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Modeling of hyperoxaluric calcium oxalate nephrolithiasis: Experimental induction of hyperoxaluria by hydroxy-L -proline
original article
http://www.kidney-international.org & 2006 International Society of Nephrology
Modeling of hyperoxaluric calcium oxalate nephrolithiasis: Experimental induction of hyperoxaluria by hydroxy-L-proline SR Khan1, PA Glenton1 and KJ Byer1 1
Department of Pathology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, USA
A number of animal models have been developed to investigate calcium oxalate (CaOx) nephrolithiasis. Ethylene glycol (EG)-induced hyperoxaluria in rats is most common, but is criticized because EG and some of its metabolites are nephrotoxic and EG causes metabolic acidosis. Both oxalate (Ox) and CaOx crystals are also injurious to renal epithelial cells. Thus, it is difficult to distinguish the effects of EG and its metabolites from those induced by Ox and CaOx crystals. This study was performed to investigate hydroxy-L-proline (HLP), a common ingredient of many diets, as a hyperoxaluria-inducing agent. In rats, HLP has been shown to induce CaOx nephrolithiasis in only hypercalciuric conditions. Five percent HLP mixed with chow was given to male Sprague–Dawley rats for 63 days, resulting in hyperoxaluria, CaOx crystalluria, and nephrolithiasis. Crystal deposits were surrounded by ED-1-positive inflammatory cells. Cell injury and death was followed by regeneration, as suggested by an increase in proliferating cell nuclear antigen-positive cells. Both osteopontin (OPN) and CD44 were upregulated. Staining for CD44 and OPN was intense in cells lining the tubules that contained crystals. Along with a rise in urinary Ox and lactate dehydrogenase, there were significant increases in 8-isoprostane and hydrogen peroxide excretion, indicating that the oxidative stress induced cell injury. Thus, HLP-induced hyperoxaluria alone can induce CaOx nephrolithiasis in rats. Kidney International (2006) 70, 914–923. doi:10.1038/sj.ki.5001699; published online 19 July 2006 KEYWORDS: calcium oxalate; hyperoxaluria; nephrolithiasis; inflammation; renal injury
Correspondence: SR Khan, Department of Pathology, Immunology and Laboratory Medicine, University of Florida, College of Medicine, Box 100275, JHMHC, Gainesville, Florida 32610-0275, USA. E-mail: [email protected] Received 24 February 2006; revised 3 May 2006; accepted 23 May 2006; published online 19 July 2006 914
Increased urinary excretion of oxalate (Ox) or hyperoxaluria is the result of either genetic (primary hyperoxaluria) or environmental factors (secondary hyperoxaluria). Enhanced absorption of Ox, secondary to many gastrointestinal diseases, ileal resection, or jejuno-ileal bypass, is called enteric hyperoxaluria.1,2 Hyperoxaluria can result in urolithiasis, nephrocalcinosis, metabolic acidosis, hematuria, pyelonephritis, hydronephrosis, and renal failure. In the case of primary hyperoxaluria, however, there is a systemic deposition of calcium oxalate (CaOx) in almost all of the body tissues including kidneys, heart, bone, cartilage, teeth, vasculature, and brain.3 Patients with primary hyperoxaluria eventually develop end-stage renal failure, usually in childhood. Patients with enteric hyperoxaluria may also develop inflammation and end-stage renal disease. Several animal models have been developed to investigate hyperoxaluria and its consequences.4 In the most common model, ethylene glycol (EG), a precursor of Ox, is given to rats in their drinking water5,6 with or without additional ammonium chloride or vitamin D. Animals develop hyperoxaluria, which leads to crystalluria and CaOx crystal deposition in the kidneys.4–6 Hyperoxaluria and crystal deposition are associated with lipid peroxidation and injury to the renal epithelium,7–9 and altered expression of various genes and production of macromolecules10–20 including bikunin, a-1microglobulin, Tamm–Horsfall protein, osteopontin (OPN), and haparan sulfate. However, EG consumption leads to multiorgan injuries and concentrations above 0.75% may produce metabolic acidosis.21–23 In addition, in the EG model, Ox may not be the only cause of injury and inflammation. Many other metabolites of EG are shown to be injurious to the renal epithelium.24 In response to various criticisms of the EG model, we decided to induce hyperoxaluria by the oral administration of hydroxy-L-proline (HLP), a physiological precursor of Ox. HLP is a derivative of the common amino acid proline and a component of collagen, thus a common ingredient of many Western diets. We treated rats with 5.2% (w/v) HLP for 10 days, which produced hyperoxaluria; however, the treatment did not result in nephrolithiasis, the deposition of CaOx crystals in the kidney.9 The addition of 10% HLP to the standard diet of pigs resulted in hyperoxaluria, CaOx Kidney International (2006) 70, 914–923
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SR Khan et al.: Hyperoxaluria and calcium oxalate nephrolithiasis
crystalluria, and nephrolithiasis at 20 days.25 Administration of 3 or 5% HLP in drinking water to genetic hypercalciuric (GH) rats for 18 weeks produced CaOx crystal deposits in their urinary space.26 GH rats generate stones of poorly crystalline calcium phosphate (CaP) in their kidneys and ureters. It was concluded that hypercalciuria and CaP crystal deposition played a significant role in the development of CaOx nephrolithiasis in these rats. We report here that the addition of 5% HLP to regular chow not only induces hyperoxaluria and crystalluria, but also promotes the formation of CaOx crystal deposits in the rat kidneys without additional hypercalciuria and CaP crystal deposition. Morphological changes induced by HLP administration and CaOx crystal deposition are similar to those seen in other animal hyperoxaluria models of CaOx nephrolithiasis. RESULTS
All rats stayed healthy and gained weight. However, over time, the treated rats gained significantly less weight (Po0.5) compared to the normal controls, even though both consumed similar amounts of food (Figure 1a and d). Treated rats drank significantly (Po0.005) more water (Figure 1b) than
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We determined the urinary excretion of calcium (Ca) and Ox ions, as they have been shown to be directly involved in CaOx supersaturation and crystallization. They have also been shown to be altered during experimental induction of hyperoxaluria and CaOx nephrolithiasis in male rats. Compared to baseline, urinary Ox excretion increased significantly in treated rats (Figure 3a), reaching the highest level by day 14. It remained considerably higher than control levels for all time periods investigated from day 7 to 63. Excretion of Ox by the control rats remained at the baseline level. On the other hand, the urinary excretion of Ca by the treated rats decreased significantly compared to the baseline and was significantly lower than the urinary excretion of Ca by the controls rats for all time periods investigated (Figure 3b).
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the controls and also produced significantly more (Po0.02) urine (Figure 1c). Creatinine levels were significantly lower in the urine of the treated rats (Figure 2a). They also demonstrated significantly lower creatinine clearance (Figure 2b). Although the pH of treated rat urine was generally lower than that of the controls (results not shown), the differences were not significant.
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Figure 1 | Food intake by the rats (g/day). (a) Compared to baseline food intake on day 0, all rats consumed significantly more food on all other days. Treated rats, however, ate less than the control rats. (b) Rats on HLP drank significantly more water than the control rats whose water consumption did not differ significantly from the baseline. (c) Rats on HLP excreted more urine than the control rats whose urinary excretion did not differ from the baseline. (d) Both control and treated rats gained significant weight compared to day 0, but treated rats gained significantly less weight compared to the controls. Kidney International (2006) 70, 914–923
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SR Khan et al.: Hyperoxaluria and calcium oxalate nephrolithiasis
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Figure 2 | Urinary creatinine and creatinine clearance. (a) Treated rats had significantly less creatinine in their urine. (b) Treated rats demonstrated significantly less creatinine clearance than the control rats.
