Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Umekawa, T.
Right arrow Articles by Khan, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Umekawa, T.
Right arrow Articles by Khan, S. R.
J Am Soc Nephrol 15:635-644, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Effect of Angiotensin II Receptor Blockage on Osteopontin Expression and Calcium Oxalate Crystal Deposition in Rat Kidneys

Tohru Umekawa*, Yuji Hatanaka*, Takashi Kurita* and Saeed R. Khan{dagger}

*Department of Urology, Kinki University School of Medicine, Osaka, Japan; and {dagger}Department of Pathology, College of Medicine, University of Florida, Gainesville, Florida

Correspondence to Dr. Saeed R. Khan, Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Box 100275, Gainesville, FL 32610-0275. Phone: 352-392-3574; Fax: 352-392-8177; E-mail: Khan{at}pathology.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Hyperoxaluria leads to calcium oxalate (CaOx) crystallization and development of tubulointerstitial lesions in the kidneys. Treatment of hyperoxaluric rats with angiotensin II (Ang II) type I receptor blocker (ARB) reduces lesion formation. Because Ang II mediates osteopontin (OPN) synthesis, which is involved in both macrophage recruitment and CaOx crystallization, it was hypothesized that ARB acts via OPN. Hyperoxaluria was induced in 10-wk-old male Sprague-Dawley rats, and they were treated with ARB candesartan. At the end of 4 wk, kidneys were examined for crystal deposits, ED-1–positive cells, and expression of OPN mRNA. PCR was used to quantify OPN, renin, and angiotensin-converting enzyme (ACE) mRNA in kidneys. RIA was used to determine renal, plasma, and urinary OPN; plasma renin; Ang II and ACE; and renal Ang II. For evaluating oxidative stress, malondialdehyde was measured. Urinary calcium, oxalate, creatinine, and albumin were also determined. Despite similar urinary calcium and oxalate levels, kidneys of hyperoxaluric rats on candesartan had fewer CaOx crystals, fewer ED-1–positive cells, reduced OPN expression, and reduced malondialdehyde than hyperoxaluric rats. Urinary albumin excretion and serum creatinine levels improved significantly on candesartan treatment. mRNA for OPN, renin, and ACE were significantly elevated in hyperoxaluric rats. OPN synthesis and production increased with hyperoxaluria but to a lesser extent in candesartan-treated hyperoxaluric rats. These results show for the first time that oxalate can activate the renal renin-angiotensin system and that oxalate-induced upregulation of OPN is in part mediated via renal renin-angiotensin system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxalate, a by-product of metabolism, is normally excreted in the urine and at low concentrations is harmless to the renal epithelial cells. However, elevated oxalate levels and/or calcium oxalate (CaOx) crystals are injurious (1–3). In addition, exposure of renal epithelial cells in vitro to high levels of oxalate and/or CaOx crystals upregulates the production of chemoattractants osteopontin (OPN) (4) and monocyte-chemoattractant protein-1 (MCP-1) (5, 6). Mild idiopathic hyperoxaluria is associated with CaOx nephrolithiasis, whereas hyperoxaluria resulting from jejunal bypass for obesity or genetic defects in oxalate metabolism is nephrotoxic and produces tubulointerstitial lesions. Tubulointerstitial damage is recognized as one of the most important risk factors for the development of chronic renal diseases and eventual renal failure (7).

All components of the renin-angiotensin system (RAS) are produced within the kidney, and intrarenal RAS plays pivotal role in renal disease progression (8). Kidneys produce both angiotensinogen and angiotensin-converting enzyme (ACE), and juxtaglomerular apparatus is the main source of circulating renin. Renin catalyzes the production of angiotensin I, which is converted to angiotensin II (Ang II) by the actions of ACE. Ang II acts through two receptors, types 1 (AT1) and 2 (AT2), and mediates many effects of the RAS, regulating numerous physiologic reactions including salt and water balance, aldosterone release, and BP (9). Oxidative stress plays a significant role in proinflammatory effects of Ang II. ACE inhibitors and AT1 receptor blockers have been shown to provide protection against Ang II–induced fibrosis and oxidative stress.

