Protective effect of dexamethasone implant on nephrotoxicity of adriamycin in rats / 中国临床药理学与治疗学

AIM: To study the establishment of adriamycin nephropathy rat model and the protective effects and mechanisms of dexamethasone implants (DEXI) through renal capsule implantation. METHODS: The adriamycin-induced nephropathy model was built by injecting Adriamycin (4 mg/kg) and Adriamycin (3.5 mg/kg) was injected again after week into tail-vein in SD rats. Renal capsule puncture was performed in model group. The excipient control group injected intra-renal capsule with drug-free excipient (1.4 mg/kg). The experimental group (2.8, 1.4, 0.7 mg/kg) was given by intrarenal capsule injection and positive drug group (0.1 mg/kg, qd × 8 w) was made by intragastric administration. The rat weight, kidney function and blood biochemical were observed and detected during the experiment. After the experiment, rat kidneys were stained with periodic acid-schiff to observe the morphological changes of mesangial and basement and sirius red to observe the renal tissue collagen fibers, expression of podocin and CD2AP were detected by immunohistochemistry. RESULTS: The blood protein content of adriamycin rats decreased, total blood cholesterol, uric acid, blood creatinine and urea nitrogen increased (P<0.05 or P<0.01), mesangium and fibers increased. The expression of Podocin protein in kidney tissue decreased and the expression of CD2AP protein increased (P<0.05 or P<0.01). DEXI increased the weight and blood protein levels of adriamycin rats, reduced blood lipids and blood uric acid levels (P<0.05 or P<0.01), improved renal function and tissue damage, and regulated the abnormal expression and distribution of Podocin and CD2AP proteins (P<0.05 or P<0.01). CONCLUSION: These results suggest that injecting adriamycin into the tail vein can establish a stable kidney disease model. DEXI renal capsule implantation can improve adriamycin nephropathy injury, and its mechanism may be related to restoration the expression and distribution of Podocin and CD2AP proteins on podocyte slit diaphragm.

El síndrome urémico hemolítico y manejo renal de proteínas / Hemolytic uremic syndrome and renal handling of proteins

El síndrome urémico hemolítico (SUH) está definido por la tríada de anemia hemolítica microangiopática, trombocitopenia e insuficiencia renal aguda. En Argentina constituye la primera causa de insuficiencia renal aguda en pediatría. Aproximadamente, del 2% al 4% de los pacientes mueren durante la fase aguda de la enfermedad, y solo un tercio del 96% restante que sobrevive lo hace con secuelas renales, como la persistencia de la proteinuria. Un individuo adulto sano filtra alrededor de 5000 mg/día de proteínas, si bien la excreción en orina es escasa (150 mg/día). La escasa cantidad de proteínas excretadas indica la presencia de un mecanismo de reabsorción a nivel del túbulo proximal. Por lo tanto, la reabsorción tubular renal desempeña un papel muy importante ya que, ante una función glomerular normal, es el principal mecanismo encargado de evitar la depleción proteica corporal. Desde hace aproximadamente 30 años se sabe que la albúmina es reabsorbida en el túbulo proximal. La reabsorción proteica se produce por un mecanismo de endocitosis mediada por el receptor dependiente de clatrina y por endocitosis de fase líquida. Clásicamente se ha descrito que el mecanismo básico del daño renal en el SUH típico y en el atípico es una microangiopatía trombótica, pero de diferentes causas. Sin embargo, debe tenerse en cuenta que la fisiopatología de esta enfermedad es más compleja de lo que se creía, ya que la alteración tubular que surge va a evolucionar en fallas en el mecanismo de endocitosis de proteínas que se suman a las eliminadas por las alteraciones a nivel de la barrera de filtración glomerular.

Hemolytic uremic syndrome (HUS) is defined by the triad of hemolytic anemia microangiopathic, thrombocytopenia and acute renal failure. In Argentina it constitutes the first cause of acute renal failure in Pediatrics. Approximately 2-4% of patients die during the acute phase of the disease, and only a third of the remaining 96% survive with renal sequelae, such as the persistence of proteinuria. A healthy adult filters around 5000 mg/day of proteins, with an excretion in urine of 150 mg/day. The little quantity of proteins excreted indicates the presence of a reabsorption mechanism at the level of the proximal tubule. Therefore, the tubular reabsorption plays a very important role since it is the main mechanism responsible for preventing the depletion of protein. For approximately 30 years, it has been known that albumin is reabsorbed in the proximal tubule. Protein reabsorption occurs by a clathrin-dependent receptor mediated endocytosis mechanism and by fluid phase endocytosis. The basic mechanism of renal damage in typical and atypical HUS has been described as a thrombotic microangiopathy, but of different causes. However, the pathophysiology of this disease is more complex than what was believed since the emerging tubular alteration will ewvolve into failures of the protein endocytosis mechanism that are added to the alterations at the level of the glomerular filtration barrier.