Ca excretion decreased precipitously within the first 2 weeks and continued to decrease thereafter, but not as significantly. There was a significant increase in lactate dehydrogenase (LDH) in the urine of treated rats compared to the baseline level, and it remained significantly high compared to controls for all time periods studied (Figure 4a). Administration of HLP was also associated with a significant increase in urinary hydrogen peroxide (H2O2) and 8-isoprostanec (8-IP) (Figure 4b and c). There was a significant increase in urinary H2O2 and 8-IP by day 7, which continued to increase till day 28. After day 28, there was a gradual but significant drop in their urinary concentration; however, the urinary concentration of both H2O2 and 8-IP stayed significantly higher than the baseline and control levels. Crystalluria
Microscopic analysis revealed that the urine of control rats was either devoid of any crystals or had CaP- and/or struvitetype crystals (Table 1). By day 7, all treated rats produced a large number of CaOx dihydrate (COD) and a few CaOx monohydrate (COM) crystals along with a few struvite and CaP crystals. Struvite crystals had the typical coffin-shaped habit and CaP crystals were generally spherulitic (Figure 5a). 916
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Figure 3 | Urinary excretion of calcium and Ox. (a) There was significant increase in urinary Ox in the urine of treated rats, reaching highest levels by day 14. Urinary Ox of controls rats remained at the baseline level. (b) Calcium excretion decreased significantly over time, compared to both the control and baseline levels.
Table 1 | Crystalluria type and score on various days Day
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0 7 14 21 28 35 42 49 56 63
Str Str Str Str Str Str Str Str Str Str
1, 1, 1, 1, 1, 1, 1, 1, 1, 1,
CaP CaP CaP CaP CaP CaP CaP CaP CaP CaP
Treated 0–1 0–1 0–1 0–1 0–1 0–1 0–1 0–1 0–1 0–1
Str Str Str Str Str Str Str Str Str Str
1, CaP 0–1 1, CaP 0–1, COM 1, COD 3 1, CaP 0–1, COM 3, COD 3 1, CaP 0–1, COM 3, COD 2 1, CaP 0–1, COM 1–3, COD 1-2 1, CaP 0–1, COM 1–3, COD 1–2 1, CaP 0–1, COM 1–3, COD 1–2 0–1, CaP 0–1, COM 1–3, COD 1–2 0–1, CaP 0–1, COM 1–3, COD 1–2 0–1, CaP 0–1, COM 1–3, COD 1–2
CaP, calcium phosphate; COD, calcium oxalate dihydrate; COM, calcium oxalate monohydrate; Str, struvite-type crystals. Drop of urine was mounted on a glass slide and examined by a light microscopy at 10. Five fields/urine sample were examined and average recorded as 0=no crystals, 1=up to 10 crystal/field, 2=10–20 crystals/field, and 3=more than 20 crystals.
COD crystals had the typical bipyramidal habit, whereas COM crystals were plate-like, often with characteristic dumbbell-shaped morphology (Figure 5b). By day 14, the number of COM and COD crystals in the urine increased, Kidney International (2006) 70, 914–923
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SR Khan et al.: Hyperoxaluria and calcium oxalate nephrolithiasis
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Figure 4 | Urinary excretion of LDH, H2O2, and 8-IP. (a) Urinary excretion of LDH by treated rats was significantly higher than the controls on all days examined. (b) There was a significant amount of H2O2 in the urine of treated rats. Highest levels were reached on day 21. (c) Urinary excretion of 8-IP by treated rats increased significantly, reaching highest levels by day 28.
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Figure 5 | Urinary crystals. (a) Spherulitic CaP in the urine of control rats. (b) COM crystals in the urine of treated rats.
whereas that of struvite decreased. Initially, both COM and COD crystals were small. With time, there was a substantial increase in the size of COM and COD crystals that appeared aggregated. Urine aspirated at the time of killing contained both small crystals and large aggregates (Figure 6a and b), often up to 100 mm in diameter. Some deposits contained both COM and COD crystals; however, most were comprised of only COM or COD crystals. Crystal twining was common. Kidney International (2006) 70, 914–923
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Figure 6 | Stone-size crystal deposits isolated from the urinary bladder of treated rats at the time of killing on day 63. (a) A COD stone demonstrating both crystal growth and aggregation. (b) CaOx stone containing both mono and dehydrate crystals.
A few spheroidal CaP crystals were seen in the urine from both the control and treated rats. Kidneys and crystal deposition
The kidneys of all treated rats were larger than those of the comparable control rats. The surface of kidneys of some of the treated rats had a speckled appearance, clearly showing the presence of crystals inside. Examination of paraffin917
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SR Khan et al.: Hyperoxaluria and calcium oxalate nephrolithiasis
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Figure 7 | Light micrograph of median section through the kidney of rat killed after 28 days of consuming HLP. CaOx crystals are birefringent under polarizing conditions. (a) Birefringent CaOx crystals are mainly present in the cortex and parts of the outer medulla. A few crystals are present at the papillary base. (b) Subsurface birefringent crystals at the papillary tip. Some crystals are also seen embedded in the pelvic floor. Apparently, a stone was present at the papillary tip occupying the pelvis all the way to the pelvic floor. Stone was lost during processing and only the crystals adherent to the kidney and pelvic floor remained. Area surrounded by the rectangle is magnified in (c) and (d). (c) White arrow points to crystals inside a collecting duct. Black arrow shows crystal deposit most likely in the loop. (d) Crystals are present in the collecting ducts located under the surface epithelium. White arrow points to a subepithelial crystal. Tubules are damaged. Papillary surface epithelium shows focal hyperplasia. Black arrow points to giant cell formation.
embedded sections by light microscopy revealed the presence of birefringent CaOx crystals (Figure 7a and b). Scanning electron microscopic (SEM) investigations showed that most crystal deposits consisted mainly of plate-like, rectangular COM crystals. Some deposits contained both COM and tetragonal bipyramidal COD crystals. Elemental analysis of the crystal deposits by energy dispersvie X-ray microanalysis produced major peak of only Ca, indicating the absence of CaP crystals (results not shown). Microscopic analysis of kidneys did not reveal major differences in the pattern of crystal deposition between rats treated for 4, 6, or 9 weeks. After 4 weeks, kidneys of all treated rats (4/4) contained CaOx crystals present in all sections of the kidneys: cortex, medulla, and papilla. Two rats demonstrated an interesting pattern of renal crystal distribution with most crystals located in the cortex and outer medulla (Figure 7a), a few in the inner medulla, and large deposits were observed on the papillary tip surface (Figure 7b). After 6 weeks, the kidneys of three out of four rats still contained CaOx crystals in the cortex, medulla, and papilla; however, in one rat, they were mainly seen in the papilla encrusting the papillary surface. After 9 weeks, even though all sections of the kidneys of all treated rats (4/4) contained some CaOx crystal deposits, they were primarily present in 918
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Figure 8 | SEM analysis of the papillary surfaces of treated rat kidneys. (a) A small protrusion on the papillary surface. Surface epithelium is mostly intact. (b) Papillary surface protuberance with stretched surface epithelium showing development of cracks. (c) Surface epithelium shows cracks with protruding crystals.
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Figure 9 | Higher magnifications of the areas in Figure 8. (a) Development of cracks on the surface. Intact cells show excellent morphology. (b) Monoclinic COM crystals mixed with cellular debris are clearly visible.
the renal papillae. Crystal deposits present in the renal papillae were mostly located in the subepithelial collecting ducts near the surface in the fornices and on the papillary tips. Owing to tissue damage during processing for light microscopic examination, the precise location of crystals within various segments of the renal tubules was difficult to determine. However, careful examination showed that after 4 weeks of treatment, crystals were mostly present in the tubular lumens of the distal tubules and collecting ducts. Crystals were also seen in both the thin and the thick limbs of the loop of Henle. After 6–9 weeks, crystals were also found in the renal interstitium, particularly of the renal papillae. Most Kidney International (2006) 70, 914–923
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SR Khan et al.: Hyperoxaluria and calcium oxalate nephrolithiasis
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OPN was also poor in the normal renal tubules, but increased in tubules of the treated rats (Figure 11b). Crystal deposits were heavily stained. Epithelial staining, however, was diffuse and not limited to only the crystal-associated sections of the tubules. DISCUSSION
Figure 10 | Immunohistochemical localization of PCNA and ED-1 in the kidneys of treated rats. Crystals have dropped out of the paraffin sections during staining. (a) A large number of epithelial cells stain positive for PCNA. (b) A number of ED-1-positive cells are present in the renal interstitium.
