Clinical Effect of Jianpi Yangyin Guse Decoction on Podocin and Nephrin and Its Protection Against Podocyte Injury in Diabetic Nephropathy Patients with Qi and Yin Deficiency / 中国实验方剂学杂志

ObjectiveTo observe the clinical effect of Jianpi Yangyin Guse decoction on patients with diabetic nephropathy （DN），and to explore its protection against podocyte injury. MethodThe enrolled 120 DN patients at stages Ⅲ and Ⅳ and diagnosed with Qi and Yin deficiency from January 2017 to January 2020 were randomly divided into observation group and control group. During the same period，20 healthy volunteers were recruited as the normal group. In addition to the basic treatment in control group，patients in the observation group were given Jianpi Yangyin Guse decoction，and the course of treatment lasted for 3 months. The traditional Chinese medicine （TCM）syndrome score，24 h urine protein （24 h UP），urine albumin-to-creatinine ratio（UACR），liver and renal functions，D-dimer， hemoglobin A1c （HbA1c）， urine podocin and nephrin and α-smooth muscle actin （α-SMA） excretion of the two groups were observed before and after treatment，and the changes were statistically analyzed and compared with those in the normal group. ResultAfter treatment，the reduction of TCM syndrome score in the observation group was more significant than that in the control group（P<0.01）. The 24 h UP level，UACR and renal function in the observation group in the 2nd and 3rd months after treatment were lower than the conditions before treatment（P<0.05）， and those in the 3rd month after treatment were decreased compared with the conditions in the control group during the same period. The levels of podocin and nephrin in each month and the α-SMA excretion in the 3rd month after treatment in the observation group were down-regulated compared with the conditions before treatment and in the control group （P<0.05）， and the observation group had reduced α-SMA excretion in the 2nd month after treatment compared with before treatment. There were no marked changes in D-dimer and liver function of the two groups before and after treatment. The level of HbA1c in the observation group was higher than that in the control group after treatment（P<0.05）. ConclusionJianpi Yangyin Guse decoction has desirable clinical efficacy in DN patients，and its mechanism may be related to reducing podocin and nephrin and α-SMA excretion levels.

Effect of Huangkui on mouse podocytes under high glucose environment / Efecto de Huangkui en podocitos de ratón en un entorno de glucosa alta

To explore a new underlying molecular mechanism of Huangkui Extract Powder (HKEP) in the alleviation of diabetic nephropathy (DN). Murine immortalized podocytes were divided into (i) normal glucose (NG, 5.6 mM), (ii) NG + HKEP (0.45 g/L), (iii) HG, and (iv) HG + HKEP (0.45 g/L) groups. MTT assay and flow cytometry were used to detect the podocyte proliferation, apoptosis and cell cycle. Cell viability was inhibited, and apoptosis increased in(iii) HG group compared with (i) NG group (p<0.05). mRNA and protein expression of nephrin and podocin significantly decreased in (iii) HG group compared with (i) NG group (p<0.05). When compared with (iii) HG group, (iv) HG + HKEP group had higher cell viability, lower apoptotic rate and higher mRNA and protein expression of nephrin and podocin (p<0.05). HKEP can attenuate HG-induced podocyte damage, which may be one of the mechanisms of HKEP for attenuating DN.

Explorar un nuevo mecanismo molecular subyacente del extracto del polvo de Huangkui (HKEP) en el alivio de la nefropatía diabética (ND). Los podocitos murinos inmortalizados se dividieron en (i) grupos de glucosa normal (NG, 5,6 mM), (ii) NG + HKEP (0,45 g/L), (iii) HG y (iv) HG + HKEP (0,45 g/L). Se utilizaron el ensayo MTT y la citometría de flujo para detectar la proliferación de podocitos, la apoptosis y el ciclo celular. La viabilidad celular se inhibió y la apoptosis aumentó en el grupo (iii) HG en comparación con el grupo (i) NG (p<0,05). La expresión de ARNm y proteínas de nefrina y podocina disminuyó significativamente en el grupo (iii) HG en comparación con el grupo (i) NG (p<0,05). En comparación con el grupo (iii) HG, el grupo (iv) HG + HKEP tuvo una mayor viabilidad celular, una tasa de apoptosis más baja y una expresión de ARNm y proteínas más altas de nefrina y podocina (p<0,05). HKEP puede atenuar el daño de los podocitos inducido por HG, que puede ser uno de los mecanismos de HKEP para atenuar la DN.

mRNA Expression of Podocyte Associated Proteins in Peripheral Blood Mononuclear Cells of Type 2 Diabetes Mellitus Patients with and without Nephropathy.

