Cell biology of pathologic renal calcification: contribution of crystal transcytosis, cell-mediated calcification, and nanoparticles.

INTRODUCTION: The earliest lesion in the kidneys of idiopathic calcium oxalate stone formers is deposition of calcium phosphate in the interstitium, termed a Randall's plaque. Yet the cellular and molecular factors leading to their formation are unknown. METHODS: The influence of urinary proteins on adhesion of preformed calcium oxalate crystals to rat continuous inner medullary collecting duct (cIMCD) cells was studied in vitro, and cIMCD cells were also exposed to calcifying media containing beta-glycerophosphate for up to 28 days. Renal tissue was obtained from a stone-forming and non-stone-forming individual at the time of nephrectomy. These nanoparticles, isolated from renal stones obtained at the time of surgical resection, were analyzed and propagated in standard cell culture medium. RESULTS: Urinary proteins influence crystal adhesion to renal epithelial cells, and this activity is abnormal in the urine of stone-forming patients. cIMCD cells assumed an osteoblastic phenotype when exposed to the calcifying medium, expressing two bone matrix proteins (osteopontin and bone sialoprotein) that were also identified in the kidney of the stone-forming patient and associated with crystal deposition. Nanoparticles were propagated from the majority of renal stones. Isolates were susceptible to selected metabolic inhibitors and antibiotics and contained conserved bacterial proteins and deoxyribonucleic acid (DNA). CONCLUSIONS: These results suggest new paradigms for Randall's plaque formation and idiopathic calcium oxalate stone disease. It seems unlikely that these events are driven solely by physical chemistry; rather, they are critically influenced by specific proteins and cellular responses, and understanding these events will provide clues toward novel therapeutic targets.

Renal cell adaptation to oxalate.

Renal manifestations of chronic hyperoxaluria include nephrolithiasis and, when extreme, interstitial scarring and progressive loss of function. Exposure of cultured renal cells to oxalate has been reported to cause cell death, as well as proliferation. The current study was performed to assess the time course and cell-type specificity of these responses. Proximal (LLC-PK(1)) and distal [cIMCD and primary human renal (HRC1)] renal epithelial cells, as well as interstitial KNRK cells, were exposed to oxalate (0.5-2.0 mM) for 24-72 h. The generation of reactive oxygen species (ROS) was measured using the fluorescent probe DCF, and cell number was determined with CyQuant reagent. HSP-70 expression was assessed via real time PCR and quantitative Western blot. In response to all oxalate concentrations (0.5-2.0 mM) and lengths of exposure (15 min-2 h), cultured proximal and distal renal epithelial cells and renal fibroblasts generated ROS. After 24 h, cells demonstrated initial cell death and decrease in cell numbers, but by 48-72 h adapted and grew, despite the continued presence of oxalate. This response was associated with increased expression of HSP-70 mRNA and protein. Renal cells in vivo may possess adaptive mechanisms to withstand chronic hyperoxaluria, including increased expression of chaperone molecules such as HSP-70.

Urinary macromolecular inhibition of crystal adhesion to renal epithelial cells is impaired in male stone formers.

BACKGROUND: Retention of microcrystals that form in tubular fluid could be a critical event in kidney stone formation. This study was performed to determine if urinary macromolecules from stone-forming (SF) individuals have reduced ability to inhibit crystal adhesion to renal cells. METHODS: A first morning whole urine (WU) sample was obtained from 24 SF subjects (17 males and 7 females) and 24 age-, race-, and sex-matched controls (C). An aliquot of urine was centrifuged and an ultrafiltrate (UF) free of macromolecules >10 kD and 10x concentrate (U(conc)) were prepared. RESULTS: Supplementing UF with increasing amounts of U(conc) to return the macromolecule concentration to 0.25x, 0.5x, or 1x of baseline progressively decreased crystal binding to cells. This effect was blunted in the male SF group compared to controls (P < 0.05, SF vs. C, for UF plus 0.25x macromolecules). No difference was apparent in the female groups. In order to identify responsible macromolecule(s), calcium oxalate monohydrate (COM) crystals were coated with U(conc) and adherent proteins then released and probed by Western blot. Coated COM crystals from male controls contained 3.5-fold more Tamm-Horsfall protein (THP) than SF subjects (P < 0.01). COM crystal coating with other proteins did not consistently differ between the groups. COM crystal coating by urinary prothrombin fragment 1 (UPTF1, P < 0.05) and crystal adhesion inhibitor (CAI) (P= 0.09) correlated with decreased crystal binding to cells, whereas coating with osteopontin (OPN) correlated with increased adhesion tendency (P < 0.05). CONCLUSION: Urinary macromolecules >10 kD coat COM crystals and block their adhesion to renal cells. This capacity appears to be blunted in male but not female SF individuals. Multiple urinary proteins may play a role in renal cell-urinary crystal interactions, and THP appears to be one of the more important ones.

