Protein liquid-liquid phase separation (LLPS), a pivotal phenomenon intricately linked to cellular processes, is regulated by various other proteins. However, there is still a lack of high-throughput methods for screening protein regulators of LLPS in target proteins. Here, we developed a CRISPR/Cas9-based screening method to identify protein phase separation regulators by integrating bimolecular fluorescence complementation (BiFC) and fluorescence-activated cell sorting (FACS). Using this newly developed method, we screened the RNA-binding proteins that regulate PABPN1 phase separation and identified the tumor suppressor QKI as a promoter of PABPN1 phase separation. Furthermore, QKI exhibits decreased expression levels and diminished nuclear localization in colorectal cancer cells, resulting in reduced PABPN1 phase separation, which, in turn, promotes alternative polyadenylation (APA), cell proliferation, and migration in colorectal cancer.
Humans ; Colorectal Neoplasms/genetics* ; RNA-Binding Proteins/genetics* ; Poly(A)-Binding Protein I/genetics* ; CRISPR-Cas Systems ; Flow Cytometry ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement

Objective To investigate the protective effect of wogonin on acute kidney injury(AKI)induced by li-popolysaccharide(LPS).Methods The model of septic-induced AKI was established on male C57BL/6J mice by a single intraperitoneal injection of LPS and normal C57BL/6J mice were used as normal control group.Twenty-four male C57BL/6 mice were randomly divided into 4 groups(6 mice in each group):normal control group(NC),normal control+wogonin(NC+WOG 12.5 mg/kg),LPS model group(LPS 10 mg/kg),LPS model+wogonin(LPS 10 mg/kg+WOG 12.5 mg/kg).After LPS intervention for 24 h,serum samples were collected to detect blood creatinine(CRE)and urea nitrogen(BUN)levels.HE staining and PAS staining were performed to observe the degree of renal pathological injury.Immunohistochemistry was performed to detect the degree of expression of inflammatory markers interleukin(IL)-1 β,IL-6 and tumor necrosis factor(TNF)-α in renal tissues.PCR was performed to detect the expression of KIM-1 and NGAL in renal tissues.Western blot was performed to detect the changes in protein expression of NF-κB signaling pathway subunits P65 and PP65 in renal tissues.Results Com-pared with NC group,CRE and BUN levels in LPS group increased(FCRE=60.90,P＜0.001,FBUN=82.13,P＜0.001);compared with LPS group,these indexes decreased in LPS+WOG group(P＜0.001).PCR test re-sults showed that compared with the NC group,the expression of KIM-1 and NGAL mRNA was significantly in-creased in LPS group(FKIM-1=146.3,P＜0.001,FNGAL=161.2,P＜0.001).In contrast,KIM-1 and NGAL mR-NA expression was decreased in the LPS+WOG group(P＜0.01).Renal histopathological examination showed that compared with the NC group,renal tissues of mice had renal tubular dilatation and inflammatory cell infiltration in LPS group;compared with LPS group,the number of tubular dilatation reduced and inflammatory cell infiltration was reduced in LPS+WOG group(FHE=721.4,P＜0.001;FPAS=518.9,P＜0.001).Immunohistochemical staining showed that the expression of IL-1 β,IL-6 and TNF-α was significantly increased in the LPS group;com-pared with the LPS groups(FIL-1β=114.6,FIL-6=108.9,FTNF α=251.6,all P＜0.001),these indexes de-creased in LPS+WOG group(all P＜0.01).Further studies using Western blot showed that the NF-κB signaling pathway of LPS-treated mice had been activated and produced a hyperphosphorylated state in comparison to the NC group(FPP65=13.02,P＜0.01),yet this pathway in the LPS+WOG group showed the opposite effect,namely attenuated activity and reduced phosphorylation when the control was LPS(P＜0.01).Conclusion WOG effec-tively blocked the NF-κB signaling pathway in the LPS-induced acute kidney injury model mice,thereby attenua-ting the inflammatory response and tissue damage in the kidneys of LPS-induced acute kidney injury mice.

