Pathogenesis of Oxalate Urolithiasis: Lessons From Experimental Studies With Rats Saeed R. Khan, PhD • Calcium oxalate (CaOx) urolithiasis in rats is induced by producing hyperoxaluria. Depending on the degree and length of hyperoxaluria, CaOx crystals may either form in the nephron or the bladder and mayor may not be retained in the kidneys. Crystals may nucleate in one part of the nephron and be retained in another part. Papillary collecting duct tubular epithelium and its basement membrane appear to be involved in crystal retention in the kidneys. © 1991 by the National Kidney Foundation, Inc. INDEX WORDS: Urolithiasis; kidney stone; crystal retention; hyperoxaluria; nephrolithiasis.
C
ALCIUM OXALATE (CaOx) urolithiasis is the most common human urinary stone disease in the western hemisphere. To understand the pathobiology of this disease, the rat has generally been used as the in vivo model, although rats seldom form urinary stones spontaneously. CaOx urolithiasis in rat is therefore experimentally induced by causing hyperoxaluria either by administration of excess oxalate or precursors of oxalate such as ethylene glycol or hydroxY-L-proline, or by nutritional manipulations as exemplified by pyridoxine deficiency. HYPEROXALURIA
Hyperoxaluria is one of the main risk factors of human idiopathic CaOx stone disease' and its experimental induction is necessary for the production of CaOx urolithiasis in rat. 2 An increase in the urinary oxalate results in a proportional increase in the urinary CaOx supersaturation, which shows a positive correlation with the number of crystals in the urine. 3 ,4 Various methods of induction have been used and have resulted in two basic types of hyperoxaluria, (1) chronic, when a rat is challenged with multiple, generally small doses of lithogen for a long period of time, and (2) acute, when a single, generally large dose of lithogen is administered. Different types of hyperoxaluria From the Department of Pathology, College of Medicine, University of Florida, Gainesville, FL. Supported by National Institutes of Health Grant No.5 POl DK20586. Address reprint requests to Saeed R. Khan, PhD, J-275, JHMHC, College of Medicine, University of Florida, Gainesvlle, FL 32610. © 1991 by the National Kidney Foundation, Inc. 0272-6386/9111704-0010$3.00/0
398
produce different crystallization patterns in the rats. In the absence of other mitigating factors, urinary CaOx crystals are not encountered in lowgrade chronic hyperoxaluria. In one experiment in which 0.25%, 0.5%, or 0.75% ethylene glycol (EG) was administered to rats through drinking water for 24 days, there was an increase in urinary oxalate of all animals, but consistent crystalluria was present only in the rats receiving 0.75% EG. On day 10 of the experiment (Table 1), urinary oxalate was 62 % higher in rats on 0.25 % EG than in normal controls, 105 % higher on 0.5 % EG, and 206% higher on 0.75%. Rats on 0.25% EG had no crystalluria and no renal crystal deposition for up to 24 days, whereas two of six rats on 0.5% EG showed transient crystalluria and one rat had crystal deposits in the renal papilla after 24 days. All rats on 0.75% EG developed crystalluria by day 12, and three of six rats had crystals in the renal papillae by day 24. Apparently crystalluria and renal crystal deposition start after urinary oxalate has reached a threshold level, which in case of male Sprague-Dawley rats appears to be at least 100 % over the normal control for induction of crystalluria and over at least 200 % of the normal control for induction of crystal deposition in the kidneys. In some of our experiments, similar administration of 0.75 % ethylene glycol did not result in consistent crystalluria or nephrolithiasis, because urinary oxalate did not cross this threshold. Renal papillary tip and calyces are the preferential sites for early crystal deposition in chronic hyperoxaluria. 2 ,5 Chances of crystal deposition in the upper nephron increase with increasing urinary oxalate and length of hyperoxaluric state. Chronic hyperoxaluria even at the low level
American Journal of Kidney Diseases, Vol XVII, No 4 (April), 1991: pp 398-401
399
HYPEROXALURIA IN CALCIUM OXALATE UROLITHIASIS
Table 1. Day 10 of Chronic Hyperoxaluria Induced by EG Administration Dose
OX INCR
Crystalluria (Total No. of Rats)
Nephrolithiasis (Total No. of Rats)
0.25% 0.5% 0.75%
63% 106% 206%
No 2 (3) 3 (3)
No No 2 (3) PAP
NOTE. N = 3 rats per group. Abbreviations: OX INCR, oxalate increase; PAp, papilla.
