The development of kidney stones requires formation of crystals followed by their retention in the kidney (1 2 3–7
Most kidney stones are predominantly composed of precipitated calcium salts, the most common of which is calcium oxalate monohydrate (5 8 9 10 11 11
HA is a high molecular mass polysaccharide (>106 Da), composed of linear polymers of a repeating disaccharide structure of alternating glucuronic acid and N-acetylglucosamine. In the kidney, HA is hardly detectable in the cortex but is abundantly present as the main component of the renal inner medullary interstitium (12 13–17 13,14,18–20 21,22 13,14 in vivo renal tubular injury and HA, OPN, and CD44 expression are involved in crystal retention.
Materials and Methods
Experimental Design
Male Wistar rats (300 to 350 g) were obtained from the Central Animal Breeding Center (Harlan, Zeist, the Netherlands) and divided into three groups (n = 9 each) receiving drinking water supplemented with 0, 0.5, or 0.75% (vol/vol) ethylene glycol (EG) for 1, 4, and 8 d. All animals had free access to standard chow. Twenty-four hours before the indicated times, rats were housed individually in metabolic cages to collect 24-h urine samples and to monitor fluid intake. Urine samples were divided into portions of 5 ml, one portion of which was acidified with 100 μl of 1 M hydrochloric acid and stored at −20°C until analysis. Animals were sedated and killed; kidneys were extracted and decapsulated; and sagittal slices were immediately fixed in either methacarn (60% methanol, 30% chloroform, 10% acetic acid) or Dubosq-Brasil fixative (47% ethanol, 11.7% H2 O, 23.5% formaldehyde, 17.6% acetic acid, and 4 mM picric acid) for 4 h, rinsed with 70% ethanol, and embedded in low-melting-point paraffin (52°C; BDH Laboratory Supplies, Poole, UK). Serum specimens were collected and frozen at −20°C until biochemical analysis. The experiments were approved by the local University Animal Committee and carried out in accordance with the Netherlands Experiments on Animals Act (1977) and the European Convention for the Protection of Vertebrate Animals Used for Experimental Purposes (Strasbourg, March 18, 1986).
Urine and Serum Biochemistry
Urinary oxalate was determined in acidified urine portions with a quantitative enzymatic colorimetric assay (Sigma Diagnostics, Dei- senhofen, Germany). For determination of urinary citrate, the ultraviolet method with the test combination of Boehringer Mannheim (Darmstadt, Germany) was used. The concentrations of calcium in urine and bicarbonate, calcium, and creatinine in serum were determined on a routine autoanalyzer system (Vitros 750 XRC). Urine samples were centrifuged at 5000 × g , and sediments were inspected by optical and polarized light microscopy (Zeiss Axioplan microscope, Oberkochen, Germany).
Tubular Morphology
Methacarn-fixed, paraffin-embedded renal tissue sections (4 μm) were stained with periodic acid-Schiff (PAS), and nuclei were counterstained with methyl green. Histologic damage was evaluated with a morphologic scoring system (Table 1 ) in proximal tubules (PT), thin limbs of Henle (TLH), distal tubules (DT; including thick ascending limbs [TAL]) and collecting ducts (CD). Tubules were morphologically inspected by a reproducible procedure, which comprised a random selection of the first tubular cross-section, followed by shifting the microscopic field over fixed distances according to a standardized pattern (×300 magnification). The cortex, outer stripe of the outer medulla (OSOM), inner stripe of the outer medulla (ISOM), and inner medulla of each kidney section were evaluated. In the cortex, PT (S1–S2) and DT were evaluated (n = 50 and 25, respectively); in the OSOM, PT (S3) and DT (TAL) were evaluated (n = 50 and 25, respectively); in the ISOM, TLH and DT (TAL) were evaluated (n = 25 each); and in the inner medulla, TLH and CD were evaluated (n = 25 each). PT and DT could be distinguished according to at least one of the following morphologic criteria: topographical localization, tubular size and form, cytoplasmic density and position of the nuclei, and presence or absence of brush border and basolateral cell aspect. Tubules in the cortex and OSOM were scored as PT only when a brush border could be identified; if not, then they were scored as DT. In the ISOM and inner medulla, TLH could be distinguished from DT (TAL) and CD by tubular size and position of the nuclei.
