Role of Autophagy in Membranous Glomerulonephropathy---From Animal to Human Model

Membranous glomerulonephropathy (MN) is the most common cause of idiopathic nephrotic syndrome in adults. The clinical course in the majority of patients appears to be indolent and slowly, although some series have suggested a higher incidence of progression to end-stage renal disease (ESRD). The incidence of ESRD is around 14% in 5 years、35% in 10 years and 40% in 15 years. However, the definite mechanisms of MN is still not clear. Recent research indicates that progressive glomerulosclerosis and podocyte injury are key determinants of glomerular diseases and ESRD. To date, the role of autophagy in glomerular disease remains under-investigated. With regards to the glomerulus, recent studies suggested that mTORC1 inhibition by rapamycin or everolimus can activate autophagy and delay or reverse glomerular diseases, such as focal segmental glomerulosclerosis, membranous nephropathy, minimal change disease, crescentic glomerulonephritis and diabetic nephropathy. Thus, autophagy may act as a major cytoprotective pathway promoting cell survival and play an essential function for tissue homeostasis. Our preliminary data have demonstrated time course dependent autophagic protein expression in MN mice model. We also hypothesize that autophagy expression might be related to signal transduction protein such as mTOR/PI3K class I, PI3K class III/BECN1 and BNIP3/HSPIN1 etc. Therefore, we proposed the three – year’s proposal to discover the relationship between autophagic proteins and its probable transduction signal pathway on MN from animal model to human. A. First year : Study the course of autophagic protein profile and therapeutic effect of autophagic modulator in MN mice 1. Investigate the time course changes (including 2, 4, 6, 8 and 12 weeks) of autophagic protein profiles in kidney tissue using western blot from MN mice. 2. Observe directly the autophagosome (eg. MDC, an autophagaic vesicle tracer in vivo) in kidney tissue slide from MN mice at different time point by using fluoresces/electronic microscope. 3. Study the role of autophagic protein and autophagosome in the pathogenesis of MN by using pre-treatment with inducer/inhibitor (rapamycin/3-MA). 4. Test the efficacy of 8 FDA-proved drugs (luspirilene, trifluoperazine, pimozide, niguldipine, nicardipine, amiodarone, loperamide and penitrem A) through autophagic protein and autophagosome assay in kidney tissue from MN mice. 5. Check the correlation between these autophagic proteins/autophagosome expression after 8 drugs pre-treatment and the change of disease severity guided by biochemical and histological index during the course of MN. B. Second year : Study the possible signal pathway which induce autophagic protein and cell type in MN mice 1. Based on the data of first year, we will study the transduction signals, such as mTOR/PI3K class I, PI3K class III/BECN1 and BNIP3/HSPIN1 signal pathway, at different time points in kidney tissue from MN mice 2. Detect the autophagic protein, autophagosome and signal proteins and then double stain with the specific cell marker [eg. podocyte - podocalyxin, RTC - Tamm-Horsfall protein (THP) etc.] in kidney tissue slide from MN mice at different time point by using fluorescencs microscope. 3. Investigate above autophagic protein, autophagosome and transduction signals at different time points using cellular models such as renal tubular cell, mesangial cell or podocyte cell lines etc. 4. Double labeling of autophagic protein/autophagosome and signal transduction protein at different time points using cellular models such as renal tubular cell, mesangial cell or podocyte cell lines etc. C. Third year : Study the autophagic protein profile and its related signal pathway of MN in human 1. Samples of urine; serum from MN and NC person and renal biopsy specimen from patient, while normal renal tissue from non-renal diseased patient (as control) will be collected for investigating the autophagic proteins expression, transduction signals, such as mTOR/PI3K class I, PI3K class III/BECN1 or BNIP3/HSPIN1 signal pathway during MN process. 2. Characterize and confirm the expression and location of specifically candidate proteins (including beclin-1, LC3I/II etc.) by using IF double staining. 3. Check the correlation between these autophagic proteins/autophagosome expression and the change of disease severity guided by biochemical and histological index in patients with MN