Background: Diabetic nephropathy is the leading causeof End-Stage Renal Disease (ESRD) emerging indeveloped as well as developing countries, with thecomplicated pathogenesis. The study of expression ofthe genes related to kidney cells e.g. podocytes has beenshown to be associated with the condition, helping in theelucidation of pathogenesis of the disease. Previouslythe gene expression associated was studied in urinesamples. Material and Methods: In the present study, itwas attempted to analyze the mRNA expression ofpodocyte related genes viz. podocalyxin, podocin andsynaptopodin in Peripheral Blood Mononuclear Cells(PBMCs) in patients with diabetes with and withoutnephropathy in comparison with healthy controls byreverse transcriptase Polymerase Chain Reaction(PCR), followed by semi-quantitative PCR. Results:The expression of Synaptopodin (SYNPO) wasincreased in diabetics than the controls, while nosignificant difference was found for Podocalixyn(PODXL) and Podocin (NPHS2). The expression ofPODXL and NPHS2 was significantly up-regulated;SYNPO was unaltered in microalbuminuric patientsthan healthy controls. PODXL and SYNPO wereincreased significantly in nephropathy subjects thancontrols, with no significant change in NPHS2. Theexpression of only PODXL was found to be upregulated in microalbuminuric patients as compared toT2DM patients without nephropathy. PODXL, SYNPOwere significantly up-regulated and NPHS2 wassignificantly down-regulated in nephropathy subjects ascompared to T2DM patients without nephropathy. Asignificant down-regulation was found for NPHS2expression in nephropathy patients than microalbuminuric patients of T2DM with nephropathy.Conclusion: The detection of gene expression of theseproteins can be used as an early marker for the detectionof development of nephropathy in T2DM patients andpreventive measures can be taken to prolong the onsetof nephropathy in these patients, increasing the lifeexpectancy.

Effect of Huangkui extract powder on the expression of nephrin and podocin in podocyte induced by high glucose / 中国基层医药

Objective To investigate the effect of Huangkui extract powder (HK) on the expression of nephrin and podocin proteins in mouse podocytes induced by high glucose,which is involved in the treatment of diabetic nephropathy (DN).Methods Cultured mouse podocytes (MPC5) were incubated in high glucose and HK at 5.6 mmol/L NG,5.6 mmol/L NG + 0.45 g/L HK,25 mmol/L HG,25 mmol/L HG + 0.45 g/L HK,respectively.The 5.6 mmol/L NG group was used as normal control.After 24 hours of intervention,we detected podocyte apoptosis by Annexin-V FITC/PI double staining,measured the mRNA and protein expression of nephrin and podocin by qRT-PCR and Western blot.Results Compared with the control group (5.6 mmol/L,NG),the apoptosis rate of podocytes in the high glucose concentration group (25 mmol/L,HG) was significantly higher [(20.39 ± 0.03) ％ vs.(17.70 ± 0.91) ％,t =2.947,P ＜ 0.05)].The apoptosis rate of podocytes in the 25 mmol/L HG + 0.45 g/L HK group was significantly lower than that in the 25 mmol/L HG group [(11.96 ± 1.11) ％ vs.(20.39 ± 0.03) ％,t =7.586,P ＜ 0.01].The results of qRT-PCR and Western blot showed that the expression of nephrin and podocin was significantly inhibited by high glucose concentration compared with the control group[(0.489 ±0.040) vs.(0.721 ±0.022),t =4.992,P ＜0.01;(0.387 ±0.014) vs.(0.778 ±0.036),t =10.050,P ＜0.01],and the expression of podocin and nephrin was increased by appropriate concentration of H K [(0.603 ± 0.013) vs.(0.489 ± 0.040),t =2.653,P＜0.05;(0.640±0.024) vs.(0.387 ±0.014),t=8.946,P＜0.01].Conclusion Podocyte apoptosis can be induced by prolonged high glucose treatment,but a certain concentration of HK can inhibit podocyte death induced by high glucose.The possible mechanism is that HK may inhibit the apoptosis of podocytes by regulating the expression of podocin and nephrin in podocytes at high glucose concentration,thus plays a protective role on podocytes.

