Pathogenesis Of Steroid-resistance

Part One: Mutations in NPHS2 in a Chinese family with steroid-resistantnephrotic syndromeObjective Primary nephrotic syndrome (PNS) is characterized by heavy proteinuria, hypoalbuminemia, edema and hyperlipidemia. It is the most common glomerular disease in childhood. Most patients respond to steroid therapy and show favorable outcomes; however, 10-20% of patients fail to respond to steroid therapy and may progress to end stage renal failure. Recent studies have demonstrated that mutations in NPHS2, which was mapped to chromosome 1q25-31 and encodes podocin, an integral memberane protein of 383 amino acids, with a single memberane domain (amino acids 105 to 121) forming a hairpin-like structure placing both N- and C-terminal domains in the cytosol of podocyte, cause autosomal recessive steroid-resistant nephrotic syndrome (SRNS). Since the identification of the NPHS2gene, different investigators in Europe, Middle East, and North America have identified NPHS2 mutations in familial SRNS. However, whether NPHS2 is the causative gene in Chinese families with SRNS is not yet known. This study aims to perform mutational analysis of the NPHS2 gene in a Chinese family with autosomal recessive SRNS. Methods Renal biopsy was performed on the proband and her sibling for routine histologic and immunohistochemical investigation and electron microscopic examination. The expression of podocin, nephrin, alpha-actinin-4 and WT1 in glomeruli of the proband was detected by indirect immunofluorescence. Peripheral blood samples were collected for genetic analysis from the proband and her parents, and 53 adults with normal urianalysis. Genomic DNA was isolated from peripheral blood leucocytes. Eight exons of NPHS2 were amplified by polymerase chain reaction (PCR). Mutational analysis was performed by denaturing high-performance liquid chromatography (DHPLC), and DNA segments with aberrant elution profiles by DHPLC were re-amplified and sequenced directly. Results Renal pathology of the proband and her sibling showed changes of focal segmental glomerulosclerosis. By immunofluorescence with P21, a polyclonal rabbit antibody against recombinant human podocin (amino acids 15-89, the N-terminal part of podocin and upstream the transmembrane domain), and P35, a polyclonal rabbit antibody against recombinant human podocin (amino acids 135-383, the C-terminal part of podocin and downstream the transmembrane domain), podocin was revealed along the glomerular capillary wall in a linear pattern in control kidney samples. The staining with P21 was positive, and that with P35 was intensely positive. In the sample from the proband, no staining with P21 antibodies was observed throughout the kidney sections. The podocin staining with P35 was uneven, non-linear, and weakly positive. The area, location, distribution, and fluorescent intensity of the staining of the glomeruli of the proband with nephrin, alpha-actinin-4,and WTl antibodies were the same as in the controls. By DHPLC, double peaks were detected in exon 4 ofNPHS2 in the proband, the proband's father and the proband's mother. The chromatograms of exon 4 of NPHS2 showed a compound heterozygous mutation of both 467₄68insT and 503G>A in the proband, a heterozygous mutation of 503G>A in her father, and a heterozygous mutation of 467₄68insT in her mother. No sequence variations in the remaining seven exons of NPHS2 were found in the proband. Sequence changes of NPHS2, 467₄68insT and 503G>A, were absent in 106 control chromosomes. Conclusion From the data provided, we draw the following conclusions. (1) For the first time we identified a novel mutation of NPHS2 (467₄68insT and 503G>A) in a Chinese family with autosomal recessive SRNS. 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. (2) NPHS2 mutation of 503G>A was novel. (3) We also detected negative staining with a specific podocin N-terminal antibody (P21) and clearly decreased staining with a specific podocin C-terminal antibody (P35). These results were contrary to those predicted from the mutated sites.Part Two: Mutations in NPHS2 in sporadic steroid-resistant nephrotic syndromein Chinese childrenObjective PNS is characterized by heavy proteinuria, hypoalbuminemia, edema and hyperlipidemia. It is the most common glomerular disease in childhood. Most patients respond to steroid therapy and show favorable outcomes; however, 10-20% of patients fail to respond to steroid therapy and may progress to end stage renal failure. Recent studies have demonstrated that mutation in NPHS2, which encodes podocin, cause