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1Department of Medicine and 3Department of Urology, University of Chicago, Chicago, Illinois; and 2Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana
Submitted 30 January 2006 ; accepted in final form 9 May 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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calcium oxalate; kidney stones; hyperoxaluria
We reasoned that tissue samples taken at earlier time points could be combined with a design that separated the effect of a high-oxalate diet from that of intestinal resection to ascertain the sequence of crystallizations that either diet or resection might produce and also provide insight into mechanisms producing the crystals. We present here the results of such a design. Both resection and diet appear to have independent effects on the pattern of renal crystallization and involve specific initial sites of crystal deposition and identifiable mechanisms for crystal production.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Male Sprague-Dawley rats, weighing 180–200 g, and acclimated to a room temperature of 25°C, with a 12:12-h light-dark cycle, were fed a standard diet until 24 h before surgery, at which time the diet was removed and they had free access to a solution containing 25 g of dextrose in 500 ml of water. Anesthesia was performed using an intraperitoneal injection of a 0.3–0.5 ml solution of ketamine (90 mg/kg), atropine (0.05 mg/kg), and xylocaine (10 mg/kg). Following the administration of anesthesia, the animals were secured and the abdominal area was shaved of fur and sterilely prepared with 70% ethanol and Betadine paint. A midline laparotomy was performed. Control rats underwent transection of the distal ileum without excision of any intestine, followed by reanastomosis. Resection rats underwent removal of the distal 40–45 cm of small intestine measured from the ileocecal valve, followed by reanastomosis of the small intestine. In all animals, hemostasis during surgery was accomplished using electrocautery with an average blood loss of <1 ml. Following completion of surgical anastomosis, 12 ml of saline were instilled in the peritoneum and the abdomen was closed using 3-0 silk sutures. The rats received buprenorphine (0.01–0.05 mg sc) every 8–12 h for the first 24 h as postoperative analgesia.
All animals were given standard rat chow (5.7% lipid; 1.2% calcium) beginning 8 h after surgery. This diet continued until the animals regained their preoperative weight, which generally occurred by 7–10 days following surgery. The animals were then placed in individual cages and fed 15 g/day of the experimental diets. Rats in group 1 underwent resection and were fed a diet containing 1% sodium oxalate, 0.02% calcium, and 18% lipids in powdered form daily. Those in group 2 were fed the same diet but were transected rather than resected. Group 3 animals were resected and fed standard rat chow. Those in group 4 were transected and fed standard rat chow. The 15-g portions of food ensured equal and complete consumption of the diet by each animal during every 24-h period and allowed for continued growth of the rats. The rats were maintained on these diets until they were killed. Five rats in each group were planned for euthanasia after 2, 4, 8, and 12 wk of the diet, giving 20 rats in each group and time category (80 rats in all). In fact, we had 6 rats at 4 wk and 8 wk in group 4 and 4 rats at 8 wk in group 1, totaling 81 rats in all.
On the day of death, all animals were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and the kidneys were perfusion fixed. The abdominal cavity was opened to expose the aorta. Thereafter, a ligature was quickly placed around the abdominal aorta just below the diaphragm but not tied and another just proximal to the origin of the common iliac vessels and tied. The ligature at the diaphragm was tied, the aorta was cannulated between the ligatures with PE-90 tubing, and the perfusion was started with a flush of 0.9% NaCl for 20 s at 130–150 mmHg. Subsequently, the kidneys were perfused with 5% paraformaldehyde in phosphate buffer for 10–20 min. Following perfusion, the kidneys were removed and placed in a vial containing the same fixative. Blood was taken by cardiac puncture just before the start of perfusion. Animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Chicago Institutional Animal Care and Use Committee.