Because RAS plays a significant role in the development of renal tubulointerstitial fibrosis, Toblii et al. (10) investigated its involvement in hyperoxaluria-induced tubulointerstitial lesion. They induced hyperoxaluria in male Sprague-Dawley rats by administering ethylene glycol (EG) and evaluated whether AT1 receptor blockade prevents the development of CaOx deposits in the kidneys. Despite similar oxalate levels, rats that received losartan, AT1 receptor blocker, showed fewer CaOx crystal deposits in the kidneys and fewer ED-1–positive cells in the interstitium. It was concluded that the beneficial effect of losartan is a result of a combination of factors including reduction in crystal formation, control of inflammation, and reduction in oxidative stress. Oxidative stress is a key component of both oxalate-induced and angiotensin-induced renal injury. Toblli et al. (11) obtained similar results when an ACE inhibitor, enalapril, was given to the hyperoxaluric rats. Results of these studies raised a number of questions. Were there any changes in the components of the renal RAS system, and how did these changes affect crystal deposition in the kidneys? Because OPN can modulate various steps of CaOx crystallization (12–18) and plays a significant role in CaOx crystal deposition in the kidneys in experimental animals (19, 20) and its tissue expression is affected by Ang II (21–23), we hypothesized that Ang II receptor antagonists may in part act through the regulation of OPN production. Therefore, we administered candesartan, an AT1 receptor antagonist (ARB), to rats with EG-induced hyperoxaluria and investigated (1) changes in renal expression of OPN, renin, and ACE mRNA; (2) changes in OPN, renin, ACE, and Ang II in the supernatant of the homogenized kidneys and/or serum and/or urine; (3) change in macrophage influx into the renal interstitium; and (4) change in CaOx crystal deposition in the rat kidneys with and without ARB.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rat Model of Urolithiasis
Hyperoxaluria was induced in 10-wk-old male Sprague-Dawley rats (280 to 300 g) by administering 1.0% EG in drinking water for 4 wk. Four groups of 10 rats each were studied: group A, untreated control animals; group B, hyperoxaluria without treatment; group C, hyperoxaluria with ARB agent candesartan (20 µg/ml in drinking water); and group D, ARB (candesartan 20 µg/ml). Experimental details on urine collection, kidney removal, and processing have been described previously (19).

Plasma and supernatant of homogenized kidney tissue samples (whole kidneys were homogenized with 3 vol of cold PBS and centrifuged at 8000 x g for 10 min) were assayed for total protein using the BioRad Protein Assay kit (BioRad Laboratories, Hercules, CA). Renin, Ang II and ACE, and serum and supernatant of homogenized kidney tissue were measured with RIA using SRL kit (SRL, Tokyo, Japan). OPN in supernatant of homogenized kidney tissue samples and in urine was measured with an ELISA kit (IBL, Gumma, Japan) as described previously (24). We used anti-rat OPN rabbit IgG as capture antibody and streptavidin-peroxide–conjugated anti-OPN IgG as the secondary antibody. Human recombinant OPN was used as the standard. Urine samples were analyzed for calcium, oxalate, and albumin. Serum and urine creatinine and serum blood urea nitrogen were also measured. Calcium concentrations in whole kidneys were measured with an atomic absorption spectrophotometer according to the manufacturer’s directions.

Reverse Transcriptase for Real-Time PCR
The mRNA levels of glyceraldehyde-3 phosphate dehydrogenase (GAPDH), OPN, renin, and ACE mRNA in whole kidney was determined using real-time PCR. Total RNA was isolated from kidney using TRIZOL Reagent (Life Technologies BRL, Grand Island, NY) according to the manufacturer’s protocol. Two micrograms of total RNA was reverse-transcribed to cDNA. In brief, 50-µl reactions contained 3 µl of 100 mM MgCl, 1.25 µl of RNase inhibitor, 5 µl of 10x PCR buffer, 10 µl of 10 mM dNTP mix, 1.3 µl of Oligo d(T), and 1.5 µl of reverse transcriptase (all from Life Technologies BRL). This mixture was incubated 60 min at 37°C, and then reaction mixture was heated to 94°C for 5 min to stop the reaction.

Primers for Real-Time PCR
Primers were designed using Primer Express software (PE Applied Biosystems, Foster City, CA) as follows: GAPDH (accession no. NM_017008), 5'-TGCCAAGTATGATGACATCAAGAA-3' (forward primer, bases 780 to 803) and 5'-AGCCCAGGATGCCCTTTAGT-3' (reverse primer, bases 831 to 850); OPN (accession no. NM_012881), 5'-TGAGACTGGCAGTGGTTTGC-3' (forward primer, bases 81 to 100) and 5'-CCACTTTCACCGGGAGACA-3' (reverse primer, bases 125 to 143); renin (accession no. NM_012642), 5'-ACCAGGGCAACTTTCACTACGT-3' (forward primer, bases 740 to 761) and 5'-ACCCCCTTCATGGTGATCTG-3' (reverse primer, bases 787 to 806); ACE (accession no. NM_012544), 5'-TTGTCTGTCACTGGAGCCTGAT-3' (forward primer, bases 2327 to 2348) and 5'-CACACCCAAAGCAATTCTTCGT-3' (reverse primer, bases 2380 to 2401).

Real-Time Quantitative PCR
PCR product was directly monitored by measuring the increase in fluorescence of dye (SYBR GREEN; PE Applied Biosystems) bound to the amplified double-stranded DNA. The parameter of threshold cycle (CT) was defined as the fractional cycle number at which fluorescence exceeds a threshold level. The comparative CT method quantifies the amount of mRNA relative to that of a reference sample, termed the calibrator, for comparison of the expression level of every unknown sample. For normalizing the relative amount of MCP-1 mRNA, GAPDH mRNA was chosen as an internal reference. The changes in expression are given by unknown samples of interest.

All PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). For each PCR run, a master mix was prepared: 1x SYBR PCR buffer; 3 mM MgCl2; 200 µM dATP, dCTP, and dGTP; 400 µM dUTP; 300 µM primer set for MCP-1 and GAPDH; and 1.25 units of AmpliTaq Gold DNA polymerase. Five milliliters of diluted (1:20) cDNA was added to 45 µl of the PCR master mix. After an initial 10-min denaturation at 95°C, the thermal cycling comprised 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min.