Tacrolimus inhibits Puromycin induced injury of podocytes by stabilizing the expression and distribution of podocin / 中华实用儿科临床杂志

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.

Effects of Tangshen 1st Capsule on levels of serum SREBP-1c and SREBP-2c and expression of IRS-1, PI3K, and podocin proteins in diabetic nephropathy rats / 中草药

Objective To observe effect and possible mechanism of Tangshen 1st Capsule on diabetic nephropathy rats. Methods Diabetic nephropathy model was established by SD rat ip injected with STZ. Dividing model rats randomly into six groups, 15 rats in model group, 15 rats in benazepril (1.04 g/kg) group, 15 rats in Tangshen 1st Capsule (9 g/kg), 15 rats in Tangshen 1st Capsule (6 g/kg), and 15 rats in Tangshen 1st Capsule (3 g/kg) group; In addition, there were 15 rats designated as normal control group. Rats in normal control group and model group were given corresponding drugs for 8 weeks. After 8 weeks, it was time to observe the blood glucose, blood lipid, cystain c, SREBP-1c, SREBP-2c, urinary albumin, expression of IRS-1, PI3K, and podocin protein levels in renal tissue. Results Compared with model group after treatment, in Tangshen 1st Capsule high-dose group and mid-dose group, blood glucose, blood lipid, cystain C, and urinary microalbumin decreased significantly (P 0.05), in high-dose group, and mid-dose group were statistically significant (P < 0.05). Conclusion Tangshen 1st Capsule have protective effects on diabetic nephropathy rats, and its mechanisms are in part associated with reducing blood glucose and blood lipid, and increasing podocin protein expression.

Identificación de variantes del gen NPHS2 en niños con síndrome nefrótico corticorresistente / NPHS2 Mutation analysis study in children with steroid-resistant nephrotic syndrome

Resumen: La podocina es una proteína localizada en el diafragma de filtración glomerular donde participa en la regulación de la filtración glomerular. Las mutaciones del gen NPHS2, que codifica a la podocina, son la principal causa de síndrome nefrótico corticorresistente (SNCR) autosómico recesivo en niños. Objetivos: Identificar mutaciones de NPHS2 en niños chilenos con SNCR, y establecer la prevalencia de las variantes más frecuentes en un grupo de adultos sanos. Pacientes y método: Análisis mutacional de NPHS2 en 34 niños chilenos con SNCR. Una vez identificadas las dos variantes de NPHS2 de mayor frecuencia, se realizó un screening de estas mutaciones en 223 adultos sanos. El análisis mutacional se realizó por secuenciación directa de los ocho exones codificantes amplificados por reacción de polimerasa en cadena. La secuenciación del DNA se realizó mediante método fluorométrico y las secuencias fueron evaluadas con el software SeqPilot. La asociación entre la presencia de variantes de NPHS2 y SNCR se calculó comparando las frecuencias alélicas entre los pacientes con SNCR y los voluntarios sanos utilizando prueba exacta de Fisher. Se consideró significativo p < 0,05. Resultados: Se detectaron mutaciones patogénicas de NPHS2 en siete de los 34 pacientes (21%) estudiados, de los cuales seis resultaron heterocigotos para p.R229Q y p.A284 V. En voluntarios sanos la prevalencia de p.R229Q fue de 2,46%. Conclusiones: Este estudio muestra que p.R229Q y p.A284 V son las variantes de NPHS2 más frecuentes en niños chilenos con SNCR. Por primera vez se describe esta asociación en niños chilenos, en base a la cual es posible proponer una estrategia de screening para estudio genético en pacientes con SNCR y sus familias. Se propone una estrategia de búsqueda de p.R229Q y p.A284 V en forma paralela o secuencial en estos pacientes.