Urine and Blood Collection and Measurements
Rats were placed in Nalgene metabolic cages (Fisher Scientific, Hanover Park, IL) 10 days before death, and daily 24-h urine samples were collected with 250–500 mg of thymol in the urine containers. The 24-h urine collections were analyzed for volume, calcium, oxalate, citrate, creatinine, phosphorus, sodium, potassium, magnesium, ammonia, and sulfate using techniques described elsewhere (1). There was no significant variation in measurements over the 10 days, so we averaged values from all urine from each rat for our analyses. At death, blood was drawn for creatinine, calcium, sodium, potassium, phosphorus, total CO2 content, uric acid, and chloride. CaOx, CaP, and uric acid supersaturations (SS) were calculated using EQUIL 2 (17).
Light Microscopic Histology
Each fixed rat kidney was dehydrated and routinely processed for embedment in paraffin. A set of 10 sections/kidney was cut at 4 µm and stained for calcium deposits by the Yasue metal substitution method or by hematoxylin and eosin (H&E) for routine histological examination. The Yasue method has been shown to be an effective stain for detecting calcium salts like CaOx, CaP, and calcium carbonate in tissue (7). Tissues were examined with a range of objectives up to a x100 oil-immersion lens.
Quantitative Microscopic Evaluations
One of us (A. Evan) assessed the extent of crystal accumulation in the cortex, medulla and papilla. Partially and fully polarized light was used to highlight birefringent crystal deposits. In the cortex, the extent of crystallization in proximal or distal tubules was semiquantitatively evaluated using a subjective scoring system, where moderate = several intraluminal crystal deposits with no cell injury, severe = many crystal deposits with cell injury, and none = no crystals or cell injury found. Each rat was classified by the highest grade of lesion detected. In the medulla, IMCD tubule plugging was noted in some rats; such rats were classified as having this lesion, which was not graded as to severity. Rats were further classified as to having free renal pelvic stones or not, without grading as to the number of stones. In other words, each rat was classified along three axes: proximal or distal tubule crystal deposit grade, IMCD deposits or not, and free pelvic stones or not. A search for extremely tiny crystals in IMCD was made using a x100 oil-immersion lens; animals were graded as having such crystals or not. When present, these tiny crystals were examined with polarizing optics for the presence of birefringence.
Data Analysis
Cross tabulation was used to compare counts of rats having specific lesions across the four diet/surgery groups and three axes of histopathological grading. Urine measurements were analyzed using a general linear model to adjust for urine creatinine, whereby the four groups were compared within each of the four time points using post hoc analysis. Serum measurements were analyzed using ANOVA with post hoc pairwise comparisons across the four diet/surgery groups. In addition, we used linear models to compare urine measurements adjusted for urine creatinine across histopathologically defined groups with or without specific crystal deposits. In several cases, we used logistic regression and discriminant analysis to determine the relationships between urine measurements and crystal deposits. All analyses used conventional statistical software (Systat, Richmond, CA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Urine oxalate and calcium, adjusted for urine creatinine, varied modestly within groups over time (Table 1); we did not formally analyze time changes as we had no questions directly related to this issue. Within each of the four time points, urine oxalate was lower and urine calcium higher in groups 3 and 4 vs. groups 1 and 2 (Table 1). Urine CaOx SS was highest in group 3 at all time points. Urine volumes differed significantly but by small magnitudes among groups; mainly, group 1 had higher volumes than the others.
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Twelve of the 81 rats showed birefringent microcrystals at the brush border of proximal tubular cells and at the apical cell surface of the distal convoluted tubules (Table 2). In seven cases, the crystals were mainly one or two in number, ranging about 1 µm in diameter (Fig. 1 c). In some tubules, multiple crystals partially filled the tubule lumen (Fig. 1d). At the light microscopic level, there was no evidence of cellular injury. We have called this the moderate cortical microcrystal lesion and found it in 1, 2, 2, and 2 rats at 2, 4, 8, and 12 wk, respectively. They were only found in animals of groups 1 and 2 receiving the high-oxalate diet (Table 2).