Morphologic Studies
Paraffin-embedded sections (4 µm thick) were stained with hematoxylin and eosin to count crystal deposits. Sections were stained with VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions for immunohistochemistry of OPN (x1000 primary antibody: rabbit anti-human OPN polyclonal antibody; IBL, Gumma, Japan) or ED-1 (x500 primary antibody: mouse anti-rat monocytes/macrophage monoclonal antibody; Serotec, Oxford, UK). The number of crystal deposits (% in total tubules) in renal tubules and ED-1–positive cells in the renal interstitium were determined by assessing randomly selected 20 fields per kidney (x200).

Detection of OPN mRNA by In Situ Hybridization
In situ hybridization to detect OPN mRNA expression was performed and analyzed as described in detail previously (19).

Renal Content of Malondialdehyde
Determination of renal content of malondialdehyde (MDA) was done in supernatants of kidney slices as previously described (24).

Statistical Analyses
Data are presented as mean ± SD, and statistical significance was calculated using a nonparametric Wilcoxon Mann-Whitney test in the case of a single comparison. For multiple comparisons, Sheffe post hoc test was used only when one-way factorial ANOVA showed a significant difference at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
There were no significant differences in body weight and volume of drinking water (Table 1) among the four groups. Rate of creatinine clearance was lower in hyperoxaluric rats of group B compared with the normal rats of group A. The clearance rate, however, improved significantly when hyperoxaluric rats were given candesartan as seen in group C rats.


View this table:
[in this window]
[in a new window]

  		Table 1. Rat body weight, volume of drinking water, and rate of creatinine clearancea
 
As expected, there was a marked increase in urinary excretion of oxalate by rats in groups B and C (Table 2). However, there were no significant differences in urinary excretion of calcium. Whereas candesartan-treated animals in group C showed a high level of oxalate excretion similar to group B, group C rats showed significantly lower amount of albumin in their urine (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2. Urinary and serum concentrations of various substancesa
 
Table 2 shows urinary concentrations of Ang II; albumin; OPN; and serum concentrations Ang II, OPN, renin, and ACE. Urinary and serum Ang II were significantly higher in groups C and D, which received candesartan. Significant elevation in urinary and serum OPN were observed in hyperoxaluric rats of group B compared with normal rats in group A. Both urinary and serum OPN were significantly reduced on candesartan treatment in group C. Serum renin was significantly increased in rats in groups B, C, and D compared with normal controls of group A. There were no significant differences in serum ACE levels between the various groups.

Hyperoxaluric rats had CaOx crystal deposits in both the cortex and the medulla (Figure 1). Crystals were seen in the tubular lumens. Crystal-containing tubules appeared dilated. There was a significant increase in both the calcium contents and the number of crystal deposits in hyperoxaluric rats of groups B and C as compared with the nonhyperoxaluric rats in groups A and D (Table 3). However, hyperoxaluric rats in group C, which received candesartan treatment, had significantly lower calcium and crystal deposits than the hyperoxaluric rats of group B, which did not receive the candesartan treatment. There was a significant increase in the number of ED-1–positive cells in the renal interstitium of hyperoxaluric rats (Figure 2) in groups B and C. However, candesartan-treated rats in group C had significantly fewer ED-1-positive infiltrates compared with rats in the group B (Figure 2, Table 3).



View larger version (171K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Section of a kidney from the rat in group B, which received only ethylene glycol (EG) for 4 wk. Note calcium oxalate (CaOx) crystals (*) in the tubular lumen. H&E staining, reduced from x200.

 

View this table:
[in this window]
[in a new window]

  		Table 3. Various renal factors at 4 weeksa
 


View larger version (137K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Kidney sections immunostained for ED-1. (A) A kidney from group B rats that received EG only for 4 wk. ED-1–positive cells, corresponding to monocytes/macrophage, are present in the renal interstitium (arrows). Most crystals were dislodged and completely disappeared during processing. (B) A kidney from group C hyperoxaluric rats that received candesartan showing reduced number of ED-1–positive cells. Reduced from x200.

 
Table 3 shows renal Ang II, OPN, and MDA levels after 4 wk of experimentation. Hyperoxaluric rats of groups B and C had significantly high levels of renal MDA, a marker of oxidative stress, which went down significantly when candesartan was provided. Levels of Ang II in the kidneys increased with hyperoxaluria in group B and nearly doubled in the candesartan-treated rats of groups C and D. Renal OPN increased in the hyperoxaluric rats of both groups B and C. Candesartan treatment of hyperoxaluric rats in group C resulted in a significant reduction in renal OPN compared with rats in group B, which did not receive the treatment.

Morphologic examination of OPN synthesis and production in kidneys by in situ hybridization (Figure 3) and immunohistochemistry (not illustrated) showed that in normal rats, expression was scanty and limited to a few cells of the loops of Henle and surface epithelium of the renal papillae in the renal calyces (not illustrated). Renal papillary tubules of normal control rats, however, were completely devoid of OPN expression. After 4 wk of treatment, however, there was the expected increase in the frequency and intensity of expression in the epithelial cells. The expression was much more prominent and spread throughout the whole kidney, including the epithelial cells lining the tubules of the inner medulla and the renal papillae (Table 4).



