Objective To investigate the role and mechanism of ubiquitin-specific protease 46 (USP46) in hypoxia/reoxygenation (H/R)-induced pyroptosis of renal tubular epithelial cells. Methods Renal tubular epithelial cells were divided into negative control siRNA group (si-CTL group), USP46 knockdown group (si-USP46 group), negative control siRNA + H/R treatment group (si-CTL+H/R group), and USP46 knockdown + H/R treatment group (si-USP46+H/R group). Flow cytometry was used to detect cell apoptosis in each group. Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to measure the messenger RNA (mRNA) expression of USP46, NOD-like receptor protein 3 (NLRP3), gasdermin D (GSDMD), interleukin (IL)-18, and IL-1β. Western blotting was used to detect the protein expression of USP46, NLRP3, GSDMD, and cleaved cysteinyl aspartate specific proteinase (C-Caspase)-1. The levels of inflammatory factors and lactate dehydrogenase (LDH) in the cell supernatants were detected, and the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the cells were detected. Co-immunoprecipitation was used to verify the interaction between USP46 and NLRP3. Results Compared with the si-CTL group, the si-CTL+H/R group exhibited increased cell apoptosis, elevated protein expression of USP46, NLRP3, GSDMD-N and C-Caspase-1, increased mRNA expression of USP46, NLRP3, GSDMD, IL-18 and IL-1β, higher levels of IL-18, IL-1β, TNF-α and LDH, and increased ROS and MDA levels (all P < 0.05). Compared with the si-CTL+H/R group, the si-USP46+H/R group showed decreased cell apoptosis, reduced protein expression of USP46, NLRP3, GSDMD-N and C-Caspase-1, decreased mRNA expression of USP46, GSDMD and IL-18, lower levels of IL-18, IL-1β, TNF-α and LDH, and decreased ROS and MDA levels (all P < 0.05). Co-immunoprecipitation results indicated that USP46 could bind to NLRP3. Conclusions Downregulation of USP46 alleviates H/R-induced pyroptosis in renal tubular epithelial cells, possibly by inhibiting USP46-dependent NLRP3 deubiquitination and promoting NLRP3 ubiquitination and degradation.

Objective To investigate the role and mechanism of spliced X-box binding protein 1 (XBP1s) in the senescence of primary renal tubular epithelial cells induced by hypoxia/reoxygenation (H/R). Methods Primary renal tubular epithelial cells were divided into the normal control group (NC group), H/R group, empty adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), empty adenovirus+H/R treatment group (Ad-shNC+H/R group) and targeted silencing XBP1s adenovirus+H/R treatment group (Ad-shXBP1s +H/R group), respectively. The expression levels of XBP1s in the NC, H/R, Ad-shNC and Ad-shXBP1s groups were measured. The number of cells stained with β-galactosidase, the expression levels of cell aging markers including p53, p21 and γH2AX, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were determined in the Ad-shNC, Ad-shNC+H/R and Ad-shXBP1s+H/R groups. Chromatin immunoprecipitation was employed to verify Sirtuin 3 (Sirt3) of XBP1s transcription regulation, and the expression levels of Sirt3 and downstream SOD2 after down-regulation of XBP1s were detected. Mitochondrial reactive oxygen species (mtROS) were detected by flow cytometry. Results Compared with the NC group, the expression level of XBP1s was up-regulated in the H/R group. Compared with the Ad-shNC group, the expression level of XBP1s was down-regulated in the Ad-shXBP1s group (both P<0.001). Compared with the Ad-shNC group, the number of cells stained with β-galactosidase was increased, the expression levels of p53, p21 and γH2AX were up-regulated, the levels of ROS, MDA and mtROS were increased, the SOD activity was decreased, the expression level of Sirt3 was down-regulated, and the ratio of Ac-SOD2/SOD2 was increased in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the number of cells stained with β-galactosidase was decreased, the expression levels of p53, p21 and γH2AX were down-regulated, the levels of ROS, MDA and mtROS were decreased, the SOD activity was increased, the expression level of Sirt3 was up-regulated and the ratio of Ac-SOD2/SOD2 was decreased in the Ad-shXBP1s+H/R group (all P<0.05). Conclusions Down-regulation of XBP1s may ameliorate the senescence of primary renal tubular epithelial cells induced by H/R, which probably plays a role through the Sirt3/SOD2/mtROS signaling pathway.

