Proposed mechanisms in renal tubular crystal retention
2007; Elsevier BV; Volume: 72; Issue: 1 Linguagem: Inglês
10.1038/sj.ki.5002272
ISSN1523-1755
AutoresCarl F. Verkoelen, Anja Verhulst,
Tópico(s)Parathyroid Disorders and Treatments
ResumoThe production of concentrated urine inevitably leads to the precipitation of poorly soluble waste salts in the renal tubular fluid. These crystallization processes are physiologic and without consequences as long as all crystals are excreted with the urine. The retention of crystals in the renal tubules, however, may lead to tubular nephrocalcinosis. Here, we present a brief survey of the possible mechanisms involved in this process, which seems to depend predominantly on the presence of regenerating/(re)differentiating cells in the renal tubules. Crystal binding to the surface of these cells can be mediated by a number of luminal membrane molecules, including acidic fragment of nucleolin-related protein, annexin-II, osteopontin, and hyaluronan. The production of concentrated urine inevitably leads to the precipitation of poorly soluble waste salts in the renal tubular fluid. These crystallization processes are physiologic and without consequences as long as all crystals are excreted with the urine. The retention of crystals in the renal tubules, however, may lead to tubular nephrocalcinosis. Here, we present a brief survey of the possible mechanisms involved in this process, which seems to depend predominantly on the presence of regenerating/(re)differentiating cells in the renal tubules. Crystal binding to the surface of these cells can be mediated by a number of luminal membrane molecules, including acidic fragment of nucleolin-related protein, annexin-II, osteopontin, and hyaluronan. The definition of the term nephrocalcinosis is not clear. It often refers to a diffuse renal parenchymal calcification demonstrable by imaging techniques (e.g., ultrasonography, computed tomography, X-ray). However, imaging, with its known limitations, is not able to show the exact location of the deposits in the kidney. From histological observations throughout literature, calcifications of the renal tissue can be found either in the tubules or in the interstitium. To prevent confusion in this review, the tubular calcifications will be called tubular nephrocalcinosis, whereas the interstitial calcifications will be called interstitial nephrocalcinosis. Interstitial nephrocalcinosis has been proposed to start in the interstitium (beneath the basement membrane) around the thin limbs of Henle and is thought to give rise to subepithelial calcifications, which are better known as Randall's plaques.1Evan A. Lingeman J. Coe F.L. et al.Randall's plaque: pathogenesis and role in calcium oxalate nephrolithiasis.Kidney Int. 2006; 69: 1313-1318Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar In comparison with nephrocalcinosis, kidney stones (nephrolithiasis) are found further on in the urological tract, that is the renal calyces and pelvis. Although nephrocalcinosis is not necessarily accompanied by nephrolithiasis, both intratubular2Finlayson B. Reid F. The expectation of free and fixed particles in urinary stone disease.Invest Urol. 1978; 15: 442-448PubMed Google Scholar and interstitial nephrocalcinosis1Evan A. Lingeman J. Coe F.L. et al.Randall's plaque: pathogenesis and role in calcium oxalate nephrolithiasis.Kidney Int. 2006; 69: 1313-1318Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar have been proposed to play a role in the development of specific varieties of kidney stones (Figure 1). Nephrolithiasis is a major health problem in the western world where it affects 12% of men and 6% of women at some point in their lives. Kidney stones are responsible for about 10% of urological hospital admissions per year and account for a significant number of visits to the hospital emergency departments.3Taylor E.N. Curhan G.C. Diet and fluid prescription in stone disease.Kidney Int. 2006; 70: 835-839Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar Although nephrolithiasis is associated with much pain and suffering, kidney stones seldom lead to loss of kidney function. Although less painful, intratubular nephrocalcinosis may constitute a much greater health risk. The incidence of tubular nephrocalcinosis is often underestimated because of the low sensitivity of radiological imaging techniques and because most crystals are lost from the renal tubules during tissue processing for routine histology.4Marengo S.R. Chen D.H. Evan A.P. et al.Continuous infusion of oxalate by minipumps induces calcium oxalate nephrocalcinosis.Urol Res. 2006; 34: 200-210Crossref PubMed Scopus (14) Google Scholar Apart from the possibility that tubular