View larger version (111K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Section of a kidney from group B rats that received EG only for 4 wk. There was pronounced positive staining in cells lining renal tubules in both renal cortex (A) and medulla (B). Most crystals were dislodged and completely disappeared during processing, leaving large holes. In situ hybridization for osteopontin (OPN), reduced from x200.

 

View this table:
[in this window]
[in a new window]

  		Table 4. Expression of OPN and OPN mRNA in kidneys: Results of immunohistochemistry and in situ hybridizationa
 
We quantified the increase in GAPDH, OPN, ACE, and renin mRNA using real-time PCR. The standard and amplification curves for GADPH, ACE, OPN, and renin indicated that assays were done in optimum conditions. Figure 4 shows calculated relative quantities of ACE, OPN, and renin mRNA. There were no significant differences in ACE mRNA between various groups. There was, however, significant upregulation of both the OPN and renin mRNA in the hyperoxaluric rats of groups B and C. Candesartan treatment was associated with significant reduction in OPN mRNA in the hyperoxaluric rats of group C but not to the levels of normal rats of group A (Figure 4).



View larger version (16K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Relative quantities of ACE, OPN, and renin. Figure shows the mean ± SD; n= 10.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Both tissue culture and animal model studies have provided evidence that renal epithelial cells are injured in the presence of high levels of oxalate and CaOx crystals (1–3). Injury is mediated by the production of reactive oxygen species causing lipid peroxidation of the cellular membranes (3, 25, 26). Renal tubular insult is a known cause of glomerular injury and hyperfiltration resulting in enhanced transglomerular protein traffic and proteinuria (27). In our experiments, hyperoxaluric rats had lower creatinine clearance than normal control rats and hyperoxaluric rats on candesartan. In addition, there was a significant reduction in albumin excretion by hyperoxaluric rats that received candesartan. Earlier studies have also demonstrated a reduction in albumin excretion by hyperoxaluric rats that received the ACE inhibitor losartan (10) or the AT1 receptor blocker enalapril (11). Similarly, correlation of urinary albumin excretion with the tubulointerstitial fibrosis was also reported (27, 28). In proteinuric rats, both ACE and angiotensin are upregulated and AT1 receptor is activated.

Results show a significant increase in the expression of renin mRNA in the kidneys and a significant increase in renin levels in the serum of the hyperoxaluric rats. Even though an increase in renin expression in kidneys has been reported in a number of experimental models, this is the first report of elevated renin expression in response to oxaluria and CaOx crystal deposition. This expression may reflect a nonspecific response to renal injury because administration of EG is known to generate a number of metabolites that can be toxic to the kidneys (29). Increased renin production upregulates the production of angiotensin. The activation of the proximal tubular RAS in streptozotocin-induced diabetic rat is mediated by enhanced expression of renin mRNA. An increase in local production of Ang II is suggested to contribute to tubulointerstitial injury in this diabetes model (30). In addition, RAS is implicated in the pathogenesis of diabetic nephropathy, because of the ability of ACE inhibitors or ARB to diminish proteinuria and retard progressive glomerulosclerosis (31, 32). Changes in renal renin expression have been seen in salt depletion and ACE inhibition (33, 34). In the renal tubules of rats with subtotal nephrectomy, expression of renin and Ang II mRNA increased in association with overexpression of TGF-{beta} (35). Ang II is a known mediator of TGF-{beta} production in a variety of cell types, including proximal tubular cells, renal interstitial fibroblasts, and mesangial and vascular smooth muscle cells. It is interesting that Toblli et al. (10, 11) found increased expression of TGF-{beta} in kidneys of hyperoxaluric rats.

AT1 receptor blockage downregulated OPN synthesis and production, suggesting an involvement of the RAS system in hyperoxaluria-induced OPN upregulation. Involvement of Ang II in renal OPN expression has been suggested by many studies. Dramatic increase in OPN protein and mRNA levels was observed in renal epithelial cells of the distal tubules and collecting ducts on Ang II infusion (22). Proximal tubular cells subjected to mechanical stretch exhibited increased mRNA expression. Significant reduction in OPN expression was found after transfection with angiotensinogen or AT1 antisense oligonucleotide (23). Ang II may stimulate the OPN expression in renal tubules either directly (36) or through induction of TGF-{beta} (37).