Objective To evaluate the effect of spliced X-box binding protein 1 (XBP1s) on hypoxia/reoxygenation (H/R) injury of mouse renal tubular epithelial cells and unravel underlying mechanism. Methods Mouse renal tubular epithelial cells were divided into adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), Ad-shNC+H/R group and Ad-shXBP1s+H/R group. The apoptosis level, mitochondrial reactive oxygen activity, mitochondrial membrane potential and mitochondrial calcium ion level were detected in each group. Chromatin immunocoprecipitation followed by sequencing (ChIP-seq) was employed to analyze the binding sites of XBP1s in regulating the inositol 1,4,5-trisphosphate receptor (ITPR) family. The expression levels of XBP1s and ITPR family messenger RNA (mRNA) and protein were determined in each group. Results Compared with the Ad-shNC group, the apoptosis level was higher, mitochondrial reactive oxygen species level was increased, mitochondrial membrane potential was decreased and mitochondrial calcium ion level was elevated in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the apoptosis level was lower, mitochondrial reactive oxygen species level was decreased, mitochondrial membrane potential was elevated, and mitochondrial calcium ion level was decreased in the Ad-shXBP1s+H/R group (all P<0.05). Compared with the Ad-shNC group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 mRNAs and proteins were down-regulated in the Ad-shXBP1s group (all P<0.05). Compared with the Ad-shNC group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 proteins were up-regulated in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 were down-regulated in the Ad-shXBP1s+H/R group (all P<0.05). ChIP-seq results showed that XBP1s could bind to the promoter and exon of ITPR1, the exon of ITPR2, and the exon of ITPR3. Conclusions XBP1s may affect mitochondria-associated endoplasmic reticulum membrane structure and function by directly regulating ITPR transcription and translation. Down-regulating XBP1s may inhibit ITPR expression and mitigate mitochondrial damage.

Objective To evaluate the effect of glycogen synthase kinase 3β (GSK3β) on hypoxia/reoxygenation (H/R)-induced injury of senescent renal tubular epithelial cell (RTEC) in aged mice and its regulatory mechanism. Methods RTEC were divided into the Young group (young RTEC with normal growth), Old group (aged RTEC induced by Etoposide), Old+Ad-shNC+H/R group [aged RTEC induced by Etoposide and then transfected with adenovirus negative control (Ad-shNC) for H/R treatment], and Old+Ad-shGSK3β+H/R group (aged RTEC induced by Etoposide and then transfected with short-hairpin RNA-expressing adenovirus with targeted silencing GSK3β for H/R treatment), respectively. Apoptosis level and mitochondrial reactive oxygen species level were detected by flow cytometry. Calcium ion level was determined by immunofluorescence staining. The expression and phosphorylation levels of GSK3β, mitochondria-associated endoplasmic reticulum membrane (MAM)-related proteins of inositol 1,4,5-trisphosphate receptor1 (ITPR1), voltage dependent anion-selective channel 1(VDAC1) and glucose-regulated protein 75 (GRP75) were detected by Western blot. The interaction between GSK3β and MAM-related proteins was analyzed by immunoprecipitation. Results Compared with the Young group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were higher in the Old group. Compared with the Old group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were higher in the Old+Ad-shNC+H/R group. Compared with the Old+Ad-shNC+H/R group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were lower in the Old+Ad-shGSK3β+H/R group, and the differences were statistically significant (all P<0.05). Compared with the Young group, the expression levels of ITPR1, GRP75 and GSK3β proteins were up-regulated, the phosphorylation levels of ITPR1 and GRP75 were increased, whereas the total protein and phosphorylation levels of VDAC1 were decreased in the Old group. Compared with the Old group, the expression level of GSK3β protein was unchanged, the total protein and phosphorylation levels of ITPR1 and GRP75 were increased, the expression level of total VDAC1 protein remained unchanged and the phosphorylation level was increased in the Old+Ad-shNC+H/R group. Compared with the Old+Ad-shNC+H/R group, the expression level of GSK3β protein was decreased, the expression levels of total ITPR1, GRP75 and VDAC1 proteins were unchanged, whereas the phosphorylation levels were decreased in the Old+Ad-shGSK3β+H/R group. Immunoprecipitation showed that GSK3β could interact with ITPR1, GRP75 and VDAC1 proteins. Conclusions The expression level of GSK3β is up-regulated in senescent RTEC. Down-regulating GSK3β expression may reduce the phosphorylation level of ITPR1-GRP75-VDAC1 complex, constrain the overload of mitochondrial calcium ion, protect mitochondrial function and mitigate cell damage during reperfusion.