Table 1: a
Proliferation was determined by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) using the PC10 mAb (DAKO, Glostrup, Denmark) as described previously (23
Crystal Retention
During the evaluation of tubular morphology, each tubule was additionally inspected by polarized light microscopy for the presence of crystals. In this way, crystal retention was assessed and sites of crystal location were correlated with tubular morphology.
Calcium deposits were also visualized by von Kossa staining. Deparaffinized Dubosq-Brasil–fixed 4-μm tissue sections were incubated in 5% silver nitrate for 45 min. Slides were rinsed in water, incubated in 1% pyrogallic acid for 3 min, rinsed in water, fixed in 5% sodium thiosulfate for 1 min, and counterstained with hematoxylin and eosin. In each sagittal kidney section, calcium deposits were quantified by counting the total number of positive stained crystals in the cortex + OSOM and ISOM + inner medulla.
HA, OPN, and CD44 Expression
Renal tissue sections were stained for HA, OPN, and CD44 as described previously (11
Expression of HA, OPN, and CD44 was quantified morphometrically with KS-400 V2.0 image analysis software in the cortex and OSOM, by measuring positive signals in 10 and six randomly chosen microscopic fields, respectively (×200 magnification). Measurements were expressed as fractional positive area of the tissue section. OPN and CD44 were also quantified in the remaining part of the medulla, by analyzing six randomly chosen microscopic fields of the ISOM and inner medulla. HA was not quantified in this region, because the well known abundant amount of HA in the interstitium of the inner medulla stains nearly the entire tissue (12
Statistical Analyses
Data are expressed as mean ± SEM. P < 0.05 was considered to be significant, using t test. Correlation analysis between total tubular PCNA expression per sagittal kidney section and the total amount of positive von Kossa renal calcium deposits per sagittal kidney section in individual rats was performed by the nonparametric Spearman’s rank order test. P < 0.01 was considered significant (two-tailed). Computations were performed using SPSS version 10.
Results
Urine and Serum Biochemistry
In the control group, the average urinary oxalate and calcium excretion was 0.95 ± 0.05 and 3.24 ± 0.79 mg/24 h, respectively (Figure 1 ), and the urinary sediment did not contain crystals (Figure 2A ). Administration of 0.5 and 0.75% EG to the drinking water induced within 24 h a significant concentration-dependent hyperoxaluria (4.37 ± 1.71 and 7.78 ± 4.46 mg/24 h, respectively). In the 0.5% EG group, oxalate gradually increased further to reach its maximum level at day 8, whereas in the 0.75% EG group, urinary oxalate reached its maximum level already at day 4. Urinary calcium was decreased at day 1 and was undetectable at day 4 and day 8 (Figure 1 ). At day 1, calcium oxalate crystals were observed in urinary sediments of both EG groups (Figure 2B ). Oxalate was determined in acidified urine portions, which dissolves crystals and therefore represents the total amount of oxalate, including oxalate precipitated with calcium. Because calcium was determined in urine that was not acidified, it represents the amount of free calcium ions. Thus, the decreased amounts of urinary calcium apparently resulted from the formation of CaOx crystals.
Figure 1.:
Urine- and serum biochemical analysis, urine production, and fluid intake. Statistical analysis by t test. *Significantly different compared with controls at the same point in time (P < 0.05).
Figure 2.:
Urinary sediment inspected by polarized light microscopy of a control rat (A) and a 0.5% ethylene glycol (EG)-treated rat (B) after 1 d. (A) Some debris was observed, but no urinary crystalline material. (B) Numerous urinary calcium oxalate crystals in the EG-treated rat are clearly visible. Magnification, ×400.