Candesartan treatment resulted in reduced CaOx crystal deposition and infiltration of ED-1-positive inflammatory cells into renal interstitium. OPN is most likely involved in both events. Free OPN in the solution inhibits the heterogeneous nucleation (16), growth (14, 15), and aggregation of CaOx crystals (17). OPN modulates the adhesion of CaOx monohydrate crystals to renal epithelial cells, a mechanism considered critical for crystal retention within the kidneys. Previous exposure of crystals but not cells to OPN blocked the adhesion (18). However, crystal exposure to urine had no effect on crystal adherence to confluent LLC-PK1 or regenerating MDCK cells (38). Because candesartan treatment also resulted in reduced OPN production, its crystallization inhibitory activity may not be operative in this model. It has been suggested, however, that surface-immobilized OPN promotes crystal adhesion. Even though OPN is a secreted and soluble protein, it can be immobilized by cross-linking to the extracellular matrix. A prominent coat of OPN is seen on luminal surfaces of specific populations of epithelial cells of gall bladder and urinary tract (39), where it is bound to integrin receptors via arginine-glycine-aspartate cell-binding domains. In normal kidneys, this layer is only faintly visible and restricted to only specific locations (5, 6, 19), but in hyperoxaluric rats, this layer becomes very conspicuous and CaOx crystals are universally found associated with OPN (19). Pretreatment of MDCK cells with polyclonal antibodies against OPN inhibited the adhesion of CaOx crystals (40, 41). Similarly, transfection of NRK52E cells with antisense OPN cDNA significantly reduced adhesion of CaOx crystals to the transfected cell (41). OPN immobilized on surface of collagen granules promoted CaOx crystal adhesion in vitro (24). On the basis of the results presented here, we conclude that blockage of AT1 receptor reduced the synthesis and production of OPN and its presence on luminal cell surfaces in the renal tubules, inhibiting crystal adherence, and deposition in the kidneys. Contrary to our results in which reduction in OPN production is associated with decline in crystal retention, results of a recent study using OPN knockout mice demonstrated lack of OPN causing crystal retention in the kidneys after EG administration (20). These differences may reflect a species-specific reaction to EG and its processing and perhaps crystallization of calcium oxalate. Administration of 1% EG to rats induces copious CaOx crystal deposition (personal observations, S.R. Khan) in the kidneys. However, there was no deposition in wild-type mice and limited deposition in OPN knockout mice. In addition, CaOx crystal deposition in the rats is associated with apoptosis (42), whereas no significant difference in apoptosis was seen between wild-type and OPN knockout mice. Moreover, administration of only 0.75% EG to rats resulted in not only increased synthesis, production, and excretion of OPN but also CaOx crystal deposition (19).

Reduced synthesis and production of OPN by candesartan-treated rats may be partially responsible for reduced infiltration of inflammatory cells into the renal interstitium. OPN is a chemoattractant for many cell types. OPN promotes migration of both T cells and macrophages in vitro in a dose-dependent manner (43, 44). Elevated OPN gene and protein expression occurs early and precedes the infiltration of monocytes/macrophages (45, 46). Notable inhibition of glomerular and tubulointerstitial accumulation of macrophages is seen in a rat model of crescentic glomerular basement membrane nephritis after administration of anti-OPN antibody (47). Because macrophages may be injurious to tubular epithelial cells, they should exhibit an increased level of injury in untreated rats. CaOx crystal deposition in rat kidneys is almost always associated with renal epithelial injury (1, 48 ), apoptosis (42), and necrosis. Injured epithelium has long been considered receptive to crystal adherence because injuries to the renal epithelial cell surfaces cause an exposure of crystal-binding molecules, including phosphatidylserine (49), hyaluronan and its ligands CD44, and OPN (50).

There is increasing evidence of a possible link between the RAS, in particular Ang II, and OPN expression and macrophage accumulation. Ang II infusion stimulates expression of OPN, macrophage infiltration, and tubulointerstitial injury (44). Blockade of the RAS by using either an ACE inhibitor or an ARB reduces tubulointerstitial injury. Furthermore, AT1 receptor activation on proximal tubular cells leads to OPN overexpression, provoking the migration of macrophages and induction of tubulointerstitial injury (23). Subtotal nephrectomy is associated with substantial upregulation of OPN expression, macrophage accumulation, and severe proteinuria (51). In a transgenic rat model of diabetic nephropathy characterized by increased synthesis and production of OPN and extensive macrophage accumulation, administration of ACE inhibitor reduced both expression of OPN and buildup of macrophages (52).

In our model, accumulation of ED-1–positive cells may additionally be mediated by MCP-1. We reported earlier that MCP-1 mRNA and protein synthesis are elevated in NRK 52E cells on exposure to oxalate ions or CaOx monohydrate crystals (5, 6 ). Glomerular macrophage recruitment in experimental diabetes is largely determined by angiotensin-stimulated MCP-1 expression as well as OPN (53). AT1 receptor mediates the induction of MCP-1 and macrophage infiltration in hypertensive nephrosclerosis (54).

Ang II is implicated in causing oxidative stress by stimulating membrane-bound NADH/NADPH oxidase, which leads to increased generation of superoxide (55–57). Ang II administration via osmotic pumps increased kidney contents of thiobarbituric acid reactive substances and upregulated heme oxygenase-1, a redox-sensitive enzyme (58). NADH/NADPH oxidase and p42/44 mitogen-activated protein kinase signaling pathways are involved in the regulation of Ang II–stimulated OPN expression in cardiac microvascular endothelial cells (21). Patients with hypertension show significantly higher MDA and lower superoxide dismutase. ACE inhibitors and ARB ameliorate these conditions (59). AT1 receptor blockage in patients with progressive chronic kidney disease and type 2 diabetes or glomerulonephritis results in reduced urinary excretion of MCP-1 and MDA (60).

