Objective To observe the changes of foot processes,expression and distribution of transient receptor potential cation channel 6 (TRPC6) in podocytes by puromycin aminonucleoside (PAN) and dexamethasone (DEX) intervention,then to investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases.Methods Podocytes cultured in vitro were divided into three group:control group,PAN stimulation group and DEX intervention group.Mouse podocyte cell line (MPC5) were cultured in 0.02％ dimethyl sulfoxide (DMSO) in control group,subjected to PAN (50 μg/ml) treatment alone or with DEX (1 μmol/L) in other two groups for 8 h,24 h,48 h.The podocyte morphology was observed and took pictures by phase-contrast microscope,then the differences of morphology and areas were analyzed.The distribution,mRNA expression and protein expression of TRPC6 were detected by indirect immunocyto-fluorescence,real-time quantitative PCR and Western blotting,respectively.Results The well-developed podocyte arborization and interconnection was formed in control group,but PAN led to significant shrinkage of podocytes (P ＜ 0.05),together with podocyte foot process retraction,effacement and loss of cell contact.DEX significantly prevented the shrinkage and apoptosis of podocytes.The apoptosis rate was significantly increased after PAN stimulated 48 h (P ＜ 0.05).Real-time quantitative PCR and Western blotting found TRPC6 mRNA and protein expression were prone to increase in PAN group compared with control group (P ＜ 0.05).The distribution of TRPC6 becamed abnormal in PAN group.DEX decreased TRPC6 mRNA and protein expression at 48 h compared with PAN group (P ＜ 0.05).The abnormal distribution of TRPC6 was also alleviated by the protection of DEX.Conclusion DEX exerts a direct action to podocyte which retains the integrity of slit diaphragm against podocyte injury,and alleviates proteinuria via stabilizing mRNA,protein expression and distribution of TRPC6.

Objective To investigate the role of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway in Dexamethasone (DEX) inhibiting podocytes apoptosis which was induced by Puromycin (PAN).Methods Mouse glomerular podocytes were cultured in vitro,and were divided into control group,dimethyl sulfoxide (DMSO) group,PAN group,DEX group,and LY294002 (inhibitor of PI3 K) group.The mRNA expression of CD2-associated protein (CD2AP) was measured by using real time fluorescent quantitative polymerase chain reaction,and intracellular distribution was detected by using indirect immunofluorescence staining.Co localization of CD2AP and p85 was detected by using confocal fluorescence microscopy.The expressions of Akt,phosphorylated (p)-Akt,glycogen synthase kinase-3β (GSK3 β) and phosphorylated (p)-GSK3β were evaluated by using Western blot.Results The expressions of CD2AP mRNA in PAN group at each time point (8 h,24 h,48 h) (1.11 ± 0.16,0.78 ±0.09,0.56 ± 0.43) were significantly lower than those in the control group (1.90 ± 0.26,2.09 ± 0.12,2.28 ±0.95),and the differences were statistically significant (all P ＜ 0.05);CD2AP distributed in foot process with uniform filament and discontinuous coarse particle around perinuclear;CD2AP and p85 distributed in cell membrane and cytoplasm evenly in control group,but accumulated in nuclei in the PAN group.The expressions of CD2AP mRNA in DEX group at each time point (8 h,24 h,48 h) (1.53 ± 0.14,2.15 ± 0.27,2.13 ± 0.15) were significantly higher than those in the PAN group,and the differences were also statistically significant (all P ＜ 0.05);the distribution density and range of CD2AP were greater than those in the PAN group,and the accumulation with p85 in nuclei decreased obviously.The expressions of p-Akt and p-GSK3β were inhibited by PAN in a dose-dependent manner (P ＜0.05).The expressions of p-Akt and p-GSK3 β were lowest after PAN stimulated at 15 min and 30 min respectively.However,the expressions of p-Akt and p-GSK3 β increased depending on the concentration of DEX (P ＜ 0.05).In addition,the expressions of p-Akt and p-GSK3 β could be blocked by LY294002 (P ＜ 0.01).Conclusion DEX can protect podocytes and inhibit podocytes apoptosis through stabilizing the expression and distribution of CD2AP.The stale expression of PI3K/Akt signaling pathway is the key factor in DEX protecting podocytes.