Effects and mechanisms of 6-week intensive training on renal function in rats / 中国应用生理学杂志

OBJECTIVE@#To study the effect of 6-week intensive training on renal function in rats and the mechanism of exercise-induced proteinuria.@*METHODS@#Thirty-six male SD rats, aged 6 weeks, were divided into two groups, including a control group(C,=12)and an overtraining group(M,=24). After the rats adapted to feeding for 4 d, group C did not carry out any exercise, and the M group did 6-week of increasing load swimming, 6 days a week, once a day. Started with the load of 1%weight at the beginning of the 4 week,and gradually increased (to 6% weight). Took a single urine from both groups 30 min after the end of the training. Blood was taken from the main ventral vein, and the bilateral kidneys were to be tested. The levels of tested urine protein, microalbumin and neutrophil gelatinase associated lipocalin(NGAL) was determined by using enzyme linked immunosorbent assaytest. The content of urine creatinine was tested with alkaline picric acid method,. The serum levels of colorimetric method to determine serum creatinine and urea nitrogen were determined by colorimetric method. The expression of Nephrin in renal tissue was detected by Western blot and the radioimmunoassay was used to test serum testosterone, corticosterone and renin-angiotensin system related index.@*RESULTS@#Compared with group C, the serum testosterone/cortisone(T/C) of group M was decreased significantly (<0.01). The urine total protein(TP), microalbumin (mAlb), microalbumin/creatinine (mAlb/CRE), NGAL, blood urea nitrogen (BUN) and serum creatinine(SCr) were increased significantly (<0.01). The abnormality of glomerular structure was obvious, and the paller scores were higher. The protein expression of Nephrin was obviously down decreased (<0.01). The renin activity (Ra) and angiotension Ⅱ (Ang Ⅱ) in renal and circulating blood were decreased significantly (<0.01).@*CONCLUSIONS@#The effects of 6-week intensive training on renal function in rats and the mechanism of exercise-induced proteinuria may be that overtraining can induce the continuous excitation of Reninrenin activity in renal and circulating blood, down-regulated the expression of Nephrin, lead to abnormality of renal structure and function, and proteinuria.

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.

Effect of Sanqi Oral Liquid on Podocytes and Slit Diaphragm-associated Molecules Expression in Diabetes Mellitus Rats After Unilateral Nephrectomy / 广州中医药大学学报

Objective To observe the effect of Sanqi Oral Liquid on the podocytes and the expression of slit diaphragm-associated molecules (including Nephrin, Podocin and CD2AP proteins) of diabetes mellitus (DM) rats after unilateral nephrectomy, so as to explore its mechanism for protecting renal function. Methods SD male rats were randomized into sham operation group, model group and Chinese medicine group. The experimental diabetes mellitus ( DM) model was given unilateral nephrectomy and intraperitoneal injection of 35 mg/kg of streptozocin (STZ). And then the model rats were given intragastric administration of Sanqi Oral Liquid (2.5 g· kg-1·d-1) and the same volume distilled water respectively for 8 weeks. After treatment, the density of podocytes and the podocytic foot process width of different groups were measured. Immunohistochemistry was used to observe the changes of the expression of Nephrin, Podocin and CD2AP in renal tissues of different groups. Results After treatment with Sanqi Oral Liquid, the density of podocytes was increased, the foot process lesion was relieved, and the expression levels of Nephrin, Podocin and CD2AP proteins were increased (P<0.05 or P<0.01 as compared with those in the model group). Conclusion The protective mechanism of Sanqi Oral Liquid for renal function of unilateral nephrectomy DM rats is possibly related with the alleviation of podocyte injury and with the regulation of the expression of Nephrin, Podocin and CD2AP proteins in podocytes.

Current concepts of the podocyte in nephrotic syndrome

Nephrotic syndrome is a disorder of the glomerular filtration barrier, and central to the filtration mechanism of the glomerular filtration barrier is the podocyte. We are starting to better understand how this cell, with its unique architectural features, fulfils its exact filtration properties. The multiprotein complex between adjacent podocyte foot processes, the slit diaphragm, is essential to the control of the actin cytoskeleton and cell morphology. Many of the proteins within the slit diaphragm, including nephrin, podocin, transient receptor potential-6 channel, and alpha-actinin-4, have been identified via genetic studies of inherited nephrotic syndromes. Signaling from slit diaphragm proteins to the actin cytoskeleton is mediated via the Rho GTPases. These are thought to be involved in the control of podocyte motility, which has been postulated as a focus of proteinuric pathways. Nephrotic syndrome is currently treated with immunosuppressive therapy, with significant adverse effects. These therapies may work in nephrotic syndrome due to specific effects on the podocytes. This review aims to describe our current understanding of the cellular pathways and molecules within the podocyte relevant to nephrotic syndrome and its treatment. With our current knowledge of the cellular biology of the podocyte, there is much hope for targeted therapies for nephrotic syndromes.