In summary, we confirm earlier results of RAS involvement in hyperoxaluria and CaOx crystal–induced renal inflammation. In addition, we provide the evidence that hyperoxaluria and CaOx crystal deposition induce renin synthesis activating the RAS system. Ang II upregulates the synthesis and production of OPN, which coats the luminal surfaces of renal tubular epithelium causing crystal adherence. Blockage of the AT1 receptor reduces OPN production and crystal adherence, thereby reducing crystal deposition within the kidneys (Figure 5). We believe that the findings presented here have clinical and therapeutic significance. Interruption of RAS by either AT1 receptor blocking or inhibition of ACE may reduce renal crystal burden in patients with primary hyperoxaluria.



View larger version (20K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Hyperoxaluria and CaOx crystal formation in renal tubules.

 

   Acknowledgments
 
Dr. Khan’s research was supported by National Institutes of Health Grant RO1 DK 053962.

Results were presented at the 2003 Annual Meeting of the American Urological Association.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Khan SR: calcium oxalate crystal interaction with renal tubular epithelium, mechanism of crystal adhesion and its impact on stone development. Urol Res 23: 71, 1995[CrossRef][Medline]
  2. Koul H, Kenington L, Nair G, Honeyman T, Menon M, Scheid CR: Ox-induced initiation of DNA synthesis in LLC-PK1 cells, a line of renal epithelial cells. Biochem Biophys Res Commun 205: 1632–1637, 1994[CrossRef][Medline]
  3. Scheid CR, Koul H, Hill WA, Luber-Narod J, Kennington L, Honeyman T, Jonassen J, Menon M: Ox toxicity in LLC-PK1 cells: Role of free radicals. Kidney Int 49: 413–419, 1996[Medline]
  4. Lieske JC, Hammes MS, Hoyer JR, Toback FG: Renal cell osteopontin production is stimulated by CaOx monohydrate crystals. Kidney Int 51: 679–686, 1997[Medline]
  5. Umekawa T, Chegini N, Khan SR: Increased expression of monocyte chemoattractant protein-1 (MCP-1) by renal epithelial cells in culture on exposure to calcium oxalate, phosphate and uric acid crystals. Nephrol Dial Transplant 18: 664–669, 2003[Abstract/Free Full Text]
  6. Umekawa T, Chegini N, Khan SR: Oxalate ions and calcium oxalate crystals stimulate MCP-1 expression by renal epithelial cells. Kidney Int 61: 105–112, 2002[CrossRef][Medline]
  7. Nath K: The tubulointerstitium in progressive renal disease. Kidney Int 54: 992–994, 1998[CrossRef][Medline]
  8. Seikaly MG, Aarnt BS Jr, Seney FD Jr: Endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat. J Clin Invest 86: 1352–1357, 1990
  9. Antus B, Exton MS, Rosivall L: Angiotensin II: A regulator of inflammation during renal disease? Int J Immunopathol Pharmacol 14: 25–30, 2001[Medline]
  10. Toblli JE, Ferder L, Stella I, De Cavanaugh EM, Angerosa M, Inserra F: Effects of angiotensin II subtype 1 receptor blockade by losartan on tubulointerstitial lesions caused by hyperoxaluria. J Urol 168: 1550–1555, 2002[CrossRef][Medline]
  11. Toblli JE, Stella I, Cavanagh E, Angerosa M, Inserra F, Feder L: Enalapril prevents tubulointerstitial lesions by hyperoxaluria. Hypertension 33: 225–231, 1999[Abstract/Free Full Text]
  12. Khan SR: Interactions between stone-forming calcific crystals and macromolecules. Urol Int 59: 59–71, 1997[CrossRef][Medline]
  13. Boskey AL, Maresca M, Ukrich W, Doty SB, Butler WT, Prince CW: Osteopontin-hydroxyapatite interactions in vitro: Inhibition of hydroxyapatite formation and growth in a gelatin-gel. Bone Miner 22: 147–159, 1993[Medline]
  14. Shiraga H, Min W, Van Dusen WJ, Clayman MD, Miner D, Terrell CH, Scherbotie JR, Foreman JW, Przysiecki C, Neilson EG, 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 U S A 89: 426–430, 1992[Abstract/Free Full Text]
  15. Hoyer JR: Uropontin in urinary calcium stone formation. Miner Electrolyte Metab 20: 385–392, 1994[Medline]
  16. Worcester EM, Beshensky AM: Osteopontin inhibits nucleation of calcium oxalate crystals. Ann N Y Acad Sci 21: 375–377, 1995
  17. Asplin JR, Arsenault D, Parks JH, Coe FL, Hoyer JR: Contribution of human uropontin to inhibition of calcium oxalate crystallization. Kidney Int 53: 194–199, 1998[CrossRef][Medline]
  18. Lieske JC, Leonardo R, Toback FG: Adhesion of calcium oxalate monohydrate crystals to renal epithelial cells is inhibited by specific anions. Am J Physiol 268: 604–612, 1995