In the 0.5% EG group at day 8 and in the 0.75% EG group at days 4 and 8, increased diuresis and fluid intake were observed compared with controls. Thus, the addition of EG led to polyuria, which may be secondary to the osmotic effect of EG excreted in the urine unchanged (24 Figure 1 ). There was no increase in serum creatinine, except for a slight increase in the low-dose EG at day 8, indicating that renal function was preserved in these rats. These biochemistry data are in accordance with earlier observations in rats treated with EG (25
EG caused a metabolic acidosis, as can be derived from the concentration-dependent decrease in urinary citrate after 4 d. This metabolic acidosis was relatively mild, however, as serum bicarbonate and calcium were not affected and a compensatory homeostatic response seemed to normalize urinary citrate after continued EG challenge (Figure 1 ).
Tubular Morphology
The relatively low concentrations of EG in the present study did not result in frank necrosis but in mild changes in tubular morphology. At day 1, tubular morphology was comparable to controls, but at days 4 and 8, different degrees of injury/regeneration were found in tubules (Figure 3 ). PT of the OSOM (S3) and, to a lesser extent, PT in the cortex (S1–S2) suffered the most morphologic damage, for the most part with loss of brush border (score 2). Tubules with flattened cells (score 3) were also observed, predominantly in DT (TAL) of the OSOM. Because a tubule with flattened cells was scored only as PT if there was still brush border recognizable, the number of PT with score 3 could actually have been higher. In TLH and CD at days 4 and 8, increased amounts of luminal cell debris (score 1) were observed, but the majority of these tubules had a normal morphology (score 0). Importantly, cell debris in the tubular lumen in the distal nephron could also have been derived from an “upstream” section of the nephron, and the tubule concerned could actually have been completely healthy.
Figure 3.:
Evaluation of tubular morphology with the scoring system of
Table 1 in proximal tubules (PT), thin limbs of Henle (TLH), distal tubules (DT; including thick ascending limbs [TAL]), and collecting ducts (CD 0 = intact tubule with normal appearance, 1 = tubule with luminal cell debris, 2 = PT: tubule with loss of brush border/DT (TAL) and CD: dilated tubule, 3 = tubule with flattened cells).
At day 1, PCNA staining was comparable to control kidneys, in which tubular expression of this protein was sparse (Figure 4A ), whereas at days 4 and 8, PCNA staining was clearly upregulated in tubules in rats that received EG (Figure 4B ).
Figure 4.:
Proliferating cell nuclear antigen (PCNA) staining in a kidney of a control rat (A) and of a rat that received 0.75% EG for 8 d (B through D). In D, periodic acid-Schiff (PAS) counterstaining was omitted. (A) Sparse PCNA staining, mainly present in the interstitium and glomeruli in a control rat. (B) Strongly upregulated PCNA in the tubules of an EG-treated rat. (C and D) Crystals associated with PCNA-positive, flattened cells. Magnifications: ×400 in A, ×200 in B, ×630 in C and D.
Crystal Retention
No crystals were found in control rat kidneys (Figure 5 ). After 1 d of 0.5 and 0.75% EG, no crystals were observed by polarized light microscopy, consistent with a marginal amount of positive von Kossa signals. At days 4 and 8, however, an increasing number of crystals were found attached to the luminal membrane of tubular epithelial cells, corresponding with a markedly increased number of positive von Kossa signals (Figure 5 ). Crystals were smaller in size than the tubular lumen and not seen intracellularly or in the interstitium.
Figure 5.:
Quantification of renal calcium deposits on von Kossa–stained tissue sections.
In Figure 6 , retention of crystals and tubular morphology is plotted in a graph. In PT, DT (TAL), and CD, crystals were not observed in histologic normal tubules (score 0) or tubules with luminal cell debris (score 1). Crystals were found adhered to tubular cells in PT with loss of brush border and DT (TAL) and CD with dilation of the tubular lumen (score 2), and in tubules with flattened cells (score 3). Crystals were not observed in TLH (data not shown). Figure 7 shows a DT with small crystals at the luminal membrane of flattened tubular epithelial cells (score 3). Furthermore, these flattened cells were PCNA positive, proliferating cells (Figure 4, C and D ). Approximately 93% of the observed crystals in PCNA-stained renal tissue sections were found to be adhered to PCNA-positive cells.
