Transcriptomic Analysis Of Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis(FSGS) is one of the most common cause of steroid resistant nephrotic syndrome(SRNS) and renal failure among adults and children. The pathophysiologic mechanism of FSGS is still unclear. The diagnosis of FSGS is based on renal biopsy. It’s characterized by focal segmental glomerulosclerosis and diffuse foot process effacement. However, the absence of glomeruli with segmental sclerosis on renal biopsy may not necessary rule out the diagnosis of FSGS because of the focal nature of this disease and the random sampling of kidney biopsy. FSGS can be divided into three categories through its etiology: primary, secondary and familial. Familial FSGS(FFSGS), also called hereditary FSGS, which accounts for 9% of non-secondary FSGS. FSGS has a clinical character of massive proteinuria, partially characterized by nephrotic range proteinuria. Treatment strategy of this part of FSGS patients mainly involved in corticosteroid and immunosuppressant.FSGS patients can be divided into 3 groups according to their response of corticosteroid, steroid sensitive, steroid dependent and steroid resistant, FSGS patients with steroid dependent or steroid resistant are prone to progress to end stage renal disease(ESRD)..Transcriptomics is a gene expression analysis based on RNA level. Previous report of transcriptomic analysis of FSGS patients, using m RNA extracted from formalin-fixed, paraffin-embedded renal specimens(biopsied renal sample including glomeruli and tubulointerstitial tissue), which may not be suitable for transcriptome analysis because FSGS is podocyte related disease, and transcriptome from tubulointerstitial tissue might result in confounding bias. Additionally, RNA integrity and quantity might be injured during sample preparation. In this study, we isolated glomeruli from biopsied kidney of FSGS patients, and Microarray analysis of transcriptome extracted from isolated glomeruli. And we aimed to search for pathogenic mechanism of FSGS from transcriptomic aspect. We inrolled sporadic FSGS patients with SSNS and SRNS, hereditary FSGS patients with COL4A3 mutation, in order to study mechanism of FSGS from varied aspect.In the first part of this study, we recruited 6 primary FSGS patients and 5 MCD patients, all the patients have not received corticosteroid and/or immunosuppressant treatment at the time when receiving kidney biopsy. And all the FSGS and MCD patients were manifested by steroid-sensitive nephrotic syndrome, excluding bias resulting from different treatment response. We isolated glomeruli from cortical regions of biopsied kidney from patients, under an inverted microscope. We compared the profiles of glomerular transcriptome between the two groups of patients, focusing on analysis of differential expression genes(DEGs). The results revealed that up-regulated DEGs in FSGS as compared to MCD patients were mostly involved in spermatogenesis, gamete generation, response to unfolded protein, response to proteins stimulus, regulation of muscle contraction. While down-regulated DEGs were mostly related to metabolic process, intracellular transport, oxidation reduction, establishment of localization in cell. We validated the expression of the top 6 up-regulated DEGs, and top 6 down-regulated DEGs in 7 addiotional FSGS patients and 7 addiotional MCD patients by RT-PCR, the expression tendency of related genes by RT-PCR analysis were consistent with Microarray data. Among these down-regulated genes, we validated that expression of membrane metalloendopeptidase(MME) protein was lower in glomeruli of FSGS patients compared with MCD patients, which indicated MME can be a potential marker that may help us to differente FSGS from MCD, and reminded us that MME may take part in the pathogenesis of FSGS.Besideds sporadic FSGS, previous study pointed out a part of FFSGS were caused by mutation of podocyte related genes. Which reminded us there was different mechanism between FFSGS patients and sporadic FSGS. Our previous study identified heterozygous COL4A3 mutations in FSGS families. In the second part of this study, we recruited 3 FFSGS patients with heterozygous COL4A3 mutation(C1616Y,G801 R,L737H) and 5 MCD patients.We compared clinical parameters of the two groups. From the results of this part, compared with MCD patients, there was an earlier onset of the disease in FFSGS patients. In addition only a small part of the FFSGS patients were manifested by nephrotic syndrome, and more patients with FFSGS were complicated with haematuria. The FFSGS patients with COL4A3 mutation has lower level of urinary protein, but a higher level of serum albumin, and a lower level of filtration rate when receiving renal biopsy. We compared the profiles of glomerular transcriptome between the two groups of patients. Analysis of DEGs revealed that up-regulated genes in FFSGS patients with COL4A3 mutation as compared to MCD patients were mostly involved in cell-cell signaling, cell surface receptor linked signal transduction, neurological system process, cognition and sensory perception, G-protein coupled receptor protein signaling pathway. While down-regulated DEGs were mostly related to metabolic and catabolic process, proteolysis and ubiquitin-dependent protein catabolic process, intracellular transport. We validated the expression of the top 6 up-regulated DEGs, and top 6 down-regulated