nephrocalcinosis may lead to nephrolithiasis, tubular nephrocalcinosis can lead to an (obstruction-induced) tubulopathy. Massive tubular nephrocalcinosis often leads to end-stage renal failure in primary hyperoxaluria5van Woerden C.S. Groothoff J.W. Wijburg F.A. et al.Clinical implications of mutation analysis in primary hyperoxaluria type 1.Kidney Int. 2004; 66: 746-752Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar and recently there is accumulating evidence suggesting that tubular nephrocalcinosis also plays an important role in chronic allograft nephropathy.6Pinheiro H.S. Camara N.O. Osaki K.S. et al.Early presence of calcium oxalate deposition in kidney graft biopsies is associated with poor long-term graft survival.Am J Transplant. 2005; 5: 323-329Crossref PubMed Scopus (76) Google Scholar, 7Memeo L. Pecorella I. Ciardi A. et al.Calcium oxalate microdeposition in failing kidney grafts.Transplant Proc. 2001; 33: 1262-1265Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 8Nankivell B.J. Borrows R.J. Fung C.L. et al.The natural history of chronic allograft nephropathy.N Engl J Med. 2003; 349: 2326-2333Crossref PubMed Scopus (1661) Google Scholar, 9Verhulst A. Asselman M. De Naeyer S. et al.Preconditioning of the distal tubular epithelium of the human kidney precedes nephrocalcinosis.Kidney Int. 2005; 68: 1643-1647Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Ultrafiltrate does not contain calcium oxalate (CaOx) crystals because of the low levels of supersaturation. The concentration of oxalate and to a lesser extent that of calcium gradually increases as a result of oxalate secretion and water reabsorption, but in physiological conditions CaOx saturation probably does not become high enough to form crystals in the proximal tubule. The upper limits of metastability most probably are reached somewhere beyond the thin limbs of Henle. Late segments of the nephron (e.g., distal tubule, collecting ducts) and the renal calyces are therefore major regions of interest for studying crystal retention, respectively, in tubular nephrocalcinosis and nephrolithiasis. The mechanisms involved in the retention of crystals in the renal calyces are relatively unexplored. At least a subset of stones seem to grow on Randall's plaques.10Evan A.P. Lingeman J.E. Coe F.L. et al.Randall's plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle.J Clin Invest. 2003; 111: 607-616Crossref PubMed Scopus (493) Google Scholar The mechanisms by which crystals become associated with these calcium phosphate-rich structures at the surface of the renal pelvic wall are unknown. The general perception is that crystals are not easily retained in the urinary tract. Crystal binding to the epithelium of the renal tubules may require specific alterations in the composition of the cell surface, but under certain conditions it is also possible that relatively large aggregated clumps of crystals are trapped in the renal tubules.11Kok D.J. Papapoulos S.E. Bijvoet O.L. Crystal agglomeration is a major element in calcium oxalate urinary stone formation.Kidney Int. 1990; 37: 51-56Abstract Full Text PDF PubMed Scopus (128) Google Scholar Because the far majority of crystal–cell interaction studies have been performed with CaOx and renal tubular cells, this review is focused on mechanisms involved in CaOx crystal binding to the renal tubular epithelium. During their transit through the nephron, crystals encounter soluble and membrane-associated glycoconjugates with affinity for crystals. Whereas only relatively small molecules or fragments of larger molecules can enter the nephron at the glomerulus, most tubular fluid glycoproteins, glycolipids, and glycosaminoglycans are synthesized by renal tubular cells. Through the interaction of cationic calcium ions with negatively charged glycoconjugates the equilibrium [Ca2+][C2O42-] product is much higher in urine than in water. These natural inhibitors of crystallization are responsible for the delay in stone-salt precipitation in a supersaturated environment. Although these glycoconjugate inhibitors play an important role in renal stone disease and compelling review articles have been devoted to this subject,12Worcester E.M. Inhibitors of stone formation.Semin Nephrol. 1996; 16: 474-486PubMed Google Scholar crystallization inhibition is beyond the scope of this review. In primary hyperoxaluria type I patients with end-stage renal failure, the rise in serum oxalate ultimately leads to oxalate crystal deposits in bone, skin, vessels, eyes, and joints (systemic oxalosis).13Bobrowski A.E. Langman C.B. Hyperoxaluria and systemic oxalosis current therapy and future directions.Expert Opin Pharmacother. 