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Figure 11 | Immunohistochemical localization of CD44 and OPN in the kidneys of treated rats. (a) CD44 staining is limited to the crystal-containing tubules and is more pronounced on baso-lateral aspects of the epithelial cells. (b) OPN staining is seen in many crystal-containing tubules. The luminal contents as well as epithelial cells are positively stained.
tubules containing crystals were dilated and showed destruction of the lining epithelium. Neighboring tubules appeared normal with no overt signs of injury. Inflammatory cells were present in the interstitium adjacent to the large crystal deposits in the cortex as well as in the papilla (Figure 7c and d). Crystal deposition in collecting ducts close to the surface resulted in the development of surface protrusions, which in some sections appeared to erupt through the urothelium and form crystalline encrustations on papillary surfaces. SEM examination of such surfaces showed the protrusions to range from small bulges with intact surface epithelium to large eruptions (Figure 8a–c) with protruding CaOx crystals and stretched surface epithelium with localized areas of cell separations (Figure 9a). Large eruptions showed remnants of epithelium at the edges and as cell debris mingled with the exposed crystals (Figure 9b). Immunohistochemical analysis of the kidneys
Staining of tubular epithelial cells for proliferating cell nuclear antigen (PCNA) was increased in kidneys of treated rats (Figure 10a). Increased staining was detected in tubules containing crystal as well as in some with no crystal deposits. Crystal deposits were surrounded by ED-1-positive cellular infiltrates in the adjacent interstitium (Figure 10b). CD44 was poorly expressed in the normal rat kidneys and highly expressed in renal tubules of treated rats (Figure 11a), particularly segments with crystal deposits. Expression of Kidney International (2006) 70, 914–923
This report describes a rat model of CaOx nephrolithiasis wherein excretion of Ox was increased by the administration of HLP.28 HLP has been used successfully to induce both hyperoxaluria and CaOx crystal deposition in pigs25 and the GH rats.26 In our rat model, as well as in the pigs, HLP was administered with the food, whereas in GH rats HLP was provided in the drinking water. As we have shown, HLP administration through diet not only increased urinary Ox, but after 4 weeks it also produced CaOx crystal deposits in the kidneys in normal male Sprague–Dawley rats. In an earlier study, intraperitoneal dispensation of HLP to male rats also resulted in crystal deposition in the kidneys.29 Reasons for these discrepancies are not clear, but the amount of lithogen given and the mode of administration may play a role. Differences in the absorption and subsequent metabolism of HLP are also likely to be involved. It should also be pointed out that administration of various amounts of HLP resulted in the development of similar levels of hyperoxaluria in both the GH rats and pigs. Crystal deposits in the GH rat kidneys were restricted to the urinary space, whereas those in the kidneys of pigs on HLP were seen in the renal tubules encrusting the renal papillae. In our study, CaOx crystals were seen in the renal tubules of both the cortex and medulla, including the ducts of Bellini of the renal papillae.30 These results of HLP-induced CaOx nephrolithiasis in normal rats and pigs are similar to EGinduced CaOx nephrolithiasis in the rats, where CaOx crystals are seen in all parts of the kidneys: cortex, medulla, and papilla. Perhaps, the urine of GH rats becomes supersaturated with respect to CaOx only after reaching the urinary space, whereas in normal rats urinary supersaturation reaches the crystallization level much earlier in the nephron. CaOx crystal deposits were seen in all parts of the kidney as well as in all segments of the renal tubules. Notably, crystal deposition was observed in the outer medulla at corticomedullary junction and in the renal fornices and papillary tips. These sites are characterized by a change in luminal diameter of the renal tubules and urine is moving from wider to the narrower space.5,30 HLP-induced hyperoxaluria and renal CaOx crystal deposition was associated with renal injury. There was a significant increase in urinary excretion of LDH, a marker of membrane permeability and damage. This increase was evident on day 7, much earlier than crystal deposition, indicating that by that day Ox levels reached a concentration high enough to injure the kidneys. By day 7, urinary excretion of H2O2 and 8-IP also increased significantly, indicating the overproduction of reactive oxygen species and the development of 919
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oxidative stress.31 Results of other studies of hyperoxaluriainduced CaOx nephrolithiasis in rats have confirmed the production of reactive oxygen species and changes in the renal cellular endogenous antioxidant defenses in association with renal injury.7,8,19,32 Recently obtained human data are also suggestive of the development of oxidative stress in kidney stone patients.33,34 Earlier studies have shown the presence of renal injury in kidneys of idiopathic stone formers.35 Morphological changes associated with CaOx crystal deposition in kidneys of the HLP-administered rats included tubular obstruction, focal dilatation, and loss of tubular epithelium. In addition, a number of tubular epithelial cells stained positive for PCNA, indicating cellular regeneration following tubular epithelial loss. These changes were mostly limited to tubular sections that contained the crystals and are similar to renal changes seen in other models of hyperoxaluric CaOx nephrolithiasis. Crystal retention within the renal tubules is promoted by renal epithelial injury, which exposes a variety of crystal adhesion molecules on epithelial surfaces, including CD44 and its ligands OPN and hyaluronic acid.36–38 OPN is upregulated in EG-induced CaOx nephrolithiasis showing highest expression in crystal-containing tubules. Renal epithelial cells exposed in vitro to both high levels of Ox and CaOx crystals produce copious amounts of OPN.39 A reduction in OPN production leads to reduced crystal attachment.40 OPN immunostaining is also increased in urothelium surrounding CaOx crystal deposits in renal fornix of the GH rats.41 Thus, the increased levels of CD44 and OPN in epithelial cells of crystal-containing tubules in HLPinduced CaOx nephrolithiasis may also be similarly involved in crystal retention. CaOx crystal deposition in kidneys of hyperoxaluric rats is commonly associated with inflammation in which neighboring interstitium becomes occupied with multinucleated giant cells and macrophages that stain positive for ED-1 antigen.42,43 Human kidneys with acute or chronic oxalosis also show inflammation and fibrosis.44–46 The presence of ED-1positive cells in the renal interstitium near HLP-induced CaOx crystal deposits provides further proof for the inflammation-inducing activity of such crystals. We have hypothesized that macrophages are recruited to the sites of crystal deposition to reduce the renal crystal burden and thus provide an early defense against stone formation.47 Other studies have provided evidence that, at least in animal models, anti-inflammatory substances such as angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists reduce the deposition of CaOx crystals in the kidneys.16,48 In our study, crystals were mostly present in the renal tubules, and in the papillae, many were lodged in subepithelially located collecting ducts. Large deposits appeared to have burst open and showed protruding CaOx crystals. Similar crystal deposits have been seen in other rat models of hyperoxaluric CaOx nephrolithiasis.5 Encrustations seen in hyperoxaluric pigs appear to be similarly formed. These 920
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intratubular deposits are reminiscent of Randall’s Type 2 plaques.49,50 Randall51,52 suggested that kidney stones develop on two types of pre-calculus lesions. The more common lesion consisting of interstitial subepithelial deposits of CaP and calcium carbonate on the pelvic aspect of the papilla was called Type 1 lesion. The infrequent lesion formed by CaP crystal deposition in the ducts of Bellini was termed Type 2 lesion. He suggested that Type 1 lesion arose from pathologic condition of the papilla, whereas Type 2 developed from excessive supersaturation and necrosis of the tubular epithelial cells. It has been suggested that plaques lose their epithelial covering, become exposed to the pelvic urine, and develop into stone niduses. Various studies have confirmed the formation of a variety of stones including CaP, CaOx, uric acid, and cystine on Type 1 lesions.51–55 Renal interstitium and epithelial cells of idiopathic stone formers remain normal and crystal matrix is positive for OPN. Brushite stones and CaOx stones developed after intestinal bypass surgery are formed in association with Type 2 lesions consisting of intraluminal CaP deposits. Renal tubules appear damaged and interstitium contains inflammatory cells. In a case where papillary tissue was removed from a patient with bilateral renal lithiasis, we found a CaOx stone lodged in a duct of Bellini (554). Biological apatite crystals were found in the nearby interstitium. Tubular cells were damaged and interstitium appeared fibrotic Conclusions