Abstract: Podocin is a protein located in the glomerular slit diaphragm where it takes part in the regulation of glomerular filtration. Mutations of the NPHS2 gene that codes podocin are the main cause of autosomal recessive steroid resistant nephrotic syndrome (SRNS). Objectives: To identify the NPHS2 mutations in Chilean children with SRNS, and to determine the prevalence of the most common variants in a group of healthy adults. Patients and methods: Mutation analysis of NPHS2 in 34 Chilean children with SRNS. Once the two most common variants of NPHS2 were identified, screening for these mutations was performed on 233 healthy adults. The mutation analysis was performed by the direct sequencing of the eight coding exons by polymerase chain reaction amplification. The DNA sequencing was performed using a fluorometric method, and then evaluated with SeqPilot™ software. The relationship between the presence of NPHS2 variants and SRNS was calculated by comparing the allele frequency between patients with SRNS and those of the healthy volunteers using the exact Fisher test. A P < .05 was considered significant. Results: Pathogenic NPHS2 mutations were detected in 7 (21%) of the 34 patients studied, of which 6 were heterozygotes for p.R229Q and p.A284 V. The presence of p.R229Q was 2.46% in the healthy volunteers. Conclusions: This study shows that p.R229Q and p.A284 V are the most frequent variants in Chilean children with SRNS. It is the first time that this relationship has been reported in Chilean children. Based on this, a screening strategy is proposed for the genetic study in patients with SRNS and their families. A parallel or sequential search strategy for p.R229Q and p.A284 V in these patients is proposed.

Protective effects of astragaloside in rats with adriamycin nephropathy and underlying mechanism / 中国天然药物

The present study was designed to determine the mechanism underlying the treatment of nephrotic syndrome using astragaloside by observing the effects of astragaloside on the expression of nephrin and podocin proteins and genes in kidneys of rats with adriamycin nephropathy. The rats were injected with adriamycin and, after successful model establishment, randomly divided into a model group, a Methylprednisolone (MP) group, and an astragaloside group. The 24-h complete urine samples were collected. Biochemical indicators were monitored, and kidney tissues were collected for pathological analysis using light microscopy and electron microscopy. The mRNA expression of nephrin and podocin was measured in the kidney tissues using the real-time qPCR, and the protein expression levels of nephrin and podocin were detected using Western blot analysis. At the end of 12 weeks of drug intervention, the urinary protein level was lower in the MP and astragaloside groups than that in the model group (P = 0.008 and P = 0.01, respectively). Serum albumin was higher in the MP and astragaloside groups than in the model group (P < 0.001 and P = 0.012, respectively). Podocytes in the MP group were nearly normal, and fusion of podocytes in the astragaloside group was significantly less than that in the control group. The nephrin and podocin mRNA and protein expression levels in the intervention groups were higher (P < 0.05) than that in the model group. Due to the increased expression of podocyte-related nephrin and podocin proteins, astragaloside maintained slit diaphragm integrity and decreased the level of proteinuria in rats with adriamycin nephropathy.

Mechanisms of bevacizumab inducing proteinuria in mice models / 医学研究生学报

Objective Bevacizumab ( BM ) is an angiogenesis inhibitor widely used in cancer therapy, but its off-target effect of proteinuria may lead to discontinuation of treatment.This study was to explore the mechanisms of BM inducing proteinuria in mice. Methods Twenty-four healthy mice were randomly divided into four groups, saline control, low-dose BM, medium-dose BM, and high-dose BM, treated by injection of normal saline and BM at 10, 35, and 60 mg per kg of the body weight, respectively, though the tail vein.At 4 weeks after injection, 24-hour urine was collected to determine the total urine protein and blood obtained from the eyeballs for biochemical analysis.Then all the mice were sacrificed and the kidneys harvested for observation of pathologic changes in the glomeruli as well as for immunohistochemistry, Western blotting, and real-time PCR analysis. Results Compared with normal saline,BM obviously elevated the level of 24-hour urine protein, with statistically significant differences between the control and the medium-and high-dose BM groups (0.23 ±0.02 vs 1.14 ±0.13 and 1.43 ±0.10, P0.05).No significant differences were observed among the four groups in the levels of Cr, BUN, AST and ALT (P>0.05).Under the optical microscope, the kidneys showed normal structures in the control group, no signifi-cant pathologic changes in the low-dose BM, and vacuolus-like alteration with atrophic glomerular endothelial cells in the medium-and high-dose BM groups.Immunohistochemical analysis demonstrated the expressions of VEGF and podocin were moderately or strongly positive in the control and low-dose BM groups, by weakly positive or negative in the medium-and high-dose BM groups.Compared with the control group, the expression of the VEGF protein in the renal tissue was significantly decreased in the high-dose BM group (0.76 ±0.09 vs 0.39 ±0.05, P0.05), and the expression of the podocin protein was significantly reduced in the medium-dose BM (0.67 ±0.07 vs 0.43 ±0.10, P0.05).The mRNA expressions of VEGF and podocin were not significantly changed in the low-dose BM group as compared with the control (1.07 ±0.61 and 1.12 ±0.09 vs 1.23 ±0.25 and 1.17 ±0.19, P>0.05) but remarkably de-creased in the medium-dose (0.82 ±0.38 and 0.71 ±0.18) and high-dose BM groups and (0.47 ±0.64 and 0.42 ±0.09) groups (P<0.01). Conclusion Bevacizumab damages glomerular filtration membrane and induce proteinuria partially by down-regulating the protein and mRNA expressions of VEGF and podocin.