Ischemia-reperfusion injury(IRI)and rejection are the main factors affecting the long-term survival of transplant kidneys.IRI or rejection reactions can cause endoplasmic reticulum stress(ERS)leading to renal injury.X-box binding protein 1(XBP1),as the main ERS-related protein regulating cell homeostasis,generates spliced XBP1 after splicing introns by inositol-requiring enzyme 1α(IRE1α),thereby affecting the expression of target genes and reshaping the cell environment.Appropriate ERS can promote cell survival.However,when the threshold is exceeded,excessive expression of XBP1 can lead to cell instability or death,thereby causing an imbalance in the renal internal environment,which is related to the pathogenesis and progression of kidney transplant-related injury.Therefore,the effective activation of XBP1 has a protective effect on stress injury and helps maintain the vitality and integrity of the renal system.This article reviews the ERS pathway IRE1α-XBP1,the role of XBP1 in renal parenchymal cells and immune cells,the role of XBP1 in renal ischemic injury and rejection,and the clinical detection and potential therapies targeting XBP1.It explores the potential value of targeting XBP1 in kidney transplant-related injury,aiming to provide new targets and directions for improving the prognosis of kidney transplantation.

Objective:To explore the protective effect of methyl eugenol (Me) on islet ischemia/reperfusion (I/R) injury and elucidate its underlying mechanism.Methods:The islets were isolated and purified from 6-8 week male BALB/c mice and divided into four groups of normal control (normal culture without any treatment), hypoxia/reoxygenation (H/R treatment), H/R+ dimethyl sulfoxide (DMSO dosing plus H/R treatment) and H/R+ Me (Me dosing plus H/R). Viability of islet cells in each group was detected by acridine orange (AO)/propidium iodide (PI) double stain.Function of islet cells (insulin secretion) was measured by enzyme-linked immunosorbent assay (ELISA). Murine islet β Min6 cells were selected for detecting the effect of Me on the proliferative activity of normal cultured and H/R treated islet cells under different concentration gradients by CCK8.Then Min6 cells were divided into four groups of normal, H/R, H/R+ DMSO and H/R+ Me.The definition of group was the same as that of primary murine islets.Flow cytometry and Hoechst 33342 nuclear stain were utilized for detecting cell apoptotic rate in each group.The protein expressions of p-JNK, p-p38, JNK, p38, Bcl-2 and Bax were detected by Western blot.And the data were processed by one-way ANOVA or t test.Results:The proportion of dead islet cells in H/R group was (29.47±2.65)% and it was significantly lower than that in normal group (7.63±1.53)%.And the inter-group differences were statistically significant ( P<0.001). The proportion of dead islet cells was (20.63±3.07)% in H/R+ Me group.It was higher than that in H/R group (29.47±2.65)% and in H/R+ DMSO group (30.13±1.50)% and inter-group difference was statistically significant ( P<0.05 & P<0.01). Under the stimulation of high glucose, the insulin secretion level of islet in H/R+ Me group was (1.76+ 0.08) mg/L, which was higher than that in H/R group and H/R+ DMSD group(1.24±0.14)mg/L and(1.27±0.05)mg/L, and the difference was statistically significant[(1.76±0.08) vs. (1.24±0.14) mg/L; (1.76±0.08) vs.(1.27±0.05) mg/L, P<0.01]. There was no significant effect on cell viability after Me dosing within a certain concentration range (0-40 μmol/L). After Me dosing (5 μmol/L), cell viability of H/R-treated Min6 cells was significantly higher than that without Me.And the difference was statistically significant[(1.19±0.03) vs.(1.00±0), P<0.01]. As compared with H/R and H/R+ DMSO groups, overall apoptotic rate declined in H/R+ Me group (Hoechst 33342 stain: 14.50%±1.05% vs. 23.30%±1.18%, 14.50%±1.05% vs. 22.77%±1.75%, P<0.001; Flow cytometry: 4.36%±0.54% vs. 21.44%±1.02%, 4.36%±0.54% vs. 21.68%±3.06%, P<0.01). The expressions of p-JNK and p-p38 were down-regulated (p-JNK: 0.77±0.06 vs. 1.03±0.05, 0.77±0.06 vs.0.93±0.04, P<0.001; p-p38: 0.80±0.05 vs. 1.01±0.08; 0.80±0.05 vs. 1.00±0.05, P<0.05) while Bcl-2/Bax ratio rose (1.62±0.13 vs. 0.72±0.10, 1.62±0.13 vs. 0.74±0.13, P<0.01). Conclusions:Me can improve the viability and function of islets and suppress the apoptosis of Min6 cells after H/R.The mechanism is correlated with JNK and p38 MAPK signaling pathways.

