Proteomic Patterns and Prediction of Glomerulosclerosis and Its Mechanisms

Abstract

Protein expression profiles linked to sclerosis in the 5/6 nephrectomy (Nx) rat model of focal segmental glomerulosclerosis were investigated. Sections of control glomeruli from normal baseline Nx tissue and nonsclerotic and sclerotic glomeruli from 12 wk after 5/6 Nx were isolated by laser capture microdissection. Protein profiles were acquired directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Classification accuracy was 99.2% for distinguishing normal versus sclerotic glomeruli and 96.7 and 97.8% for nonsclerotic versus normal and sclerotic glomeruli, respectively. The proteomic pattern of the nonsclerotic glomeruli was more similar to sclerotic than normal glomeruli (P < 0.0001). Thymosin ␤4, a protein with relevant interactions with plasminogen activator inhibitor-1, angiogenesis, and wound healing, was identified as a key differentially expressed protein. P rogression of focal segmental glomerulosclerosis (FSGS) from early injury to overt sclerosis involves injury to all glomerular cell types (1,2), through multiple complex mechanisms. With the advancement of proteomic techniques (3,4), simultaneous examination of hundreds of proteins related to kidney disease holds promise in unraveling novel underlying mechanisms of progression and thus identifying possible targets for intervention in FSGS.To obtain protein expression profiles from a localized area, laser capture microdissection (LCM) has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for tissue protein profiling (5,6). LCM is an important tool in biologic research, enabling the isolation of specific cell populations from a heterogeneous tissue section (7). The technique of direct protein profiling from the laser capture microdissected sample using MALDI MS is fast, sensitive, and accurate. This technique has been applied in several studies, including those of human breast carcinoma (5), mouse epididymis (8), and human lung carcinoma (9). This technique enables the determination of protein expression profiles from even minute tissue structures within limited samples.The focal segmental nature of sclerosis in FSGS raises the question of whether at a given time point the remaining nonsclerotic glomeruli are already programmed to sclerotic pathways or, alternatively, whether these remaining nonsclerotic glomeruli have less prosclerotic activation and thus may be more susceptible to therapy. We therefore sought to examine proteomic profiles from the nonsclerotic glomeruli in FSGS and compare profiles from them with normal glomeruli and glomeruli with established sclerosis. We aimed first to establish the sensitivity of LCM and MALDI MS techniques to differentiate sclerosis from normal and, second, to investigate whether nonsclerotic glomeruli have a prosclerotic phenotype at the protein level. We also sought to identify key differentially expressed protein markers to advance our understanding of mechanisms and possible intervention in progressive renal disease. Materials and ...

The kidney in diabetes: dynamic pathways of injury and repair. The Camillo Golgi Lecture 2007

“…In addition, the healing capacities may be influenced by the inflammatory responses [69]. Newer high-throughput research tools [67] applied to this area could improve our understanding of healing processes, which may use signalling pathways similar to those involved in the original injury [70]. Thus, subtle manipulations of these injury/repair processes could lead to major benefits in terms of disease prevention and treatment.…”

Identification of Early Intestinal Neoplasia Protein Biomarkers Using Laser Capture Microdissection and MALDI MS

“…The HPLC fractions containing the proteins with the target molecular weight were found using MALDI MS and digested using trypsin. The tryptic peptides were sequenced using reverse-phase capillary LC-MS/MS with an LCQ Deca instrument (ThermoFinnigan, San Jose, CA) as described previously (10).…”

Протеоміка Та Мас-Спектрометрія З Матрично-Активованою Лазерною Десорбцією/Іонізацією Як Сучасний Діагностичний Інструмент При Захворюваннях Нирок

“…However, they also noted that non-sclerotic glomeruli within FSGS were in fact more similar to sclerotic glomeruli than completely normal glomeruli from a proteomic standpoint, hypothesizing that the early onset of sclerotic processes can be detected by molecular analyses at an earlier stage [20]. Employing a mouse model, Kaneko et al studied the pathogenesis of IgA nephropathy (IgAN).…”