2006; 7: 1887-1896Crossref PubMed Scopus (28) Google Scholar The contact with CaOx crystals of many eukaryotic cells, including endothelial cells,14Falasca G.F. Ramachandrula A. Kelley K.A. et al.Superoxide anion production and phagocytosis of crystals by cultured endothelial cells.Arthritis Rheum. 1993; 36: 105-116Crossref PubMed Scopus (42) Google Scholar fibroblasts,15Hasselbacher P. Stimulation of synovial fibroblasts by calcium oxalate and monosodium urate monohydrate. A mechanism of connective tissue degradation in oxalosis and gout.J Lab Clin Med. 1982; 100: 977-985PubMed Google Scholar leukocytes,16Elferink J.G. Riemersma R.C. Calcium oxalate crystal-induced cytolysis in polymorphonuclear leukocytes and erythrocytes.Agents Actions. 1980; 10: 439-444Crossref PubMed Scopus (8) Google Scholar erythrocytes,16Elferink J.G. Riemersma R.C. Calcium oxalate crystal-induced cytolysis in polymorphonuclear leukocytes and erythrocytes.Agents Actions. 1980; 10: 439-444Crossref PubMed Scopus (8) Google Scholar lung cells,17Yoshida K. The correlation between tissue injury and calcium oxalate crystal production in rat's lung with experimental Aspergillus niger infection].Kansenshogaku Zasshi. 1998; 72: 621-630Crossref PubMed Scopus (6) Google Scholar and renal proximal tubular cells18Schepers M.S. van Ballegooijen E.S. Bangma C.H. et al.Crystals cause acute necrotic cell death in renal proximal tubule cells, but not in collecting tubule cells.Kidney Int. 2005; 68: 1543-1553Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar,19Koul H.K. Menon M. Chaturvedi L.S. et al.COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells.J Biol Chem. 2002; 277: 36845-36852Crossref PubMed Scopus (58) Google Scholar leads to inflammation-mediated membranolysis and acute necrotic cell death. Cell culture studies suggest that the response of renal tubular cells to CaOx crystals is nephron segment-specific. Proximal tubular cells, which in normal physiological conditions do not encounter crystals, avidly bind crystals to the brush border after which the crystals penetrate the plasma membrane and enter the cell leading to inflammation-mediated cell death.18Schepers M.S. van Ballegooijen E.S. Bangma C.H. et al.Crystals cause acute necrotic cell death in renal proximal tubule cells, but not in collecting tubule cells.Kidney Int. 2005; 68: 1543-1553Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 19Koul H.K. Menon M. Chaturvedi L.S. et al.COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells.J Biol Chem. 2002; 277: 36845-36852Crossref PubMed Scopus (58) Google Scholar, 20Hackett R.L. Shevock P.N. Khan S.R. Madin–Darby canine kidney cells are injured by exposure to oxalate and to calcium oxalate crystals.Urol Res. 1994; 22: 197-203Crossref PubMed Scopus (107) Google Scholar However, the handling of CaOx crystals by renal distal or collecting tubular cells is quite different. Crystals bind to regenerating/(re)differentiating cells in subconfluent cultures or in healing scrape wounds, but not to differentiated cells organized in functional confluent monolayers.21Sorokina E.A. Wesson J.A. Kleinman J.G. An acidic peptide sequence of nucleolin-related protein can mediate the attachment of calcium oxalate to renal tubule cells.J Am Soc Nephrol. 2004; 15: 2057-2065Crossref PubMed Scopus (37) Google Scholar, 22Farell G. Huang E. Kim S.Y. et al.Modulation of proliferating renal epithelial cell affinity for calcium oxalate monohydrate crystals.J Am Soc Nephrol. 2004; 15: 3052-3062Crossref PubMed Scopus (31) Google Scholar, 23Verkoelen C.F. van der Boom B.G. Houtsmuller A.B. et al.Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture.Am J Physiol. 1998; 274: F958-F965PubMed Google Scholar Even when crystals bind to regenerating/(re)differentiating Madin–Darby canine kidney (MDCK)-I cells in a wound, they are not taken up and do not induce inflammation.24Schepers M.S. Asselman M. Duim R.A. et al.Pericellular matrix formation by renal tubule epithelial cells in relation to crystal binding.Nephron Exp Nephrol. 2003; 94: e103-e112Crossref PubMed Scopus (9) Google Scholar,25Schepers M.S. Duim R.A. Asselman M. et al.Internalization of calcium oxalate crystals by renal tubular cells: a nephron segment-specific process?.Kidney Int. 2003; 64: 493-500Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Taken into account that these results are predominantly obtained from cell culture studies using a limited number of cell lines assumed to be representative for the nephron segments involved, they collectively suggest that CaOx crystals are relatively inert to cells in areas where their presence is common (e.g., renal collecting ducts, lower urinary tract), whereas they cause acute inflammation-mediated cell death in areas where they are found only under extreme or pathological circumstances (e.g., blood, renal proximal tubule) (Table 1).Table 1Cellular response to calcium oxalate crystalsTissueInflammationConditionErythrocytes63Wiessner J.H. Mandel G.S. Mandel N.S. Membrane interactions with calcium oxalate crystals: variation in hemolytic potentials with crystal morphology.J Urol. 1986; 135: 835-839Crossref PubMed Scopus (28) Google Scholar+OxalosisaPrimary hyperoxalurias.Leukocytes16Elferink J.G. Riemersma R.C. Calcium oxalate crystal-induced cytolysis in polymorphonuclear leukocytes and erythrocytes.Agents Actions. 