Modulation of proliferating renal epithelial cell affinity for calcium oxalate monohydrate crystals.

Adhesion of urinary crystals to distal tubular cells could be a critical event that triggers a cascade of responses ending in kidney stone formation. Monolayer cultures of distal nephron-derived MDCKI cells were used as a model to study crystal-cell interactions. COM crystal adhesion reached a peak 2 d after plating and progressively fell thereafter. The decline in crystal binding was accelerated by prostaglandin E(2) (PGE(2)) supplementation and delayed by blockade of PG production. Crystals avidly adhered to cells that migrated in to repair a scrape wound made in the monolayer and after a transient hypoglycemic insult. Exposure of MDCKI cells to uric acid crystals and soluble uric acid was also associated with increased crystal adhesion. Treatment of physically or hypoglycemically injured cells with trypsin or neuraminidase reduced crystal binding to baseline levels, suggesting that increased exposure of cell surface glycoproteins mediated the effect, whereas PGE(2) treatment blunted crystal binding to regenerating cells. Furthermore, when cells were grown in the presence of synthetic d-mannosamine analogues that can modify the conformation of cell surface sialoglycoconjugates, crystal binding to proliferating cells was decreased, whereas blockade of N-glycosylation with tunicamycin increased crystal adhesion to these cells. Therefore, COM crystal binding is enhanced to growing renal cells, synthesis of N-glycosylated cell surface proteins is essential to downregulate crystal binding to cells, and this response is modulated by physiologic signals such as PGE(2). Sialic acid residues seem to mediate crystal adhesion to growing cells, either directly or via linkage to other crystal-binding molecules. Subtle renal injury and subsequent nephron repair could be a factor promoting crystal adhesion and favoring calculus formation.

Extract from Herniaria hirsuta coats calcium oxalate monohydrate crystals and blocks their adhesion to renal epithelial cells.

PURPOSE: The interaction of calcium oxalate crystals with renal epithelial cells is a critical event in kidney stone formation. In this study we assessed the effect of aqueous extract from Herniaria hirsuta on the adhesion of calcium oxalate monohydrate (COM) crystals to cultured renal cells. MATERIALS AND METHODS: Madin Darby canine kidney cells were used as a model for studying the adhesion of radioactive COM crystals in the presence and absence of plant extract. RESULTS: COM crystal binding to cells was inhibited by extract in a concentration dependent manner. Prior exposure of crystals but not cells to extract blocked crystal binding, suggesting that plant molecules can coat and exert their effect at the crystal surface. Crystal attachment appeared related to membrane fluidity since crystal adhesion increased at higher vs lower temperatures (37C vs 0C) and Herniaria extract altered crystal adhesion only under conditions of increased fluidity (increased temperature). Extract also displaced a significant portion of prebound crystals without apparent effects on cell function or the morphology of preexisting calcium oxalate crystals. Herniaria extract exerted no adverse or toxic effect on cells, which proliferated normally in its presence even at relatively high concentrations. CONCLUSIONS: Our current data suggest a mechanism whereby Herniaria hirsuta extract used in traditional medicine might prevent and possibly eliminate preexisting kidney stones. Further characterization of the active compound(s) could identify a new candidate drug for patients with nephrolithiasis.

Renal epithelial cells constitutively produce a protein that blocks adhesion of crystals to their surface.

Attachment of newly formed crystals to renal tubular epithelial cells appears to be a critical step in the development of kidney stones. The present study was undertaken to identify autocrine factors released from renal epithelial cells into the culture medium that inhibit adhesion of calcium oxalate crystals to the cell surface. A 39-kDa glycoprotein that is constitutively secreted by renal cells was purified by gel filtration chromatography. Amino acid microsequencing revealed that it is novel and not structurally related to known inhibitors of calcium oxalate crystallization. Hence, it was named crystal adhesion inhibitor, or CAI. Immunoreactive CAI was detected in diverse rat tissues, including kidney, heart, pancreas, liver, and testis. Immunohistochemistry revealed that CAI is present in the renal cell cytosol and is also on the plasma membrane. Importantly, CAI is present in normal human urine, from which it can be purified using calcium oxalate monohydrate crystal affinity chromatography. CAI could be an important defense against crystal attachment to tubular cells and the subsequent development of renal stones in vivo.

The effect of ions at the surface of calcium oxalate monohydrate crystals on cell-crystal interactions.