Síndrome nefrótico resistente a corticoides en pediatría, actualización / Steroid-resistant nephrotic syndrome in pediatrics, an update

El síndrome nefrótico (SN) constituye la glomerulopatía más frecuente en pediatría. El pilar del tratamiento con-tinúa siendo la terapia con corticoides. Dependiendo de la respuesta se clasifica en síndrome nefrótico corti-coresistente (SNCR) y corticosensible. La mayoría de los pacientes con SNCR tienen glomeruloesclerosis focal y segmentaria, asociada con 50% de riesgo de enfermedad renal terminal, por lo que se recomienda biopsia renal. Es importante realizar pruebas genéticas, ya que ciertas mutaciones resultan en corticorresistencia, siendo la mutación del gen NPHS2 (podocina) la más relacionada. Este artículo es una revisión de la literatura mundial y nacional acerca del SNCR en pediatría, enfatizando en nuevos enfoques de diagnóstico y tratamiento

Nephrotic syndrome (NS) is the most frequent glomerulopathy in pediatrics. The mainstay of treatment continues to be corticosteroid therapy. Depending on the response, it is classified as corticosteroid nephrotic syndrome (SNCR) and corticosensitive syndrome. Most patients with SNCR have focal and segmental glomerulosclerosis, associated with a 50% risk of end-stage renal disease, and renal biopsy is recommended. It is important to perform genetic tests, since certain mutations result in corticoresistance, with the mutation of NPHS2 gene (podocin) being the most related. This article is a review of the global and national literature on SNCR in pediatrics, emphasizing new approaches to diagnosis and treatment.

Expression and significance of podocyte molecules mRNA in Doxorubicin-induced nephrotic rats / 中华实用儿科临床杂志

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.

Effect of berberine on the expression of nephrin, podocin and intergrin α3β1 in diabetic nephropathy rats / 中国药理学通报

Aim To investigate the effect of berberine on the expression of nephrin, podocin and intergrinα3β1 in diabetic nephropathy ( DN ) rat model, and further probe in to the renoprotective effects of berber-ine and its potential mechanisms. Methods The rat model of DN was induced by intraperitoneal injection of streptozotocin ( STZ ) after fed with high-sugar and high-fat diet for six weeks. The rats were assigned into 6 groups randomly: normal control group, DN model group, BBR (50,100 and 200 mg·kg-1 ) treatment group and enalaprilat positive control group ( 1 mg · kg-1 ) . The distribution and expression of kidney podocyte related proteins nephrin, podocin and interg-rinα3β1 were detected by immunohistochemical meth-od following electron microscopy observation ( × 1000 ) and high magnification observation( × 400) and West-ern blot. Results The podocyte related protein neph-rin, podocin and intergrin α3β1 were mainly distribu-ted in podocyte, but slightly different. Compared with normal control group, the expresion of podocyte related protein nephrin, podocin and intergrin α3β1 was de-creased obviously; compared with model group, BBR (100 and 200 mg·kg-1 ) treatment group could sig-nificantly suppress the abnormalities of pathological changes of the kidney and upregulate the expression levels of podocyte specific protein nephrin, podocin and intergrin α3β1 in the kidney of diabetic rats with nephropathy. Conclusions Berberine could alleviate the abnormalities of kidney pathological changes and proteinuria production in the DN model rats, which may be related to the upregulation of the expression of the podocyte proteins nephrin, podocin and intergrinα3β1.