1980; 10: 439-444Crossref PubMed Scopus (8) Google Scholar+Endothelium14Falasca G.F. Ramachandrula A. Kelley K.A. et al.Superoxide anion production and phagocytosis of crystals by cultured endothelial cells.Arthritis Rheum. 1993; 36: 105-116Crossref PubMed Scopus (42) Google Scholar+Lung64Wiessner J.H. Henderson Jr, J.D. Sohnle P.G. et al.The effect of crystal structure on mouse lung inflammation and fibrosis.Am Rev Respir Dis. 1988; 138: 445-450Crossref PubMed Scopus (52) Google Scholar+Joint65Mandel N.S. The structural basis of crystal-induced membranolysis.Arthritis Rheum. 1976; 19: 439-445Crossref PubMed Scopus (46) Google Scholar+Renal proximal tubule19Koul H.K. Menon M. Chaturvedi L.S. et al.COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells.J Biol Chem. 2002; 277: 36845-36852Crossref PubMed Scopus (58) Google Scholar+EGbPoisoning/experimental animals treated with EG.Renal distal tubule18Schepers M.S. van Ballegooijen E.S. Bangma C.H. et al.Crystals cause acute necrotic cell death in renal proximal tubule cells, but not in collecting tubule cells.Kidney Int. 2005; 68: 1543-1553Abstract Full Text Full Text PDF PubMed Scopus (90) Google ScholarcCrystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury.-Healthy urinary tractcCrystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury.Renal collecting ducts18Schepers M.S. van Ballegooijen E.S. Bangma C.H. et al.Crystals cause acute necrotic cell death in renal proximal tubule cells, but not in collecting tubule cells.Kidney Int. 2005; 68: 1543-1553Abstract Full Text Full Text PDF PubMed Scopus (90) Google ScholarcCrystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury.-Renal calycescCrystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury.-PyelumcCrystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury.-UretercCrystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury.-Bladder66Pantazopoulos D. Karagiannakos P. Sofras F. et al.Effect of drugs on crystal adhesion to injured urothelium.Urology. 1990; 36: 255-259Abstract Full Text PDF PubMed Scopus (10) Google ScholarcCrystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury.-UrethracCrystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury.-EG, ethylene glycol.a Primary hyperoxalurias.b Poisoning/experimental animals treated with EG.c Crystals are commonly present in these areas. If crystals are damaging in these areas, all individuals should suffer from crystal-induced urinary tract injury. Open table in a new tab EG, ethylene glycol. Phosphatidylserine (PS) is the only anionic amino acid-containing glycerophospholipid in animal cells. It may comprise up to 20% of the total phospholipid in the plasma membrane where it is located entirely on the inner monolayer surface of the plasma membrane. PS plays a role in the regulation of physiological cell death (apoptosis). The normal distribution of PS on the inner leaflet of the membrane bilayer is disrupted during apoptosis because of stimulation of the enzyme scramblase. Enrichment of the cell membrane with PS or apoptotic cell death leads to increased levels of CaOx crystal binding. Crystal binding was also higher after PS was translocated to the outer leaflet of the lipid bilayer under the influence of a calcium ionophore. This increase in crystal binding could be blocked by PS-specific annexin-V. These results suggested that apoptosis is one of the mechanisms by which CaOx crystals bind to the renal tubular epithelium.26Bigelow M.W. Wiessner J.H. Kleinman J.G. et al.Surface exposure of phosphatidylserine increases calcium oxalate crystal attachment to IMCD cells.Am J Physiol. 1997; 272: F55-F62PubMed Google Scholar This was one of the first attempts to identify molecules possibly involved in renal crystal retention. To date, no evidence has been presented that stone formers express more apoptotic cells in the kidney than non-stone formers. N-acetyl neuraminic acid is the terminal sugar residue of oligosaccharide side-chains of cell surface glycoconjugates that provides the cell surface with most of its electronegative charge. As calcium crystals can be viewed as large cationic macromolecules, N-acetyl neuraminic acid is a potential crystal-binding molecule. CaOx crystals avidly bound to BSC-1 cells (African green monkey cells) and this binding was significantly lower after digesting N-acetyl neuraminic acid from the cell surface with neuraminidase (sialidase), suggesting that crystal binding depends on sialic acid residues of membrane glycoproteins and glycolipids.27Lieske J.C. Leonard R. Swift H. et al.Adhesion of calcium oxalate monohydrate crystals to anionic sites on the surface of renal epithelial cells.Am J Physiol. 1996; 270: F192-F199PubMed Google Scholar Later, it was found that collecting duct MDCK-I cells without affinity for CaOx crystals contained more sialic acid residues than cells with affinity for crystals, thereby questioning the role for sialic acid as crystal-binding molecule.28Verkoelen C.F. van der Boom B.G. Kok D.J. et al.Sialic acid and crystal binding.Kidney Int. 2000; 57: 1072-1082Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Basement membranes play fundamental roles in differentiation, proliferation, survival, and migration of cells during embryonic development and wound healing. Members of the collagen IV family are present in all basement membranes and are characterized by their ability to form complex, covalently linked structural scaffolds required for basement membrane assembly. Rat renal collecting tubular cells and CaOx crystals formed collagen IV-positive clumps. It was proposed that collagen IV serves as binding molecule for crystals after denudation of the renal tubular basement membrane.29Kohri K. Kodama M. Ishikawa Y. et al.Immunofluorescent study on the interaction between collagen and calcium oxalate crystals in the renal tubules.Eur Urol. 1991; 19: 249-252PubMed Google Scholar Nucleolin is a major nucleolar protein of exponentially growing cells and shuttles between cytoplasma and nucleus.30Nigg E.A. Nucleocytoplasmic transport: signals, mechanisms and regulation.Nature. 1997; 386: 779-787Crossref PubMed Scopus (914) Google Scholar Rat inner medullary collecting duct (IMCD) cells were biotinylated after which all cell surface biotinylated proteins were lysed and purified using calcium phosphate and CaOx affinity chromatograhy. One of the most prominent bands was a 110-kDa calcium-binding glycoprotein. Amino-acid sequencing and DNA cloning demonstrated a close resemblance to nucleolin, which is why it was designated nucleolin-related protein (NRP).31Sorokina E.A. Kleinman J.G. Cloning and preliminary characterization of a calcium-binding protein closely related to nucleolin on the apical surface of inner medullary collecting duct cells.J Biol Chem. 1999; 274: 27491-27496Crossref PubMed Scopus (37) Google Scholar Next, they showed that NRP is randomly expressed over the plasma membrane of proliferating IMCD cells, reaching peak levels in late stages of mitosis to decline gradually to undetectable levels with epithelial maturation. Compared with intact monolayers, regenerating cells in scrape wounds express higher levels of NRP and bind more crystals. The acidic fragment (AF) of NRP was proposed as the active binding region for CaOx crystals. Apical membrane staining of the AF was higher in cells transfected with the AF of NRP and these cells bound more crystals than cells transfected with an empty vector. Conditioned medium of human embryonic kidney cells infected with viruses expressing the AF of NRP inhibited CaOx binding to IMCD cells, in contrast to conditioned medium produced by human embryonic kidney cells infected with wild-type viruses. Crystal binding could be mediated by the AF of NRP at the plasma membrane and inhibited by the secreted AF of NRP.21Sorokina E.A. Wesson J.A. Kleinman J.G. An acidic peptide sequence of nucleolin-related protein can mediate the attachment of calcium oxalate to renal tubule cells.J Am Soc Nephrol. 2004; 15: 2057-2065Crossref PubMed Scopus (37) Google Scholar Annexin-II (Ax-II) is a calcium- and phospholipid-binding protein and substrate for protein tyrosine kinases. Ax-II is actively involved in DNA synthesis and cell proliferation, which may explain its role in wound healing. The same method that was used for the identification of NRP in IMCD was applied to find crystal-binding molecules at the surface of MDCK-I cells. Proteins isolated with CaOx affinity chromatography were trypsin-digested, separated, and microsequenced. Peptide mixtures were subjected to mass spectrometry and protein identification confirmed by Western blotting. These studies revealed Ax-II as the most prominent apical membrane crystal-binding protein. Preincubating the cells with anti-Ax-II antibodies inhibited CaOx crystal binding.32Kumar V. Farell G. Deganello S. et al.Annexin II is present on renal epithelial cells and binds calcium oxalate monohydrate crystals.J Am Soc Nephrol. 