Objective To observe the expression and distribution of α-actin-4 mRNA through puromycin aminonueleoside(PAN) injury podocyte,and discuss the relation between α-actin-4 and podocyte damage.Methods Podocytes were cultured in vitro,and 2 groups were set up:control group and PAN stimulation group.The control group was cultured with concentration of 100 mL/L FBS RPMI 1640 nutrient solution culture,while the PAN group was cultivated to PAN(50 mg/L) treatment,and cell morphology,extraction of total RNA of α-actin-4 were observed in 8 h,24 h and 48 h.The podocyte morphology was observed and pictures were taken through phase-contrast microscope,then the differences of morphology and areas between the 2 groups were analyzed.The distribution and mRNA expression of α-actin-4 were detected by indirect immunocyto-fluorescence and real-time quantitative PCR,respectively.Results The well-developed podocyte arborization was formed after the in vitro induction,and the PAN treatment led to the podocyte foot process retraction and effacement together with the mouse podocyte cell line shrinkage and the loss of cell contact.The above time point α-actin-4 mRNA expressions between the 2 groups were compared,and there was no significant difference in 8 h (P ＞ 0.05),but significant difference was found in 24 h,48 h,α-actin-4 higher mRNA expression,with statistical significance(all P ＜0.01).α-actin-4 in the control group had thin filaments evenly distributed in the cytoplasm,but a radioactive distribution in foot process.In the experimental group,α-actin-4 pressure silk fiber was shorter,with disordered arrangement,and PAN stimulus after 24 h,α-actin-4 distribution in cytoplasm was decreased significantly,while cytoplasmic distribution was missing after 48 h.Conclusions The abnormal of distribution and mRNA expression of α-actin-4 is time-related to the PAN injury podocyte,and α-actin-4 is an important part of podocyte damage mechanism.

Objective To observe the effect of puromycin aminonucleoside(PAN) on the expressions of CD2associated protein (CD2AP) and phosphatidylinositol 3-kinase (PI3 K) p85 in induced podocytes,and to explore the significance of abnormal expression of CD2AP in apoptosis of podocytes in vitro.Methods Mouse podocytes were injected into the control group and the PAN treatment group (PAN group).The cells were cultured for 8 h,24 h and 48 h respectively.The podocyte morphology was observed by phase-contrast microscope.The apoptosis ratio of podocytes was determined with flow cytometry.The mRNA expression of CD2AP was detected by real time fluorescent quantitative polymerase chain reaction.The protein expressions of CD2AP and PI3K p85 were detected by Western blot,respectively.The distributions and the relationships between CD2AP and PI3K p85 in the podocytes were detected by confocal fluorescence microscopy.Results PAN treatment led to significant shrinkage of podocytes with decreasing distribution tendency,podocyte foot process retraction as well as effacement and loss of cell contact.Compared with the control group,the apoptosis rate was increased at 24 h and 48 h[(7.52 ± 1.35)％,(17.09 ±2.53)％ vs (4.32 ±0.81％,(6.81 ± 1.34)％] in PAN group and the differences were significant (t =4.98,8.79,all P ＜ 0.05) ;CD2AP mRNA expression tended to decrease at each time point,and the differences were significant (0.34 ± 0.12,0.46 ±0.21,0.47 ± 0.10 vs 0.62 ± 0.23,0.97 ± 0.14,0.96 ± 0.23),and there were statistical differences (t =2.64,4.95,4.79,all P ＜ 0.05) ;CD2AP and PI3K p85 protein expressions tended to decrease at 24 h (0.36 ± 0.12,0.61 ± 0.20 vs 0.64 ±0.23,0.97 ±0.31) and 48 h(0.27 ± 0.15,0.48 ± 0.20 vs 0.51 ± 0.20,0.84 ± 0.35),and the differences were significant(t =2.64,2.31,2.35,2.40,all P ＜ 0.05).In the control group,the distribution of CD2AP was uniformly filamentary structure in cytoplasm and nucleus of podocytes,after PAN treatment the distribution of CD2AP changed to discontinuous coarse granular concentrated in the perinuclear.Immunocyte fluorescence showed that the distribution and the relationships between CD2AP and PI3K p85 in the podocytes became abnormal.Conclusions When the apoptosis of podocytes was induced by PAN,the expression and distribution of CD2AP were abnormal.CD2AP may play an important role in the survival of podocytes though the PI3K signaling pathway.