Protective effect of ACTH4-10 on adriamycin-induced podocyte injury / 中华肾脏病杂志

Objective To observe the influence of adrenocorticotropic hormone (ACTH4-10) in the changes of podocyte proliferation,apoptosis and expression of nephrin and podocin on adriamycin (ADR)-induced podocyte injury and investigate the protective effect of ACTH4-10.Methods All podocytes were randomly divided into following groups:normal control,ADR-induced group and ACTH4-10 intervention group (low,middle and high concentration).Normal control group was not treated,ADR-induced group was induced to set the model of podocyte injury by ADR (1 μmol/L) for 24 hours and ACTH4-10 intervention groups were intervened by 1 μg/L,10 μg/L and 100 μg/L ACTH4-10 for 1 hours respectively,prior to setting the model of podocyte injury.Cell counting kit (CCK-8) was used to detect the multiplication of podocytes and TUNEL apoptosis detection kit was used to detect podocyte apoptosis.Real-time PCR and Western blotting were used to examine the expression of nephrin and podocin.Results Compared with control group,podocyte proliferation and expression of nephrin and podocin was decreased significantly in ADR-induced group (P ＜ 0.05),meanwhile podocyte apoptosis was increased obviously (38.14％ vs 5.12％).Compared with ADR-induced group,podocyte proliferation and expression of nephrin and podocin was increased generally with concentration of ACTH4-10.Although podocyte apoptosis rates (20.45％,17.39％,11.02％) were increased in ACTH4-10 intervention group (low,middle and high concentration) while comparing with normal control group,podocyte apoptosis decreased obviously while comparing with ADR-induced group.Conclusions ACTH4-10 can stabilize the expression of nephrin and podocin on slid diaphragm,and has the protective effect on podocyte injury induced by ADR,while the effect depends on the concentration of ACTH4-10.

Danshao GranularⅢimproves hematuresis and proteinuria and increases nephrin and podocin expressions in rats with Henoch-Sch?nlein purpura nephritis / 医学研究生学报

Objective Few reports are seen about the effects of cooling blood and removing stasis on nephrin and podocin . This study was to evaluate the therapy of cooling blood and removing stasis for Henoch -Sch?nlein purpura nephritis ( HSPN) and its ac-tion mechanisms by observing the effects of Danshao Granular Ⅲ on hematuresis , proteinuria and the expressions of nephridial nephrin and podocin in HSPN rats . Methods Twenty-one SD male rats were e-qually randomized to a blank control , an HSPN model , and a Dan-shao group.At 13 weeks after modeling , the animals in the model group were treated intragastrically with distilled water , while those in the Danshao group with Danshao Granular Ⅲtwice daily for 4 weeks. Then, the urinary red blood cell ( RBC) count was examined , the 24 h urinary protein quantity determined , the glomerular mesangial changes observed under the light microscope , the protein expres-sions and distributions of nephrin and podocin detected by indirect immunofluorescence , and their mRNA expressions determined by re-al-time PCR. Results The urinary RBC count and 24 h urine protein quantity were significantly higher in the HSPN model than in the blank control group (26.5/HP vs 0.3/HP and [2.214 ±1.090]g/24 h vs [0.624 ±0.354]g/24 h, both P0.05).The distributions of nephrin and podocin were improved after Danshao treatment .The mRNA expressions of nephrin and podocin were markedly higher in the Danshao group than in the HSPN models (0.530 ±0.089 vs 0.117 ±0.021 and 0.490 ±0.160 vs 0.033 ±0.025, P<0.05). Conclusion Danshao Granular Ⅲ, with its main action mechanisms of cooling blood and removing stasis , can effectively reduce urinary RBC count and urinary protein quantity and improve the symptoms of HSPN in rats .