DEGs in the same patients by RT-PCR, the results showed expression tendency of related genes were consistent with Microarray data. Since collagen 4 is the major component of GBM, and in clinical practice, we found a portion of FSGS patients pathologically characterized by segmental thinning GBM, the mechanism and chinical value of this change is still not clear. Our previous study found FSGS patients with heterozygous COL4A3 mutation can manifest by segmental thin GBM, but not diffuse GBM thinning. We compared clinical and pathological parameters of FSGS patients with segmental thin GBM, and FSGS patients with normal GBM, aimed to find clinical, pathological and prognostic difference between the two groups. We compared the clinical and pathological features and the prognosis of 63 FSGS patients with segmental thin GBM, and 60 FSGS patients with normal GBM. The results showed comparing with FSGS patients with normal GBM, patients with segmental thin GBM had a higher urine protein excretion rate and a lower serum albumin level, has a lower level of filtration rate and serum hemoglobulin,. However, there were no significant difference of percentage of patients with nephrotic syndrome, hematuria, or pathological character or treatment response when comparing the two groups..Treatment strategy of FSGS mainly involved corticosteroid and immunosuppressant. Accrording to previous data, one in third of FSGS patients had poor response to corticosteroid, but the reason is unknown. In the third part of the study, we recruited 6 primary FSGS patients with SSNS and 3 primary FSGS patients with SRNS. We compared the profiles of glomerular transcriptome between the two groups of patients using Microarray analysis. Analysis of DEGs revealed that up-regulated DEGs in FSGS-SRNS patients as compared to FSGS-SSNS patients were mostly involved in cellular amino acid metabolic process, and ion transport. While down-regulated DEGs were mostly related to anatomical structure morphogenesis, regulation of DNA-dependent transcriptomic process, negative regulation of MAP kinase antivity. We validated the expression of the top 6 up-regulated genes and a selected panel of up-regulated genes(AK4、NPHS2、TRPC6), and STRA6, which was down-regulated in FSGS-SRNS patients compared with FSGS- SSNS patients, by real-time PCR, the expression tendency of related genes by RT-PCR analysis were consistent with Microarray data. Immunohistochemical staining of human kidney biopsy specimens demonstrated that protein expression of STRA6 was also significantly decreased in kidneys of FSGS-SRNS patients ompared with FSGS-SSNS patients. Our studies suggest that low expression of STRA6 in FSGS patients may be related to development of SRNS, and we found that STRA6 can be a potential clinical biomarkers predicting FSGS patients with different treatment response and STRA6 may involved in the development of FSGS patients with SRNS.Totally speaking, our study recruited primary FSGS-SSNS, hereditary FSGS(with COL4A3 heterozygous mutation), FSGS-SRNS patients; and MCD patients as the control group. We obtained transcriptomic fingerprint of FSGS patients via transcriptomic analysis. And we found greatly distinct glomerular transcriptomes between primary FSGS-SSNS patients, hereditary FSGS and MCD patients; and largely diversed glomerular transcriptomes between FSGS-SRNS patients and FSGS-SSNS patients. Among the differentially expressed genes, genes involved in permatogenesis, response to unfolded protein, metabolic process, were differentially expressed in glomerular transcriptomes comparing FSGS-SSNS patients with MCD patients; Genes take part in cell-cell signaling, cell surface receptor linked signal transduction, cognition and sensory perception, G-protein coupled receptor protein signaling pathway; and genes related to metabolic and catabolic process, proteolysis and ubiquitin-dependent protein catabolic process, intracellular transport were differentially expressed in glomerular transcriptomes comparing hereditary FSGS(with COL4A3 heterozygous mutation) with MCD patients; Genes involved in cellular amino acid metabolic process, ion transport, anatomical structure morphogenesis, regulation of DNA-dependent transcriptomic process were differentially expressed comparing FSGS-SRNS patients with FSGS-SSNS patients.Our studies found that expression of MME gene and protein were significantly lower in glomeruli of primary FSGS patients compared with MCD patients, which indicate analysis of expression of MME between primary FSGS and MCD patients may help us to differente these two diseases, and MME may take part in development of FSGS. Expression of STRA6 gene and protein were also significantly decreased in glomeruli of FSGS-SRNS patients ompared with FSGS-SSNS patients. suggest that STRA6 can be a potential clinical biomarkers predicting FSGS patients with different treatment response and STRA6 may involved in the development of FSGS patients with SRNS.We found a part of sporadic FSGS patients were manifested by segmental GBM thinning, we further analyzed clinical, pathological and prognostic differentiation between FSGS patients with and without segmental GBM thinning, and we concluded FSGS patients with segmental GBM thinning has lower level of glomerular filtration rate, higher level of urinary protein, and lower level of serum albumin, which indicated clinical manifestation is more severe.