  19. Khan SR, Johnson JM, Peck AB, Cornelious JG, Glenton PA: Expression of osteopontin in rat kidneys: Induction during ethylene glycol induced calcium oxalate nephrolithiasis. J Urol 168: 1173–1181, 2002[CrossRef][Medline]
  20. 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 inhibitors of calcium oxalate formation and retention in renal tubules. J Am Soc Nephrol 14: 139–147, 2003[Abstract/Free Full Text]
  21. Xie Z, Pimental DR, Lohan S, Vesertriger A, Oligavko C, Colucci WS, Singh K: Regulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: Role of p42/44 mitogen-activated protein kinase and reactive oxygen species. J Cell Physiol 188: 132–138, 2001[CrossRef][Medline]
  22. Giachelli CM, Pichler R, Lombardi D, Denhardt DT, Alpers CE, Schwartz SM, Johnson RJ: Osteopontin expression in angiotensin II-induced tubulointerstitial nephritis. Kidney Int 45: 515–524, 1994[Medline]
  23. Ricardo SD, Franzoni DF, Roesener CD, Crisman JM, Diamond JR: Angiotensinogen and AT(1) antisense inhibition of osteopontin translation in rat proximal tubular cells. Am J Physiol Renal Physiol 278: 708–716, 2000
  24. Umekawa T, Iguchi M, Kurita T: The effect of osteopontin immobilized collagen granules in the seed crystal method. Urol Res 29: 282–286, 2001[CrossRef][Medline]
  25. Thamilselvan S, Hackett RL, Khan SR: Lipid peroxidation in ethylene glycol induced hyperoxaluria and CaOx nephrolithiasis. J Urol 157: 1059–1063, 1997[CrossRef][Medline]
  26. Khand FD, Gordge MP, Robertson WG, Noronha-Dutra AA, Hothersall JS: Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells. Free Radic Biol Med 32: 1339–1350, 2002[CrossRef][Medline]
  27. Abbate M, Zoja C, Corna D, Capitanio M, Bertani T, Remuzzi G: In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation. J Am Soc Nephrol 9: 1213–1224, 1998[Abstract]
  28. Remuzzi G, Ruggenenti P, Perico N: Chronic renal diseases: Renoprotective benefits of renin-angiotensin system inhibition. Ann Inern Med 136: 604–615, 2002[Abstract/Free Full Text]
  29. 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[Medline]
  30. Zimpelmann J, Kumar D, Levine DZ, Wehbi G, Imig JD, Navar LG, Burns KD: Early diabetes mellitus stimulates proximal tubule renin mRNA expression in the rat. Kidney Int 58: 2320–2330, 2000[CrossRef][Medline]
  31. Wolf G, Muller E, Stahl RA, Ziyadeh FN: Angiotensin II-induced hypertrophy of cultured murine proximal tubular cells is mediated by endogenous transforming growth factor-beta. J Clin Invest 92: 1366–1372, 1993
  32. Kagami S, Border WA, Miller DE, Noble NA: Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells. J Clin Invest 93: 2431–2437, 1994
  33. Tank JE, Henrich WL, Moe OW: Regulation of glomerular and proximal tubule renin mRNA by chronic changes in dietary NaCl. Am J Physiol 273: F892–F898, 1997
  34. Moe OW, Ujiie K, Star RA, Miller RT, Widell J, Alpern RJ, Henrich WL: Renin expression in proximal tubule. J Clin Invest 91: 774–779, 1993
  35. Gilbert RE, Wu LL, Kelly DJ, Cox A, Wilkinson-Berka JL, Johnson CI, Cooper ME: Pathological expression of renin and angiotensin II in the renal tubules after subtotal nephrectomy, implications for the pathogenesis of tubulointerstitial fibrosis. Am J Pathol 155: 429–440, 1999[Abstract/Free Full Text]
  36. Diamond JR, Kreisberg R, Evans R, Nguyen TA, Ricarda SD: Regulation of proximal tubular osteopontin in experimental hydronephrosis in the rat. Kidney Int 54: 1501–1509, 1998[CrossRef][Medline]
  37. Malyankar UM, Almeida M, Johnson RJ, Pichler RH, Giachelli CM: Osteopontin regulation in cultured rat renal epithelial cells. Kidney Int 51: 1766–173, 1997[Medline]
  38. Schepers MS, ven der Broom BG, Romjin HC, Verkeolen CF: Urinary crystallization inhibitors do not prevent crystal binding. J Urol 167: 1844–1847, 2002[CrossRef][Medline]
  39. Brown LF, Berse B, Van de Water L, Papadopoulos-Sergiou A, Perruzzi CA, Manseau EJ, Dvorak HF, Senger DR: Expression and distribution of osteopontin in human tissues: Widespread association with luminal epithelial surfaces. Mol Biol Cell 3: 1169–1180, 1992[Abstract]
  40. Yamate T, Kohri K, Umekawa T, Ishikawa Y, Iguchi M, Kurita T: Interaction between osteopontin on Madin-Darby canine kidney cell membrane and calcium oxalate crystals. Urol Int 62: 81–86, 1999[CrossRef][Medline]
  41. Yasui T, Fujita K, Asai K, Kohri K: Osteopontin regulates adhesion of calcium oxalate crystals to renal epithelial cells. Int J Urol 9: 100–108, 2002[CrossRef][Medline]