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In six rats, occasional IMCD lumens were plugged with a mass of crystals (Fig. 3). Cell injury was always present and consisted of vacuolization to frank necrosis (Fig. 3). Four of the six rats were at 12 wk; the other two were at 4 and 8 wk, respectively, on the experimental diets. Four of the animals (Table 2) were in group 3 (3 at 12 wk, 1 at 4 wk), one was in group 1 (8 wk) and one was in group 2 (12 wk). In other words, five of the six animals with this lesion were resected, and four of the five resected animals with the lesion were eating normal chow. The one sham animal with this lesion ate the high-oxalate diet (group 2, Table 2). Cell numbers are small enough that statistical analysis would not be appropriate. Of special note, all crystals were birefringent except for those in the one animal from group 1 having this lesion (Fig. 3d), whose crystals were a mixture of birefringent and nonbirefringent. This same animal (Fig. 3c) also produced IMCD plugs of purely birefringent crystals. Several of these plugged IMCD showed changes in the adjacent interstitium, which included increased staining and/or fibrosis suggestive of an inflammatory response .
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Of the 12 animals with cortical microcrystals, only 1 also had IMCD plugging (8 wk, group 1). This was the only animal with lesions at both sites and was the one whose IMCD crystals were both birefringent and nonbirefringent (Fig. 3, c and d). In addition, this was the only animal with suburothelial birefringent crystals (Fig. 4) associated with urothelial proliferation. In other words, a single animal, resected and fed the high-oxalate diet, went on to develop a mixture of lesions whereas none of the others did. We note here that this combination of lesions occurred in animals we reported elsewhere (15) who were resected and fed the high-oxalate diet and studied at a longer interval than 12 wk; in other words, this one animal is like those at a later time stage.
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Having found birefringent masses of IMCD crystals, we hypothesized that their precursor might be very tiny crystals of the same type that attach to apical cell surfaces. To test this idea, we inspected the IMCD cells from kidneys of all 81 animals in this series using a x100 oil-immersion lens. We found (Fig. 5) crystals that appeared as single or multiple birefringent dots. These resemble the cortical microcrystals except in being much smaller. These were found in the six animals with IMCD plugs but in none of the other 75 rats.
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Stones were found in seven animals (1, 3, and 3 rats with stones at 2, 8, and 12 wk, respectively). All but two rats were in group 1 (Table 2). There was no particular cosegregation of stones with either IMCD plugging (2/6 associated with stones) or cortical microcrystals (4/12 associated with stones). Two animals that formed a stone had neither microcrystals nor IMCD plugging, and one animal with a stone had both lesions.
Urine Correlates of Crystal Deposits
Our purpose here is to attempt a mechanistic explanation for specific lesions in terms of driving forces. We studied all rats at exactly the same time points, with equal numbers of animals (apart from the small exceptions noted in MATERIALS AND METHODS) in each group by time point cell (
5/cell). Because crystallizations form over time, and CaOx deposits are especially most unlikely to dissolve, the mass of a deposit must respond to time-averaged driving forces such as SS. Therefore, we have considered all four time points as their average and attempted to analyze those values in relation to the specific lesions.
Cortical microcrystals. Because this lesion occurred exclusively with a high-oxalate diet, mainly in resected animals, we reasoned that high oxalate absorption and subsequent renal oxalate excretion might be involved in their pathogenesis. To test this hypothesis, we created a general linear model with urine oxalate excretion rate (mg/day) as the dependent variable, creatinine excretion as a covariate, and the presence of severe, moderate, and no microcrystals as a factor. Adjusted values of oxalate excretion were 14 ± 1, 11 ± 0.7, and 4.9 ± 0.2 mg/day with severe, moderate, and no tubule lesion, respectively (P < 0.001 for all 3 comparisons). On the other hand, urine CaOx SS showed no relationship to the microcrystals; group 1 animals had a value of 12 ± 1 compared with 12 ± 1 for group 4 animals that had no microcrystals and 23 ± 1 for group 3 that also had no microcrystals (Table 1). This analysis supports the hypothesis that oxalate absorption and renal excretion are involved with cortical microcrystal formation, but that CaOx SS itself is not.