Objective To investigate the correlation between serum bicarbonate level and cardiovascular events in peritoneal dialysis(PD)patients.Methods The data of PD patients who underwent PD catheterization and were followed up regularly until March 31,2023 were retrospectively collected.The included patients were divided into low bicarbonate group and normal bicarbonate group according to the time-averaged serum bicarbonate level.The incidence of cardiovascular events(including coronary heart disease,heart failure,stroke,peripheral vascular dis-ease,death related to cardiovascular surgery or death due to aneurysm dissection or rupture,fatal pulmonary em-bolism,or death from other or unknown cardiovascular causes)was compared between the two groups and the risk factors for cardiovascular events were analyzed.Results At the end of follow-up,a total of 110 PD patients were included,and 34 patients had cardiovascular events.Compared with the normal bicarbonate group,the low bicar-bonate group had a higher incidence of cardiovascular events.Univariate Cox regression analysis showed that the risk of cardiovascular events in the low bicarbonate group was 4.197 times higher than that in the normal bicarbon-ate group(95％CI=2.115-8.331,P＜0.001).After adjusting for multiple confounding factors,the risk of car-diovascular events in the low bicarbonate group was 3.506 times higher than that in the normal bicarbonate group(95％CI=1.709-7.193,P=0.001).The results of multivariate competing risk model showed that the risk of cardiovascular events in the low bicarbonate group was 3.801 times higher than that in the normal bicarbonate group(95％CI=1.920-7.525,P＜0.001).Conclusion Low serum bicarbonate level is closely related to the oc-currence of cardiovascular events in patients with PD,and it is an independent risk factor for cardiovascular events in patients with PD.

Objective: To investigate the effect of lansoprazole(LPZ) on cisplatin-induced acute kidney injury in mice and renal tubular epithelial cell injury. Methods: C57BL/6J mice and renal tubular epithelial cells were divided into normal control(NC) group,lansoprazole(LPZ) group,cisplatin(CIS) group and cisplatin+lansoprazole(CIS+LPZ) group.In animal experiments,LPZ(25 mg/kg) was dissolved in normal saline,and the mice were intraperitoneally injected with LPZ for three consecutive days followed by cisplatin(20 mg/kg).Mice were fed normally for 3 days,and serum samples were collected to detect serum creatinine(CRE) and blood urea nitrogen(BUN) levels.HE staining and PAS staining were used to observe renal pathology,and transmission electron microscopy was used to observe renal ultrastructure.Western blot and immunohistochemistry were used to detect the changes in the expression levels of KIM-1,NGAL and pyroptosis-related proteins.In the cell experiment,the cells were treated with cisplatin(20 μmol/L) and LPZ(5 μmol/L) for 24 hours,and the expressions of KIM-1,NGAL and pyroptosis-related factors were detected by Western blot and Real-time PCR. Results: Compared with the NC group,CRE and BUN levels increased in the CIS group,and LPZ treatment aggravated the serological indicators of the mice(P<0.001).Histopathological examination showed that compared with the CIS group,the LPZ+CIS group had obvious renal tubular dilatation,inflammatory cell infiltration and glycogen deposition in the renal tissue(P<0.001).Electron microscopy showed swelling of mitochondria,increased membrane density and decreased or absent mitochondrial crista in CIS group,which were aggravated by LPZ+CIS group.In vivo and in vitro experiments confirmed that LPZ treatment promoted CIS-induced acute kidney injury in mice and increased the expression levels of kidney injury factors KIM-1,NGAL and key factors of renal pyroptosis(Caspase 1,GSDMD,NLRP3 and IL-18) in renal tubular epithelial cells(P<0.001). Conclusion Lansoprazole promotes cisplatin-induced acute kidney injury by enhancing pyroptosis of renal tubular epithelial cells.