autosomal-recessive SRNS. Since the identification of the NPHS2 gene, various investigators in Europe, Middle East, and North America have demonstrated that an NPHS2 mutation is a frequent cause of sporadic SRNS, and occurs in 10.5-28% of children with the syndrome. PNS is also the most frequent glomerular disease in Chinese children, of which approximately 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 Peripheral blood samples were collected for genetic analysis from 23 Chinese children with sporadic SRNS and 53 adults with normal urianalysis. Genomic DNA was isolated from peripheral blood leucocytes. Eight exons oï¿¡NPHS2 were amplified by PCR. Mutational analysis was performed by DHPLC, and DNA segments with aberrant elution profiles by DHPLC were re-amplified and sequenced directly. Results By DHPLC, 6 aberrant elute profiles were revealed in exons 1,2,4 and 8 in some patients with sporadic SRNS, and 5 aberrant elute profiles in some controls, whereas a single peak was revealed in exons 3, 5, 6 and 7 of the NPHS2 gene in all of the patients and controls. The chromatogram of exon 8 of the NPHS2 gene showed a heterozygous missense mutation of L361P (1082T>C) in 1 of 23 children withsporadic SRNS, whereas a sequence change of NPHS2, 1082T>C, was absent in 106 control chromosomes. In addition, seven NPHS2 polymorphisms, -51G>T, 288OT, IVS3-46OT, IVS3-21OT, IVS7-74OC, 954T>C and 1038A>G, were also detected in some patients and some controls. There was no significant difference in the genotypic and allelic frequencies of -51OT, 288OT, IVS3-46OT, IVS3-21OT, IVS7-74G>C, 954T>C and 1038A>G of NPHS2 between the patients and the controls (P>0.05). Conclusion From the data provided, we draw the following conclusions. (1) For the first time we detected NPHS2 mutation in 1 of 23 Chinese children with sporadic SRNS (4.3%). The result demonstrates that NPHS2 mutation also is present in Chinese sporadic SRNS. Our investigation supports the necessity of searching for mutations in NPHS2 in Chinese children with sporadic SRNS. (2) NPHS2 mutation 1082T>C was novel. (3) Three novel NPHS2 polymorphisms, -51G>T> IVS3-46OT fP IVS3-21OT, were identified. (4) There is no association between the seven NPHS2 polymorphisms (-51G>T, 288C>T, IVS3-46OT, IVS3-21OT, IVS7-74G>C, 954T>C and 1038A>G) and SRNS in Chinese children.Part Three: Expression of V165X and R168H mutant podocinsin mammalian cellsObjective Podocin, a plasma memberane anchored stomatin-like protein of 383 amino acids, with a single memberane domain (amino acids 105 to 121) forming a hairpin-like structure placing both N- and C-terminal domains in the cytosol, is expressed in lipid raft at the insertion site of the slit diaphragm of podocytes of the kidney. It is shown to target to the plasma memberane, where it facilitates nephrin signaling, and to form homo-oligomers involving the N- and C-terminal cytoplasmic domains. Podocin plays a crucial role in the establishment of the glumerular barrier as confirmed by the occurrence of massive proteinuria and death in a few days after birth in Nphs2-I- mice. Mutations in NPHS2, which encodes podocin, are associated with familial and sporadic cases of SRNS. In previous study, we detected negative staining with a specific podocin N-terminal antibody (P21) and clearly decreased staining with a specific podocin C-terminal antibody (P35) in the renal sample from the proband with a compound heterozygous NPHS2 mutation of both V165X (467₄68insT) and R168H (503G>A) of a Chinese family with SRNS, which were contrary to those predicted from the mutated sites. As a further step in understanding the consequences of two NPHS2 mutations, V165X and R168H, we studied the in vitro expression of the V165X and R168H mutant podocins in transfected human embryonic kidney cells (HEK293). Methods We isolated the human podocin cDNA by reverse transcription PCR (RT-PCR) and built constructs of cDNAs encoding the wild-type, V165X and R168H mutant podocins. The wild-type, V165X and R168H mutant cDNAs in the expression plasmids were transfected into HEK293 cells with Lipofectamin 2000. Subsequently, we studied the subcellular localization of the wild-type, mutant podocins using immunofluorescence staining with P21 and P35,double-immunolabling and confocal microscopy. Results Constructs of cDNAs encoding the wild-type, V165X and R168H mutant podocins were confirmed by sequencing. The fluorescence intensities of the wild-type, V165X and R168H mutant podocins with P21 were positive, so were those of the wild-type and R168H mutant podocins with P35, whereas the staining