It has been suggested that EG is not a useful agent to induce CaOx nephrolithiasis in animal models because it can produce severe life-threatening metabolic acidosis, has multiorgan toxicity, and its metabolites glycoaldehyde and glyoxylate are also highly toxic. HLP, a common amino acid, is considered a better alternative but so far in the normal male rats it has produced only hyperoxaluria. HLP administration to GH rats produced CaOx crystals in the kidneys, but they were limited to the urinary space. Results of our study show that renal and urinary changes seen during the development of CaOx nephrolithiasis in HLP-treated rats are similar to those seen in rats provided with EG. Porcine model of CaOx nephrolithiasis showed similar morphological changes in the kidneys. Other rat models of CaOx nephrolithiasis, regardless of the type of hyperoxaluria-inducing agent and means of delivery, have shown similar renal injury in the presence of hyperoxaluria and CaOx crystal deposition (Table 2). Thus, renal pathology seen in animals with experimental CaOx nephrolithiasis is perhaps a result of cellular exposure to high Ox and CaOx crystals because the damage was mostly focal and seen in the tubules that were full of CaOx crystals. Crystal deposits seen in the renal papillae of rats with experimentally induced hyperoxaluria are similar to Randall’s Type 2 lesions in that both are intratubular, plug the collecting ducts, damage the tubular epithelium, and induce Kidney International (2006) 70, 914–923
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SR Khan et al.: Hyperoxaluria and calcium oxalate nephrolithiasis
Table 2 | CaOx nephrolithiasis in rats Induction technique
Crystal deposition
Renal and urinary changes
Strengths
EG in drinking water4,5,11–17
Intraluminal in renal tubules of both cortex and medulla; crystals deposit in association with cellular degradation products; plaques, and stones at papillary tips
Easy to induce consistent hyperoxaluria, crystalluria, and CaOx nephrolithiasis
HLP in drinking water9,41
No crystals in the renal tubules; crystal aggregates in the renal fornices and pelvis
HLP mixed with food
Intraluminal in renal tubules of both cortex and medulla; plaques, and stones at papillary tips Intraluminal in renal tubules of both cortex and medulla
Necrotic and apoptotic renal injury; interstitail inflammation; increased synthesis and urinary excretion of OPN, bikunin, MCP-1, a-1-microglobulin, hyperoxaluria, enzymuria, membranuria, and CaOx crystalluria Kidneys appear normal; hyperoxaluria, enzymuria, and CaOx crystalluria; increased synthesis of OPN by papillary surface epithelial cells Hyperoxaluria and CaOx crystalluria; signs of renal injury and inflammation in association with the crystals Hyperoxaluria, CaOx crystalluria, and upregulation of TNF receptor kidney injury marker and OPN Hyperoxaluria, hypercalcalciuria, enzymuria, hypocitraturia, and CaOx crystalluria
Implantation of osmotic mini-pumps filled with Ox56 Vitamin B-6-deficient diet57 Glycolic acid in diet58
Ileal resection and feeding of Ox59
Intraperitoneal administration of Ox or HLP29,50
CaOx crystals intraluminal in tubules of medulla; plaques on papillary tips; stones in renal fornices, pelvis, ureters, and bladder CaOx crystals in the tubules of renal cortex and medulla; stones in renal pelvis CaOx mixed with CaP and Ca carbonate crystals; intraluminal in both cortex and medulla; interstitial in the papilla; plaques on papillary surface CaOx crystal deposition in all sections of the kidneys, initially intraluminal and then also seen in the interstitium; papillary plaques of CaOx
Simple; more physiological than the administration of EG or some other Ox precursors Same as above
Reliable and consistent hyperoxaluria and CaOx nephrolithiasis
Hyperoxaluria
Tubular obstruction and interstitial inflammation; hyperoxaluria and hypocitraturia
Models nephrolithiasis after ileal resection or bypass surgery
Tubular obstruction and dilatation; interstitial inflammation; crystal formation in association with membrane fragments; hyperoxaluria, CaOx crystalluria, enzymuria, and membranuria
Easy to accomplish crystal deposition in the kidneys for investigating crystal cell interactions
Ca, calcium; CaOx, calcium oxalate; CaP, calcium phosphate; EG, ethylene glycol; HLP, hydroxy-L-proline; MCP, monocyte chemoattractant protein; OPN, osteopontin; Ox, oxalate; TNF, tumor necrosis factor.
interstitial inflammation. Eruption of crystals deposited in the subepithelial collecting ducts leads to the formation of a stone nidus. The difference between human and rat Type 2 lesions is that in the humans, lesions are generally apatite, but a human case with intratubular CaOx crystals has been reported. MATERIALS AND METHODS Animal protocol Male Sprague–Dawley rats (120–130 g) were purchased from Harlan. After 1 week of acclimatization to our animal facilities, they were divided into two groups, control and experimental. Both had easy access to food and water, and their consumption was recorded daily. Food was ground, moistened with water, and formed into small balls, which were individually weighed. Control rats received normal rat chow, whereas experimental animals were given chow mixed with 5% HLP (weight/weight HLP/chow) purchased from ICN Biochemicals (Aurora, OH, USA). Food and water consumption were regularly monitored. Rats were weighed weekly to check their growth. Urine was collected daily for determination of crystalluria and weekly for the analyses of urinary ions and markers of cell injury and lipid peroxidation. Four control rats and four experimental rats were killed after 4, 6, and 9 weeks. Their bladder urine was aspirated and examined for the presence of crystals. Their kidneys were Kidney International (2006) 70, 914–923
processed for localization of crystals and various other morphological analyses. Urine collection and analysis Once a week, 24-h urine collection was made with 0.02% sodium azide to prevent bacterial growth. Rats were placed in metabolic cages for the collection of urine. After determining urinary volume and pH, urine was aliquoted for various assays. For the determination of Ox, an aliquot of 24 h urine samples from control and experimental subjects was acidified by the addition of 1 M HCl (20:1 dilution) and kept frozen in closed vials until analyzed. Just before analysis, the samples were thawed and brought to room temperature, mixed, and Ox determinations were carried out in triplicate. Analyses were performed using a Dionex DX100 gradient ion chromatography system equipped with a 0.4 25 cm AS10A anion exchange analytical column containing an AG10A guard column. Urinary Ca and creatinine were also determined using methods described in our previous studies. Serum creatinine was measured at the time of killing. Centrifuged urine supernatants were used to determine LDH. LDH was analyzed on the spectrophotometer by measuring the rate of decrease of absorbance of nicotinamide adenine dinucleotide (reduced form) at 340 nm using pyruvate as the substrate. To investigate the development of oxidative stress, we determined urinary excretion of H2O2, a reactive oxygen species, and 8-IP, a product of lipid peroxidation. H2O2 quantification was carried out 921
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SR Khan et al.: Hyperoxaluria and calcium oxalate nephrolithiasis
using a colormetric assay obtained from Pierce (PeroXoquant Quantitative Peroxide Assay Kit, Pierce Biotechnology Inc., Rockford, IL, USA: catalogue no. 23280). Determination of urinary isoprostane was carried out using a kit from Oxford Biomedical Research (Urinary Isoprostane, Cayman Chemical Company, Ann Arbor, MI, USA: catalogue no. EA85). Urine was examined by light microscopy to analyze crystalluria. Selected urine samples and bladder urine collected at the time of killing were filtered through 0.2 mm nucleopore filter for examination and analysis,27 with an SEM and energy dispersive X-ray microanalysis.
14.
Kidney tissue processing Four rats from the normal control group and four from the experimental group were killed after 4, 6, and 9 weeks. Details of tissue preparation for light and SEM analyses are described in our earlier publications.8,9,11–14 The presence of CaOx crystal was scored on a basis of 0–4 þ .
19.
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17. 18.
20. 21.
22.