2003; 14: 289-297Crossref PubMed Scopus (84) Google Scholar The role of Ax-II in crystal binding was supported in an experimental cell culture model for Dent's disease.33Carr G. Simmons N.L. Sayer J.A. Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells.Cell Mol Life Sci. 2006; 63: 367-377Crossref PubMed Scopus (29) Google Scholar Dent's disease is a genetic disorder associated with low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and renal failure. This disease is caused by mutations in a renal chloride channel gene, CLCN5. Crystal binding was studied to IMCD cells in which clc-5 was disrupted by antisense clc-5 or by overexpression of truncated clc-5. The disruption of clc-5 resulted in the translocation of Ax-II from the cytoplasm to the luminal cell surface, which was accompanied with increased levels of crystal binding that could be blocked with Ax-II antibodies.33Carr G. Simmons N.L. Sayer J.A. Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells.Cell Mol Life Sci. 2006; 63: 367-377Crossref PubMed Scopus (29) Google Scholar Osteopontin (OPN) is a phosphorylated glycoprotein with crystal-binding properties.34Lieske J.C. Hammes M.S. Hoyer J.R. et al.Renal cell osteopontin production is stimulated by calcium oxalate monohydrate crystals.Kidney Int. 1997; 51: 679-686Abstract Full Text PDF PubMed Scopus (89) Google Scholar The role of this protein in the development of renal calcifications is controversial. High OPN levels are found in urine, hence urinary OPN is sometimes called uropontin. In healthy people, the urinary excretion of uropontin is about 4000 μg/day.35Min W. Shiraga H. Chalko C. et al.Quantitative studies of human urinary excretion of uropontin.Kidney Int. 1998; 53: 189-193Abstract Full Text PDF PubMed Scopus (66) Google Scholar In another study, 20 mg/g creatinine was reported.36Gang X. Ueki K. Kon S. et al.Reduced urinary excretion of intact osteopontin in patients with IgA nephropathy.Am J Kidney Dis. 2001; 37: 374-379Abstract Full Text PDF PubMed Scopus (23) Google Scholar In contrast to urine, normal renal tissue does not contain much OPN and is even undetectable by Western blotting.37Rittling S.R. Feng F. Detection of mouse osteopontin by western blotting.Biochem Biophys Res Commun. 1998; 250: 287-292Crossref PubMed Scopus (42) Google Scholar Consequently, it seems that OPN is produced in the renal tubules for export to the urine. In vitro, OPN acts as an inhibitor of crystal nucleation, growth, aggregation, and retention.38Shiraga H. Min W. VanDusen W.J. et al.Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily.Proc Natl Acad Sci USA. 1992; 89: 426-430Crossref PubMed Scopus (387) Google Scholar,39Asplin J.R. Arsenault D. Parks J.H. et al.Contribution of human uropontin to inhibition of calcium oxalate crystallization.Kidney Int. 1998; 53: 194-199Abstract Full Text PDF PubMed Scopus (117) Google Scholar In addition, OPN favors formation of CaOx dihydrate over CaOx monohydrate crystals. CaOx dihydrate is less adherent to renal epithelial cells than CaOx monohydrate.40Wesson J.A. Worcester E.M. Wiessner J.H. et al.Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules.Kidney Int. 1998; 53: 952-957Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar In conclusion, there are many indications suggesting that OPN is an important inhibitor of nephrolithiasis. A crystal-inducing diet indeed produced tubular nephrocalcinosis in OPN knockout mice, but not in wild-type mice.41Wesson J.A. Johnson R.J. Mazzali M. et al.Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules.J Am Soc Nephrol. 2003; 14: 139-147Crossref PubMed Scopus (232) Google Scholar Whereas OPN is moderately expressed in healthy kidneys, its expression is strongly upregulated in damaged kidneys where OPN also becomes expressed at the luminal surface of the cells lining the renal distal tubule. Taking into account the affinity of OPN for CaOx crystals, this suggests that crystals can be retained by OPN in the renal tubules of damaged kidneys. A reduced expression of OPN at the surface of MDCK cells indeed resulted in lower levels of crystal binding.42Yamate T. Kohri K. Umekawa T. et al.Osteopontin antisense oligonucleotide inhibits adhesion of calcium oxalate crystals in Madin-Darby canine kidney cell.J Urol. 1998; 160: 1506-1512Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar In our cell culture, animal, and clinical studies, OPN was always expressed at the luminal surface of crystal binding cells together with hyaluronan (HA) and CD44 (see below). The colocalization of crystals and OPN at the cell surface, however, does not prove that the crystals actually bind to OPN. Furthermore, in a recent study investigating the chronology of HA, OPN and CD44 expression on the one hand and crystal retention on the other did not support a promoter role for OPN in crystal retention (unpublished results). HA is a linear glycosaminoglycan composed of multiple units of glucuronic acid and N-acetylglucosamine (GlcUA-GlcNAc-)n. During dynamic morphogenetic processes such as inflammation, wound healing, embryonic development, and cancer, hydrated coats of HA around cells provide the microenvironment conducive to tissue remodeling.43Toole B.P. Hyaluronan is not just a goo!.J Clin Invest. 