of V165X mutant podocin with P35 was negative or weakly positive. The evident distribution changes of 165X and R168H mutant podocins were revealed by double-immunolabling. The staining for wild-type podocin was distributed around nuclei and mainly on the cell memberane surface in a filamentous pattern, whereas 165X and R168H mutant podocins staining localized predominantly around nuclei with a loss of surface expression. Conclusion From the data provided, we draw the following conclusions. (1) We succeeded in building the constructs of cDNAs encoding the wild-type, V165X and R168H mutant podocins and detecting the expression of the wild-type and two mutant podocins in HEK293 cells. (2) The staining for wild-type podocin was distributed around nuclei and mainly on the cell memberane surface in a filamentous pattern, whereas 165X and R168H mutant podocins staining localized predominantly around nuclei with a loss of surface expression, which support the pathogenic role of the two NPHS2 mutations, 467₄68insT and 503G>A. (3) In vitro expression of V165X and R168H mutant podocins also is contrary to in vivo expression of the two mutated genes, which imply that the expression and control of mutated gene in eukaryotic cells is very complex.Part Four: Variations of NR.3C1 in children with steroid-resistantnephrotic syndromeObjective PNS is characterized by heavy proteinuria, hypoalbuminemia, edema and hyperlipidemia. It is the most common glomerular disease in childhood. Most patients respond to steroid therapy and show favorable outcomes; however, 10-20% of patients fail to respond to steroid therapy and may progress to end stage renal failure. Previous studies have demonstrated that genetic variations of glucocorticoid receptor gene (NR3C1) are associated with glucocorticoid sensitivity. Ye et al. from our research group have identified two NR3C1 haplotypes, Haplotype 3, [1374A>G + IVSG-68_IVSG-63delAAAAAA + IVSH-9OG + 2382OT], and Haplotype 5, [1896OT + 2166OT + 2430T>C], and have found that the risk of developing steroid resistance for nephrotic children with the Haplotype 3 and the Haplotype 5 revealed 4.41 and 2.26 times more than those without the two haplotypes; however, the statistical significance analysis (P values = 0.09 and 0.22, respectively) did not support the association between the two haplotypes and steroid resistance in nephrotic children, speculating the cause may be small size of samples. As a further step in studying the association between the two haplotypes and steroid resistance in nephrotic children, in this study we added the size of samples and examined NR3C1 variation in the patients with NS. Furthermore, we established means of amplifing segments of NR3C1 mRNA using RT-PCR, which will help to detect mRNA expression of NR3C1 in peripheral blood lymphocytes of nephrotic children with the Haplotype 3 or the Haplotype 5. Methods Peripheral blood samples were collected, for genetic analysis from 6 children with steroid-sensitive nephrotic syndrome (SSNS) and 1 case with SRNS. Genomic DNA was isolated from peripheral blood leucocytes. Four exons (exons 3, 6, 8 and 9alpha) of NR3C1 were amplified by PCR. Mutationalanalysis was performed by DHPLC, and DNA segments with aberrant elution profiles by DHPLC were re-amplified and sequenced directly. With odds ratio (OR) and Fisher's exact test, the statistical significance reanalysis was performed again. In addition, peripheral blood lymphocytes were isolated from peripheral blood sample collected from an adult volunteer. Total RNA were isolated from the peripheral blood lymphocytes. We amplified segments of NR3C1 alpha mRNA, beta mRNA and ACTB mRNA using RT-PCR. Results By DHPLC, 3 aberrant elute profiles were revealed in exons 6, 8 and 9alpha of NR3C1 in one patient with SSNS. The chromatograms of exons 6, 8 and 9alpha of NR3C1 showed 1896OT> 2166C>T and 2430T>C of NR3C1, which is the Haplotype 5. OR of the Haplotype 3 and the Haplotype 5 were 4.824 and 2.469 (P values = 0.070 and 0.092, respectively). RT-PCR products of NR3C1 alpha mRNA, beta mRNA and ACTB mRNA were confirmed by agarose gel electrophoresis and sequencing. Conclusion From the data provided, we draw the following conclusions. (1) The risk of developing steroid resistance for sporadic nephrotic children with the Haplotype 3 and the Haplotype 5 showed 4.824 and 2.469 times more than those without the two haplotypes, respectively. Further study in larger cohorts is required to verify our preliminary results. (2) We succeed in establishing means of amplifing segments of NR3C1 mRNA using RT-PCR.