Objective:To compare the long-term clinical efficacy of simultaneous pancreas-kidney transplantation (SPK) in the treatment of type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) with end-stage renal disease (ESRD) , and to explore whether T2DM with ESRD can be an indication for SPK.Methods:A total of 62 cases of SPK performed in our center were retrospectively analyzed. Based on the same baseline principle, the patients were divided into T1DM group (30 cases) and T2DM (32 cases) according to the primary disease of diabetes.Results:There was no significant difference in male gender, preoperative hemoglobin or HbA1c between the two groups. Compared with T2DM group, both of the age at surgery [ (31.8±5.2) years vs (49.5±5.7) years, P<0.001] and body mass index [ (21.8±1.3) kg/m 2 vs (25.0±3.8) kg/m 2, P<0.001] were lower in T1DM group. Compared with T2DM, the insulin dosage in the T1DM was higher, and there was no C-peptide release, and the difference was statistically significant. The patients were followed up for 5 years. There were no significant differences in fasting blood glucose, HbA1c, C-peptide, serum creatinine or eGFR between the two groups at 1, 3 and 5 years after surgery. The incidence of pulmonary infection, BK virus infection and acute rejection of transplanted kidney in T1DM group was lower than that in T2DM group, but the difference was not statistically significant. At 1 and 3 years after transplantation, the survival rates of recipients, transplanted pancreas and kidneys were 100% in both groups. In the 5th year, one patient in T2DM group died of acute myocardial infarction at 48 months after surgery, but the renal and pancreatic grafts functioned well. The survival rate of recipients in T1DM group (100%) was higher than that in T2DM group (96.9%) on 5 years after surgery ( P=0.333) , but the difference was not statistically significant. After excluding the deceased recipients, the transplanted pancreas and kidneys in both groups survived. Conclusions:There is no significant difference in the short and long-term clinical outcomes between patients with T2DM and ESRD who received SPK and those with T1DM and ESRD. T2DM with ESRD can be an indication for SPK, but a large sample size study is still needed to further improve the selection criteria.

Objective:We employ different regimens of induction therapy in living donor ABO-incompatible kidney transplantation(ABOi-KT) recipients to compare their clinical outcomes during 6 months post-KT.Methods:A retrospective analysis was conducted for the relevant clinical data of 41 ABOi-KT recipients from June 2018 to September 2022.Thirteen recipients on induction therapy of anti-human T lymphocyte porcine immunoglobin(pATG)were enrolled in pATG group; 19 recipients on induction therapy of basiliximab in basiliximab group; 9 recipients on induction therapy of rabbit anti-human thymocyte immunoglobulin(rATG)in rATG group.Differences in age, gender, body mass index(BMI), dialysis modality/duration, sideness of donor kidney, frequency of blood group antibody treatment, dose of rituximab, basic blood group antibody titers of IgG/IgM, and the gender and BMI of recipient's donor were compared for three groups.Immune status was assessed by comparing absolute lymphocyte count before pre-treatment and within 6 months post-KT in recipients under different induction regimens among 3 groups by one-way analysis of variance.Transplant kidney function was assessed by comparing the levels of serum creatinine, estimated glomerular filtration rate(eGFR)and serum urea nitrogen using one-way analysis of variance.The incidence of delayed graft function(DGF), acute rejection(AR)and infection was compared among three groups.Results:Regarding baseline profiles, except for donor age pATG group[(60.23±6.10)years]versus basiliximab group[(51.95±6.97)years]was statistically significant( P=0.002), the differences in the remaining parameters were not statistically significant among three groups(all P>0.05). At Day 1/3/7/10/14 post-KT, absolute lymphocyte counts were(0.17±0.07)×10 9/L, (0.27±0.14)×10 9/L, (0.85±0.40)×10 9/L, (1.05±0.56)×10 9/L and(1.10±0.56)×10 9/L in pATG group and(0.69±0.04)×10 9/L, (0.18±0.21)×10 9/L, (0.57±0.44)×10 9/L, (0.67±0.45)×10 9/L and(0.81±0.46)×10 9/L in rATG group respectively.They were all higher than those in basiliximab group[(0.46±0.18)×10 9/L, (0.67±0.26)×10 9/L, (1.29±0.48)×10 9/L, (1.56±0.49)×10 9/L, (1.75±0.53)×10 9/L]and the differences were statistically significant(all P<0.05). No statistically significant difference existed in absolute lymphocyte count among 3 groups before pre-treatment and after Day 21 post-KT(all P>0.05). At Week 1/2/4/12/24 post-KT, the differences in serum levels of creatinine and urea nitrogen were not statistically significant( P>0.05). At Month 1/3 post-KT, eGFR was(47.24±14.51)and(49.94±14.31)ml·min -1·(1.73 2) -1 in rATG group and they were lower than(67.36±21.60)and(65.00±14.67)ml·min -1·(1.73 2) -1 in basiliximab group with a statistically significant difference( P<0.05). However, at Week 1/2/24 post-KT, no statistically significant difference existed in eGFR among 3 groups( P>0.05). In ATG, basiliximab and rATG groups, DGF(1 case, 1 case, 1 case), AR(2 cases, 2 cases, 1 case)and infection(4 cases, 7 cases, 3 cases)occurred during 6 months post-KT. Conclusions:Through a limited sample of single centers, no statistically significant difference existed in graft function recovery for ABOi-KT recipients on induction therapies of pATG, basiliximab and rATG.And DGF, AR and infections occurred in all three groups.However, there were little inter-group differences.

