Article pubs.acs.org/crystal
Effect of Urinary Macromolecules on L‑Cystine Crystal Growth and Crystal Surface Adhesion Trinanjana Mandal, Alexander G. Shtukenberg, Anthony C. Yu, Xiao Zhong, and Michael D. Ward* Department of Chemistry and the Molecular Design Institute, New York University, 100 Washington Square East, New York, New York 10003-6688, United States S Supporting Information *
ABSTRACT: L-Cystine is the primary crystalline component of L-cystine kidney stones, which are a consequence of cystinuria, a genetic disorder that results from mutation in the SLC3A1 or the SLC7A9 gene. Urinary macromolecules present in the cellular matrix are thought to play a role in the pathogenesis of kidney stones affecting crystal nucleation, growth, crystal−crystal aggregation, and adhesion to epithelial cells. The effect of six prevalent urinary constituentsosteopontin, Tamm−Horsfall protein, albumin, apotransferrin, chondroitin sulfate, and lysozymeon crystallization kinetics and adhesion events on the L-cystine (0001) surface was investigated with real-time in situ atomic force microscopy (AFM). These additives did not significantly change crystal morphology and crystallization yield, although slight reductions in step velocities were observed at nanogram per milliliter and microgram per milliliter concentrations. Chemical force microscopy performed with AFM tips decorated with terminal carboxylate, amino, and cysteine moieties revealed that macromolecular additives reduce the binding affinity of the {0001} face of L-cystine toward these groups. Collectively, these observations suggest that these macromolecules may actually mitigate L-cystine crystal growth and aggregation into stones.
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INTRODUCTION Cystinuria is an autosomal recessive disorder caused by mutation in either the SLC3A1 gene on chromosome 2 or SLC7A9 gene on chromosome 19, which results in defective reabsorption of cystine, ornithine, lysine, and arginine amino acids in the proximal tubule of the nephron and epithelial cells of the gastrointestinal tract.1 The relatively low solubility of Lcystine under the physiological conditions of the kidney and bladder, compared with the other amino acids, leads to the formation of L-cystine crystals and their stones,2 which are aggregates of hexagonal plates of L-cystine crystals (Figure S1, Supporting Information). The mechanism of stone formation involves several key steps, including nucleation of the main crystalline component, crystal growth, aggregation, and attachment to renal epithelial cells.3 Calcium oxalate monohydrate (COM), the major constituent of calcium oxalate kidney stones, has been studied most extensively in this regard.4−15 Hydrophilic urinary proteins containing a number of carboxylate and phosphate groups, such as osteopontin and Tamm−Horsfall protein, were reported to interact strongly and selectively with various faces of calcium oxalate monohydrate, thereby inhibiting crystal growth while altering crystal habit.4,5,7,15 The face-selective binding of the additives has been attributed to a dependence on surface densities of Ca2+ and C2O42− ions, orientations of C2O42− ions at exposed crystal faces, and stereochemical matching of macromolecules into steps with specific heights.5,7,10,15 Extensive studies have been reported on protein−calcium oxalate crystal interaction, but opposing © 2015 American Chemical Society
views exist regarding the role of proteins, specifically whether they promote5,11,12 or inhibit13,14 aggregation and crystal adhesion. Tamm−Horsfall protein has been reported to undergo a role reversal from being an aggregation inhibitor to promoter of stone formation with change in concentration.16,17 Previously reported analyses of the organic matrix in various kidney stonescalcium oxalate, uric acid, and struvitereveal the presence of commonly occurring urinary proteins, such as human serum albumin, transferrin, and immunoglobulin.18 To the best of our knowledge, there have not been any studies exploring the role of common urinary macromolecules in cystine stone formation. Herein we describe the influence of several common urinary macromoleculesosteopontin, Tamm−Horsfall protein, apotransferrin, albumin, chondroitin sulfate, and lysozyme (Table 1)and their effect on L-cystine crystal growth kinetics, crystal morphology, and crystal surface adhesion. As the face-selective adsorption of biomolecules on specific crystal faces is often masked at high concentrations,19 crystal growth measurements were performed at nanograms per milliliter and micrograms per milliliter biomolecule concentrations, within the range known to exist in urine.20 Real-time in situ atomic force microscopy (AFM) experiments were conducted to study the influence of additives on the {0001} face micromorphology and crystallization kinetics. AFM probes Received: October 2, 2015 Revised: December 4, 2015 Published: December 18, 2015 423
DOI: 10.1021/acs.cgd.5b01413 Cryst. Growth Des. 2016, 16, 423−431
Crystal Growth & Design
Article
Table 1. Macromolecule Additives macromolecule
MW (KDa)
osteopontin (OPN)
44
Tamm−Horsfall protein (THP) apotransferrin (Apt)a human serum albumin (HSA) chondroitin sulfate A (ChS)b lysozyme (Lsz)
68 80 66.5 46.3 14.3
pI 3.5 3.5 5.5 4.4 6.8 11.4
concn in urine (μg/mL)
no. of cysteine residues
∼4
2
20−25
