negatively correlated with ultrafiltration of peritoneal dialysis (r=-0.365, P<0.05). Partial correlation analysis showed that the peritoneal thickness was positively correlated with the solute transport index D/Pcr (r=0.539, P<0.05) and the peritoneal Ccr (r=0.338, P<0.05). Binary logistic regression results showed that peritoneal thickening was a risk factor affecting peritoneal transport function (OR=1.175, 95%CI 1.009-1.369, P<0.05). Conclusions There is a positive correlation between the peritoneal membrane thickness and the baseline solute transport index in patients with non-diabetic peritoneal dialysis. Peritoneal thickening is a risk factor affecting peritoneal transport function.

Objective To investigate the causes and outcomes in the children who did not immediately receive glucocorticoids therapy after initial diagnosis of primary nephrotic syndrome (PNS). Methods The clinical data of PNS patients not immediately receiving glucocorticoids therapy after initial diagnosis at the Department of Nephrology, the Children's Hospital, Zhejiang University School of Medicine from January 1, 2005 to December 31, 2014 were retrospectively analyzed. Results A total of 1 431 cases were initially diagnosed with PNS, including 1 061 males and 370 females. Among them, 130 cases did not receive conventional glucocorticoids treatment immediately, accounting for 9.1%. Of whom, 75 cases were found showing spontaneous remission after symptomatic treatment; 23 cases were directly treated with adrenocorticotropic hormone (ACTH), one case with mycophenolate mofetil (MMF), and 31 cases not given glucocorticoids or immunosuppressants because of parental refusal. Among 75 cases with spontaneous remission, 16 cases were found in sustained remission; 39 cases were treated with glucocorticoids and 6 cases with ACTH at relapse; 14 cases were lost. Among 29 cases using ACTH, 7 cases were found in sustained remission. Among the 31 cases who refused glucocorticoids or immunosuppressants therapy, one died. The case treated with MMF, later were given with halved hormone because of no-effective response. Conclusions Spontaneous remission is found in a small proportion of PNS patients at first-onset, but most subsequently relapse. Hormone therapy should be routinely given unless remission has occurred before application. Some children's parents refuse hormone therapy, and need further communication. Some cases initially treated with ACTH are found in sustained remission, which should be further observed and studied to clear the efficacy and safety of ACTH.

Objective To analyze the clinicopathological features in diabetic kidney disease (DKD) and non-diabetic kidney disease (NDKD) patients, and provide reference for patients who will receive renal biopsy with diabetes mellitus complicated with chronic kidney disease. Methods The patients with type 2 diabetes mellitus complicated with chronic kidney disease who underwent renal biopsy were collected through the database at the Nanfang Hospital of Southern Medical University from February 2002 to June 2018. According to the results of renal biopsy, they were divided into DKD group and NDKD group (including DKD+NDKD). The clinical manifestations and pathological types were compared between the two groups. Results A total of 507 patients were eventually included in the study. There were 114 cases (22.5%) with DKD and 393 cases (77.5%) with NDKD. Pathologically, the most common pathological types of NDKD were membranous nephropathy (30.0%) and IgA nephropathy (19.1%). Among NDKD patients, 5.6% patients had DKD combing with NDKD. In term of the clinical manifestations, DKD patients had a longer history of diabetes (>1 year, 76.3% vs 36.1%, P<0.001), higher quantity of urinary protein [3.69(1.70, 6.74) g/24 h vs 2.21(0.91, 4.97) g/24 h, P<0.001], higher serum creatinine [117.5(85.8, 194.5) μmol/L vs 89.0(68.0, 143.8) μmol/L, P<0.001] than NDKD patients. But the hemoglobin [(105.07±20.85) g/L vs (124.41±25.02) g/L, P=0.002] and cholesterol [(5.69±1.87) mmol/L vs (6.43±2.75) mmol/L, P=0.001] in DKD patients were lower than those in NDKD patients. Logistic regression analysis showed that diabetes mellitus history (OR=4.162, 95%CI 1.717-10.098, P=0.002）, higer systolic pressure (every 1 mmHg, OR=1.028, 95%CI 1.011-1.045, P=0.001）, history of antihypertensive medication (OR=3.141, 95%CI 1.496-6.591, P=0.002), diabetic retinopathy (OR=5.561, 95%CI 2.361-13.100, P<0.001) and higher glycated hemoglobin level (every 1%, OR=1.680, 95%CI 1.333-2.118, P<0.001) were related factors of DKD, while hematuria (OR=2.781，95%CI 1.334-5.798，P=0.006) and higher hemoglobin level (every 1 g/L, OR=1.022, 95%CI 1.008-1.037, P=0.002) were related factors of NDKD. Conclusions There are differences in clinical manifestations and pathological types between DKD and NDKD. The history of diabetes, antihypertensive medication, fundus examination, higher of proteinuria and glycosylated hemoglobin may predict DKD, while hematuria and higher level of hemoglobin may have certain guiding significance for the diagnosis of NDKD. The indication of renal biopsy in patients with diabetes mellitus complicated with chronic kidney disease should include comprehensive clinical manifestations.