Effects of Zuogui Jiangtang Yishen Decoction on Expression of Nephrin and Podocin in Podocyte of MKR Mice with Diabetic Nephropathy / 中国中医药信息杂志

Objective To investigate the effects of Zuogui Jiangtang Yishen decoction (ZGJTYS) on the expression of nephrin and podocin in podocyte of MKR mice with diabetic nephropathy (DN), and explore its mechanism. Methods Forty MKR mice (8 weeks old) were randomly divided into four groups as follows:negative control group (group A), DN model group (group B), ZGJTYS group (group C) and positive control group (group D, Gliquidone and benazepri). All mice from group B, C and D were received high-fat diet feed and unilateral nephrectomy. Four weeks after operation, all mice were received drug intervention, and four weeks later, all mice were put to death. The levels of UmAlb were observed by ELISA, the serum BUN and Cr by biochemical, and the FBG by electrochemical detection method. The nephrin and podocin mRNA expression were measured by RT-PCR and the protein expression by western blotting. The morphological structure changes of the podocytes were observed by transmission electron microscopes. Results As compared with group A, FBG, BUN, SCr and urine UmAlb in the mice of group B increased significantly (P<0.01), the expression level of nephrin and podocin mRNA and protein were markedly decreased (P<0.01). After intervention of drugs, all biochemical indicators above in the mice of group C and D significantly decreased (P<0.01), the expression level of nephrin and podocin mRNA and protein were markedly increased (P<0.01, P<0.05), compared with group B. The renal pathological lesions of group C and D were significantly improved compared with group B. Conclusion ZGJTYS decoction exerts reno-protective effect via reinstating nephrin and podocin expression to repair the damaged podocyte.

The expression of Podocin in glomerular podocytes of obesity rats and its significance / 中华内分泌外科杂志

Objective To discuss the mechanism of obesity related glomerularpathology,we detect the expression of Podocin in glomerular podocytes of obesity rats induced by high fat diet.Methods The rats were divided into 2 groups:normal control group(Con group) and obesity group(Ob group).At the 14th week of molding,the body weight,body long,tail long,kidney weight,uria microalbumin,RBP,uria Cre,blood glucose,cholesterol,triglyceride,HDL-C,UA,Cre,BUN,and insulin were measured.The Lee's index,insulin resistance index,insulin sensitive index,Ccr and relative kidney weight were calculated.The tissue structures of the kidney of both groups were studied under light microscope and transmission electro microscope.The expression of Podocin in podocyte of rats' kidney was measured by immunohistochemistry and western blot.Results ①The difference had no statistical significance between the 2 groups in gender,weight,body length,tail length and Lee's index at the beginning (P ＞ 0.05).But after 14 weeks,the weight,body length and Lee's index of Ob group were significantly higher than those of Con group(P ＜0.05).②At the 14th week,the fasting plasma glucose of Ob group was higher than that of Con group (P ＜ 0.05),and the high density lipoproteins (HDL) of Ob group was lower than that of Con group(P ＜ 0.05).However,the difference had no statistical significance in urine acid (UA),serum Cre and blood urea nitrogen (BUN) between the 2 groups (P ＞ 0.05).③The urine protein and endogenous creatinine clearance rate(Ccr) in Ob group were higher than those of Con group (P ＜ 0.05,P ＜0.01).④The relative kidney weight of Ob group was lower than that of Con group(P ＜ 0.05).⑤For rats in Ob group,glomerular hypertrophy,the Bowman' capsule stricture,abnormality in the segmental capillary wall and extraglomerular mesangial cell were observed under light microscope.The glomerular podocytes became broader and foot process of podocyte in Ob group became microvillus-like in Ob group under transmission electro microscope.⑥Immunohistochemistry and western blot showed that the expression of Podocin in podocyte of kidney decreased in Ob group.Conclusions The expression of Podocin decreased in podocyte of kidney in obesity rats.At the same time,the ultrastructure of podocytes was changed.Those may be one of the reasons for protein urine related to obesity.

NPHS2 Mutations in Indian Children with Sporadic Early Steroid Resistant Nephrotic Syndrome.

We examined the frequency and spectrum of podocin NPHS2 mutations in Indian children with sporadic steroid resistant nephrotic syndrome (SRNS). Of 25 children screened, only one (4%) had a pathogenic mutation resulting in a stop codon. The allele and genotype frequencies of the four known single nucleotide polymorphisms detected in the cohort were similar to that of controls. This finding emphasizes the need to screen for mutations in other genes involved in the pathogenesis of SRNS.