Figure 6.:
Retention of crystals in relation to tubular morphology. In PT, DT (including TAL), and CD, crystals were not observed in tubules with score 0 (normal appearance) or score 1 (luminal cell debris). Crystals were exclusively retained at the luminal surface of tubular cells in PT, DT, and CD with score 2 (loss of brush border in PT and dilated tubules in DT and CD) and score 3 (tubules with flattened cells).
Figure 7.:
PAS and methyl green–stained renal tissue section of a rat treated with 0.75% EG for 4 d by optical (A) or polarized (B) light microscopy. (A) A dilated DT is shown with flattened epithelial cells (score 3). (B) In the lumen of this DT, crystals that are smaller than the tubular lumen are clearly visible and located at the luminal side of the injured/regenerating flattened epithelium. Magnification, ×1000.
Correlation analysis was performed between the total amount of tubular PCNA expression and the amount of positive von Kossa calcium deposits in sagittal kidney sections of individual rats (Figure 8 ). Tubular cell regeneration was associated with crystal adhesion/retention (Spearman’s Rho = 0.819; P < 0.0001).
Figure 8.:
Correlation analysis was performed between the total amount of tubular PCNA expression and the amount of positive von Kossa calcium deposits in sagittal kidney sections of individual rats (Spearman’s Rho = 0.819, P < 0.0001).
HA, OPN, and CD44 Expression
HA in the cortex and OSOM of control rats was scarcely observed in the interstitium and around a few glomerular capsules. After 1 d, HA expression in EG-treated rats was comparable to controls (Figure 9 ). After 4 and 8 d, however, HA was upregulated in a focal pattern throughout the cortex and OSOM of EG-treated rats (Figure 9 ), primarily in the interstitium but also at the luminal membrane of tubular cells (Figure 10A ). Strikingly, in the majority of cases, crystals were found at the luminal surface of HA-expressing cells (Figure 10A ).
Figure 9.:
Quantification of renal hyaluronan (HA), osteopontin (OPN), and CD44 expression using computerized image analysis software (KS400). Data are expressed as fractional positive area (%). Statistical analysis by t test. *Significantly increased compared with controls at the same point in time (P < 0.05).
Figure 10.:
(A) The outer stripe of the outer medulla (OSOM) stained for HA of a rat with 0.75% EG in the drinking water for 8 d. HA is upregulated in the interstitium as well as at the apical membrane of tubular epithelial cells. A crystal is shown close to cells that express HA at their luminal membrane. (B and C) The OSOM stained for OPN after 8 d of 0.75% EG, showing increased typical Golgi apparatus immunostaining in PT and at the apical cell membrane in DT. (C) Crystals visualized with polarized light microscopy. Retained crystals stained for OPN are smaller than the tubular lumen and closely associated with apical OPN-expressing epithelium. (D) The cortex stained for CD44 after 8 d of 0.75% EG. CD44 is upregulated at basolateral and apical tubular cell membranes. Polarized light microscopy shows birefringent crystals in the lumen of a tubule closely associated with the surface of flattened cells positive for CD44. Magnification, ×1000.
OPN was expressed in a limited number of distal tubular epithelial cells of control rats. After 1 d of EG, OPN expression was not different from controls, except for a small increase in the OSOM of the moderate-dose EG group (Figure 9 ). At days 4 and 8, however, OPN was significantly upregulated in the kidneys of EG-treated rats (Figure 9 ). Both PT and DT showed a rise in OPN immunostaining (Figure 10, B and C ). The classical OPN expression pattern was observed, with Golgi apparatus immunostaining in PT and at the apical membrane in DT, allowing differentiation between PT and DT (previously described after renal ischemia/reperfusion) (19,26 Figure 10, B and C ).
CD44 was hardly expressed in the rat kidney, except for some interstitial cells and glomeruli. After 1 d of EG, CD44 expression was not significantly different from controls (Figure 9 ). After 4 and 8 d of EG, however, CD44 in the cortex, OSOM, and medulla was markedly upregulated (Figure 9 ). CD44 expression was prominent in a focal pattern along basolateral and apical tubular cell membranes (Figure 10D ). Crystals were observed frequently closely at sites where cells expressed CD44 at their luminal membrane (Figure 10D ).