Objective To observe the formation of autophagosome,the expression and distribution of autophagy-related protein LC3-Ⅰ,LC3-Ⅱ and Beclin-1 in adriamycin nephropathy rats at different pathological periods,to explore the relationship between autophagy and renal tissue injury,the occurrence of proteinuria,the progression of renal disease.Methods Sixty normal male SD rats were randomly divided into control group (n=30) and model group (n=30),the rats in model group were injected with adriamycin(6.5 mg/kg) via tail-vein for one time,while the rats in control group were injected with saline.Urine protein quantitation of 24 hour,the levels of serum albumin and total cholesterol were measured serially at the 2,4,6,8,10 weeks.The changes of kidney tissue pathology were detected after HE,PAS and Masson staining by light microscope.The formation of autophagy were detected by transmission electron microscopy,the localization and distribution of LC3-Ⅰ,LC3-Ⅱ and Beclin-1 were detected by indirect immunofluorescence staining in kidney tissue,the autophagy-related proteins LC3-Ⅰ,LC3-Ⅱ and Beclin-1 expression was detected by Western blotting.Results In model group,urinary protein began to increase at the first two weeks,serum albumin decreased at the same time,and total cholesterol increased in the four weeks.There was a statistically significant difference compared with the control group (P ＜ 0.01).The Scr and BUN were increased slightly at the four weeks in model group,and showed the deterioration of renal function after the eight weeks.There was a statistically significant difference compared with the control group (P ＜ 0.01).Mesangial cell proliferation,mitochondrial swelling and foot process broadening and integration appeared early in the model group,while foot process disappearing and nuclear pyknosis were observed in the late by transmission electron microscope;Renal pathology gradually changed from mesangial proliferation to focal segmental glomerulosclerosis (FSGS) by light microscope.A low expression of autophagy was detected in renal tissue of control group rats by transmission electron microscopy and immunofluorescence microscope;in model group,with the progression of disease,the autophagy was significantly enhanced and maintained at a high level.With the progression of disease,the autophagyrelated proteins LC3-Ⅰ,LC3-Ⅱ and Beclin-1 was significantly enhanced in the model group than the control group (P＜0.05).Conclusion Autophagy is involved in renal tissue injury and the occurrence of proteinuria,closely related to the progression of renal disease.

Objective To observe the expression of mRNA of podocin,nephrin,CD2AP and α - actin - 4 in Doxorubicin - induced nephrotic(ADN)rats,and explore the possible mechanisms of podocyte molecule during the de-velopment of proteinuria. Methods Forty - eight Sprague - Dawley(SD)rats were divided into ADN model group(in-jected with 6. 5 mg/ kg Doxorubicin in tail vein,n = 24)and control group(injected with saline solution in tail vein,n =24). After the nephropathy model was established,6 rats were killed at the end of 1st ,2nd ,4th ,6th week in each group. The changes of the following indicators were observed:(1)24 - hour urinary protein,serum albumin and cholesterol were detected;(2)mRNA expression of nephrin,podocin,CD2AP and α - actin - 4 in cortex of kidney were examined by real time fluorescence quantification PCR. Results The model group came out massive proteinuria(15. 66 ± 1. 50) mg/ 24 h,(45. 98 ± 1. 45)mg/ 24 h,(65. 58 ± 4. 68)mg/ 24 h,(82. 83 ± 8. 43)mg/ 24 h in 1,2,4,6 weeks respec-tively,hypoalbuminemia(27. 4 ±2. 5)g/ L,(23. 6 ±2. 9)g/ L,(20. 6 ±1. 5)g/ L,(6. 9 ± 2. 3)g/ L in 1,2,4,6 weeks respectively and hypercholesterolaemia(2. 00 ± 0. 25)mmol/ L,(2. 16 ± 0. 44)mmol/ L,(4. 02 ± 0. 81)mmol/ L, (7. 54 ± 1. 12)mmol/ L in 1,2,4,6 weeks respectively,and the differences of proteinuria,plasma albumin and total cholesterol compared with control group at each time point had statistical significance(all P ﹤ 0. 01). Compared with the control group,podocin mRNA expression in the model group decreased at the end of 1st week(10. 56 ± 3. 62),de-creased significantly at the end of 2nd week(20. 44 ± 9. 03),and decreased at the end of 4th week(2. 19 ± 0. 18)com-pared with the control group;nephrin mRNA expression decreased at the end of 1st week(2. 41 ± 1. 10)and reached to the peak value,decreased at the end of 4th week(0. 52 ± 0. 18);CD2AP mRNA expression did not change significantly in the 1st week(4. 17 ± 0. 79),increased at the end of 2nd week(6. 74 ± 1. 53),reached to the peak value at the end of 4th week(6. 91 ± 1. 13),but did not change significantly at the end of 6th week(4. 04 ± 0. 82);α - actin - 4 mRNA ex-pression did not change significantly at the end of 1st week(1. 75 ± 0. 48),decreased at the end of 2nd week(2. 01 ± 0. 55),reached to the peak value at the end of 4th week(2. 24 ± 0. 81),but did not change significantly at the end of 6th week(1. 39 ± 0. 18). Compared with the control group,the difference had statistical significance( all P ﹤ 0. 05). Conclusion The abnormal expression of podocyte molecules mRNA in ADN rats may be an important molecular mechanism in the development of proteinuria.