High Glucose and Advanced Glycosylation Endproducts (AGE) Modulate the CD2AP Expression in Glomerular Epithelial Cells (GEpC) / 대한신장학회잡지

BACKGROUND: Regardless of the underlying diagnosis, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the foot processes and componental changes in slit diaphragm. We examined the molecular basis for this alteration of the podocyte phenotype, involving quantitative and distributional changes especially on CD2AP as a candidate regulating the modulation of pathogenic changes in the barrier to protein filtration. METHODS: To investigate whether high glucose and AGE induce podocyte cytoskeletal changes, we cultured rat GEpC under normal (5 mM) or high glucose (HG, 30 mM) and AGE- or BSA-added conditions and examined the distribution of CD2AP by confocal microscope and measured the change of CD2AP expression by Western blotting and RT-PCR. RESULTS: We found that CD2AP moved from peripheral to inner cytoplasm in the HG condition by confocal microscopy. In Western blotting, administration of high glucose or AGE decreased the CD2AP productions by 36.9% (p<0.05) and 16.0% (p< 0.05), respectively. Furthermore, both high glucose and AGE decreased the amount of CD2AP more significantly by 64.6% compared to those of control (p<0.01). Such changes was not seen in osmotic control. In RT-PCR, administration of high glucose, AGE or both high glucose and AGE decreased the expression of CD2AP mRNA by 44.9%, 27.9%, and 29.3% (p<0.05), respectively, compared to that of control. CONCLUSION: We could find that HG induce the inward translocation of CD2AP molecule and HG and AGE suppress the production of CD2AP at transcriptional and partly translational level. We suggest that these changes may explain the structural and functional changes of podocytes in diabetic conditions.

Expression of Nephrin in Puromycin Aminonucleoside Nephrosis in Rat / 대한신장학회잡지

BACKGROUND: Nephrin, a recently identified protein, could be a slit diaphragm component and it has been suggested to play a crucial role in maintaining the glomerular filtration barrier. It has been reported that mutations in the nephrin gene lead to congenital nephrosis. However, the expression of nephrin in acquired glomerular disease has not yet been fully clarified. We induced nephrotic-range proteinuria in experimental animal and performed morphologic analysis with immunoelectron microscopy. This study was designed to examine the expression and distribution of nephrin in acquired glomerular disease and to suggest a role of nephrin in pathogenesis of proteinuria. METHODS: Twenty-three rats were divided into 3 experimental groups and control(n=6). 17 rats of experimental groups had intravenous injection of puromycin aminonucleoside(PAN) singly, and were sacrificed at 1 week(n=5), 2 weeks(n=6) and 3 weeks(n= 6) later. The expression of nephrin was observed by immunoelectron microscopy employing the polyclonal antibody against nephrin and gold particle. For quantifications, the gold particles were counted from photographs. RESULTS: The average length of foot process in 1 week group(2,307+/-524 nm) was far greater than that of control(317+/-45 nm). The average number of total gold particles per unit length(10,000 nm) of the GBM was reduced at 1 week(4.4+/-1.3), compared with control(12.1+/-3.9). Also, the average number of junctional gold particles at 1 week(1.7+/-0.5) was decreased compared with control(6.7+/-2.2). No difference was observed in the number of junctional gold particles per slit diaphragm among groups. But, there were significant differences in the distribution of gold particles among groups. Gold particles were seen more frequently at apical plasma membrane and cytoplasm in 1 week group, whereas those were observed prominently at junctions in control. CONCLUSION: These data show that the expression of nephrin was decreased with effacement of foot process in PAN induced nephrosis rat. However, nephrin was preserved at not-damaged slit diaphragm. And the distribution of nephrin was changed in PAN nephrosis. Further studies for nephrin production and redistribution should be needed to understand pathogenesis of nephrotic syndrome.

Adaptive Reaction of Actin Cytoskeleton in Podocyte Structure of Glomerulus / 实用儿科临床杂志

Actin cytoskeleton in podocyte is a complicated network structure,and the stability of this structure depend on many proteins which located in slit diaphragm,the apical membrane domain and the basal membrane domain with the stimulus of mechanical stress,the actin cytoskeleton can be adaptive regulated to maintain the normal function of glomerulus,and several signal pathways involve in the process,such as RhoA/Rho kinase signal pathway and TRPC6.