  42. Miyazawa K, Suzuki K, Ueda Y, Katsuda S: Demonstration of apoptosis and its related genes in rat tubular epithelium of calcium oxalate crystal formation. In: Urolithiasis 2000, Proceedings of the 9th International Symposium on Urolithiasis, Cape Town, South Africa, University of Cape Town, 2001, pp 253–254
  43. O’Regan A, Berman JS: Osteopontin: A key cytokine in cell mediated and granulomatous inflammation. Int J Exp Pathol 81: 373–390, 2000[CrossRef][Medline]
  44. Giachelli CM, Lombardi D, Johnson RJ, Murry CE, Almeida M: Evidence for a role of osteopontin in macrophage infiltration in response to pathological stimuli in vivo. Am J Pathol 152: 353–358, 1998[Abstract]
  45. Cao Z, Cooper ME: Role of angiotensin II in tubulointerstitial injury. Semin Nephrol 21: 554–562, 2001[CrossRef][Medline]
  46. Ophascharoensuk V, Giachelli CM, Gordon K: Obstructive uropathy in the mouse: Role of osteopontin in interstitial fibrosis and apoptosis. Kidney Int 56: 571–580, 1999[CrossRef][Medline]
  47. Yu XQ, Nikolic Paterson DJ, Mu W, Giachelli CM, Atkins RC, Johnson RJ, Lan HY: A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat. Proc Assoc Am Physicians 110: 50–64, 1998[Medline]
  48. Khan SR, Coskrell CA, Finlayson B, Hackett RL: Crystal retention by injured urothelium of the rat urinary bladder. J Urol 132: 153–157, 1984[Medline]
  49. 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: S441–S445, 1999
  50. Verhulst A, Asselman A, Persy VP, Schepers MSJ, Helbert MF, Verkoelen CF, de Broe ME: Crystal retention capacity of cells I 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 13: 107–115, 2003
  51. Yu XQ, Wu LL, Huang XR, Yang N, Gilbert RE, Cooper ME, Johnson RJ, Lai KN, Lan HY: Osteopontin expression in progressive renal injury in remnant kidney: Role of angiotensin II. Kidney Int 58: 1469–1480, 2000[CrossRef][Medline]
  52. Kelly DJ, Wilkinson-Berka JL, Ricardo SD, Cox AJ, Gilbert RE: Progression of tubulointerstitial injury by osteopontin-induced macrophage recruitment in advanced diabetic nephropathy of transgenic (mRen-2) 27 rats. Nephrol Dial Transplant 17: 985–991, 2002[Abstract/Free Full Text]
  53. Kato S, Luyckx VA, Ots M, Lee KW, Ziai F, Troy JL, Brenner BM, Mac Kenzie HS: Renin-angiotensin blockade lowers MCP-1 expression in diabetic rats. Kidney Int 56: 1037–1048, 1999[CrossRef][Medline]
  54. Hilgers KF, Hartner A, Porst M, Mai M, Witmann M, Hugo C, Ganten D, Geiger H, Veelekn R, Mann JF: Monocyte chemoattractant protein-1 and macrophage infiltration in hypertensive kidney injury. Kidney Int 58: 2408–2419, 2000[CrossRef][Medline]
  55. Knight JA: Free radicals: Their history and current status in aging and disease. Ann Clin Lab Sci 28: 331–346, 1998[Abstract]
  56. James EA, Galceran JM, Raij L: Angiotensin II induces superoxide anion production by mesangial cells. Kidney Int 54: 775–784, 1998[CrossRef][Medline]
  57. Hanken T, Chroeder R, Stahl RAK, Wolf G: Angiotensin II mediated expression of p27 and induction of cellular hypertrophy in renal tubular cells depend on generation of oxygen radicals. Kidney Int 54: 1923–1933, 1998[CrossRef][Medline]
  58. Haugen EN, Croatt AJ, Nath KA: Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro. Kidney Int 58: 144–152, 2000[CrossRef][Medline]
  59. Baykal Y, Yilmaz MI, Celik T, Gok F, Rehber H, Akay C, Kocar IH: Effects of antihypertensive agents, alpha receptor blockers, beta blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers and calcium channel blockers on oxidative stress. J Hypertens 21: 1207–1211, 2003[CrossRef][Medline]
  60. Agarwal R: Proinflammatory effects of oxidative stress in chronic kidney disease: Role of additional angiotensin II blockade. Am J Physiol Renal Physiol 284: F863–F869, 2003[Abstract/Free Full Text]
Received for publication August 15, 2003. Accepted for publication November 14, 2003.




This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
T. Umekawa, K. Byer, H. Uemura, and S. R. Khan
Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate monohydrate and brushite crystal-induced upregulation of MCP-1 in NRK 52E cells
Nephrol. Dial. Transplant., May 1, 2005; 20(5): 870 - 878.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Umekawa, T.
Right arrow Articles by Khan, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Umekawa, T.
Right arrow Articles by Khan, S. R.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP