Macropinocytosis is the Major Mechanism for Endocytosis of Calcium Oxalate Crystals into Renal Tubular Cells
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ORIGINAL PAPER
Macropinocytosis is the Major Mechanism for Endocytosis of Calcium Oxalate Crystals into Renal Tubular Cells Rattiyaporn Kanlaya • Kitisak Sintiprungrat Sakdithep Chaiyarit • Visith Thongboonkerd
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Ó Springer Science+Business Media New York 2013
Abstract During an initial phase of kidney stone formation, the internalization of calcium oxalate (CaOx) crystals by renal tubular cells has been thought to occur via endocytosis. However, the precise mechanism of CaOx crystal endocytosis remained unclear. In the present study, MDCK renal tubular cells were pretreated with inhibitors specific to individual endocytic pathways, including nystatin (lipid raft/caveolae-mediated), cytochalasin D (actindependent or macropinocytosis), and chlorpromazine (CPZ; clathrin-mediated) before exposure to plain (non-labeled), or fluorescence-labeled CaOx monohydrate (COM) crystals. Quantitative analysis by flow cytometry revealed that pretreatment with nystatin and CPZ slightly decreased the crystal internalization, whereas the cytochalasin D pretreatment caused a marked decrease in crystal uptake. Immunofluorescence study and laser-scanning confocal microscopic examination confirmed that the cytochalasin D-pretreated cells had dramatic decrease of the internalized crystals, whereas the total number of crystals interacted with the cells was unchanged (crystals could adhere but were not internalized). These data have demonstrated for the first time that renal tubular cells endocytose COM crystals mainly via macropinocytosis. These novel findings will be useful for
Electronic supplementary material The online version of this article (doi:10.1007/s12013-013-9630-8) contains supplementary material, which is available to authorized users. R. Kanlaya K. Sintiprungrat S. Chaiyarit V. Thongboonkerd (&) Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, and Center for Research in Complex Systems Science (CRCSS), Mahidol University, 6th Floor SiMR Building, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand e-mail: [email protected]; [email protected]
further tracking the endocytosed crystals inside the cells during the course of kidney stone formation. Keywords Calcium oxalate Crystal Endocytosis Internalization Macropinocytosis
Introduction Calcium oxalate (CaOx) is the major causative crystalline composition of kidney stones, which lead to a common health problem around the globe [1]. Many lines of evidence from in vivo investigations and clinical studies have demonstrated that CaOx crystals can deposit inside tubular lumen [2] and renal interstitium [3, 4] during the course of kidney stone development and then aggravate renal tissue injury and dysfunction [5]. By adhesion, crystals can be retained on apical surface of renal tubular cells, which subsequently internalize the adherent crystals most likely by endocytosis [6, 7]. The internalized or endocytosed crystals are then translocated to basolateral site of renal tubular cells and finally to the interst
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