Screening For Mutations And Proteomic Study In Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a disease characterizedmorphologically by segments of sclerosis in some glomeruli1. It is considered one ofthe most important causes of glomerulonephritis and nephrotic syndrome in adults.FSGS has been reported in5.6~37.8%of the biopsy-proven primaryglomerulonephritis2-5. Though the prevalence of FSGS varies among different areas, itis known to be increasing these years6-8. The typical clinical manifestations of FSGSinclude proteinuria, hematuria, hypertension, and progression to end-stage renaldisease(ESRD). The prognosis is relatively poor.5-year and10-year renal survivalrates are83.5%and43.8%respectively after the diagnosis of FSGS9. RefractoryFSGS with massive proteinuria and who are unresponsive to steroid therapy oftenhave a poor outcome;moreover, up to50%of these patients progress to ESRD within3-8years5. Thus, FSGS has been a hotspot for basic and clinical researchesthroughout the world.FSGS is categorized as primary and secondary10. The latter includes familialcases11. Currently, genetic variations in the podocyte genes are generally believed tobe the major cause of familial FSGS. Although it is known that among the podocytegenes associated with this disease, ACTN4, TRPC6and INF2gene mutations canlead to adult-onset familial FSGS12, few studies have reported the mutation rates ofACTN4gene and TRPC6gene in Chinese familial FSGS patients.The pathogenesis of primary FSGS which accounts for80%of the total cases isstill not very clear13.The development of proteomic techniques provide a new methodand possibility to find out the key points in the pathogenesis of FSGS and may lead tothe discovery of new therapeutic targets. These years,using isobaric tags for relativeand absolute quantitation(iTRAQ) together with2D-nano-HPLC-MS/MS, peptides can be accurately identified and quantified14. Comparing with2-dimensionalelectrophoresis， the method mentioned above has advantages in repetitiveness,sensitivity, labeling effciency and quantification15-17. Nowadays, proteomics has beenwidely used in the diagnosis of certain diseases, investigations of pathogenesis,evaluation of disease activities and prediction of prognosis18-23. Among various ofbody fluids, urine because of its easy obtaining, relatively stable nature and closerelationship with urinary system in anatomy has been an ideal specimen for theproteomic studies of kidney diseases.In the first part of this study，we performed a screening of TRPC6and ACTN4mutations in a large cohort of adult onset familial FSGS families(80families). Amongthe80probands, two index patients from two unrelated families were found to have amissense heterozygous change,p.Q889K in TRPC6and no mutations were found inthe ACTN4gene. Twenty-two SNPs of ACTN4and fourteen SNPs of TRPC6werealso found in this group. Thus, the observed disease-causing mutation rates of TRPC6and ACTN4were2.5%and0%in this cohort of Chinese patients with adult-onsetfamilial FSGS. This study supplement the data of TRPC6and ACTN4mutation ratesin adult-onset familial FSGS in China.In the second part of the study,200proteins were identified and quantified using4plex iTRAQ together with2D-nano-HPLC-MS/MS and the differences of urinaryproteome among primary FSGS, MCD patients and normol controls were compared.The identified proteins included enzymes, growth factors, transcription regulatoryfactors, receptors, transporters, ion channels etc located in cytoplasm, plasmamembranes, nuclei and extracellular spaces. Further study with Ingenuity PathwayAnalysis（IPA）software identified paxillin signaling, actin cytoskeleton signaling areamong the top canonical pathways, which indicated these two signaling pathwaysmight be involved in the pathogenesis of FSGS. Urinary vinculin which wasidentified to be increased in FSGS patients comparing with normal controls by2D-nano-HPLC-MS/MS was confirmed to be increased by urinary ELISA tests.In the third part of the study, we found an up-regulation of vinculin protein inpodocytes24hours after stimulated with adriamycin. Furthermore, FSGS patients had a higher fluorescence intensity in their frozen kidney biopsy specimens than patientswith glomerular minor lesions. These results indicate vinculin may play an importantrole in the pathogenesis of FSGS. No correlation was found between theconcentration of urinary vinculin and the clinical biochemical indicators includingurinary protein, serum albumin and serum creatinine.In summary, the mutation rates of TRPC6gene and ACTN4gene were very lowin Chinese adult-onset FSGS families, which indicate they may not be the mostcommon causes for the disease.The development of proteomic techniques contributesto the researches in FSGS.Vinculin may take part in the pathogenesis of FSGS.