Immunohistochemistry To confirm that HLP-induced hyperoxaluria follows similar patterns and produces similar outcomes, OPN and CD44 were localized immunohistochemically using specific antibodies. Cell proliferation was determined by immunohistochemical staining for PCNA. Migration of M/M into the interstitium was determined by immunohistochemical staining with ED-1 antibodies. ACKNOWLEDGMENTS
This study was supported by NIH Grant No. RO1 DK 065658 and UF Center for the Study of Lithiasis.
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http://www.kidney-international.org & 2006 International Society of Nephrology
Modeling of hyperoxaluric calcium oxalate nephrolithiasis: Experimental induction of hyperoxaluria by hydroxy-L-proline SR Khan1, PA Glenton1 and KJ Byer1 1
Department of Pathology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, USA
A number of animal models have been developed to investigate calcium oxalate (CaOx) nephrolithiasis. Ethylene glycol (EG)-induced hyperoxaluria in rats is most common, but is criticized because EG and some of its metabolites are nephrotoxic and EG causes metabolic acidosis. Both oxalate (Ox) and CaOx crystals are also injurious to renal epithelial cells. Thus, it is difficult to distinguish the effects of EG and its metabolites from those induced by Ox and CaOx crystals. This study was performed to investigate hydroxy-L-proline (HLP), a common ingredient of many diets, as a hyperoxaluria-inducing agent. In rats, HLP has been shown to induce CaOx nephrolithiasis in only hypercalciuric conditions. Five percent HLP mixed with chow was given to male Sprague–Dawley rats for 63 days, resulting in hyperoxaluria, CaOx crystalluria, and nephrolithiasis. Crystal deposits were surrounded by ED-1-positive inflammatory cells. Cell injury and death was followed by regeneration, as suggested by an increase in proliferating cell nuclear antigen-positive cells. Both osteopontin (OPN) and CD44 were upregulated. Staining for CD44 and OPN was intense in cells lining the tubules that contained crystals. Along with a rise in urinary Ox and lactate dehydrogenase, there were significant increases in 8-isoprostane and hydrogen peroxide excretion, indicating that the oxidative stress induced cell injury. Thus, HLP-induced hyperoxaluria alone can induce CaOx nephrolithiasis in rats. Kidney International (2006) 70, 914–923. doi:10.1038/sj.ki.5001699; published online 19 July 2006 KEYWORDS: calcium oxalate; hyperoxaluria; nephrolithiasis; inflammation; renal injury
Correspondence: SR Khan, Department of Pathology, Immunology and Laboratory Medicine, University of Florida, College of Medicine, Box 100275, JHMHC, Gainesville, Florida 32610-0275, USA. E-mail: [email protected] Received 24 February 2006; revised 3 May 2006; accepted 23 May 2006; published online 19 July 2006 914
Increased urinary excretion of oxalate (Ox) or hyperoxaluria is the result of either genetic (primary hyperoxaluria) or environmental factors (secondary hyperoxaluria). Enhanced absorption of Ox, secondary to many gastrointestinal diseases, ileal resection, or jejuno-ileal bypass, is called enteric hyperoxaluria.1,2 Hyperoxaluria can result in urolithiasis, nephrocalcinosis, metabolic acidosis, hematuria, pyelonephritis, hydronephrosis, and renal failure. In the case of primary hyperoxaluria, however, there is a systemic deposition of calcium oxalate (CaOx) in almost all of the body tissues including kidneys, heart, bone, cartilage, teeth, vasculature, and brain.3 Patients with primary hyperoxaluria eventually develop end-stage renal failure, usually in childhood. Patients with enteric hyperoxaluria may also develop inflammation and end-stage renal disease. Several animal models have been developed to investigate hyperoxaluria and its consequences.4 In the most common model, ethylene glycol (EG), a precursor of Ox, is given to rats in their drinking water5,6 with or without additional ammonium chloride or vitamin D. Animals develop hyperoxaluria, which leads to crystalluria and CaOx crystal deposition in the kidneys.4–6 Hyperoxaluria and crystal deposition are associated with lipid peroxidation and injury to the renal epithelium,7–9 and altered expression of various genes and production of macromolecules10–20 including bikunin, a-1microglobulin, Tamm–Horsfall protein, osteopontin (OPN), and haparan sulfate. However, EG consumption leads to multiorgan injuries and concentrations above 0.75% may produce metabolic acidosis.21–23 In addition, in the EG model, Ox may not be the only cause of injury and inflammation. Many other metabolites of EG are shown to be injurious to the renal epithelium.24 In response to various criticisms of the EG model, we decided to induce hyperoxaluria by the oral administration of hydroxy-L-proline (HLP), a physiological precursor of Ox. HLP is a derivative of the common amino acid proline and a component of collagen, thus a common ingredient of many Western diets. We treated rats with 5.2% (w/v) HLP for 10 days, which produced hyperoxaluria; however, the treatment did not result in nephrolithiasis, the deposition of CaOx crystals in the kidney.9 The addition of 10% HLP to the standard diet of pigs resulted in hyperoxaluria, CaOx Kidney International (2006) 70, 914–923
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crystalluria, and nephrolithiasis at 20 days.25 Administration of 3 or 5% HLP in drinking water to genetic hypercalciuric (GH) rats for 18 weeks produced CaOx crystal deposits in their urinary space.26 GH rats generate stones of poorly crystalline calcium phosphate (CaP) in their kidneys and ureters. It was concluded that hypercalciuria and CaP crystal deposition played a significant role in the development of CaOx nephrolithiasis in these rats. We report here that the addition of 5% HLP to regular chow not only induces hyperoxaluria and crystalluria, but also promotes the formation of CaOx crystal deposits in the rat kidneys without additional hypercalciuria and CaP crystal deposition. Morphological changes induced by HLP administration and CaOx crystal deposition are similar to those seen in other animal hyperoxaluria models of CaOx nephrolithiasis. RESULTS
All rats stayed healthy and gained weight. However, over time, the treated rats gained significantly less weight (Po0.5) compared to the normal controls, even though both consumed similar amounts of food (Figure 1a and d). Treated rats drank significantly (Po0.005) more water (Figure 1b) than
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We determined the urinary excretion of calcium (Ca) and Ox ions, as they have been shown to be directly involved in CaOx supersaturation and crystallization. They have also been shown to be altered during experimental induction of hyperoxaluria and CaOx nephrolithiasis in male rats. Compared to baseline, urinary Ox excretion increased significantly in treated rats (Figure 3a), reaching the highest level by day 14. It remained considerably higher than control levels for all time periods investigated from day 7 to 63. Excretion of Ox by the control rats remained at the baseline level. On the other hand, the urinary excretion of Ca by the treated rats decreased significantly compared to the baseline and was significantly lower than the urinary excretion of Ca by the controls rats for all time periods investigated (Figure 3b).
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the controls and also produced significantly more (Po0.02) urine (Figure 1c). Creatinine levels were significantly lower in the urine of the treated rats (Figure 2a). They also demonstrated significantly lower creatinine clearance (Figure 2b). Although the pH of treated rat urine was generally lower than that of the controls (results not shown), the differences were not significant.
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Figure 1 | Food intake by the rats (g/day). (a) Compared to baseline food intake on day 0, all rats consumed significantly more food on all other days. Treated rats, however, ate less than the control rats. (b) Rats on HLP drank significantly more water than the control rats whose water consumption did not differ significantly from the baseline. (c) Rats on HLP excreted more urine than the control rats whose urinary excretion did not differ from the baseline. (d) Both control and treated rats gained significant weight compared to day 0, but treated rats gained significantly less weight compared to the controls. Kidney International (2006) 70, 914–923
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Figure 2 | Urinary creatinine and creatinine clearance. (a) Treated rats had significantly less creatinine in their urine. (b) Treated rats demonstrated significantly less creatinine clearance than the control rats.