Objective To investigate the effect of Puromycin(PAN)and Tacrolimus(FK506)on the expre-ssion of podocin in podocytes,and discuss the mechanism of FK506 in improving proteinuria caused by the damage of glomerular podocytes.Methods Mouse podocyte were cultured and divided into 3 groups,which were control group, PAN group and FK506 group,respectively.The changes of each group after 8 hours,24 hours and 48 hours of treatment were detected by using phase-contrast microscope,and the expression and distribution of protein and mRNA were tested by adopting Western blot,quantitative real-time PCR and immunofluorescence technique.Results Compared with the control group(8.54 ± 0.25,8.27 ± 0.07,7.45 ± 0.13)at each time point(8 hours,24 hours and 48 hours),the soma size of the PAN group(6.41 ± 0.22,4.96 ± 0.09,3.76 ± 0.16)was reduced.But in the FK506 group(7.67 ± 0.06, 6.62 ± 0.04,5.57 ± 0.27),it was increased at each time point(8 hours,24 hours and 48 hours)compared with the PAN group.The podocytic process and the intercellular connection disappeared,and the distribution was significantly scattered.The mRNA(0.87 ± 0.15,0.78 ± 0.15,0.58 ± 0.12)and protein(0.82 ± 0.02,0.62 ± 0.03,0.50 ± 0.02) expressions of podocin increased in FK506 group and mRNA(0.63 ± 0.12,0.56 ± 0.01,0.48 ± 0.02),protein (0.71 ± 0.03,0.46 ± 0.01,0.34 ± 0.02)were observably reduced in PAN group at each time point(8 hours,24 hours and 48 hours)and showed abnormal distribution in PAN group,compared with the control group[podocin mRNA (1.22 ± 0.15,1.18 ± 0.06,0.87 ± 0.30),protein(0.86 ± 0.03,0.87 ± 0.03,0.61 ± 0.07)].Conclusions PAN attenuates the mRNA and protein expression of podocin by damaging podocytes,inversely,while FK506 protects poto-cyte injury by stabilizing the mRNA and protein expression of podocin,which maybe involve inhibition of proteinuria.It can be used as a target for the study and treatment of kidney diseases.

Objective To explore the relationship between posterior reversible encephalopathy syndrome (PRES) and the treatment of immunosuppressants such as cyclosporine A (CsA) and tacrolimus (FK506) in children with nephrotic syndrome.Methods The clinical data of nephrotic syndrome children with PRES caused by immunosuppressants who were hospitalized in Guangzhou First People's Hospital from June 2014 to May 2017 were collected.Their clinical characteristics,imaging features,treatments and prognosis were analyzed.Results A total of 23 children were enrolled,including 13 children with CsA and 10 children with FK506.In the concurrent of PRES 20 cases were in the activity stage of nephrotic syndrome,with large amounts of urinary protein,obvious edema,hypoalbuminemia and hyperlipidemia;while 3 cases were in the remission of nephrotic syndrome.The main clinical symptoms of PRES were hypertension,headache,epileptic attack,consciousness disorder,visual disorder and so on.Sixty-nine point six percent of children were using high dose immunosuppressive agents,and 78.3％ had high drug concentration.The cranial magnetic resonance imaging (MRI) results of 17 patients showed that they had T1 weighted (T1WI) hypointense,T2 weighted (T2WI) and fluid-attenuated inversion recovery (FLAIR) images hyperintense,as well as iso-and slight hypointense of diffusion-weighted image (DWI) in parietal-occipital regions or complicated with frontal lobes or basal nuclei region.Computer tomography (CT) examinations of 6 cases showed low-density focus of the occipital lobes.Children were relieved muscular spasm,debased intracranial hypertension,improved circulation,discontinued or reduced immunosuppressants at the onset of PRES.After these treatments,21 patients' symptoms and signs disappeared within one week;two patients suffered convulsions 2 times in one week,but recovered after one month.After three months 5 children had MRI and CT re-examination and it showed that their brain lesions disappeared.Conclusions PRES may be related to the dose and blood concentration of immunosuppressive agents.The immunosuppressants for nephrotic syndrome children should be increased gradually with low initiating doses.Physicians need to be precautious to prevent the occurrence of PRES once neurological symptoms occur.

