High Calcium Enhances Calcium Oxalate Crystal Binding Capacity of Renal Tubular Cells via Increased Surface Annexin A1 but Impairs Their Proliferation and Healing

Abstract

Hypercalciuria is associated with kidney stone formation and impaired renal function. However, responses of renal tubular cells upon exposure to high-calcium environment remain largely unknown. We thus performed a proteomic analysis of altered proteins in renal tubular cells induced by high-calcium and evaluated functional significance of these changes. MDCK cells were maintained with or without 20 mM CaCl(2) for 72 h. Cellular proteins were then analyzed by two-dimensional electrophoresis (2-DE) (n = 5 gels derived from 5 independent culture flasks per group). Spot matching and quantitative intensity analysis revealed 20 protein spots (from a total of 700) that were differentially expressed between the two groups. These altered proteins were then identified by Q-TOF-MS and MS/MS analyses, including those involved in calcium binding, protein synthesis, carbohydrate metabolism, lipid metabolism, cell proliferation, mitosis regulation, apoptosis, cell migration, oxidative stress, and ion transport. Protein network analysis and functional validation revealed that high-calcium-exposed cells had 36.5% increase in calcium oxalate monohydrate (COM) crystal-binding capacity. This functional change was consistent to the expression data in which annexin A1 (ANXA1), a membrane-associated calcium-binding protein, was markedly increased on the apical surface of high-calcium-exposed cells. Pretreatment with anti-ANXA1 antibody could neutralize this increasing crystal-binding capacity. Moreover, high-calcium exposure caused defects in cell proliferation and wound healing. These expression and functional data demonstrate the enhanced crystal-binding capacity but impaired cell proliferation and wound healing in renal tubular cells induced by high-calcium. Taken together, these phenomena may contribute, at least in part, to the pathogenic mechanisms of hypercalciuria-induced nephrolithiasis and impaired renal function. Our in vitro study offers several candidates for further targeted functional studies to confirm their relevance in hypercalciuria and kidney stone disease in vivo.

Caffeine prevents kidney stone formation by translocation of apical surface annexin A1 crystal-binding protein into cytoplasm: In vitro evidence

“…annexin A1, α-enolase, heat shock protein 90 (HSP90)) on apical membranes of renal tubular epithelial cells as potential crystal receptors essential for COM crystal adhesion on apical surface of the cells, intratubular COM crystal retention, and subsequently, stone formation712131415. Interestingly, expression of these potential COM crystal receptors could be altered by stone modulators or risk factors.…”

Unravelling Pathophysiology of Crystalline Nephropathy in Ceftriaxone-Associated Acute Kidney Injury: A Cellular Proteomic Approach

“…The expression of annexin A1 (ANXA1 gene) in Cef-Ca-treated MDCK cells was validated by Western blot analysis because of its involvement in a predicted function of altered proliferation and wound healing, together with its characteristic of calcium binding [21] and playing roles in heat stress response [22]. Unexpectedly, annexin A1 was not detected in renal tubules by 4 of 5 antibodies used in Human Protein Atlas (www.proteinatlas.org/­ENSG00000135046-ANXA1/tissue).…”

“…Another set of proteome change that should be considered is the downregulation of annexin A1, annexin A2, and alpha-enolase, since they are recognized as calcium-binding molecules and characterized on distal tubular cell surface as receptors of calcium oxalate crystalline (the major component of adult kidney stones) [21, 30, 31]. Of these proteins, annexin A1 shows evidence of serving as an important player in the heat stress response [22, 32], and thus best fits the proposed pathophysiology of ceftriaxone-associated AKI in this study.…”

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

“…The mechanism of kidney stone disease is generally started with crystallization due to supersaturation of calcium and oxalate ions in renal tubular fluid [23,36,37]. Subsequent events include crystal adhesion on renal tubular cell surface [38,39], crystal growth [40,41], crystal aggregation [42,43], and finally, stone formation.…”

“…In the present study, we focused our attention on the pathogenic mechanisms of COM kidney stone formation, which has been widely accepted to initiate at the tubular segment of the distal nephron [48][49][50][51]. MDCK cells, which were originated from renal cortex and showed many characteristics of renal tubular epithelial cells [15,16], have been widely used for in vitro investigations of COM kidney stone disease [23,37,[52][53][54]. Therefore, MDCK cells were selected for our present study, whereas other renal cell types that would definitely be beneficial for other stone models and other kidney diseases were not included.…”