IMCD lumen plugging. We reasoned that IMCD tubule fluid composition must approximate that of the final urine and that therefore urine CaOx SS should be the principle driver of such plugging. In addition, urine citrate is a known endogenous inhibitor of CaOx crystal formation (4) that we quantified here, and therefore we reasoned that low values might correlate with plugging. In a general linear model with CaOx SS as dependent, plugging or not as a factor, and urine citrate as an independent variable, both were significant (P < 0.001). Adjusted least square mean values for CaOx SS were 21 ± 2 vs. 15 ± 0.5, plugging vs. no plugging (P < 0.001), suggesting a straightforward mechanism wherein high CaOx SS drives IMCD plugging. Urine citrate excretion, adjusted for urine creatinine excretion, was actually higher in animals with plugging, 26 ± 2 vs. 22 ± 1 mg/day, plugging vs. no plugging, respectively (P = 0.011). The fact that urine citrate was higher in animals with IMCD plugging speaks against a loss of this particular inhibitor as an important mechanism.
To further distinguish mechanisms for plugging, we performed a logistic regression with plugging or not as a dependent and urine calcium and oxalate excretions, CaOx SS, and urine creatinine excretion as predicting variables. Urine calcium and CaOx SS both entered (P < 0.01 for both), whereas neither urine oxalate nor creatinine excretions entered. To confirm this result, we performed a linear discriminant analysis with plugging or not as a dependent and the same variables as potential classifiers; once again, only urine calcium excretion and CaOx SS entered (F = 11 and 7, respectively, P < 0.001), and together these gave a 73% overall predictive accuracy. Urine oxalate and creatinine excretions did not enter. Finally, we created a linear model in which CaOx SS was the dependent variable, and urine calcium, oxalate and creatinine excretions and plugging or not were covariates. Of these, plugging and urine calcium were significant correlates of CaOx SS. All three analyses converge: high CaOx SS and high urine calcium are related to IMCD plugging, urine oxalate has no effects, and it is high urine calcium that appears to determine the high CaOx SS in the animals with plugging. The fact that urine calcium excretion and CaOx SS are both independently correlated with plugging suggests that calcium may be playing a role apart from that of merely increasing CaOx SS. We explored the possibility that higher urine calcium might be related to deposits via CaP SS, but CaP SS did not enter in a subsequent logistic or discriminant analysis designed to test its role. Similarly, urine volume did not enter in either of these tests.
Protein-crystalline aggregates (stones). One might expect that formation of stones would follow CaOx SS given that the stones were found in the renal pelvis and may well have formed in either the urine or IMCD lumens. This conjecture is incorrect. CaOx SS did not differ (15 ± 2 vs. 15 ± 0.5, stone vs. no stone). However, oxalate excretion (mg/day), corrected for urine creatinine, was higher in those animals that formed a stone (12 ± 1 vs. 6 ± 0.2, P < 0.001). Because CaOx SS differences cannot explain stone formation, it is not clear why the higher oxalate excretion and stone formation are associated. It is also not clear why stone formation and IMCD plugging should be associated with such divergent urine chemical findings: i.e., hyperoxaluria and high CaOx SS, respectively.