Discussion
In the present study, we investigated the role of HA, OPN, and CD44 in retention of crystals in renal tubules damaged by EG. The EG model is extremely suitable for this purpose since this agent not only is toxic to the nephron but also generates urinary CaOx crystals (25,27–30
EG, which itself is not nephrotoxic, is metabolized in the liver to several intermediates, including glycoaldehyde, glycolate, glyoxylate, and oxalate (27 28,29 25,31 27 Figures 4, C and D, 7, A and B, and 10, A, B, C, and D ).
In urolithiasis research, animals are usually treated with relatively high concentrations of EG for several weeks (25,30,31 25,31,32 Table 1 ), and PCNA was used to assess cell proliferation. PCNA is an auxiliary protein for DNA polymerase δ and required for both DNA replication and DNA repair. In proliferating cells, it is upregulated in cell nuclei mainly during the S-phase (DNA synthesis phase) of the cell cycle (33 23 23 Figure 4B ) are dedifferentiated proliferating cells.
HA is a high molecular mass polysaccharide found in many tissues, where it performs a great range of biologic functions (34,35 12 13 14,17 15 16 36,37 10,11,38,39 1 ) crystals bind to HA-expressing cells at subconfluence but not to cells in confluent cultures that do no longer express HA; (2 ) metabolic labeling studies showed that the surface of proliferating cells contains substantially higher levels of radiolabeled HA; (3 ) crystal binding could be decreased by Streptomyces hyaluronidase, an enzyme that specifically digests HA; and (4 ) during wound healing, HA-binding protein binds to migrating and proliferating flattened cells in damaged areas but not to cells in intact monolayers (10 in vivo .
The glycoprotein OPN is widely distributed in the body and has been implicated in several physiologic and pathologic processes, including cell adhesion, migration, signaling, inflammation, and biomineralization (18,40,41 18,19,42 31 18,43 44–46 44 Figure 10, B and C ). This is in agreement with a previous study, in which it was demonstrated that urinary inhibitors of crystallization were unable to prevent the attachment of crystals to regenerating renal tubular cells (47 et al. (44), the role of OPN remains controversial.
CD44 is a ubiquitous transmembrane glycoprotein that is involved in many processes including inflammation (48 21,22 49–51 13,14 11
The present study supports in vivo for the first time the concept that crystal retention is associated with HA expressed at the luminal surface of injured/regenerating cells. However, we cannot rule out the importance of other crystal-binding molecules, including sialic acid–containing glycoproteins (52 53 54 55 56 32 57 58 2
Although the expression of HA, OPN, and CD44 by injured/regenerating tubular epithelial cells most likely is aimed to reestablishment of the epithelial barrier integrity and restoration of renal function, a negative side effect could be that it turns a non–crystal-binding epithelium into a crystal-binding one, thereby setting the stage for crystal retention (Figure 11 ). The clinical relevance of this concept was recently reinforced by observations in kidneys of preterm neonates, in which the development of nephrocalcinosis is common, showing that HA and OPN are abundantly expressed at the luminal membrane of proliferating tubular cells (unpublished results).
Figure 11.:
Paradigm of crystal retention. A schematic representation of a cross-section of a distal tubule is shown. (A) Under normal conditions, crystals do not bind to renal tubular epithelial cells and are harmlessly excreted in the urine. (B) Crystal retention is preceded by a stressor injuring the epithelium. (C) During the process of tubular epithelial regeneration and repair, flattened epithelial cells express HA, OPN, and CD44 at their apical membrane. HA is a cell surface crystal-binding molecule. Because these regenerating dedifferentiated tubular epithelial cells are susceptible to crystal binding, crystal retention may ensue.
In conclusion, the results obtained in this study support the concept that the expression of HA, OPN, and CD44 by injured/regenerating tubular cells is a prerequisite for retention of crystals in the kidney.
