Pathophysiological correlates of two unique renal tubule lesions in rats with in - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:34 |只看该作者 |倒序浏览 |打印

作者：Elaine Worcester, Andrew Evan, Sharon Bledsoe, Mark Lyon, Mark Chuang, Marcello Orvieto, Glenn Gerber, and Fredric Coe作者单位：1 Department of Medicine and 3 Department of Urology, University of Chicago, Chicago, Illinois; and 2 Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana
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      【摘要】. p) O* t0 x! G2 ]9 ?0 R  q$ V; Y4 N
   Rats with small bowel resection fed a high-oxalate diet develop extensive deposition of calcium oxalate (CaOx) and calcium phosphate crystals in the kidney after 4 mo. To explore the earliest sites of renal crystal deposition, rats received either small bowel resection or transection and were then fed either standard chow or a high-oxalate diet; perfusion-fixed renal tissue from five rats in each group was examined by light microscopy at 2, 4, 8, and 12 wk. Rats fed the high-oxalate diet developed birefringent microcrystals at the brush border of proximal tubule cells, with or without cell damage; the lesion was most common in rats with both resection and a high-oxalate diet (10/19 with the lesion) and was significantly correlated with urine oxalate excretion ( P < 0.001). Rats with bowel resection fed normal chow had mild hyperoxaluria but high urine CaOx supersaturation; four of these rats developed birefringent crystal deposition with tubule plugging in inner medullary collecting ducts (IMCD). Two rats fed a high-oxalate diet also developed this lesion, which was correlated with CaOx supersaturation, but not oxalate excretion. Tissue was examined under oil immersion, and tiny birefringent crystals were noted on the apical surface of IMCD cells only in animals with IMCD crystal plugging. In one animal, IMCD crystals were both birefringent and nonbirefringent, suggesting a mix of CaOx and calcium phosphate. Overall, these animals demonstrate two distinct sites and mechanisms of renal crystal deposition and may help elucidate renal lesions seen in humans with enteric hyperoxaluria and stones. " i: b  X% W. g% t& D" T" c
      【关键词】 calcium oxalate kidney stones hyperoxaluria/ ^7 K+ L& c, E6 O3 \7 R- O7 }
   ELSEWHERE WE HAVE SHOWN THAT small bowel resection combined with a high-oxalate, high-fat diet produces a range of renal crystal deposits and injury that includes inner medullary collecting duct (IMCD) obstruction, suburothelial crystal deposition, papillary crystallization with necrosis, tubular ectasia, cortical scarring, interstitial inflammation, urothelial proliferation, and loose pelvic stones ( 15 ). Crystals were a mixture of calcium oxalate (CaOx) and calcium phosphate (CaP) as apatite. The multiplicity of lesions made the identification of a coherent pathogenetic sequence difficult. In addition, the combination of intestinal resection and marked diet oxalate loading prevented us from sorting out their individual contributions to renal crystal deposition. Finally, our earliest tissue samples were obtained at 4 mo after the surgery, when histological changes were already quite advanced.; J3 N( i) U. s
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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.( v* [5 j& l- y* @. S. y

MATERIALS AND METHODS# r+ ^& Z! w! M8 k: @
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Animals and Surgical Techniques
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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
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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.
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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  [" g* ]/ Z: U1 ]4 e: o
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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 CO 2 content, uric acid, and chloride. CaOx, CaP, and uric acid supersaturations (SS) were calculated using EQUIL 2 ( 17 ).

Light Microscopic Histology
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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 x 100 oil-immersion lens.* j, I1 }( j6 q2 B6 Y. Z
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Quantitative Microscopic Evaluations
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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 x 100 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.1 @* O8 ?+ j3 s3 C& q

Data Analysis( K! w: i; z* q/ O0 r( Z

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)./ U% E5 y" J7 K- X' ^" m

RESULTS! {8 T' N1 V0 M0 W2 c2 s! ^

Urine Chemistries Relevant to CaOx SS
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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.# k2 E+ {: d: F5 E