Objective: To investigate the effects of Tacrolimus(FK506) and Puromycin aminonucleoside(PAN) on apoptosis and expression of α-actinin-4 mRNA and protein in mouse glomerular podocytes in order to explore the protective effect of FK506 on podocytes. Methods: Mouse glomerular podocytes were cultured in vitro, and the control group, PAN group and FK506 group were established.After 8 h, 24 h and 48 h of treatment, the cell morphology was observed and the apoptosis rate was detected.The cells were collected by real-time PCR and Western blot was used to detect the mRNA and protein expression of α-actinin-4. Results: The cell body area of the PAN group was significantly smaller than that of the control group, and the cell area of the FK506 group was larger than that of the PAN group.There was no significant difference in the rate of podocyte apoptosis between those groups at 8 h (all P>0.05). At 24 h and 48 h, the apoptotic rate of podocytes in PAN group[(8.21±0.41)%, (16.32±0.17)%] were significantly higher than those in the control group[(4.28±0.35)%, (6.27±0.28)%], and the differences were significant (all P<0.05). The apoptosis rate of podocytes in FK506 group[(6.26±0.24)%, (13.32±0.24)%] were significantly lower than those in PAN group, and the differences were significant (all P<0.05). At 8 h, there was no significant difference in the expression of α-actinin-4 mRNA and protein(all P>0.05). The expression of mRNA (2.42±0.21, 3.78±0.25) and protein(0.77±0.04, 1.22±0.10) in the PAN group was significantly higher than mRNA(1.50±0.22, 2.15±0.15) and protein(0.44±0.03, 0.83±0.07) in the control group at 24 h and 48 h, and the differences were significant (all P<0.01). The expression of mRNA (1.65±0.24, 1.70±0.32) and protein (0.52±0.05, 0.56±0.07) in FK506 group was significantly lower than that of PAN group, and the differences were significant (all P<0.05). Conclusions FK506 can effectively inhibit the damage of PAN on podocytes and stabilize the expression of α-actinin-4, which provides a basis for the clinical application of FK506 in the treatment of glomerular diseases.

Objective To analyze the clinical phenotypes and mutation types of children with X-linked Alport syndrome(XLAS)with mutations in COL4A5 gene,and to explore the relationship between children with XLAS and nephrotic syndrome nephritic type.Methods Thirty-two children with COL4A5 gene mutations detected by second-generation sequencing and finally diagnosed with Alport syndrome at Guangzhou Red Cross Hospital affiliated with Jinan University and the First People's Hospital of Guangzhou between April 2016 and April 2023 were included,and their clinicopathological features and gene mutation characteristics were retrospectively analyzed.Results The mean age of onset of disease in children with XLAS was(3.68±2.07)years old,the mean age at diagnosis(6.56±2.95)years old,12 cases(37.5%)started with isolated hematuria,8 cases(25%)started with hematuria and proteinuria,12 cases(37.5%)started with nephrotic syndrome nephritic phenotype,and the positive family history of the children was found in 11 cases(34.4%),ocular lesions were found in 3 cases(9.37%),ear lesions in 6 cases(18.75%),and 7 cases(21.87%)were found to have developed chronic kidney disease(CKD)in the later follow-up.21 children underwent renal tissue puncture biopsy,and electron microscopy showed thinning of the basement membrane(diffuse or segmental)in 13 cases(61.9%),and uneven thickness of the basement membrane in 8 cases(38.09%);light microscopy showed thinning of the basement membrane in 13 cases(61.9%);light microscopy showed thinning of the basement membrane in 8 cases(38.09%);and light microscopy showed thinning of the basement membrane in 3 cases(11.5%).(38.09%);light microscopy:focal segmental glomerulosclerosis(FSGS)in 2 cases(9.52%),mesangial proliferative glomerulonephritis(Ms PGN)in 11 cases(52.38%),and minimal change disease(MCD)in 8 cases(38.09%).The type of mutation was categorized as missense mutation in 12 cases(37.5%),shear site mutation in 9 cases(28.12%),nonsense mutation in 6 cases(18.75%),deletion mutation in 3 cases(9.37%),and code shift mutation in 2 cases(6.25%).Genetic mutations were present in 22 cases(68.75%);spontaneous mutations were present in 10 cases(27.02%).Conclusions Children with XLAS have atypical clinical manifestations and pathologic features in the early stage of the disease,and the progress is slow,and some of them are easy to be misdiagnosed as nephrotic syndrome nephritis type in the early stage,so it is important to improve the genetic test for this disease as early as possible,and to make reason-able drug choices to predict the prognosis scientifically.