M.A. and A.V. contributed equally to this work.
This study was supported by the Oxalosis and Hyperoxaluria Foundation (Grant No. 2749036).
We express our gratitude to Dr. C.M. Giachelli (University of Washington, Seattle, WA) for the generous gift of the OP189 antiserum. We thank Simonne Dauwe for excellent technical assistance, as well as the biochemistry laboratory of the Antwerp University Hospital (Dr. V. van Hoof and C. Verstraten). We thank Frank van der Panne and Dirk de Weerdt for contribution of the photographs.
1. Finlayson B, Reid F: The expectation of free and fixed particles in urinary stone disease. Invest Urol 15: 442–448, 1978
2. Asselman M, Verkoelen CF: Crystal-cell interaction in the pathogenesis of kidney stone disease. Curr Opin Urol 12: 271–276, 2002
3. King JS Jr: Etiologic factors involved in urolithiasis: A review of recent research. J Urol 97: 583–591, 1967
4. Khan SR, Shevock PN, Hackett RL: Acute hyperoxaluria, renal injury and calcium oxalate urolithiasis. J Urol 147: 226–230, 1992
5. Mandel N: Mechanism of stone formation. Semin Nephrol 16: 364–374, 1996
6. Baggio B, Gambaro G, Ossi E, Favaro S, Borsatti A: Increased urinary excretion of renal enzymes in idiopathic calcium oxalate nephrolithiasis. J Urol 129: 1161–1162, 1983
7. Kumar S, Sigmon D, Miller T, Carpenter B, Khan S, Malhotra R, Scheid C, Menon M: A new model of nephrolithiasis involving tubular dysfunction/injury. J Urol 146: 1384–1389, 1991
8. Verkoelen CF, van der Boom BG, Kok DJ, Houtsmuller AB, Visser P, Schroder FH, Romijn JC: Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture. Kidney Int 55: 1426–1433, 1999
9. Verkoelen CF, van der Boom BG, Houtsmuller AB, Schroder FH, Romijn JC: Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture. Am J Physiol 274: F958–F965, 1998
10. Verkoelen CF, Van Der Boom BG, Romijn JC: Identification of hyaluronan as a crystal-binding molecule at the surface of migrating and proliferating MDCK cells. Kidney Int 58: 1045–1054, 2000
11. Verhulst A, Asselman M, Persy VP, Schepers MS, Helbert MF, Verkoelen CF, De Broe ME: 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 14: 107–115, 2003
12. Knepper MA, Saidel GM, Hascall VC, Dwyer T: Concentration of solutes in the renal inner medulla: Interstitial hyaluronan as a mechano-osmotic transducer. Am J Physiol Renal Physiol 284: F433–F446, 2003
13. Sibalic V, Fan X, Loffing J, Wuthrich RP: Upregulated renal tubular CD44, hyaluronan, and osteopontin in kdkd mice with interstitial nephritis. Nephrol Dial Transplant 12: 1344–1353, 1997
14. Lewington AJ, Padanilam BJ, Martin DR, Hammerman MR: Expression of CD44 in kidney after acute ischemic injury in rats. Am J Physiol Regul Integr Comp Physiol 278: R247–R254, 2000
15. Feusi E, Sun L, Sibalic A, Beck-Schimmer B, Oertli B, Wuthrich RP: Enhanced hyaluronan synthesis in the MRL-Fas(lpr) kidney: Role of cytokines. Nephron 83: 66–73, 1999
16. Wells A, Larsson E, Hanas E, Laurent T, Hallgren R, Tufveson G: Increased hyaluronan in acutely rejecting human kidney grafts. Transplantation 55: 1346–1349, 1993
17. Johnsson C, Tufveson G, Wahlberg J, Hallgren R: Experimentally-induced warm renal ischemia induces cortical accumulation of hyaluronan in the kidney. Kidney Int 50: 1224–1229, 1996
18. Xie Y, Sakatsume M, Nishi S, Narita I, Arakawa M, Gejyo F: Expression, roles, receptors, and regulation of osteopontin in the kidney. Kidney Int 60: 1645–1657, 2001