Objective To investigate the clinical and pathological features and risk factors of primary IgA nephrop-athy(IgAN)with foot process effacement(FPE).Methods The clinical and Oxford-type pathological data of 300 patients diagnosed with primary IgAN by renal biopsy were retrospectively analyzed,and the influential factors of IgAN foot process fusion were analyzed by multivariate logistic regression with significant clinical indicators.Results 300 cases of adult IgAN diagnosed by renal puncture biopsy were included,the extensive FPE group accounted for 15％.156 cases(52％)were female and 144 cases(48％)were male.The median age of onset was 36(30,50)years.The median duration of renal biopsy was 8(1,24)months.The proportion of nephrotic syndrome in the extensive fusion group was higher(55.6％vs 6.7％,P＜0.05).There was no significant difference in the propor-tion of IgAN with hypertension among different degrees of FPE(P=0.092),but the systolic and diastolic blood pressure in the extensive FPE group were higher than those in the mild FPE group(P＜0.05).The levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-c),and 24-hour urinary protein in the extensive FPE group were higher(P＜0.05),but the levels of albumin,estimated glomerular filtration rate(eGFR)and hyperuricemia were low(P＜0.05).There were no significant differences in hemoglobin,creatinine,and uric acid levels between the two groups(P＞0.05).There was a higher proportion of endothelial cell prolifera-tion(E1),segmental sclerosis or adhesion(S1),and cellular or cellular fibrous crescents(C1-2)in the extensive FPE group(P＜0.05),while there was no significant difference in tubular atrophy or interstitial fibrosis(T1-2)between the two groups(P＞0.05).Univariate logistic regression analysis showed that diastolic blood pressure,eGFR,TC,TG,and LDL-c were associated with extensive FPE in IgAN patients.Multivariate logistic regression a-nalysis showed that elevated TC(OR=2.135,95％CI 1.095-4.164,P=0.026)was an independent risk factor for extensive fusion of the foot process.Conclusion Compared with mild FPE,IgAN patients with extensive FPE had higher rates of blood pressure,blood lipid,nephrotic syndrome and hyperuricemia,slightly worse renal func-tion,and more severe pathological manifestations of the Oxford-type.Elevated TC is an independent risk factor for extensive FPE in IgAN patients.

Objective : To investigate the effects of carnosine (CAR) on streptozotocin (STZ) induced renal feroptosis and inflammation in diabetic mice . Methods : Type 1 diabetes mice model were induced by STZ , and normal C57 mice were used as normal control group . The C57 mice were divided into 5 groups (6 - 8 mice in each group) : normal control group (NC) , normal control + carnosine group (NC + CAR) , STZ model group (STZ) ,STZ model + carnosine group (STZ + CAR) , STZ model + ferroptosis inhibitor group (STZ + Fer⁃1) . After feeding the mice for 16 weeks , serum samples were collected to detect blood creatinine ( CRE) and urea nitrogen ( BUN) levels . The urine of mice was collected to detect the 24 - hour urinary albumin level . HE staining and PAS staining were performed to observe the degree of renal pathological injury . Real⁃time PCR was used to detect the expression of interleukin (IL) Ⅳ1β , IL⁃6 , monocyte chemotactic protein 1(MCP⁃1) and tumor necrosis factor⁃α (TNF⁃α ) in mouse kidney tissue . The expression of reactive oxygen species(ROS) in mouse kidney was detected by immunofluorescence . Morphology of renal mitochondria was observed by transmission electron microscopy . The protein expression levels of glutathione peroxidase 4(GPX 4) and long⁃chain lipoacyl⁃CoA synthetase 4( ACSL4) , which are ferroptosis indexes , were detected by Western blot . The contents of malondialdehyde ( MDA) , glutathione ( GSH) and Fe2 + in mouse kidney tissue were determined . Results : Compared with NC group , CRE and BUN levels in STZ group increased (P < 0. 001) ; and ompared with STZ group , these indexes decreased in STZ + CAR group (P < 0. 001 , P < 0. 01) . Renal histopathological examination showed that compared with NC group , renal tubule dilatation , inflammatory cell infiltration and glycogen deposition significantly increased in STZ group ; and compared with STZ group , tubule dilatation , inflammatory cell infiltration and glycogen deposition decreased in STZ + CAR group . Electron microscope results showed that the renal mitochondria in STZ group were swollen , membrane density increased , mitochondrial ridges decreased or absent , renal tubule dilation was improved significantly in the STZ + CAR group and STZ + Fer⁃1 group , and inflammatory cell infiltration was reduced . Real⁃time PCR test results showed that compared with NC group , mRNA expression levels of inflammatory factor (IL⁃1β , IL⁃6 , MCP⁃1 and TNF⁃α ) increased in STZ group (P < 0. 001 or P < 0. 01) ; and mRNA expressions of IL⁃1β , IL⁃6 , MCP⁃1 and TNF⁃α were decreased in STZ + CAR group compared with STZ group (P < 0. 01 or P < 0. 05) . Immunofluorescence results showed that compared with NC group , ROS level in kidney tissue of mice in STZ group increased (P < 0. 001) ; and compared with STZ group , the expression of ROS in kidney tissue of STZ + CAR group decreased while ROS expression in STZ + CAR group decreased ( P < 0. 01) . Compared with NC group , GPX4 expression and GSH content in kidney of STZ group decreased ( P < 0. 001) , and ACSL4 protein expression and MDA and Fe2 + contents increased (P < 0. 01 or P < 0. 001) , GPX4 expression and GSH content increased (P < 0. 01) , ACSL4 protein expression and MDA and Fe2 + content decreased in STZ + CAR group (P < 0. 01 or P < 0. 001) . Conclusion CAR inhibits ferroptosis and inflammation in the kidney in diabetic mice induced by STZ , and improved renal pathological injury in diabetic mice .

