Large-scale Identification of Calcium Oxalate Monohydrate Crystal-binding Proteins on Apical Membrane of Distal Renal Tubular Epithelial Cells

Abstract

Adhesion of calcium oxalate monohydrate (COM) crystals onto apical surface of renal tubular epithelial cells is a crucial mechanism for crystal retention, leading to kidney stone formation. Various proteins on apical membrane may bind to COM crystals; however, these crystal-binding proteins remained unidentified. The present study therefore aimed to identify COM crystal-binding proteins on apical membrane of distal renal tubular epithelial cells. Madin-Darby Canine Kidney (MDCK) cells were cultivated to be polarized epithelial cells and apical membrane was isolated from these cells using a peeling method established recently. Enrichment and purity of isolated apical membrane were confirmed by Western blot analysis for specific markers of apical (gp135) and basolateral (Na(+)/K(+)-ATPase) membranes. Proteins derived from the isolated apical membrane were then resuspended in artificial urine and incubated with COM crystals. The bound proteins were eluted, resolved by SDS-PAGE, and analyzed by Q-TOF MS and MS/MS, which identified 96 proteins. Among these, expression and localization of annexin II on apical surface of MDCK cells were confirmed by Western blot analysis, immunofluorescence staining, and laser-scanning confocal microscopic examination. Finally, the function of annexin II as the COM crystal-binding protein was successfully validated by COM crystal-binding assay. This large data set offers many opportunities for further investigations of kidney stone disease and may lead to the development of new therapeutic targets.

In vitro evidence of the promoting effect of testosterone in kidney stone disease: A proteomics approach and functional validation

“…COM crystal adhesion on the renal tubular cell surface is an important initiating step in kidney stone formation. Our previous study demonstrated a number of potential COM crystal receptors on renal tubular cell membranes, which can bind to COM crystals [44]. Interestingly, one of such potential COM crystal receptors is α-enolase, of which cellular level was increased upon testosterone treatment in our present study (Figs.…”

Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor

“…Our previous expression proteomics study identified α-enolase as a COM crystal-binding protein on apical membranes of renal tubular epithelial cells by quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS)4. Additional supportive evidence has demonstrated that expression level of α-enolase was increased and decreased after renal tubular cells were treated with stone aggravators1213 and inhibitor14, respectively.…”

“…Recently, a number of potential COM crystal-binding molecules and/or proteins expressed on the apical membranes of renal tubular epithelial cells have been identified234. After renal tubular cell injury by numerous inducers, the injured renal tubular cells showed increased expression of COM crystal-binding molecules/proteins in concordance with the enhanced COM crystal binding on the cell surfaces5.…”

“…Our previous expression proteomics study successfully identified a large number of COM crystal-binding proteins isolated from apical membranes of MDCK renal tubular epithelial cells4. Among them, a glycolytic enzyme α-enolase was also identified by mass spectrometry in the COM crystal-bound fraction.…”

Prospects for proteomics in kidney stone disease

“…chemical-based precipitation and centrifugation-based methods) have been proven to be problematic and ineffective for isolation/purification of apical membrane proteins by contaminations of other cellular compartments, particularly basolateral membranes [9]. Using the apical membrane peeling method, a recent MS-based proteomics study successfully identified almost a hundred of potential CaOx crystal receptors on apical membrane of renal tubular cells that may be the targets for prevention of the stone formation/ recurrence [10]. In addition, another method known as membrane protein 'shaving' is particularly useful for the development of drug targets and defining potential protein receptors [11].…”