Table 1. Urinary determinants of CaOx supersaturation% C$ e2 `9 [5 a& O( r- A
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Cortical Microcrystals, a4 H% H5 C. L2 D/ I9 |) _9 }6 o
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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. 1 d ). 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 ).- V; ?8 z  Y6 ~' K1 C5 x
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Table 2. Crystal lesions found in kidneys
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Fig. 1. Initial site of crystal formation. Photomicrographs in a and b are from an animal in group 4 that was transected and fed normal rat chow for 8 wk, whereas c and d are from animals in group 1 that were resected and fed a diet containing 1% sodium oxalate, 0.02% calcium for 2 ( c ) and 8 ( d ) wk. The single arrowheads in a show the brush border of several normal-appearing proximal tubules. Note the renal capsule on the left. The double arrowheads in b point out an inner medullary collecting duct (IMCD) near the papillary tip. In c, a birefringent microcrystal (arrow) is seen at the brush border of a normal-appearing proximal tubule that was stained with hematoxylin and eosin (H&E). In d, a larger crystal (arrow) stained with Yasue is seen at the brush border of another normal-appearing proximal tubule. This crystal is stained dark brown to black and was birefringent. The inset shows the crystal mass near, if not touching, the brush border of a normal-appearing proximal tubule cell. Magnification: x 850 ( a ); x 900 ( b ); x 1,000 ( c ); x 900 ( d ); x 2,300 ( inset, d ).

An additional five animals had the same proximal and distal convoluted tubule lumen birefringent crystals, but the crystals filled a significant fraction of the tubule lumens, and there was evidence of damage to the adjacent tubular cells that included vacuolization to frank necrosis and atrophy ( Fig. 2 ). There was no associated interstitial fibrosis suggestive of an inflammatory response. We have called this the severe cortical microcrystal lesion. Crystals associated with damage were found only at 8 and 12 wk (3 and 2 rats, respectively), and all rats with this lesion were in group 1 ( Table 2 ). Overall, it is apparent from Table 2 that 10 of the 12 rats with cortical microcrystals were in group 1 ( X 2 = 31, P # ]: G9 o, D8 {

Fig. 2. Crystal formation with cell injury. These images are from a group 1 animal that was resected and fed a diet containing 1% sodium oxalate, 0.02% calcium for 8 wk. In a a large crystal (arrow) in the lumen of a proximal tubule is shown, where b shows a similar crystal (arrow) in a distal tubule. In the inset in a, note the cellular injury (arrowheads) only to those cells near the crystal. The inset in b shows several very thin cells suggestive of an atrophic change. Both crystals were stained with Yasue and were birefringent. Magnification: x 500 ( a ); x 1,500 ( inset, a ); x 500 ( b ); x 1,500 ( inset, b ).

IMCD Lumen Plugging

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. 3 d ), whose crystals were a mixture of birefringent and nonbirefringent. This same animal ( Fig. 3 c ) 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., H# a+ f: m! w3 w2 u( I, d8 g

Fig. 3. Inner medullary collecting duct plugging. These images are from animals that were resected but on normal chow ( group 3 ) for 4 ( a ) and 12 wk ( b ) or resected and fed a diet containing 1% sodium oxalate, 0.02% calcium ( group 1 ) for 8 wk ( c and d ). The H&E-stained medullary tissue in a shows 3 adjacent medullary collecting ducts (arrows) plugged with birefringent crystals. Note the increased staining pattern of the interstitial tissue around each of these plugged collecting ducts ( inset, a ). In b a Yasue-stained crystalline plug (arrow) in an IMCD that has lost most of its lining cells is shown. Another tubule just beneath the plugged IMCD shows some Yasue-stained crystals (long arrows) and a loss of cells. Also note Yasue-positive material in the interstitial space (arrowhead). In c and d, 2 separate medullary collecting ducts with crystalline plugs in different regions of the papilla are shown. The tissue in c was stained with H&E; the single arrows define the sites of crystal around the tubular lumen, whereas the double arrows clearly show a site of extensive cell necrosis. The crystals in d (arrow) were stained with Yasue and contained a mixture of birefringent and nonbirefringent species. Note the ring of interstitial fibrosis (partly defined by a rectangle) around the plugged collecting duct in d suggestive of an inflammatory response. Magnification: x 100 ( a ); x 300 ( inset, a ); x 1,200 ( b ); x 1,400 ( c ); x 1,200 ( d ).7 i/ ^3 k9 T+ }9 O8 g7 W
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Combined Lesions