19. Persy VP, Verstrepen WA, Ysebaert DK, De Greef KE, De Broe ME: Differences in osteopontin up-regulation between proximal and distal tubules after renal ischemia/reperfusion. Kidney Int 56: 601–611, 1999
20. Xie Y, Nishi S, Iguchi S, Imai N, Sakatsume M, Saito A, Ikegame M, Iino N, Shimada H, Ueno M, Kawashima H, Arakawa M, Gejyo F: Expression of osteopontin in gentamicin-induced acute tubular necrosis and its recovery process. Kidney Int 59: 959–974, 2001
21. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B: CD44 is the principal cell surface receptor for hyaluronate. Cell 61: 1303–1313, 1990
22. Weber GF, Ashkar S, Glimcher MJ, Cantor H: Receptor-ligand interaction between CD44 and osteopontin (Eta-1). Science 271: 509–512, 1996
23. Nouwen EJ, Verstrepen WA, Buyssens N, Zhu MQ, De Broe ME: Hyperplasia, hypertrophy, and phenotypic alterations in the distal nephron after acute proximal tubular injury in the rat. Lab Invest 70: 479–493, 1994
24. Hewlett TP, Jacobsen D, Collins TD, McMartin KE: Ethylene glycol and glycolate kinetics in rats and dogs. Vet Hum Toxicol 31: 116–120, 1989
25. Khan SR: Animal model of calcium oxalate nephrolithiasis. In: Calcium Oxalate in Biological Systems,edited by Khan SR, Boca Raton, CRC Press, 1995, pp 343–359
26. Verhulst A, Persy VP, Van Rompay AR, Verstrepen WA, Helbert MF, De Broe ME: Osteopontin synthesis and localization along the human nephron. J Am Soc Nephrol 13: 1210–1218, 2002
27. Schrier RW: Ethylene glycol toxicity. In: Diseases of the Kidney and Urinary Tract, 7th ed., Philadelphia, Lippincott, Williams & Wilkins 2001, pp 1316–1326
28. Poldelski V, Johnson A, Wright S, Rosa VD, Zager RA: Ethylene glycol-mediated tubular injury: Identification of critical metabolites and injury pathways. Am J Kidney Dis 38: 339–348, 2001
29. Roberts JA, Seibold HR: Ethylene glycol toxicity in the monkey. Toxicol Appl Pharmacol 15: 624–631, 1969
30. Lyon ES, Borden TA, Vermeulen CW: Experimental oxalate lithiasis produced with ethylene glycol. Invest Urol 4: 143–151, 1966
31. Khan SR, Johnson JM, Peck AB, Cornelius JG, Glenton PA: Expression of osteopontin in rat kidneys: Induction during ethylene glycol induced calcium oxalate nephrolithiasis. J Urol 168: 1173–1181, 2002
32. Moriyama MT, Glenton PA, Khan SR: Expression of inter-alpha inhibitor related proteins in kidneys and urine of hyperoxaluric rats. J Urol 165: 1687–1692, 2001
33. Kelman Z: PCNA: Structure, functions and interactions. Oncogene 14: 629–640, 1997
34. Tammi MI, Day AJ, Turley EA: Hyaluronan and homeostasis: A balancing act. J Biol Chem 277: 4581–4584, 2002
35. Toole BP, Wight TN, Tammi MI: Hyaluronan-cell interactions in cancer and vascular disease. J Biol Chem 277: 4593–4596, 2002
36. Noble PW: Hyaluronan and its catabolic products in tissue injury and repair. Matrix Biol 21: 25–29, 2002
37. Chen WY, Grant ME, Schor AM, Schor SL: Differences between adult and foetal fibroblasts in the regulation of hyaluronate synthesis: Correlation with migratory activity. J Cell Sci 94: 577–584, 1989
38. Verkoelen CF, Van Der Boom BG, Kok DJ, Schroder FH, Romijn JC: Attachment sites for particles in the urinary tract. J Am Soc Nephrol 10 [Suppl 14]: S430–S435, 1999
39. Verkoelen CF, van der Boom BG, Kok DJ, Romijn JC: Sialic acid and crystal binding. Kidney Int 57: 1072–1082, 2000
