目的：筛选人类糖皮质激素受体基因（NR3C1）的多态性，并分析其在激素耐药型、激素敏感型肾病综合征患儿以及参照人群（随机抽取的脐血标本）中的分布，以研究NR3CI多态性与肾病综合征患儿激素耐药的关系。方法：提取39例激素耐药型、67例激素敏感型肾病综合征患儿以及64例参照人群血基因组DNA，PCR扩增NR3CI中编码人类糖皮质激素受体全都功能区的第2-9α外显子，以变性高效液相色谱（DHPLC）分析检测PCR产物，对洗脱曲线异常者进行DNA浏序。结果：在总计170份基因组DNA样本中，DHPLC分析发现12种多态性，均经DNA测序证实。另外，有3组多态性呈紧密连锁的单倍型（[198G>A+200G>A]，[1374A>G+IVSG-68-IVSG-63delAAAAAA +IvsH-9C>G +2382C>T]，[1896C>T+2166c>T +2430T>C]）。后2种单倍型为首次报道，它们在激素耐药型肾病综合征组的基因型频率（10.3%和15.4%）明显高于敏感型肾病综合征（1.5%和7.5%），2种单倍型的OR值分别为7.54和2.26。其余多态性在各组中出现频率相对较低。结论：在NR3CI基因中筛选出12处多态性；而且新发现的2种多位点紧密连锁的单倍型可能与肾病综合征患儿发生糖皮质激素耐药有关。

We report a 9-year-old boy with repeated fractures of the tibia from age 6 months and microscopic hematuria from age 2 years. His maternal family has a history of nephritis and his paternal family has neurofibromatosis type-I (NF-I). The boy's renal biopsy revealed an irregular attenuation and splitting of the glomerular basement membrane. The skin biopsy was stained with monoclonal antibody against the a5 chain of type IV collagen; the epidermal basement membrane was negative in the boy and segmentally positive in the boy's mother. We conclude that the patient inherited Alport syndrome from his mother and NF-I from his father. We postulate this was a chance association and that this case does not suggest any relationship between the two diseases.

Since the identification of the NPHS2 gene, which encodes podocin, several groups from European, Middle Eastern, and North American countries have reported NPHS2 mutations in families with steroid-resistant nephrotic syndrome (SRNS) or focal segmental glomerulosclerosis (FSGS). Families with SRNS have also been reported in China with a population of more than 1.3 billion. However, to our knowledge, there is no mutational analysis of the NPHS2 gene in familial SRNS or FSGS in China. We identified a novel mutation of NPHS-(467-468insT and 503G>A) in a Chinese family with autosomal recessive SRNS using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing techniques. The results demonstrate that there is also NPHS2 mutation in Chinese familial SRNS. Therefore, Chinese SRNS patients with a familial history of NS should also be screened for possible mutations of NPHS2. We also detected clearly decreased staining with a specific podocin C-terminal antibody (P35) and negative staining with a specific podocin Nterminal antibody (P21). These results were contrary to those predicted from the mutated sites. Further studies are needed to explore the mechanism and impact of the mutant gene on the expression and localization of the relevant protein.

Recently, the novel podocyte proteins podocin, nephrin, and aactinin-4 have been identified in three congenital/family nephritic syndromes, respectively. Further studies showed that these podocyte proteins were involved in some acquired nephrotic syndromes and various experimental models of proteinuria. However, the molecular interactions among these podocyte proteins remain unclear. In this study, to investigate the molecular interactions among podocin, nephrin, and aactinin-4, we reconstructed the RNA interference (RNAi) expression vector, pSilencer 2.1-U6, specifically targeting podocin mRNA, and it was transfected into the mouse podocyte clone (MPC5). Immunofluorescence staining, double-immunolabeling, confocal microscopy, semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blotting were used to detect the distribution and expression of podocin, nephrin, a-actinin-4, and glyseraldehyde-3-phosphate dehydrogenase (GAPDH)/b-actin. The fluorescence intensity of podocin and nephrin decreased obviously, along with the evident distribution change from the cell membrane surface to the nucleus circumference in podocyte. In relation to GAPDH, the mRNA reductions of podocin and nephrin were observed by about 65% and 70%, respectively. The expression of podocin protein was too low to be detected in the interference group. In relation to b-actin, the protein level of nephrin decreased by about 78%. The distribution and the mRNA and protein level of aactinin showed no appreciable change. Alpha-actinin localized mainly in the cytoplasm and also extended to the processes. Thus, the significant decreased expression of nephrin along with the redistribution were detected with the knockdown of podocin mRNA, whereas the expression and distribution of aactinin-4 showed no change. These results suggest that podocin may interact directly with nephrin, but not with aactinin.

Background. Since the identification of the NPHS-gene, various investigators have demonstrated that an NPHS2 mutation is a frequent cause of sporadic steroid-resistant nephrotic syndrome (SRNS), and occurs in 10.5–28% of children with the syndrome. Idiopathic nephrotic syndrome (INS) is also the most frequent glomerular disease in Chinese children, of which 20% of cases show steroid resistance. To our knowledge, however, whether or not NPHS2 is the causative gene in Chinese sporadic SRNS has not been established. This study aims to examine mutations in NPHS2 in Chinese children with sporadic SRNS. Methods. We examined 23 Chinese children with sporadic SRNS for mutations in NPHS2. The mutational analysis of NPHS2 was performed by polymerase chain reaction, denaturing high-performance liquid chromatography and DNA sequencing. Results. A heterozygous missense mutation of L361P in exon 8 of NPHS2 was detected in one of 23 children with sporadic SRNS, whereas it was not found in 53 controls. We also identified seven NPHS2 polymorphisms, -51G>T, 288C>T, IVS3-46C>T, IVS3-21C>T, IVS7-74G>C, 954T>C and 1038A>G, in some patients and controls. There was no significant difference in the genotypic and allelic frequencies of these polymorphisms between the patients and controls. Conclusion. The results demonstrate that NPHS-mutations are also present in Chinese sporadic SRNS. Our investigation supports the necessity of searching for mutations in NPHS2 in Chinese children with sporadic SRNS.