Other Analyses
Urine pH rose with time in group 3 and was higher than in the other groups by 12 wk, and groups 3 and 4 had higher urine pH values than groups 1 and 2 (Table 3). Urine ammonia excretions were higher in groups 1 and 2 than in groups 3 and 4. The combined results are compatible with worse diarrhea in group 1 and lower serum potassium values in groups 1 and 2 vs. groups 3 and 4 (Table 4). Serum values do not otherwise contribute significantly to the conclusions of this work, except that serum creatinine values were not consistently different among groups. Because of higher pH and urine calcium, CaP SS was higher in groups 3 and 4 (Table 3). As expected, weight gain and gain of muscle mass as evidenced by urine creatinine excretion were greatest in group 4 (Table 5).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To the best of our knowledge, isolated tiny birefringent crystals localized to proximal tubule brush-border and distal tubule apical membrane have not been described before. Although we could not perform crystallographic or transmission electron microscopic analysis because of their sparseness and small size, we have every reason to postulate that they are CaOx. Khan et al. (9) have described what may well be the next stage of our lesion: crystal aggregates still attached to the brush border partly filling the proximal tubule lumen. Neither we nor they found evidence of cell injury at this stage. In their experiments, lesions were present at 30 min after intraperitoneal injections of high doses of sodium oxalate, whereas we found our most minimal lesions after 2–12 wk of a high-oxalate diet. We presume that both groups have found similar lesions because both have administered excess oxalate and that because our experiment provides oxalate at a lower rate, we were able to identify an earlier stage of this lesion. It would be valuable in future experiments to attempt to identify the nature of these crystals.
What we have termed severe cortical microcrystal deposition, crystal aggregates present along with cell injury, resembles what Khan et al. (9) described at 1 h after intraperitoneal oxalate loading. Once again, our experiments are offset in time, so that we observed cell injury only after 8 wk of a high-oxalate diet, and we found a milder degree of cell injury as well. Altogether, our findings are novel only insofar as we appear to have identified an earlier precursor to a lesion seen by others and, in identifying it, have the opportunity to link its origin to the brush border proper as opposed to the bulk tubule lumen fluid.
Oxalate is freely filtered at the glomerulus but can also gain access to the proximal tubule via secretion (11). The proximal tubule is the site of multifunctional anion exchangers that can mediate oxalate secretion in exchange for chloride, sulfate, or other anions (12). Sat-1 (SLC26A1) is a sulfate/oxalate/bicarbonate exchanger found in the basolateral membrane of proximal tubule cells (8). A related protein, CFEX (SLC26A6), is the primary candidate for the apical proximal convoluted tubule oxalate transporter (14). There are some data for the existence of an oxalate/chloride exchanger in the distal tubule as well (16). Activity of these exchangers could result in very high local concentrations of oxalate near the brush border of proximal or distal tubules that could drive microcrystal formation.
The alternative, high lumen fluid CaOx SS driven by oxalate secretion, is less attractive as a cause for the earliest crystallizations we found. Because of water extraction, high proximal tubule fluid oxalate concentrations would be transformed into vastly higher IMCD concentrations, so that IMCD crystallization would be very likely, yet we found no animals with both early microcrystals and IMCD-obstructing plugs. As further support for our suggested mechanism, tubule microcrystals were strongly associated with the highest urine oxalate excretion rates, and not with the highest urine CaOx SS.
IMCD Lumen Plugging
Heretofore, isolated IMCD lumen plugging has not been observed in experimental models of hyperoxaluria and nephrolithiasis. Yamaguchi et al. (18) describe plugging but associated with papillary tip necrosis, whereas we find only necrosis of IMCD epithelial cells, but not wholesale necrosis of the papillary tip.
In other models, plugging is a later stage that follows deposits in the outer medulla and cortex (10). We presume that the particular rate of oxalate absorption and excretion in our animals along with effects of intestinal resection have provided ideal conditions for producing isolated IMCD plugging.
Our physiological measurements clearly identify high urine CaOx SS as an almost certain mechanism for IMCD plug formation. Because most of the plugging occurred in resected animals eating normal chow, and no plugging occurred in transected animals eating the same chow, we know that resection itself must be a prime mechanism. Our analysis indicates the high CaOx SS is mainly from high urine calcium, presumably on a background of mildly increased urine oxalate from resection. The higher urine calcium in the animals in group 3 vs. group 4 is of unclear origin at this time. Urine ammonia is higher in resected than transected chow-fed rats, suggesting that alkali loss from resection is increasing urine calcium via compensation for metabolic acidosis (3). As well, growth was slower in group 3 vs. group 4 animals, and possibly, as is the case in male vs. female rats (2), a lower axial growth rate permits higher urine calcium losses. This is still an unresolved issue. We presume that the higher urine oxalate following resection vs. transection is due to increased oxalate absorption as is found in human enteric hyperoxaluria (5).