40. Hudkins KL, Giachelli CM, Cui Y, Couser WG, Johnson RJ, Alpers CE: Osteopontin expression in fetal and mature human kidney. J Am Soc Nephrol 10: 444–457, 1999
41. Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, Johnson R, Hughes J: Osteopontin—A molecule for all seasons. QJM 95: 3–13, 2002
42. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS: Osteopontin as a means to cope with environmental insults: Regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 107: 1055–1061, 2001
43. Persy VP, Verhulst A, Ysebaert DK, De Greef KE, De Broe ME: Reduced postischemic macrophage infiltration and interstitial fibrosis in osteopontin knockout mice. Kidney Int 63: 543–553, 2003
44. Wesson JA, Johnson RJ, Mazzali M, Beshensky AM, Stietz S, Giachelli C, Liaw L, Alpers CE, Couser WG, Kleinman JG, Hughes J: Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules. J Am Soc Nephrol 14: 139–147, 2003
45. Yamate T, Kohri K, Umekawa T, Iguchi M, Kurita T: Osteopontin antisense oligonucleotide inhibits adhesion of calcium oxalate crystals in Madin-Darby canine kidney cell. J Urol 160: 1506–1512, 1998
46. Lieske JC, Hammes MS, Hoyer JR, Toback FG: Renal cell osteopontin production is stimulated by calcium oxalate monohydrate crystals. Kidney Int 51: 679–686, 1997
47. Schepers MS, van der Boom BG, Romijn JC, Schroder FH, Verkoelen CF: Urinary crystallization inhibitors do not prevent crystal binding. J Urol 167: 1844–1847, 2002
48. Pure E, Cuff CA: A crucial role for CD44 in inflammation. Trends Mol Med 7: 213–221, 2001
49. Lesley J, Hyman R, English N, Catterall JB, Turner GA: CD44 in inflammation and metastasis. Glycoconj J 14: 611–622, 1997
50. Lesley J, English NM, Gal I, Mikecz K, Day AJ, Hyman R: Hyaluronan binding properties of a CD44 chimera containing the link module of TSG-6. J Biol Chem 277: 26600–26608, 2002
51. Zohar R, Suzuki N, Suzuki K, Arora P, Glogauer M, McCulloch CA, Sodek J: Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration. J Cell Physiol 184: 118–130, 2000
52. Lieske JC, Leonard R, Swift H, Toback FG: Adhesion of calcium oxalate monohydrate crystals to anionic sites on the surface of renal epithelial cells. Am J Physiol 270: F192–F199, 1996
53. Wiessner JH, Hasegawa AT, Hung LY, Mandel NS: Oxalate-induced exposure of phosphatidylserine on the surface of renal epithelial cells in culture. J Am Soc Nephrol 10 [Suppl 14]: S441–S445, 1999
54. Kohri K, Kodama M, Ishikawa Y, Katayama Y, Matsuda H, Imanishi M, Takada M, Katoh Y, Kataoka K, Akiyama T, et al.: Immunofluorescent study on the interaction between collagen and calcium oxalate crystals in the renal tubules. Eur Urol 19: 249–252, 1991
55. Sorokina EA, Kleinman JG: 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 274: 27491–27496, 1999
56. Wesson JA, Worcester EM, Wiessner JH, Mandel NS, Kleinman JG: Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules. Kidney Int 53: 952–957, 1998
57. Iida S, Peck AB, Byer KJ, Khan SR: Expression of bikunin mRNA in renal epithelial cells after oxalate exposure. J Urol 162: 1480–1486, 1999
58. Nishio S, Hatanaka M, Takeda H, Iseda T, Iwata H, Yokoyama M: Analysis of urinary concentrations of calcium phosphate crystal-associated proteins: α2-HS-glycoprotein, prothrombin F1, and osteopontin. J Am Soc Nephrol 10 [Suppl 14]: S394–S396, 1999