Objective To explore the role and mechanism of lysine methyltransferase SET8 in calcification induced by high phosphorus in vascular smooth muscle cells (VSMCs). Methods (1) Male SD rats were selected for in vivo experiments and randomly divided into sham operation group and chronic renal failure vascular calcification group. The thoracic aorta was taken and calcification was detected by von Kossa staining. The expression of SET8 and Caspase-3 was detected by immunohistochemistry. (2) VSMCs were randomly divided into normal group and high phosphorus group (10 mmol/L β-glycerophosphate). Cellular calcification was detected by O-cresol hydrazide complex colorimetric assay and alizarin red staining. Apoptosis was detected by flow cytometry. The expressions of SET8, AKT and Caspase-3 were detected by RT-PCR and Western blotting. (3) In order to further verify the role of SET8 in the apoptosis of VSMCs, liposome transfection was used, and cells were divided into three groups: SET8-shRNA group, empty plasmid group and normal control group. Cellular calcification was detected by O-cresol hydrazide complex colorimetric assay and alizarin red staining. Apoptosis was detected by flow cytometry. The expressions of SET8, AKT and Caspase-3 were detected by RT-PCR and Western blotting. Results (1) In vivo experiments, compared with the sham operation group, vascular calcium deposition in the chronic renal failure vascular calcification group was significantly increased (P<0.05). Immunohistochemistry results showed that SET8 expression was significantly decreased and Caspase-3 was significantly increased in the vascular calcification group (both P<0.05). Correlation analysis showed that SET8 was negatively correlated with vascular calcification and Caspase-3 (r=-0.948, P<0.01; r=-0.961, P<0.01). (2) In vitro, the calcium deposition in the high-phosphorus group was significantly higher than that in the normal group (P<0.05). The results of flow cytometry showed that the number of apoptosis in the high-phosphorus group was significantly higher than that in the normal group (P<0.05). RT-PCR and Western blotting showed that, compared with the normal group, the mRNA and protein expression of SET8 and AKT in the high-phosphorus group decreased, and the mRNA and protein expression of Caspase-3 increased (all P<0.05). (3) After interference of SET8 gene expression, calcification and apoptosis of VSMCs significantly increased, AKT mRNA and protein expression decreased, and Caspase-3 mRNA and protein expression increased (all P<0.05). Conclusions SET8 can inhibit vascular calcification. One of the possible mechanisms is to inhibit the expression of Caspase-3 via promoting AKT activation, thereby inhibiting the apoptosis of VSMCs, and then participating in the regulation of VSMCs calcification induced by high phosphorus.

Objective To study the effect of matrine on the expression of transcription factor Snail2 in peritoneal mesothelial cells epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1). Methods Human peritoneal mesothelial cells were stimulated by TGF-β1 and treated with matrine. The experiment was divided into six groups (control group, TGF-β1-induced group (5 ng/ml), TGF-β1+0.4 mg/ml matrine intervention group, TGF-β1+0.6 mg/ml matrine intervention group, TGF-β1+0.8 mg/ml matrine intervention group and TGF-β1+1.0 mg/ml matrine intervention group). The expressions of Snail2, E-cadherin, α-smooth muscle actin (α-SMA), Fibronectin and collagen (Col)Ⅲ were detected by real-time fluorescence quantitative PCR and Western blotting. The protein phosphorylation levels of Smad2, Smad3 and extracellular signal-regulated kinase (ERK)1/2 were detected by Western blotting. Results The mRNA and protein expressions of Snail2, α-SMA, Fibronectin and ColⅢ were up-regulated after being stimulated by TGF-β1 (5 ng/ml) in human peritoneal mesothelial cells, while the mRNA and protein expression of E-cadherin was down-regulated. TGF-β1 (5 ng/ml) could up-regulate the protein phosphorylation levels of Smad2, Smad3 and ERK1/2. Matrine (0.4, 0.6, 0.8, 1.0 mg/ml) could down-regulate the mRNA and protein expression levels of Snail2, α-SMA, Fibronectin and ColⅢ after being stimulated by TGF-β1 in human peritoneal mesothelial cells. Matrine could down-regulate the protein phosphorylation of ERK1/2, and up-regulate the mRNA and protein expression levels of E-cadherin with treatment of TGF-β1 in human peritoneal mesothelial cells. Conclusions TGF-β1 can induce EMT of human peritoneal mesothelial cells. Matrine may inhibit TGF-β1-induced EMT of human peritoneal mesothelial cells by down-regulating the expression of Snail2 through the ERK1/2 signaling pathway.