Objective:To explore the safety and feasibility of optimized pathological evaluation system of donor's kidney and modified surgery during adult dual kidney transplantation(DKT)and evaluate its effectiveness to provide more alternative protocols for kidney transplantation from extended criteria donors.Methods:DKT was performed in 10 recipients using the same protocol from June 2019 to May 2021.And retrospective reviewing was performed for clinical data, including characteristics of donors and recipients, optimized pathological evaluation system, modified surgery, treatment regimens, complications and follow-ups.Results:There were 8 male and 2 female donors with an age of(57.9±12.8)years and BMI(24.1±4.1)kg/m 2.The percentage of DCD was 70% and DBD 30%.The serum creatinine before procurement was 107.6(93.3-163.5)μmol/l.Zero-point puncture biopsy was performed for both kidneys and optimized pathological evaluation system was implemented(Banff criteria & Remuzzi score). The pathological results indicated that glomerular sclerosis for left and right kidneys were 2.0(1.5-2.0)and 1.5(1.0-2.0). And Remuzzi score for left and right kidneys were(4.4±1.2)and(3.6±1.5)points respectively.All recipients were male with an age of(43.1±9.0)years and BMI(22.2±1.9)kg/m 2.All PRAs were negative pre-operation.Modified surgery was performed in all recipients(two kidneys were implanted outside iliac vessels without patch and artery of superior kidney was anastomosed to internal iliac artery). Operative duration was(195±54.3)min and serum creatinine before discharge 125.0(102.0-199.0)μmol/L.Renal dynamic scintigraphy indicated that glomerular filtration rate was(30.0±8.2)ml/min for left kidney and(29.2±13.9)ml/min for right kidney.MRA results indicated that morphologies of renal arteries and veins were regular.The time between operation and discharge was(22.4±4.7)days.Compared with SKT, serum creatinine before discharge of DKT was lower and DGF incidence of DKT was higher without statistical significance.The time between operation and discharge was longer for DKT than that for SKT( P<0.05). The complications consisted of 20% donor derived infection(DDI)and 50% DGF.And there was no surgical complication associated with vessels and ureter.Renal function remained stable during 6-month follow-ups. Conclusions:Optimized pathological evaluation system of donor's kidney and modified surgery during adult dual kidney transplantation are both safe and feasible.The postoperative function of transplanted dual kidney is successfully restored.However, long-term follow-ups are required for evaluating its effectiveness.

Objective: To analyze the clinical characteristics of one living-related kidney transplant recipient infected with 2019 coronavirus disease (COVID-19) . Method: The clinical diagnosis and treatment of one relative renal transplant recipient after the occurrence of COVID-19 were analyzed retrospectively, including the course of onset, clinical manifestations, blood routine test, renal function, lung CT scan, nucleic acid detection, outpatient and inpatient therapies and outcomes. Result: The case was diagnosed as COVID-19 (severe type) with influenza A virus infection. The clinical symptoms were gradually relieved and the lung lesions were absorbed through the treatment of reduce and stop taking immunosuppressant, antiviral therapy of abidol/oseltamivir, prevention of bacterial infection, hormone anti-inflammatory, oxygen inhalation, nutritional support and adequate rest. Conclusion This case present typical characteristics of COVID-19 in epidemiological investigation, clinical manifestation, examination, pulmonary imaging and etiology. After comprehensive treatment including reduce and stop immunosuppressive therapy, clinical cure was achieved. The long-term effect of COVID-19 on this immunosuppressive patient remains follow-up.