Ca excretion decreased precipitously within the first 2 weeks and continued to decrease thereafter, but not as significantly. There was a significant increase in lactate dehydrogenase (LDH) in the urine of treated rats compared to the baseline level, and it remained significantly high compared to controls for all time periods studied (Figure 4a). Administration of HLP was also associated with a significant increase in urinary hydrogen peroxide (H2O2) and 8-isoprostanec (8-IP) (Figure 4b and c). There was a significant increase in urinary H2O2 and 8-IP by day 7, which continued to increase till day 28. After day 28, there was a gradual but significant drop in their urinary concentration; however, the urinary concentration of both H2O2 and 8-IP stayed significantly higher than the baseline and control levels. Crystalluria
Microscopic analysis revealed that the urine of control rats was either devoid of any crystals or had CaP- and/or struvitetype crystals (Table 1). By day 7, all treated rats produced a large number of CaOx dihydrate (COD) and a few CaOx monohydrate (COM) crystals along with a few struvite and CaP crystals. Struvite crystals had the typical coffin-shaped habit and CaP crystals were generally spherulitic (Figure 5a). 916
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Figure 3 | Urinary excretion of calcium and Ox. (a) There was significant increase in urinary Ox in the urine of treated rats, reaching highest levels by day 14. Urinary Ox of controls rats remained at the baseline level. (b) Calcium excretion decreased significantly over time, compared to both the control and baseline levels.
Table 1 | Crystalluria type and score on various days Day
Control
0 7 14 21 28 35 42 49 56 63
Str Str Str Str Str Str Str Str Str Str
1, 1, 1, 1, 1, 1, 1, 1, 1, 1,
CaP CaP CaP CaP CaP CaP CaP CaP CaP CaP
Treated 0–1 0–1 0–1 0–1 0–1 0–1 0–1 0–1 0–1 0–1
Str Str Str Str Str Str Str Str Str Str
1, CaP 0–1 1, CaP 0–1, COM 1, COD 3 1, CaP 0–1, COM 3, COD 3 1, CaP 0–1, COM 3, COD 2 1, CaP 0–1, COM 1–3, COD 1-2 1, CaP 0–1, COM 1–3, COD 1–2 1, CaP 0–1, COM 1–3, COD 1–2 0–1, CaP 0–1, COM 1–3, COD 1–2 0–1, CaP 0–1, COM 1–3, COD 1–2 0–1, CaP 0–1, COM 1–3, COD 1–2
CaP, calcium phosphate; COD, calcium oxalate dihydrate; COM, calcium oxalate monohydrate; Str, struvite-type crystals. Drop of urine was mounted on a glass slide and examined by a light microscopy at 10. Five fields/urine sample were examined and average recorded as 0=no crystals, 1=up to 10 crystal/field, 2=10–20 crystals/field, and 3=more than 20 crystals.
COD crystals had the typical bipyramidal habit, whereas COM crystals were plate-like, often with characteristic dumbbell-shaped morphology (Figure 5b). By day 14, the number of COM and COD crystals in the urine increased, Kidney International (2006) 70, 914–923
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Figure 4 | Urinary excretion of LDH, H2O2, and 8-IP. (a) Urinary excretion of LDH by treated rats was significantly higher than the controls on all days examined. (b) There was a significant amount of H2O2 in the urine of treated rats. Highest levels were reached on day 21. (c) Urinary excretion of 8-IP by treated rats increased significantly, reaching highest levels by day 28.
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Figure 5 | Urinary crystals. (a) Spherulitic CaP in the urine of control rats. (b) COM crystals in the urine of treated rats.
whereas that of struvite decreased. Initially, both COM and COD crystals were small. With time, there was a substantial increase in the size of COM and COD crystals that appeared aggregated. Urine aspirated at the time of killing contained both small crystals and large aggregates (Figure 6a and b), often up to 100 mm in diameter. Some deposits contained both COM and COD crystals; however, most were comprised of only COM or COD crystals. Crystal twining was common. Kidney International (2006) 70, 914–923
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Figure 6 | Stone-size crystal deposits isolated from the urinary bladder of treated rats at the time of killing on day 63. (a) A COD stone demonstrating both crystal growth and aggregation. (b) CaOx stone containing both mono and dehydrate crystals.
A few spheroidal CaP crystals were seen in the urine from both the control and treated rats. Kidneys and crystal deposition
The kidneys of all treated rats were larger than those of the comparable control rats. The surface of kidneys of some of the treated rats had a speckled appearance, clearly showing the presence of crystals inside. Examination of paraffin917
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Figure 7 | Light micrograph of median section through the kidney of rat killed after 28 days of consuming HLP. CaOx crystals are birefringent under polarizing conditions. (a) Birefringent CaOx crystals are mainly present in the cortex and parts of the outer medulla. A few crystals are present at the papillary base. (b) Subsurface birefringent crystals at the papillary tip. Some crystals are also seen embedded in the pelvic floor. Apparently, a stone was present at the papillary tip occupying the pelvis all the way to the pelvic floor. Stone was lost during processing and only the crystals adherent to the kidney and pelvic floor remained. Area surrounded by the rectangle is magnified in (c) and (d). (c) White arrow points to crystals inside a collecting duct. Black arrow shows crystal deposit most likely in the loop. (d) Crystals are present in the collecting ducts located under the surface epithelium. White arrow points to a subepithelial crystal. Tubules are damaged. Papillary surface epithelium shows focal hyperplasia. Black arrow points to giant cell formation.
embedded sections by light microscopy revealed the presence of birefringent CaOx crystals (Figure 7a and b). Scanning electron microscopic (SEM) investigations showed that most crystal deposits consisted mainly of plate-like, rectangular COM crystals. Some deposits contained both COM and tetragonal bipyramidal COD crystals. Elemental analysis of the crystal deposits by energy dispersvie X-ray microanalysis produced major peak of only Ca, indicating the absence of CaP crystals (results not shown). Microscopic analysis of kidneys did not reveal major differences in the pattern of crystal deposition between rats treated for 4, 6, or 9 weeks. After 4 weeks, kidneys of all treated rats (4/4) contained CaOx crystals present in all sections of the kidneys: cortex, medulla, and papilla. Two rats demonstrated an interesting pattern of renal crystal distribution with most crystals located in the cortex and outer medulla (Figure 7a), a few in the inner medulla, and large deposits were observed on the papillary tip surface (Figure 7b). After 6 weeks, the kidneys of three out of four rats still contained CaOx crystals in the cortex, medulla, and papilla; however, in one rat, they were mainly seen in the papilla encrusting the papillary surface. After 9 weeks, even though all sections of the kidneys of all treated rats (4/4) contained some CaOx crystal deposits, they were primarily present in 918
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Figure 8 | SEM analysis of the papillary surfaces of treated rat kidneys. (a) A small protrusion on the papillary surface. Surface epithelium is mostly intact. (b) Papillary surface protuberance with stretched surface epithelium showing development of cracks. (c) Surface epithelium shows cracks with protruding crystals.
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Figure 9 | Higher magnifications of the areas in Figure 8. (a) Development of cracks on the surface. Intact cells show excellent morphology. (b) Monoclinic COM crystals mixed with cellular debris are clearly visible.
the renal papillae. Crystal deposits present in the renal papillae were mostly located in the subepithelial collecting ducts near the surface in the fornices and on the papillary tips. Owing to tissue damage during processing for light microscopic examination, the precise location of crystals within various segments of the renal tubules was difficult to determine. However, careful examination showed that after 4 weeks of treatment, crystals were mostly present in the tubular lumens of the distal tubules and collecting ducts. Crystals were also seen in both the thin and the thick limbs of the loop of Henle. After 6–9 weeks, crystals were also found in the renal interstitium, particularly of the renal papillae. Most Kidney International (2006) 70, 914–923
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a
b
OPN was also poor in the normal renal tubules, but increased in tubules of the treated rats (Figure 11b). Crystal deposits were heavily stained. Epithelial staining, however, was diffuse and not limited to only the crystal-associated sections of the tubules. DISCUSSION
Figure 10 | Immunohistochemical localization of PCNA and ED-1 in the kidneys of treated rats. Crystals have dropped out of the paraffin sections during staining. (a) A large number of epithelial cells stain positive for PCNA. (b) A number of ED-1-positive cells are present in the renal interstitium.