Magnesium is an abundant ion in biologic systems, including renal tubular fluid; however, the precise role of magnesium during the interaction of calcium oxalate crystals with cells has not been previously defined. In addition, the respective roles of calcium and hydrogen ions during the cell-crystal bonding interaction remain poorly defined. Here we report an atomic level three-dimensional study of a single crystal of calcium oxalate monohydrate (COM; whewellite) which was bathed in a solution of magnesium hexahydrate for 1 year. Magnesium was not incorporated into the structure of whewellite to any significant degree. Instead, COM accepted magnesium primarily as an adsorbate in a binding configuration which, as a surface phenomenon, is controlled by localized charge effects. The effect of magnesium and calcium on the efficiency of calcium oxalate crystal binding to renal cells was also investigated. When present in supraphysiologic concentrations (greater than 0.1 M), magnesium progressively inhibited adhesion of pre-formed COM crystals to cultured renal cells. Therefore, even though magnesium does not incorporate into the crystal structure of calcium oxalate, magnesium can exert important surface effects and change the interaction of pre-formed COM with molecules anchored on the cell surface. Similarly, binding was nearly blocked when the exogenous calcium concentration was > or =0.1 M (supraphysiologic range), although in lower concentrations (within the physiologic range) exogenous calcium promoted crystal adhesion. Finally, the ambient hydrogen ion concentration also influenced calcium oxalate crystal interactions with renal cells, with maximal binding occurring at a pH of 4. Therefore, hypercalciuria and/or an acidic urine could each promote renal stone formation via increased crystal adhesion to renal cells, a previously under-appreciated potential mechanism.

Whole urinary proteins coat calcium oxalate monohydrate crystals to greatly decrease their adhesion to renal cells.

PURPOSE: Adhesion of urinary crystals to renal tubular cells could be a critical event that triggers a cascade of responses ending in kidney stone formation. We clarified the role of urinary macromolecules during calcium oxalate monohydrate (COM) crystal adhesion to cells. MATERIALS AND METHODS: To assess COM crystal binding to cells in the presence of whole urine and fractions thereof we used monolayer cultures of distal nephron derived Madin-Darby canine kidney, type I cells as a model system. RESULTS: COM crystal adhesion to cells was decreased in the presence of whole urine compared with an ultrafiltrate prepared by passing urine through a 10 kDa cutoff membrane. Supplementing the ultrafiltrate with urinary concentrate containing proteins greater than 10 kDa returned crystal adhesion to low levels, similar to whole urine. Macromolecules in whole urine acted to decrease binding to cells by coating crystals and 4 proteins previously implicated in the pathogenesis of nephrolithiasis were detected on coated crystals (bikunin, osteopontin, prothrombin fragment 1 + 2 and Tamm-Horsfall glycoprotein). Crystals precipitated and grown in whole urine also bound less avidly to cells than crystals precipitated in artificial urine. CONCLUSIONS: This study confirms that macromolecules present in whole urine can coat crystals and, thereby, block their adhesion to renal tubular cells. Preventing crystal retention in the kidney could be an important mechanism whereby these macromolecules protect against kidney stones.

Annexin II is present on renal epithelial cells and binds calcium oxalate monohydrate crystals.

Attachment of newly formed crystals to renal epithelial cells appears to be a critical step in the development of kidney stones. The current study was undertaken to identify potential calcium oxalate monohydrate (COM) crystal-binding proteins on the surface of renal tubular cells. Apical membranes were prepared from confluent monolayers of renal epithelial cells (MDCKI line), and COM crystal affinity was used to isolate crystal-binding proteins that were then subjected to electrophoresis and electroblotting. Microsequencing of the most prominent COM crystal-binding protein (M(r) of 37 kD) identified it as annexin II (Ax-II). When exposed proteins on the surface of intact monolayers were biotinylated and then isolated using streptavidin agarose beads, Ax-II was detected, suggesting that at least a portion is exposed on the apical cell surface. Ax-II was not completely extracted by 0.1 M Na(2)CO(3), suggesting that at least a portion of cellular Ax-II is an intrinsic membrane-bound protein. Using confocal immunofluorescence microscopy, Ax-II was visualized together with Caveolin-1 (Cav-1) on the apical membrane of intact MDCKI cells. Cells pretreated with a monoclonal anti-Ax-II antibody bound significantly fewer COM crystals, whereas anti-LDL receptor antibody did not decrease COM binding, further suggesting a functional role for Ax-II during adhesion of crystals to intact cells. These results suggest that Ax-II avidly binds COM crystals and is present on the apical surface of MDCKI cells. Therefore, in the intact nephron, Ax-II could mediate adhesion of COM crystals to cells, and altered exposure of Ax-II on the surface of renal tubular cells could promote crystal retention and possibly kidney stone formation.