Objective : This study aimed to assess the incidence ，etiological characteristic ，potential risk factors and clinical outcomes of multidrug-resistant (MDR) infection in patients with peritoneal dialysis associated peritoni- tis (PDAP) ． Methods : The data of PDAP patients admitted in recent 3 years in our center were collected retro- spectively to analyze the distribution of pathogenic bacteria and drug resistance．According to whether the pathogen- ic bacteria were MDR or not，they were divided into MDR group and non-MDR group．The data of the two groups were compared，and the risk factors and clinical outcomes of MDR infection were analyzed. Results : PDAP pa- tients were mainly infected with gram-positive bacteria． Staphylococcus was the most common pathogen isolated from the peritoneal fluid in the MDR group，accounting for 60. 6% (43 /71) ，and streptococcus was the most com- mon pathogen isolated in the non-MDR group，accounting for 42. 0% (21 /50) ．Multivariate analysis showed that the use of wide-spectrum antibiotics for more than 5 days within 3 months before the onset of PDAP ( OR = 3. 634， P = 0. 033) ，gram-positive bacterial infection ( OR = 3. 741，P = 0. 031 ) ，higher lipoprotein ( a) level ( OR = 1. 003，P = 0. 003) ，and higher platelet-to-albumin ratio ( OR = 1. 198，P = 0. 038) were independent risk factors for MDR infection in PDAP patients．The incidence of refractory peritonitis in MDR group (29. 6% ) was higher than that in non-MDR group ( 12. 0% ) ( χ2 = 5. 229 ，P = 0. 022 ) ． The incidence of recurrent peritonitis ( 16. 9% ) was also higher than that in the non-MDR group (4. 0% ) ( χ2 = 4. 773，P = 0. 029) ． Conclusion The main pathogen of MDR infection is staphylococcus in PDAP patients． Compared with the non-MDR group ，the MDR group has a higher proportion of wide-spectrum antibiotics used for more than 5 days within 3 months before the onset of PDAP，a higher proportion of gram-positive bacterial infection，higher lipoprotein ( a) level，higher platelet-to-albumin ratio，and an increased risk of refractory peritonitis and recurrent peritonitis.

Objective : To investigate the expression of Ca2 + -independent phospholipase A2 β (iPLA2 β) in human renal tubular epithelial cells (HK-2) induced by high glucose(HG) ，the relationship between iPLA2 β and ferroptosis and the protective mechanism of HG treated HK-2 cells． Methods: The HK-2 cells were treated with 30 mmol /L glucose，the overexpression model was constructed by transfection of iPLA2 β plasmid．Ferrostatin-1 ( Fer1) (an inhibitor of ferroptosis) and erastin (an activator of ferroptosis) were used as controls．After 36 hours of intervention，the kit detected the levels of superoxide (SOD) ，malonaldehyde(MDA) and iron in HK-2 cells．DCF immunofluorescence was used to detect intracellular reactive oxygen species ( ROS) ．The expression of ACSL4， GPX4，LPCAT3，TFR1 in HK-2 cells were measured by Western blot. Results : The expression of iPLA2 β downregulated in HG-induced injury of HK-2 cells．The levels of ROS and MDA in HK-2 cells increased，while the levels of GSH and SOD decreased．The expression of ACSL4，LPCAT3 and TFR1 decreased，and the expression of GPX4 increased in HK-2 cells．However，these indexes were improved after Fer-1 intervention．iPLA2 β overexpression could reduce the injury of HK-2 cells via attenuation of KIM-1． Further research revealed that iPLA2 β overexpression inhibited oxidative stress and ferroptosis in HK-2 cells injury induced by high glucose．Meanwhile， the improvement effect of iPLA2 β on HG-induced HK-2 cells damage could be eliminated by erastin． Conclusion iPLA2 β prevents HG-induced injury of HK-2 cells via regulating ferroptosis.

Abstract: To investigate the role and mechanism of macrophage-to-myofibroblast transition(MMT) induced by exosomes from high glucose-treated renal tubular epithelial cells. Methods: Human renal tubular epithelial cells(HK2) were divided into glucose control group(5.5 mmol/L D-glucose) and high glucose group(30.0 mmol/L D-glucose) and cultured for 48 hours. The supernatant was collected by ultracentrifugation and identified by transmission electron microscopy and Western blot. PKH67 labelled exosomes were used to stimulate THP-1 macrophages. Laser scanning confocal microscopy was used to observe the phagocytosis process. The expression of inducible nitric oxide synthase(iNOS), α-SMA and CD206 was detected by flow cytometry and Western blot to determine the best concentration and time point. Laser scanning confocal microscopy was used to detect the fluorescence co-expression of rabbit polyclonal to mannose receptor(CD206), α-smooth muscle actin(α-SMA), collage type-Ⅳ(Col-IV) and fibronectin(FN). The expression of tumor growth factor-beta 1(TGF-β1), interleukin(IL)-6,IL-10 was separately detected by real-time quantitative PCR(qRT-PCR) and enzyme-linked immunosorbent assay(ELISA). The expression of TGF-β1, mothers against decapentaplegic homolog 3(Smad3), phosphorylated-smad 3(p-Smad3) in THP-1 macrophages was detected by Western blot. Results: The expression of cluster of differentiation 63(CD63)and tumor susceptibility gene 101 protein(TSG101)was positive, while the Calnexin protein was negative in the supernatant, confirming that the specimen was exosomes with high purity. THP-1 macrophages could internalize each group of exosomes. 40 mg/L exosomes for 96 hours were the best experimental condition. After being stimulated by high-glucose-exosomes, the percentage of M1 macrophages would climax in 24 hours, and the rate of M2 macrophages would climax in 96 hours. Immunofluorescence staining showed that exosomes released by HG-treated HK2 induced CD206, α-SMA, Col-IV and FN accumulation in cultured THP-1 macrophages. Compared with normal-glucose-exosomes, high-glucose-exosomes increased the expression of TGF-β1, IL-10 in M2 macrophages and decreased the expression of IL-6(allP<0.05). Moreover, TGF-β1 and p-Smad3 proteins expression also increased significantly(allP<0.05). Conclusion Exosomes secreted by renal tubular epithelial cells in high glucose environment can induce M2 macrophages transdifferentiate into myofibroblasts, and its mechanism may be related to activation of TGF-β1/Smad3 signal pathway.

Objective : To explore the occurrence and related factors of liver fibrosis in patients with End-stage re- nal disease (ESRD) ． Methods : A total of 83 ESRD patients were included in the study．Transient elastography was used to diagnose whether hepatic fibrosis occurred or not．According to the occurrence of hepatic fibrosis or not，the included patients were divided into a non-hepatic fibrosis group(n = 37) and a hepatic fibrosis group(n = 46) ．The demographic data and clinical laboratory indexes of the two groups were compared．Statistically signifi- cant variables were selected and included in the multivariate Logistics stepwise regression analysis to explore the in- fluencing factors of liver fibrosis in ESRD patients. Results : The prevalence of liver fibrosis in ESRD patients was 55. 42% ．Compared with the non-hepatic fibrosis group，the hepatic fibrosis group had lower white blood cells，e- rythrocyte sedimentation rate，complement C3 and C4(P＜0. 05) ．Multivariate Logistics analysis showed that com- plement C4 level ( OR = 0. 930，95% CI : 0. 872 －0. 992，P = 0. 028 ) and higher lactate dehydrogenase ( LDH) level ( OR = 1. 016，95% CI : 1. 005 －1. 027，P = 0. 004) were the independent influencing factors of liver fibrosis in ESRD patients． Conclusion The probability of liver fibrosis in ESRD patients is high．Serum complement C4 and LDH are independent influencing factors of liver fibrosis． Dynamic monitoring of serum complement C4 and LDH levels is conducive to target liver fibrosis in ESRD patients.