a
b
Figure 11 | Immunohistochemical localization of CD44 and OPN in the kidneys of treated rats. (a) CD44 staining is limited to the crystal-containing tubules and is more pronounced on baso-lateral aspects of the epithelial cells. (b) OPN staining is seen in many crystal-containing tubules. The luminal contents as well as epithelial cells are positively stained.
tubules containing crystals were dilated and showed destruction of the lining epithelium. Neighboring tubules appeared normal with no overt signs of injury. Inflammatory cells were present in the interstitium adjacent to the large crystal deposits in the cortex as well as in the papilla (Figure 7c and d). Crystal deposition in collecting ducts close to the surface resulted in the development of surface protrusions, which in some sections appeared to erupt through the urothelium and form crystalline encrustations on papillary surfaces. SEM examination of such surfaces showed the protrusions to range from small bulges with intact surface epithelium to large eruptions (Figure 8a–c) with protruding CaOx crystals and stretched surface epithelium with localized areas of cell separations (Figure 9a). Large eruptions showed remnants of epithelium at the edges and as cell debris mingled with the exposed crystals (Figure 9b). Immunohistochemical analysis of the kidneys
Staining of tubular epithelial cells for proliferating cell nuclear antigen (PCNA) was increased in kidneys of treated rats (Figure 10a). Increased staining was detected in tubules containing crystal as well as in some with no crystal deposits. Crystal deposits were surrounded by ED-1-positive cellular infiltrates in the adjacent interstitium (Figure 10b). CD44 was poorly expressed in the normal rat kidneys and highly expressed in renal tubules of treated rats (Figure 11a), particularly segments with crystal deposits. Expression of Kidney International (2006) 70, 914–923
This report describes a rat model of CaOx nephrolithiasis wherein excretion of Ox was increased by the administration of HLP.28 HLP has been used successfully to induce both hyperoxaluria and CaOx crystal deposition in pigs25 and the GH rats.26 In our rat model, as well as in the pigs, HLP was administered with the food, whereas in GH rats HLP was provided in the drinking water. As we have shown, HLP administration through diet not only increased urinary Ox, but after 4 weeks it also produced CaOx crystal deposits in the kidneys in normal male Sprague–Dawley rats. In an earlier study, intraperitoneal dispensation of HLP to male rats also resulted in crystal deposition in the kidneys.29 Reasons for these discrepancies are not clear, but the amount of lithogen given and the mode of administration may play a role. Differences in the absorption and subsequent metabolism of HLP are also likely to be involved. It should also be pointed out that administration of various amounts of HLP resulted in the development of similar levels of hyperoxaluria in both the GH rats and pigs. Crystal deposits in the GH rat kidneys were restricted to the urinary space, whereas those in the kidneys of pigs on HLP were seen in the renal tubules encrusting the renal papillae. In our study, CaOx crystals were seen in the renal tubules of both the cortex and medulla, including the ducts of Bellini of the renal papillae.30 These results of HLP-induced CaOx nephrolithiasis in normal rats and pigs are similar to EGinduced CaOx nephrolithiasis in the rats, where CaOx crystals are seen in all parts of the kidneys: cortex, medulla, and papilla. Perhaps, the urine of GH rats becomes supersaturated with respect to CaOx only after reaching the urinary space, whereas in normal rats urinary supersaturation reaches the crystallization level much earlier in the nephron. CaOx crystal deposits were seen in all parts of the kidney as well as in all segments of the renal tubules. Notably, crystal deposition was observed in the outer medulla at corticomedullary junction and in the renal fornices and papillary tips. These sites are characterized by a change in luminal diameter of the renal tubules and urine is moving from wider to the narrower space.5,30 HLP-induced hyperoxaluria and renal CaOx crystal deposition was associated with renal injury. There was a significant increase in urinary excretion of LDH, a marker of membrane permeability and damage. This increase was evident on day 7, much earlier than crystal deposition, indicating that by that day Ox levels reached a concentration high enough to injure the kidneys. By day 7, urinary excretion of H2O2 and 8-IP also increased significantly, indicating the overproduction of reactive oxygen species and the development of 919
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oxidative stress.31 Results of other studies of hyperoxaluriainduced CaOx nephrolithiasis in rats have confirmed the production of reactive oxygen species and changes in the renal cellular endogenous antioxidant defenses in association with renal injury.7,8,19,32 Recently obtained human data are also suggestive of the development of oxidative stress in kidney stone patients.33,34 Earlier studies have shown the presence of renal injury in kidneys of idiopathic stone formers.35 Morphological changes associated with CaOx crystal deposition in kidneys of the HLP-administered rats included tubular obstruction, focal dilatation, and loss of tubular epithelium. In addition, a number of tubular epithelial cells stained positive for PCNA, indicating cellular regeneration following tubular epithelial loss. These changes were mostly limited to tubular sections that contained the crystals and are similar to renal changes seen in other models of hyperoxaluric CaOx nephrolithiasis. Crystal retention within the renal tubules is promoted by renal epithelial injury, which exposes a variety of crystal adhesion molecules on epithelial surfaces, including CD44 and its ligands OPN and hyaluronic acid.36–38 OPN is upregulated in EG-induced CaOx nephrolithiasis showing highest expression in crystal-containing tubules. Renal epithelial cells exposed in vitro to both high levels of Ox and CaOx crystals produce copious amounts of OPN.39 A reduction in OPN production leads to reduced crystal attachment.40 OPN immunostaining is also increased in urothelium surrounding CaOx crystal deposits in renal fornix of the GH rats.41 Thus, the increased levels of CD44 and OPN in epithelial cells of crystal-containing tubules in HLPinduced CaOx nephrolithiasis may also be similarly involved in crystal retention. CaOx crystal deposition in kidneys of hyperoxaluric rats is commonly associated with inflammation in which neighboring interstitium becomes occupied with multinucleated giant cells and macrophages that stain positive for ED-1 antigen.42,43 Human kidneys with acute or chronic oxalosis also show inflammation and fibrosis.44–46 The presence of ED-1positive cells in the renal interstitium near HLP-induced CaOx crystal deposits provides further proof for the inflammation-inducing activity of such crystals. We have hypothesized that macrophages are recruited to the sites of crystal deposition to reduce the renal crystal burden and thus provide an early defense against stone formation.47 Other studies have provided evidence that, at least in animal models, anti-inflammatory substances such as angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists reduce the deposition of CaOx crystals in the kidneys.16,48 In our study, crystals were mostly present in the renal tubules, and in the papillae, many were lodged in subepithelially located collecting ducts. Large deposits appeared to have burst open and showed protruding CaOx crystals. Similar crystal deposits have been seen in other rat models of hyperoxaluric CaOx nephrolithiasis.5 Encrustations seen in hyperoxaluric pigs appear to be similarly formed. These 920
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intratubular deposits are reminiscent of Randall’s Type 2 plaques.49,50 Randall51,52 suggested that kidney stones develop on two types of pre-calculus lesions. The more common lesion consisting of interstitial subepithelial deposits of CaP and calcium carbonate on the pelvic aspect of the papilla was called Type 1 lesion. The infrequent lesion formed by CaP crystal deposition in the ducts of Bellini was termed Type 2 lesion. He suggested that Type 1 lesion arose from pathologic condition of the papilla, whereas Type 2 developed from excessive supersaturation and necrosis of the tubular epithelial cells. It has been suggested that plaques lose their epithelial covering, become exposed to the pelvic urine, and develop into stone niduses. Various studies have confirmed the formation of a variety of stones including CaP, CaOx, uric acid, and cystine on Type 1 lesions.51–55 Renal interstitium and epithelial cells of idiopathic stone formers remain normal and crystal matrix is positive for OPN. Brushite stones and CaOx stones developed after intestinal bypass surgery are formed in association with Type 2 lesions consisting of intraluminal CaP deposits. Renal tubules appear damaged and interstitium contains inflammatory cells. In a case where papillary tissue was removed from a patient with bilateral renal lithiasis, we found a CaOx stone lodged in a duct of Bellini (554). Biological apatite crystals were found in the nearby interstitium. Tubular cells were damaged and interstitium appeared fibrotic Conclusions