The reno-protective effect of a phosphoinositide 3-kinase inhibitor wortmannin on streptozotocin-induced proteinuric renal disease rats

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 +/- 0.56 mg/day, whereas wortmannin group was 1.77 +/- 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 +/- 10.82 mg/g, whereas wortmannin group was 20.27 +/- 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 +/- 0.02), podocin (87.81 +/- 0.03) and Rac1/Cdc42 (86.12 +/- 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 +/- 0.03), podocin (53.40 +/- 0.06) and Rac1/Cdc42 (54.05 +/- 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.

The correlation between NPHS2 polymorphism and IgA nephropathy in northern Chinese patients / 中华内科杂志

Objective To examine the polymorphism in NPHS2 gene of IgA nephropathy in northern Chinese patients and to investigate the possible association of the NPHS2 polymorphism with the development of IgA nephropathy, as well as its clinical and histologic manifestations.Methods The polymorphism of NPHS2 was analyzed by direct DNA sequencing in 32 northern Chinese patients with IgA nephropathy (16 with heavy proteinuria and 16 with isolated hematuria).According to preliminary results, a total of 537 IgA nephropathy patients were genotyped for the NPHS2 C357T polymorphism by PCR combined with restriction fragment length polymorphism (PCR-RFLP). We collected clinical and histologic manifestations for gene analysis in patients with IgA nephropathy, such as age, sex, urine protein excretion and so on.ResultsEight NPHS2 polymorphisms (-931A＞T, -601C ＞T, 19G＞T, 171A＞G, 357C ＞ T, IVS3-21C ＞ T, 1023C ＞ T and 1107A ＞ G) were identified.The preliminary results of gene sequencing showed that the frequency of 357T allele in nephrotic syndrome group was obviously lower than isolated hematuria group (0.038 vs 0.125, P ＜0.05).In 537 IgA nephropathy patients with clinical and histologic data, the average urinary protein excretion in the patients with the 357CT/TT genotype was less (P =0.023).The incidence of urinary protein of more than 3.5 g/d was significantly lower in patients with T allele and TT/CT genotype, respectively (P =0.017 and 0.011).The logistic regression analysis indicated that, even after adjusting for the effect of hypertension and age of patients, the CT/II genotype of NPHS2 C357T was an independent protective factor for the urinary protein excretion more than 3.5 g/d(P =0.012,OR = 0.485, 95％ CI 0.275-0.84).ConclusionsEight NPHS2 polymorphisms were identified in northern Chinese IgA nephropathy patients. The frequencies of NPHS2 T allele and TT/CT genotype were the protective factors for urinary protein, especially with that of more than 3.5 g/d.

Dexamethasone inhibits puromycin aminonucleoside-induced injury of mouse glomerular podocytes via stabilizing the expression and distribution of podocin in vitro / 中华肾脏病杂志

Objective To observe the effects of puromycin aminonucleoside (PAN) and dexamethasone (DEX) on the expression and distribution of pedocin in vitro, and to explore the possible mechanism of DEX in improving proteinuria. Methods Mouse podecyte cells (MPCs) in control group were cultured with RPMI-1640 plus 0.02% DMSO, and were subjected to PAN treatment alone (PAN group) or PAN plus DEX (DEX group) for 8, 24,48 hours respectively. The pedocyte morphology was observed by phase-contrast microscope, and was analyzed by Image J. The distribution, mRNA and protein expression of podocin were detected by indirect immunocytofluorescence, semi-quantitative RT-PCR and Western blot, respectively. Results The well-developed arborization and interconnection of podocytes were found in control group. PAN treatment led to significant shrinkage of pedocytes with decreased distribution at 43% of control group at 8 h, 10% at 24 h and 5.7% at 48 h (P<0.01), respectively, together with podocyte foot process retraction as well as effacement and loss of cell contact. RT-PCR revealed podoein mRNA expression prone to decrease. Western blot showed podoein protein expression was significantly decreased and immunocytochemistry revealed podoein expression was disappeared in the cellular membrane after PAN treatment. DEX significantly prevented the shrinkage of podcytes, with decreased area at 43.9% of control at 8 h, 26.2% at 24 h and 29.6% at 48 h (P<0.05), respectively, and up-regulated the mRNA and protein expression of podocin at 48 h (P<0.05). The abnormal distribution of podocin was also alleviated by DEX. Conclusion DEX exerts a direct action on podocyte via stabilizing mRNA, protein expression and distribution of podocin, which may be associated with the improvement of proteinuria.