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./ W3 L# H' {; @
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Fig. 4. Suburothelial crystal deposition with urothelial proliferation. These serial photomicrographs are from an animal that was resected and fed a diet containing 1% sodium oxalate, 0.02% calcium ( group 1 ) for 8 wk. It is the same animal as in Fig. 3, c and d. The double arrows in a and b point out a line of Yasue-stained crystals located beneath the urothelium. In b, the urothelium (arrow) adjacent to several regions of Yasue-positive material shows extensive proliferation. Magnification: x 200 ( a and b ).* b3 R  n# T; j+ F

Tiny IMCD Apical Membrane Crystals9 L1 v/ K& ?) J: }) f( K  d3 M$ k
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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 x 100 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.( h8 t. @. a, e$ B

Fig. 5. Single crystal formation in IMCD. These 2 photomicrographs are from an animal that was resected and fed a diet containing 1% sodium oxalate, 0.02% calcium ( group 1 ) for 8 wk ( a ) or resected but on normal chow ( group 3 ) for 12 wk ( b ). A pair ( a ) of tiny Yasue-positive crystals (arrow) is seen at the apical cell surface of an IMCD, along with a single similar crystal in b (arrow). By polarizing optics, these crystals were birefringent. Magnification: x 1,500 ( a and b ).

Stones9 j5 h9 I* s' t+ R6 i; e8 q
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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.$ U+ o4 `7 `" C; T& p, V1 l

Urine Correlates of Crystal Deposits% l* h0 h4 t$ ]5 H, `

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.: H, |0 K5 V& ?/ H8 O/ a( {$ x& h

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
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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 / K1 f/ G# m* F% Q* r% p) H
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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 . c7 ]# z. K1 Z8 P2 D

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
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Other Analyses+ L! b: ^0 l) E; a

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 ).! n% v& t+ `0 e; u6 j1 X
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Table 3. Adjusted values for urine measurements related to calcium phosphate supersaturation* e1 q9 }& z9 w/ z/ T1 }
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Table 4. Serum values2 k' q, j" P  }- x
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Table 5. Adjusted urine nutritional parameters/ _8 t+ n) M, i
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DISCUSSION

Cortical Microcrystals. s' b7 y, k5 e5 m' f$ W. g

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.- y7 U  t* s# k- O$ y' p5 _, h  H
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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.) |3 b, K' W9 i, l

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.
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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.
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IMCD Lumen Plugging
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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.! X0 d* W/ o/ j" b( B
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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.# d5 |9 o: p: q
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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 ).
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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- v/ }% t' `: f- [+ e  X+ U

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.
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Stones
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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 Hyperoxaluria1 i1 t1 |: {( S2 d
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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 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.. i. y3 ~9 l( L/ p$ g# f/ P
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GRANTS

This work was supported by National Institutes of Health Grant PO1-56788 and by the Department of Urology, University of Chicago.

ACKNOWLEDGMENTS
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We acknowledge the expert assistance of Dr. Yasushi Nakagawa in these studies.
      【参考文献】4 G! X9 K& T" s! J( B* ]9 q, d
Bushinksy DA, Asplin JR, Grynpas MD, Evan AP, Parker WR, Alexander KM, and Coe FL. Calcium oxalate stone formation in genetic hypercalciuric stone forming rats. Kidney Int 61: 975-987, 2002.

- Q* Q/ _7 l! H" W% _

Bushinsky DA, Favus MJ, and Coe FL. Elevated 1,25(OH) 2 D 3, intestinal absorption, and renal mineral conservation in male rats. Am J Physiol Renal Fluid Electrolyte Physiol 246: F140-F145, 1984.

# \3 X* ]; ?5 g; m& r9 J/ F6 v5 I

Bushinsky DA, Favus MJ, Schneider AB, Sen PK, Sherwood LM, and Coe FL. Effects of metabolic acidosis on PTH and 1,25(OH) 2 D 3 response to low calcium diet. Am J Physiol Renal Fluid Electrolyte Physiol 243: F570-F575, 1982.: l1 |4 P! h7 h$ H) t% F
3 O- R/ `( N  x- Z
1 g- Z" N/ L! ]+ j+ ]- m' B
! d, {. J% _' [" H( s
Coe F and Parks JH. Defenses of an unstable compromise: crystallization inhibitors and the kidney?s role in mineral regulation. Kidney Int 38: 625-631, 1990.