We ourselves, in an earlier study, have observed IMCD plugging in resected rats eating a high-oxalate diet, but our earliest time point was 4 mo after surgery, at which time plugging was accompanied by papillary tip crystallization and necrosis, as seen by others (15). Collecting duct ectasia and cortical scarring with mineral deposition were also present. At the shorter time intervals here, we found plugging in only one high oxalate resected rat and papillary necrosis in none, pointing to the crucial effects of time itself. On the other hand, even at the shorter time intervals we were able to produce IMCD plugging by a combination of resection with normal chow, pointing to the crucial effects of resection.
In our prior work, we showed that crystal deposits in the tubule lumens were a mixture of CaOx and apatite. Our present study did not identify apatite crystal deposits (by their lack of birefringence) except in one high oxalate resected rat; nor did we find the extensive cell injury that accompanied deposits at 4–7 mo in high oxalate resected rats. We presume this, too, is an effect of the shorter time intervals and offer a speculation concerning the mechanism. Rats in groups 3 and 4 have obviously higher urine pH and calcium levels than in groups 1 and 2, and therefore much higher CaP SS levels (Tables 1 and 3). On the other hand, we observed in our prior work apatite IMCD deposits precisely in the rats that correspond to group 1 here. Possibly, CaOx crystals form in animals in group 1, attach to and damage IMCD cells, and over time damage reduces acidification, leading to high tubule fluid pH in some IMCD. Apatite crystals would then be favored.
Tiny IMCD Apical Membrane Crystals
Our present data offer some, albeit slight confirmation of this hypothesis. In animals with IMCD plugging, we found, when looking with an oil-immersion objective, tiny birefringent crystals that we presume are the precursors of the large and easily recognized IMCD plugs. They suggest a pathogenetic scheme that begins with membrane attachment and anchoring of tiny crystals that can grow over time to occlude the tubule lumens. Such crystals might also be taken up into cells as has been described in hyperoxaluria (13). We found no evidence thus far of intracellular crystals during our survey. The fact that these crystals are found in all animals with IMCD plugs and in no animals without IMCD plugs suggests that they are a necessary first step. Given that high CaOx SS is present in IMCD plugging animals, the crystals probably form as water is extracted and tubule fluid SS increases.
Stones
Like everyone who has worked on similar models, we found stones in the renal pelvis, by which we mean protein crystal aggregates not attached to the kidney, free in the renal pelvis. They formed mainly in the high-oxalate diet resected rats but were seen in one animal each in the sham high-oxalate diet and resected chow groups. Our goal in this study was not to elucidate their pathogenesis. They are not correlated with any particular renal histopathology, or with high CaOx SS.
Relationship to Human Enteric Hyperoxaluria
We found IMCD apatite plugging as the sole lesion in humans with jejunolileal (JI) bypass for obesity and CaOx stones (6). In our resected normal chow animals, we found IMCD plugging with CaOx. We have been unable to understand why human JI bypass patients form IMCD apatite plugs because their urine pH is generally too low to support CaP stone formation. Possibly, the present experiment is an important clue. JI bypass patients may begin with CaOx plugging, that causes IMCD cell injury and local acidification disorders that lead to higher lumen pH and transformation of the CaOx to apatite over time. At high pH values, >7, apatite could have a lower solubility than CaOx and replace CaOx by lowering local calcium concentrations sufficiently to dissolve it. This is purely speculative. A critical test of this idea would be to study the temporal evolution of IMCD plugging in our rat model. In addition, the tiny IMCD microcrystals found in all rats with IMCD plugs may form in humans, leading to cell injury and reduced acidification, and later formation of massive apatite crystal deposits. The critical experiment by which to test this is beyond the scope of the present work.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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