2000; 106: 335-336Crossref PubMed Scopus (139) Google Scholar In MDCK-I cells it was observed that CaOx crystals bind to non-regenerating/(re)differentiating cells in subconfluent or healing cultures, but not to intact confluent monolayers. Evidence for HA involvement in crystal binding is multiple: (1) in both confocal laser-scanning microscopy and metabolic-labeling studies (using [3H]glucosamine as precursor for HA biosynthesis), it was observed that HA is expressed and produced by cells in subconfluent or healing cultures, but hardly by growth-inhibited cells in confluent cultures, (2) crystal binding to proliferating MDCK-I cells was decreased by Streptomyces hyaluronidase, an enzyme that hydrolyzes HA, but no other GAGs or proteins, (3) crystals bind to HA immobilized on plastic dishes.44Verkoelen C.F. Van der Boom B.G. Romijn J.C. Identification of hyaluronan as a crystal-binding molecule at the surface of migrating and proliferating MDCK cells.Kidney Int. 2000; 58: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar To confirm these findings in a human-cell model, these studies were repeated in primary cultures of human renal tubular cells. Crystal binding occurred in subconfluent cultures and was absent in confluent cultures. Confocal laser-scanning microscopy demonstrated the expression of HA (and OPN) and the cell surface HA receptor CD44 at the apical membrane of proliferating tubular cells, whereas at confluence CD44 was expressed at the basolateral membrane and HA (and OPN) were no longer detectable. In addition, a particle exclusion technique revealed that proliferating cells were surrounded by HA-rich pericellular matrices or 'cell coats' extending several microns from the cell surface. Disintegration of these coats with hyaluronidase significantly decreased the cell surface affinity for crystals.24Schepers M.S. Asselman M. Duim R.A. et al.Pericellular matrix formation by renal tubule epithelial cells in relation to crystal binding.Nephron Exp Nephrol. 2003; 94: e103-e112Crossref PubMed Scopus (9) Google Scholar,45Verhulst A. Asselman M. Persy V.P. et al.Crystal retention capacity of cells in the human nephron: involvement of CD44 and its ligands hyaluronic acid and osteopontin in the transition of a crystal binding- into a nonadherent epithelium.J Am Soc Nephrol. 2003; 14: 107-115Crossref PubMed Scopus (96) Google Scholar In the next step our in vitro observations were tested in a rat model of renal calcification.46Asselman M. Verhulst A. De Broe M.E. et al.Calcium oxalate crystal adherence to hyaluronan-, osteopontin-, and CD44-expressing injured/regenerating tubular epithelial cells in rat kidneys.J Am Soc Nephrol. 2003; 14: 3155-3166Crossref PubMed Scopus (168) Google Scholar Rats were fed ethylene glycol, a nephrotoxic precursor of oxalate, giving rise to tubular epithelial damage and crystalluria. The results of this study suggested that crystal retention does not depend on the amount of crystals formed, but on the presence of a tubular epithelium with crystal-binding phenotype. This phenotype is characterized by tubular cell regeneration (which occurs alongside tubular damage) going along with luminal expression of HA OPN and CD44. Our experimental in vitro and in vivo data were recently supported by clinical observations in preterm and transplanted human kidneys. Kidneys of preterm infants are immature and contain tubules with flattened differentiating (developing) cells that abundantly express HA and OPN at the luminal cell side.9Verhulst A. Asselman M. De Naeyer S. et al.Preconditioning of the distal tubular epithelium of the human kidney precedes nephrocalcinosis.Kidney Int. 2005; 68: 1643-1647Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar In this context, it is worth mentioning that kidneys of preterm-born infants presents an incidence of tubular nephrocalsinosis as high as 60%, probably leading to a prolonged decrease in renal function.47Ezzedeen F. Adelman R.D. Ahlfors C.E. Renal calcification in preterm infants: pathophysiology and long-term sequelae.J Pediatr. 