Research Analysis of SLC12A3 Gene Mutation and Molecular Mechanism in Gitelman Syndrome / 罕见病研究

ObjectiveThis study aimed at conducting retrospective analysis of the clinical symptoms and genetic mutations in 20 children with Gitelman syndrome treated at the Affiliated Children′s Hospital of Nanjing Medical University from August 2015 to November 2022 and also explored the molecular mechanism of the pathogenic high-frequency mutation D486N in the Chinese population.MethodsWe collected the clinical manifestations, growth and development status, laboratory examination results, and SLC12A3 gene variations of the patients. We distinguished the wild-type and mutant SLC12A3 genes overexpressed in human embryonic kidney 293T cells (HEK293T). We used protein immunoblotting to detect the expression level of NCC, and used immunofluorescence techniques to examine the subcellular localization of NCC. In addition, we investigated the impact of the high-frequency SLC12A3 gene mutation D486N on NCC protein expression and localization.ResultsIn the 20 patients with Gitelman syndrome, all of them had hypokalemia. We indemnified twenty-six SLC12A3 gene mutations, 13 of which are missense mutation, 1 of which synonymous mutation, 1 nonsense mutation, 4 frameshift mutation, and 7 splicing site mutation. Among them, four mutations (p.T235K, c.1096-1G > A, p.A464A, and c.2660+1_2660+2insT) were novel mutations.ConclusionsWe found the preliminary evidence that the high-frequency mutation D486N in the Chinese population affected the expression of total and membrane-bound NCC protein and influenced the membrane localization of NCC protein. The findings of this study provides experimental evidence for genetic counseling, diagnosis, and treatment of Gitelman syndrome.

Clinical analysis of antineutrophil cytoplasmic antibody-associated vasculitis in 13 children / 中华肾脏病杂志

Objective:To investigate the clinical manifestations, pathological characteristics, treatment and prognosis of anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) in 13 children.Methods:The clinical and pathological data of 13 cases of AAV in children′s Hospital of Nanjing Medical University from June 2000 to December 2021 were retrospectively analyzed.Results:Among the 13 cases, 12 cases were diagnosed with microscopic polyangiitis (MPA) and 1 case was granulomatosis with polyangiitis (GPA), including 10 females and 3 males. The onset age ranged from 3 years and 11 months to 13 years and 10 months. The most frequently involved organ was the kidney (12 cases, 92.3%), followed by respiratory system (7 cases, 53.8%), skin (5 cases, 38.5%), digestive system (4 cases, 30.8%), nervous system (4 cases, 30.8%) and cardiovascular system (3 cases, 23.1%). There were 10 cases with orthotic anemia, 7 cases with positive antinuclear antibody, and 3 cases with mildly decreased complement C3. Among the 12 children with renal impairment, 9 cases were accompanied by abnormal renal function at the beginning of the disease. Renal biopsy was classified according to the Berden as follows: sclerotic in 5 cases, crescentic 3 cases, focal in 2 cases and mixed in 2 cases. All children were treated with glucocorticoid combined with immunosuppressant. During the follow-up time from 8 months to 128 months, 4 cases acquired complete remission, 8 cases achieved partial remission and 1 case recurred after complete remission, and 7 cases progressed to chronic kidney disease stage 5. Three children with complete remission underwent repeated renal biopsy, including 2 cases of mixed type and 1 case of crescent type initially, and all changed to focal type.Conclusions:AAV in children occurs mainly in school-age female, and most of AAV in children is MPA. The clinical manifestations are various. Most of them have renal damage and anemia, and lung damage is also common. Patients with skin purpura onset may be misdiagnosed as Henoch-Schonlein purpura, and AAV with ANA positive or complement reduction should exclude systemic lupus erythematosus. Once the renal function is abnormal in AAV, especially estimated glomerular filtration rate<60 ml·min -1·(1.73 m 2) -1 and the pathological classification is sclerotic type or crescent type, it is difficult to reverse even after active treatment. Early diagnosis and treatment are very important for AAV.

New advance in hypophosphatemic rickets / 中华实用儿科临床杂志

Hypophosphatemic rickets(HR) is a bone mineralization disorder caused by phosphate wasting, including hypophosphatemia, bone abnormalities and short stature.X-linked hypophosphatemia(XLH) is the most common inherited disease related to phosphate metabolism, which might result in elevated levels of fibroblast growth factor 23 (FGF23). FGF23 plays an important role in the disease mechanism, so a human anti-FGF23 antibody is developed as a potential treatment for XLH.In many clinical trials, subcutaneous Burosumab increased serum phosphorus levels in pediatric and adult patients with XLH.With the development of phase 1-3 clinical trials of Burosumab in children and adults with XLH, the efficacy and safety of Burosumab is proven to be superior to that of phosphate and calcitriol used in traditional therapy.This review aims to investigate the physiopathology and treatment of HR and to enhance the recognition of HR.

Analysis of clinical diagnosis and treatment of 20 children with hypophosphatemic tickets / 中华实用儿科临床杂志

Objective To analyze the clinical diagnosis and treatment data of 20 children with hypophosphatemic rickets (HR) in order to improve the clinical diagnosis and treatment of HR.Methods The retrospective analysis of clinical data of 20 cases with HR who were hospitalized at Children's Hospital of Nanjing Medical University from May,2010 to April,2016 was performed to summarize the clinical characteristics.All patients were analyzed for the phosphate regulating gene with homologies to endopeptidase on the X chromosome(PHEX) gene by direct sequencing.If no mutations were detected,multiplex ligation-dependent probe amplification analysis was performed.Results All of the 20 cases with HR showed different degrees of growth retardation and typical X-ray rickets.After treatment,the clinical features were improved.Height standard deviation score (HSDS) was improved significantly with longer treatment time,and the difference was statistically significant(P =0.027).There was a correlation between the blood phosphorus fluctuation and secondary hyperparathyroidism(P ＜ 0.05).Nineteen cases had PHEX gene mutations.Truncating mutations was the most frequent mutation type,and 4 new mutations were found.Conclusions Clinical characteristics,laboratory test results and X-ray examination are important clinical index for the diagnosis of HR,and PHEX gene test can be used as an important auxiliary diagnostic tool.Early diagnosis and treatment can significantly improve the clinical manifestations of the patients.

Clinicopathological features and prognosis of minimal change nephropathy with IgA deposition in children / 中华实用儿科临床杂志

Objective To analyze the clinicopathological features and prognosis of minimal change nephropa-thy with IgA deposition(MCD-IgA)in children.Methods The clinical and pathological data of 10 cases in Chil-dren's Hospital of Nanjing Medical University from January 2010 to December 2015 with MCD-IgA were retrospective-ly analyzed,and 24 cases of minimal change nephrotic syndrome(MCD-NS)and 21 cases of IgA nephropathy clini-cally manifested with nephrotic syndrome(NS-IgAN)were selected as controls.Results (1)Clinical manifesta-tions:there were no significant differences in age,gender,incidence of hematuria,level of 24 hours urine protein,serum albumin and cholesterol levels and elevated serum IgA ratio in MCD-IgA compared with MCD-NS group.Compared with MCD-IgA and MCD-NS,NS -IgAN group showed older age of onset[(8.6 ± 2.1)years vs.(4.8 ± 2.4) years,(4.0 ± 1.6)years],higher level of serum albumin[(22.8 ± 4.3)g/L vs.(19.0 ± 1.9)g/L,(16.8 ± 3.0) g/L],and lower level of serum total cholesterol[(7.9 ± 1.9)mmol/L vs.(9.9 ± 2.7)mmol/L,(9.8 ± 2.1)mmol/L], and all the differences were significant(all P<0.05).NS-IgAN group was all associated with gross hematuria.(2) Pathology:the light microscope lesions in MCD-IgA and MCD-NS group were mild,but it was usually associated with severe histologic lesions in NS-IgAN,such as endocapillary proliferation,segmental sclerosis,crescent formation,tuft necrosis and chronic tubulointerstitial lesions;in MCD -NS group,immunofluorescence was negative. In MCD -IgA group,IgA deposition intensity was weak(less than + +),and 3 cases(30.0%)were accompanied with C3deposi-tion.In NS-IgAN group,IgA deposition intensity was stronger(more than + + +),and most of the cases were accom-panied with C3and other immunoglobulins deposition.Under electron microscope,both MCD-IgA and MCD-NS showed wide foot process effacement,and a small amount of mesangial electron dense deposit was detected in 9 cases of MCD-IgA.In NS-IgAN group,large amount of electron dense deposit was found in the mesangial region,and only 8 cases (38.0%)showed more than 50% of foot process effacement.(3)Prognosis:in MCD-IgA group,9 patients were ster-oid-dependent or frequently relapsed,1 case showed steroid-resistance,6 patients required additional agents.Except 1 case lost,with an average of(61.5 ± 28.8)months were followed up,8 patients achieved complete remission;In MCD-NS group,20 cases were steroid-dependent or frequently relapsed,4 cases were steroid-resistant,23 cases re-quired additional immunosuppressive agents.Followed up for an average of(36.4 ± 12.5)months,22 cases(91.7%) achieved complete remission;In NS-IgAN group,all cases were steroid-resistant and combined with cyclophospha-mide treatment;followed up for an average of(38.6 ± 15.2)months,19 cases(90.5%)achieved complete remission. Conclusions The clinical manifestations and prognosis of MCD-IgA were similar to MCD-NS,but the clinical and pathological findings of MCD-IgA were different from those of NS-IgAN.It is deduced that the nature of MCD-IgA is still a MCD,and that the IgA deposition may be nonspecific.

Clinical characters and risk factors for Henoch - Schonlein Purpura combined with cardiac damage in children / 中华实用儿科临床杂志

Objective To summarize the clinical characteristics and laboratory test results of children with Henoch - Schonlein purpura(HSP),and further to analyze the risk factors for HSP combined with cardiac damage. Methods The clinical and laboratory tests findings from 707 children diagnosed as HSP at Nanjing Children's Hospi-tal were retrospectively analyzed,who were recruited from November 2011 to December 2012. The possible risk factors for HSP with cardiac damage in children were recorded,including gender,age,predisposing causes,gastrointestinal symptoms,joint pain,kidney disorders,serum electrolytes,anti - streptolysin 〝O〝 test,erythrocyte sedimentation rate, and complement level were summarized. Chi - square test and Logistic regression were performed to analyze the risk fac-tors of cardiac damage in children with HSP. Results Among 707 cases,192(27. 2% )patients were combined with car-diac damage,115 male and 77 female,and the proportion of men to women was 1. 00: 0. 67;age ranged from 11 months to 15 years and 4 months(6 years and 5 months for median age),6 patients ﹤ 3 years old occupying 3. 1% ,103 patients≥3 - 7 years old occupying 53. 7% ,82 patients≥7 - 14 years old occupying 42. 7% ,1 patient≥14 years old occupying 0. 5% ,and the age of onset in preschool and school age. Electrocardiogram(ECG)abnormalities were found in 190 patients,the main manifestations including long Q - T interval,ST - T segment falling down and sinus bradycar-dia,and one or more items of abnormal myocardial enzymes existed in 24 cases;echocardiography was performed in 35 cases of children,but no abnormality was detected,no obvious symptoms such as flustered or chest tightness or precor-dial distress. Statistical analysis showed that gender,predisposing causes,mixed HSP,complement level were related to the incidence of cardiac damage in children with HSP(P ﹤ 0. 05). Furthermore binary Logistic regression identified that in male patients,the ratio of X1 vs OR ratio was 0. 654(95% CI 0. 462 - 0. 926,P ﹤ 0. 05),for predisposing causes,the ratio of X2 vs OR ratio was 2. 63(95% CI 1. 838 - 3. 765,P ﹤ 0. 001),for mixed HSP,the ratio of X3 vs OR ratio was 2. 452(95% CI 1. 301 - 4. 621,P ﹤ 0. 01),which were independent factors for cardiac damage in chil-dren with HSP. Conclusions ECG and/ or myocardial enzyme spectrum abnormalities are the main clinical ma-nifestations of cardiac damage in children with HSP. Male patients,predisposing causes of the respiratory tract infec-tion,mixed HSP and hypocomplementemia were high risk factors in the development of cardiac damage,which require special consideration clinically,and earlier ECG and myocardial enzymes examination,early diagnosis and treatment are necessary to avoid the occurrence of severe cases.

Clinical and immunological features of 45 children with lupus nephritis / 临床儿科杂志

Objective To analyze the clinical and immunological features of 45 pediatric patients with lupus nephritis (LN). Methods Forty-five LN patients were included in this study. Clinical, pathological data and immunological parameters were retrospectively analyzed. Results Forty-five LN patients had 6 males and 39 females, with the mean onset age of (10.9 ± 2.8) years. Acute nephritis was the most common type, accounting for 42.2%. Nephrotic syndrome accounted for 31.1%. Renal biopsy showed class II (17.8%), III (4.4%), IV (48.9%), V (2.2%), V+III (6.7%)and V+IV (13.3%)in 42 cases. The remis-sion rate reached 91.1%in the early therapeutic stage, and 15.0%patients recurred after 24-month follow-up. Conclusions The clinical manifestations of LN children are diverse. The renal pathology is complex. The clinical manifestations in part of the chil-dren are not consistent with renal pathology.

Mitochondrial dysfunction in the process of cisplatin-induced acute kidney injury in mice / 中华肾脏病杂志

Objective To assess the characteristics of different doses of cisplatin-induced acute kidney injury,further to understand mitochondrial dysfunction and its role in acute kidney injury (AKI).Methods Male C57BL/6J mice were first randomly divided into two groups:control group (n =6) and AKI group (n =12).Then,AKI group was subsequently divided into other two groups according to different dose of cisplatin (10 mg/kg or 20 mg/kg).AKI group received intraperitoneal injection of cisplatin.All mice were sacrificed after 72 h of injection.Renal biochemical function,renal pathological changes,renal injury markers,kidney mitochondrial function and structural changes were observed.Results (1) After 72 hours of injection,the AKI group performed significant kidney injury changes compared to control group,thereinto 20 mg/kg group was more serious than 10 mg/kg group.With the cisplatin dose increasing,renal function markers such as serum creatinine,urine protein gradually increased.(2)Kidney biopsy showed tubular structural damage,the formation of protein casts,kidney injury molecule-1 (KIM-1) gradually increased(P ＜ 0.05).(3)Electron microscopy found tubular mitochondrial structural damage,mtDNA copy number decreased,the level of peroxisome proliferatoractivated receptor-gamma coactivator-1alpha (PGC-1α),ATP synthase β decreased(P ＜ 0.05),and Western blotting manifested cytochrome C was released from mitochondria to the cytoplasm.These data all exhibited significant difference between different groups(P ＜ 0.05).Conclusions Cisplatin induces acute kidney injury in dose-dependent manner.Mitochondrial dysfunction participates in kidney injury,and is also related to the kidney pathological damage.

Detection of urinary α1-antitrypsin for predicting response to glucocorticoid therapy in children with primary nephrotic syndrome / 中华肾脏病杂志

Objective To detect the α1-antitrypsin (AAT) concentration in urine samples of children with primary nephrotic syndrome (PNS) before initiation of glucocorticoid treatment,in order to verify whether it could predict the response to glucocorticoid-based therapy.Methods Forty-three children diagnosed as PNS initially were chosen as subjects,namely steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS) depending on reaction to glucocorticoid therapy four weeks later,and 15 healthy children serving as normal control.The mid stream of the first morning urine samples were collected from children before taking glucocorticoid.ELISA kit was used to quantify the urinary AAT concentration which was revised by urine creatinine further.The data of urine AAT/Cr were expressed as median with interquartile range.Data analysis was performed using the SPSS 17.0.Results AAT was absent in urine samples of normal healthy children,and there were no statistic differences of the AAT concentrations in urine between children with SSNS and SRNS [(30.4+4.5) mg/L vs (31.8+4.6) mg/L,t=-1.0,P=0.33].The level of urine AAT/Cr in children with SRNS was higher than that in children with SSNS [0.049(0.028-0.073) vs 0.028(0.022-0.036),Z=2.4,P=0.02].Among the laboratory parameters of the two subgroups before taking glucocortiod,the levels of platelet,blood white cell count,serum globulin,urine white cell count,urine red cell count,urine IgG and urine α1-microglobulin were significantly different (P＜0.05).Three parameters that included urine AAT/Cr (OR=6.81 × 1028,P=0.O05),serum globulin (OR=1.69,P=0.01) and urine α1-microglobulin (OR=1.05,P=0.009) further entered the logistic regression model to predict the SRNS independently.The ROC curve based on the level of the urine AAT/Cr was constructed,and the area under the curve (AUC) was 0.72.When the cutoff value of urine AAT/Cr was 0.035,the sensitivity and specificity of the urine AAT/Cr prediction were 68％ and 75％ respectively (Youden' s index 0.43).The AUC that based on the logistic regression model which included urine AAT/Cr,serum globulin and urine α1-mieroglobulin was improved to 0.94,and the sensitivity and specificity of the model prediction were 95％ and 83％ respectively (Youden' s index 0.78).There was no significant difference of the urine AAT/Cr level among the different pathological types of the children undergoing renal biopsy.Conclusions There are no statistic differences of the AAT concentrations in urine between children with SSNS and SRNS.The level of urine AAT/Cr is significantly higher in the SRNS than that in the SSNS which can be as a candidate biomarker to predict the response to glucocorticoid-based therapy.It has a better prediction efficacy based on the model which includes urine AAT/Cr,serum globulin and urine α1-microglobulin.

Early autophagy activation inhibits podocytes from apoptosis induced by aldosterone / 中华肾脏病杂志

Objective To explore the protection of early autophagy activation on podocyte injury induced by aldosterone.Methods In vitro cultured mouse podocyte clones (MPC5) were treated with aldosterone for 6,12,24,48 h respectively.Apoptosis of podocytes was detected by Annexin V combined with flow cytometry.After 24 h treatment with aldosterone,the existence of apoptotic body and autophagosome was observed by electron microscopy.The protein expressions of LC3,caspase-3 and nephrin were examined by Western blotting.The mRNA expression of Beclin-l was detected by real-time PCR.Results The induction of apoptosis and autophagy by aldosterone in podocytes was in timedependent mannner.After 24 h treatment with aldosterone,the apoptosis was increased by 26.5％ (P ＜ 0.05)and the expression of nephrin was decreased by 28.0％ (P ＜ 0.05) compared to control group.Aldosterone remarkably induced the expression of Beclin-1 at 6 h and promoted the transformation of LC3-Ⅰ to LC3-Ⅱat 12 h (P ＜ 0.05).Compared to simple aldosterone treatment,the apoptosis rate of podocyte was increased by 39.0％ (P ＜ 0.05) and the expression of nephrin was declined by 19.5％ (P ＜ 0.05) after 3-methyladenine (3-MA) pre-treatment.Conclusions Aldosterone can induce autophagy and apoptosis in podocytes.Autophagy occurs earlier (12 h) than apoptosis (24 h).The occurrence of autophagy can inhibit the apoptosis,so the autophagy pathway may be a new research topic of glomerular disease treatment.

Oxidative stress-dependent Ras-ERK activation involves in aldosterone-induced mesangial cell proliferation / 中华肾脏病杂志

Objective To investigate the role of oxidative stress-dependent Rasextracellular signal-regulated kinase (ERK1/2) signaling in aldosterone (ALDO)-induced mesangial cell proliferation. Methods The incorporation of 3H-thymidine (3H-TdR) and cell count were used as the measure of mesangial cell (MC) proliferation.Western blotting was used to detect the activation of Ki-RasA,c-Raf,MEK1/2,ERK1/2 and PI3K. Results Aldosterone significantly induced human mesangial cell proliferation,and anti-oxidant N-Acetylcysteine (NAC),catalase,and super oxide dismutase (SOD) significantly inhibited ALDO-induced mesangial cell proliferation (P＜0.01,respectively).Stimulation by ALDO for 3 h,Ki-RasA,c-Raf,MEK1/2,and ERK1/2 activity increased by 4.05-, 3.62-, 4.52-, and 3.40-fold compared with control group (P ＜0.01,respectively).NAC almost completely blocked ALDO-induced Ki-RasA,c-Raf,MEK1/2,and ERK1/2 activation (P＜0.01,respectively).Ki-RasA siRNA dose-dependently inhibited Ki-RasA expression, ALDO-induced Ki-RasA activation, and mesangial cell proliferation (P ＜0.01,respectively).c-Raf inhibitor GW5074 and MEK1/2 inhibitor PD98059 also reduced ALDO-induced mesangial cell proliferation by 65％ respectvely (P＜0.01).Ki-RasA siRNA had no effect on ALDO-induced PI3K phosphorylation.Combining LY294002 and PD98059 completely blocked ALDO-induced mesangial cell proliferation (P＜0.01). Conclusions ALDO-induced Ki-RasA-c-Raf-MEK-ERK signaling activation is dependent on reactive oxygen species (ROS) production,which mediates ALDO-induced mesangial cell proliferation.Inhibition of both ERK1/2 and PI3K signaling simultaneously completely blocks ALDO-induced mesangial cell proliferation.

ROS-EGFR-JNK-AP-1 signaling pathway involves in angiotensin Ⅱ-induced human mesangial cells proliferation / 中华肾脏病杂志

Objective To elucidate whether Ang Ⅱ indnces the proliferation of mesangial cells through ROS-EGFR-JNK-AP-1 signaling pathway. Methods The incorporation of 3H-thymidine (3H-TdR) and cell count were used to measure mesangial cell (MC) proliferation. ROS production was determined by DCFDA fluorescence. EGFR and JNK activation was assayed by Western blot. Results Ang Ⅱ significantly enhanced ROS production in mesangial cells, which was up-regulated by 2.26 folds of control group after incubation with Ang Ⅱ for 60 min. Ang Ⅱ induced EGFR phosphorylation in dose- and time-dependent manner, with the peak (3.96 folds increase) at 30 min. EGFR phosphorylation was significantly blocked by AT1R antagonist losartan, antioxidant NAC, and NADPH oxidase inhibitor apocynin and DPI. EGFR antagonist AG1478 significantly inhibited Ang Ⅱ-induced mcsangial cell proliferation. Losartan, NAC, apocynin, DPI, and AG1478 ahnost abolished Ang Ⅱ-induced JNK activation. Conclusions ROS-EGFR-JNK-AP-1 signaling pathway is involved in Ang Ⅱ-induced mesangial cell proliferation. Apocynin and AG 1478 may be used as new therapy.

Expression of cyclooxygenase-2 in IL-1?-stimulated mesangial cells is medicated by NF-?B/I?B signal pathway / 中国病理生理杂志

AIM: To investigate the role of NF-?B/I?B signal pathway in the regulation of (cyclooxygenase-2) (COX-2) expression in human mesangial cells (HMC). METHODS: The PGE_2 concentration in supernatants of HMC was measured by radioimmunoassay. COX-2 mRNA and protein expression were determined by RT-PCR and Western blot. Electrophoretic mobility shift assay (EMSA) and Western blot were used to detect the activity of NF-?B and degradation of I?B. RESULTS: IL-1? significantly upregulated COX-2 expression and PGE_2 production in HMC. Significant up-regulation of NF-?B activation, nuclear translocation of p65 subunit, and degradation of I?B ? and I?B ? were observed in IL-1?-induced HMC. CONCLUSION: Expression of COX-2 in IL-1?-induced HMC is mediated by NF-?B/I?B signal pathway. [