Dobbins JW and Binder HJ. Importance of the colon in enteric hyperoxaluria. N Engl J Med 296: 298-301, 1997.8 ?0 s) \/ ]2 b' A& t

Evan AP, Lingeman JE, Coe FL, Parks JH, Bledsoe SB, Shao Y, Sommer AJ, Paterson RF, Kuo RL, and Grynpas M. Randall?s plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle. J Clin Invest 111: 607-616, 2003.

( K; i$ m1 L: u6 d! R$ }
Evan AP, Lingeman JE, Coe FL, Parks JH, Bledsoe SB, Shao Y, Sommer AJ, Paterson RF, Kuo RL, and Grynpas M. Randall?s plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle. J Clin Invest 111: 607-616, 2003.

& J- u/ ^; r) y1 U" T5 J( t
Karniski LP, Lotscher M, Fucentese M, Hilfiker H, Biber J, and Murer H. Immunolocalization of sat-1 sulfate/oxalate/bicarbonate anion exchanger in the rat kidney. Am J Physiol Renal Physiol 275: F79-F87, 1998.

9 |) F# ]7 t$ |6 g" N1 j/ d3 W
Khan SR, Finlayson B, and Hackett RL. Histologic study of the early events in oxalate induced intranephronic calculosis. Invest Urol 17: 199-202, 1979.
, k- e! |2 B" M0 |9 P6 q) f
8 p" u! Y- k0 e2 \/ A/ z
+ F) ^( p; K! O0 x- e
Khan SR and Hackett RL. Retention of calcium oxalate crystals in renal tubules. Scanning Microsc 5: 707-711, 1991.

8 k7 H6 O  N9 k

Knight TF, Sansom SC, Senekjian HO, and Weinman EJ. Oxalate secretion in the rat proximal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 240: F295-F298, 1981.) K; f1 @, E! n( Y% z: q

Kuo SM and Aronson PS. Oxalate transport via the sulfate/HCO 3 exchanger in rabbit renal basolateral membrane vesicles. J Biol Chem 263: 9710-9717, 1988.9 }7 D3 n- r( _5 }* D
& K5 _$ p' y; s5 d- ^
3 {. z; A  E( q/ |
6 p. V4 u4 A  E  z: L
Lieske JC, Spargo BH, and Toback FG. Endocytosis of calcium oxalate crystals and proliferation of renal tubular epithelial cells in a patient with type 1 primary hyperoxaluria. J Urol 148: 1517-1519, 1992.

" g. i7 R$ M" L' `
0 u  n; a! k8 x# l5 U2 ^9 F( N3 n
Mount DB and Romero MF. The SLC26 gene family of multifunctional anion exchangers. Pflügers Arch 447: 710-721, 2004.% ]+ v* d; c  O4 J6 ?# G

& E" Y5 C4 |! c7 g9 X* [
0 n9 a4 K2 h+ \. k5 w$ h
O?Connor RC, Worcester EM, Evan AP, Meehan S, Kuznetsov D, Laven B, Sommer AJ, Bledsoe SB, Parks JH, Coe FL, Grynpas M, and Gerber GS. Nephrolithiasis and nephrocalcinosis in rats with small bowel resection. Urol Res 33: 105-115, 2005.! D2 e1 {5 f- L2 b3 ~  p' m
" O/ V) W+ R4 c* j; ^5 B# ~

Wang T, Agulian SK, Geibisch G, and Aronson PS. Effects of formate and oxalate on chloride absorption in rat distal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 264: F730-F736, 1993.
; x2 e, l* t8 b+ z
0 K5 Z  o6 V' P. U- ]$ {6 u
0 d' x# P9 `! X- p8 u9 ]
Werness PG, Brown CM, Smith LH, and Finlayson B. Equil 2: a basic computer program for the calculation of urinary saturation. J Urol 134: 1242-1244, 1985.! W+ ^  I5 F* J9 c

8 X$ z( G4 f: u& K$ m0 c2 P
# w2 T- @" ^8 L* E0 C
Yamaguchi S, Wiessner JH, Hasegawa AT, Hung LY, Mandel GS, and Mandel NS. Study of a rat model for calcium oxalate crystal formation without severe renal damage in selected conditions. Int J Urol 12: 290-298, 2005.

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