It has been suggested that EG is not a useful agent to induce CaOx nephrolithiasis in animal models because it can produce severe life-threatening metabolic acidosis, has multiorgan toxicity, and its metabolites glycoaldehyde and glyoxylate are also highly toxic. HLP, a common amino acid, is considered a better alternative but so far in the normal male rats it has produced only hyperoxaluria. HLP administration to GH rats produced CaOx crystals in the kidneys, but they were limited to the urinary space. Results of our study show that renal and urinary changes seen during the development of CaOx nephrolithiasis in HLP-treated rats are similar to those seen in rats provided with EG. Porcine model of CaOx nephrolithiasis showed similar morphological changes in the kidneys. Other rat models of CaOx nephrolithiasis, regardless of the type of hyperoxaluria-inducing agent and means of delivery, have shown similar renal injury in the presence of hyperoxaluria and CaOx crystal deposition (Table 2). Thus, renal pathology seen in animals with experimental CaOx nephrolithiasis is perhaps a result of cellular exposure to high Ox and CaOx crystals because the damage was mostly focal and seen in the tubules that were full of CaOx crystals. Crystal deposits seen in the renal papillae of rats with experimentally induced hyperoxaluria are similar to Randall’s Type 2 lesions in that both are intratubular, plug the collecting ducts, damage the tubular epithelium, and induce Kidney International (2006) 70, 914–923
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Table 2 | CaOx nephrolithiasis in rats Induction technique
Crystal deposition
Renal and urinary changes
Strengths
EG in drinking water4,5,11–17
Intraluminal in renal tubules of both cortex and medulla; crystals deposit in association with cellular degradation products; plaques, and stones at papillary tips
Easy to induce consistent hyperoxaluria, crystalluria, and CaOx nephrolithiasis
HLP in drinking water9,41
No crystals in the renal tubules; crystal aggregates in the renal fornices and pelvis
HLP mixed with food
Intraluminal in renal tubules of both cortex and medulla; plaques, and stones at papillary tips Intraluminal in renal tubules of both cortex and medulla
Necrotic and apoptotic renal injury; interstitail inflammation; increased synthesis and urinary excretion of OPN, bikunin, MCP-1, a-1-microglobulin, hyperoxaluria, enzymuria, membranuria, and CaOx crystalluria Kidneys appear normal; hyperoxaluria, enzymuria, and CaOx crystalluria; increased synthesis of OPN by papillary surface epithelial cells Hyperoxaluria and CaOx crystalluria; signs of renal injury and inflammation in association with the crystals Hyperoxaluria, CaOx crystalluria, and upregulation of TNF receptor kidney injury marker and OPN Hyperoxaluria, hypercalcalciuria, enzymuria, hypocitraturia, and CaOx crystalluria
Implantation of osmotic mini-pumps filled with Ox56 Vitamin B-6-deficient diet57 Glycolic acid in diet58
Ileal resection and feeding of Ox59
Intraperitoneal administration of Ox or HLP29,50
CaOx crystals intraluminal in tubules of medulla; plaques on papillary tips; stones in renal fornices, pelvis, ureters, and bladder CaOx crystals in the tubules of renal cortex and medulla; stones in renal pelvis CaOx mixed with CaP and Ca carbonate crystals; intraluminal in both cortex and medulla; interstitial in the papilla; plaques on papillary surface CaOx crystal deposition in all sections of the kidneys, initially intraluminal and then also seen in the interstitium; papillary plaques of CaOx
Simple; more physiological than the administration of EG or some other Ox precursors Same as above
Reliable and consistent hyperoxaluria and CaOx nephrolithiasis
Hyperoxaluria
Tubular obstruction and interstitial inflammation; hyperoxaluria and hypocitraturia
Models nephrolithiasis after ileal resection or bypass surgery
Tubular obstruction and dilatation; interstitial inflammation; crystal formation in association with membrane fragments; hyperoxaluria, CaOx crystalluria, enzymuria, and membranuria
Easy to accomplish crystal deposition in the kidneys for investigating crystal cell interactions
Ca, calcium; CaOx, calcium oxalate; CaP, calcium phosphate; EG, ethylene glycol; HLP, hydroxy-L-proline; MCP, monocyte chemoattractant protein; OPN, osteopontin; Ox, oxalate; TNF, tumor necrosis factor.
interstitial inflammation. Eruption of crystals deposited in the subepithelial collecting ducts leads to the formation of a stone nidus. The difference between human and rat Type 2 lesions is that in the humans, lesions are generally apatite, but a human case with intratubular CaOx crystals has been reported. MATERIALS AND METHODS Animal protocol Male Sprague–Dawley rats (120–130 g) were purchased from Harlan. After 1 week of acclimatization to our animal facilities, they were divided into two groups, control and experimental. Both had easy access to food and water, and their consumption was recorded daily. Food was ground, moistened with water, and formed into small balls, which were individually weighed. Control rats received normal rat chow, whereas experimental animals were given chow mixed with 5% HLP (weight/weight HLP/chow) purchased from ICN Biochemicals (Aurora, OH, USA). Food and water consumption were regularly monitored. Rats were weighed weekly to check their growth. Urine was collected daily for determination of crystalluria and weekly for the analyses of urinary ions and markers of cell injury and lipid peroxidation. Four control rats and four experimental rats were killed after 4, 6, and 9 weeks. Their bladder urine was aspirated and examined for the presence of crystals. Their kidneys were Kidney International (2006) 70, 914–923
processed for localization of crystals and various other morphological analyses. Urine collection and analysis Once a week, 24-h urine collection was made with 0.02% sodium azide to prevent bacterial growth. Rats were placed in metabolic cages for the collection of urine. After determining urinary volume and pH, urine was aliquoted for various assays. For the determination of Ox, an aliquot of 24 h urine samples from control and experimental subjects was acidified by the addition of 1 M HCl (20:1 dilution) and kept frozen in closed vials until analyzed. Just before analysis, the samples were thawed and brought to room temperature, mixed, and Ox determinations were carried out in triplicate. Analyses were performed using a Dionex DX100 gradient ion chromatography system equipped with a 0.4 25 cm AS10A anion exchange analytical column containing an AG10A guard column. Urinary Ca and creatinine were also determined using methods described in our previous studies. Serum creatinine was measured at the time of killing. Centrifuged urine supernatants were used to determine LDH. LDH was analyzed on the spectrophotometer by measuring the rate of decrease of absorbance of nicotinamide adenine dinucleotide (reduced form) at 340 nm using pyruvate as the substrate. To investigate the development of oxidative stress, we determined urinary excretion of H2O2, a reactive oxygen species, and 8-IP, a product of lipid peroxidation. H2O2 quantification was carried out 921
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using a colormetric assay obtained from Pierce (PeroXoquant Quantitative Peroxide Assay Kit, Pierce Biotechnology Inc., Rockford, IL, USA: catalogue no. 23280). Determination of urinary isoprostane was carried out using a kit from Oxford Biomedical Research (Urinary Isoprostane, Cayman Chemical Company, Ann Arbor, MI, USA: catalogue no. EA85). Urine was examined by light microscopy to analyze crystalluria. Selected urine samples and bladder urine collected at the time of killing were filtered through 0.2 mm nucleopore filter for examination and analysis,27 with an SEM and energy dispersive X-ray microanalysis.
14.
Kidney tissue processing Four rats from the normal control group and four from the experimental group were killed after 4, 6, and 9 weeks. Details of tissue preparation for light and SEM analyses are described in our earlier publications.8,9,11–14 The presence of CaOx crystal was scored on a basis of 0–4 þ .
19.
15.
16.
17. 18.
20. 21.
22.
Immunohistochemistry To confirm that HLP-induced hyperoxaluria follows similar patterns and produces similar outcomes, OPN and CD44 were localized immunohistochemically using specific antibodies. Cell proliferation was determined by immunohistochemical staining for PCNA. Migration of M/M into the interstitium was determined by immunohistochemical staining with ED-1 antibodies. ACKNOWLEDGMENTS
This study was supported by NIH Grant No. RO1 DK 065658 and UF Center for the Study of Lithiasis.
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25. 26.
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