1988; 113: 532-539Abstract Full Text PDF PubMed Scopus (89) Google Scholar,48Downing G.J. Egelhoff J.C. Daily D.K. et al.Kidney function in very low birth weight infants with furosemide-related renal calcifications at ages 1–2 years.J Pediatr. 1992; 120: 599-604Abstract Full Text PDF PubMed Scopus (74) Google Scholar Hence, in this clinical setting we may reasonably assume that crystal retention results from exposure of the differentiating epithelium to periods of crystalluria, possibly caused by furosemide treatment in combination with a high-protein diet. Transplanted kidneys recover from ischemic injury and are chronically stressed by nephrotoxic immunosuppressive agents and several other potential nephrotoxins. Intratubular microcalcifications, not seen in implantation biopsies, are encountered in up to 40% of the renal allografts after 1 year and has been associated with poor long-term graft survival.6Pinheiro H.S. Camara N.O. Osaki K.S. et al.Early presence of calcium oxalate deposition in kidney graft biopsies is associated with poor long-term graft survival.Am J Transplant. 2005; 5: 323-329Crossref PubMed Scopus (76) Google Scholar Sequential protocol biopsies showed increased intratubular expression of HA and OPN preceding the retention of crystals, which were formed under the influence of renal tubular acidosis-induced hypocitraturia49Stapenhorst L. Sassen R. Beck B. et al.Hypocitraturia as a risk factor for nephrocalcinosis after kidney transplantation.Pediatr Nephrol. 2005; 20: 652-656Crossref PubMed Scopus (48) Google Scholar and hyperoxaluria resulting from the release of oxalate from body stores.50Mehler K. Stapenhorst L. Beck B. et al.Hyperoxaluria in patients with primary distal renal tubular acidosis.Pediatr Nephrol. 2003; 18: 722-723PubMed Google Scholar This demonstrates that the retention-prone epithelial phenotype precedes crystal adhesion leading to tubular nephrocalcinosis. The clinical implications of these findings become even more important knowing that several disorders are associated with tubular nephrocalcinosis, such as patients with Crohn's disease,51Worcester E.M. Chuang M. Laven B. et al.A new animal model of hyperoxaluria and nephrolithiasis in rats with small bowel resection.Urol Res. 2005; 33: 380-382Crossref PubMed Scopus (9) Google Scholar interstinal bypass,52Evan A.P. Coe F.L. Lingeman J.E. et al.Insights on the pathology of kidney stone formation.Urol Res. 2005; 33: 383-389Crossref PubMed Scopus (50) Google Scholar primary hyperoxaluria,53Milliner D.S. Wilson D.M. Smith L.H. 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Jones G.K. et al.Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene.J Clin Invest. 1997; 100: 1693-1707Crossref PubMed Scopus (306) Google Scholar Bartter's syndrome,58Anderson LaMJ The origin, frequency and significance of microscopic calculi in the kidney.Surg Gynecol Obstet. 1946; 82: 275-283PubMed Google Scholar cystic fibrosis59Soleimani M. Burnham C.E. Physiologic and molecular aspects of the Na+:HCO3-cotransporter in health and disease processes.Kidney Int. 2000; 57: 371-384Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Sjörgen syndrome,60Moutsopoulos H.M. Cledes J. Skopouli F.N. et al.Nephrocalcinosis in Sjogren's syndrome: a late sequela of renal tubular acidosis.J Intern Med. 1991; 230: 187-191Crossref PubMed Scopus (54) Google Scholar and acute phosphate nephropathy.61Markovich D. Aronson P.S. Specificity and Regulation of Renal Sulfate Transporters.Annu Rev Physiol. 2007; 69: 361-375Crossref PubMed Scopus (61) Google Scholar,62Verkoelen C.F. Crystal retention in renal stone disease: a crucial role for the glycosaminoglycan hyaluronan?.J Am Soc Nephrol. 2006; 17: 1673-1687Crossref PubMed Scopus (74) Google Scholar The general picture emerging from these data is that CaOx crystals preferentially bind to regenerating/(re)diffentiating renal tubular cells. Several molecules have been proposed as crystal-binding substance, including NRP, annexin-II, osteopontin, and HA. Although their relative contribution to crystal binding remains to be determined, these molecules are exclusively expressed at the luminal surface of regenerating renal tubular cells. Our next assignment is to find out if this phenotype is expressed in other disorders that are associated with nephrocalcinosis, in addition to the kidneys of preterm infants and renal allograft recipients. We also have to investigate further the possible relationship between tubular nephrocalcinosis and nephrolithiasis. Finally, it will be interesting to find out if the crystal-binding molecules described in this review not only play a role in tubular nephrocalcinosis, but also in interstitial nephrocalcinosis and nephrolithiasis.
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