and in the absence of crystalluria and nephrolithiasis can result in renal tubular injury. 6 Acute hyperoxaluria induced by a single intraperitoneal injection of sodium oxalate resulted in CaOx crystalluria and CaOx crystal deposition in the kidneys within 15 minutes. 7 The degree and the length of both hyperoxaluria (Table 2) and crystalluria depended on the severity of the challenge. In acute hyperoxaluria, crystals appeared first in the proximal tubules and then in the rest of nephron. At the low dose of 3 mg sodium oxalate per 100 g of rat body weight, crystals appeared to lie free in the tubular lumina, and tubular epithelial cells appeared normal. After 3 hours of the challenge, no crystals were observed anywhere in the kidneys. However, at higher doses of 5, 7, or 9 mg sodium oxalate per 100 g body weight, crystals were found associated with flocculent eosinophilic cellular debris. Renal tubules were dilated and demonstrated distinct cellular necrosis. There were numerous cells with dividing nuclei. Epitheliallining of the loop of Henle was often denuded, exposing the basement membrane. Crystals were retained in the renal papilla and were there even after 3 days. These observations indicate that at low doses of acute lithogenic challenge, crystals form in the renal tubules and quickly move down the nephron with the urinary flow, without causing detectable changes in the kidneys. But, crystals formed as a result of the higher dose lithogenic challenge are retained in the tubules. Apparently this retention causes renal tubular injury resulting in epithelial necrosis and release of cellular degradation products, including membranes. During early stages of the experimental CaOx nephrolithiasis, crystals are almost always found in the lumina of renal tubules. It is much later, in some cases even after the lithogenic challenge is removed and the urinary oxalate has reached a pla-
teau, that some crystals are found to be interstitially located. These crystals are mostly cortical. It appears that crystals retained in the tubules secondarily become interstitial. CRYSTAL NUCLEATION
In a low- to medium-grade chronic hyperoxaluria, nucleation of CaOx crystals appears to be heterogeneous, because in these situations, both urinary oxalate and CaOx supersaturation are not high enough for the homogeneous nucleation. For homogeneous nucleation to occur, urinary CaOx relative supersaturation should exceed 80,8 and that would require more than 20 mmol/L of oxalate in the urine of male Sprague-Dawley rats. Normal urinary oxalate is less than 1 mmollL. In experimentally induced high-grade chronic hyperoxaluria, where urinary oxalate may reach more than 20 mmollL, homogeneous nucleation is a distinct possibility. Similarly, in acute hyperoxaluria, where urinary oxalate can increase manyfold within a few minutes, initial crystal nucleation can be homogeneous. Even though urine is not a clean system and virtually all nucleation is claimed to be heterogeneous, conditions favoring homogeneous nucleation of both CaOx and calcium phosphate can exist in the urine.' A low-grade chronic hyperoxaluria on its own does not result in urinary crystal formation in the rats. However, additional membrane shedding from the renal tubular epithelium can induce crystallization. Subcutaneous injections of gentamicin sulfate result in membrane shedding from the proximal tubules. These membranes move downstream with the urine and have been detected in the bladder urine. When rats on 0.25% EG with lowgrade hyperoxaluria were exposed to subcutaneous administration of gentamicin sulfate, they developed CaOx crystalluria. 9 It appears that similar to calcium phosphate nucleation, CaOx nucleation is also aided by cellular membranes and their Table 2. Urinary Oxalate (mmollL) After Intraperitoneal Administration of Sodium Oxalate at 3 mg/100 9 or 7 mg/100 9 Rat Body Weight Time
3 mg/100 9
Control
2.66 ± 1.12 0.3 1st 6 hours 1.02 ± 0.48 0.49 2nd 6 hours Next 12 hours 0.6 ± 0.11 0.48 Next 24 hours 0.65 ± 0.1 0.62
± ± ± ±
0.15 0.0 0.09 0.12
7 mg/100 9
4.87 2.48 1.37 1.48
± ± ± ±
1.36 0.49 0.33 0.41
400
SAEED R. KHAN
lipids. Recent in vitro studies lO have also shown that renal proximal tubular brush border membrane and their lipids, as well as the urinary stone matrix and their lipids, are capable of nucleating CaOx crystals from a metastable CaOx solution that would otherwise not support it. CRYSTAL RETENTION AND STONE FORMATION
It is now widely accepted that retention of incipient stones is essential for the development of stone disease. Studies of experimental CaOx urolithiasis have demonstrated that crystal retention in upper nephron is generally caused by high-grade chronic or acute hyperoxaluria, resulting in a large number of crystals being deposited in the tubules in a short period of time. Diameter of the proximal tubular lumen is approximately 25 p.m, that of thin loop even less. But crystal deposits of up to 30 J.tm in the proximal tubules of rat kidneys occur within 1 hour of sodium oxalate injection. These crystals are unable to pass through the lumen because of their aggregated mass. On the other hand, crystal retention in the papilla, particularly the papillary tip, appears to be accomplished by crystal fixation to the tubular epithelium. II Crystals are seen anchored to the tubular basement membrane. The mechanisms involved are not well understood, but in studies of crystal! epithelium interaction I2 where a monolayer of renal papillary epithelial cells was exposed to CaOx and apatite crystals, the crystals were found to preferentially adhere to clumps of rounded cells. It was suggested that these cells lose their attachment to the basement membrane, resulting in their rounding up and exposure of molecules that bind crystals. Thus, crystal binding molecules may be of basement membrane or basolateral membrane origin. Gill et al demonstrated increased retention of CaOx crystals in rat urinary bladder I3 after its urothelium was experimentally injured by treatment with 0.1 N HCI or 5% Triton X 100. It was suggested that the retention was effected by removal of normal protective coating of the urothelium or by release of a gluing substance by the injured cells. Electron microscopic examination of similarly injured urothelium l4 demonstrated that such chemical treatments denuded the urothelium to its basement membrane. Crystal retention and aggregation was facilitated by being trapped in the cellular degradation products. Exposed basement
membrane also appeared to play a role in this phenomenon. DISCUSSION AND CONCLUSIONS
Even though concentration and rate of excretion is higher in rats than in man, IS. 16 CaOx urolithiasis is not a spontaneous phenomenon in rats.2 However, the physiology and metabolism of oxalate in rats and humans are similar. 15 . 16 In addition, rat kidneys exhibit a similar oxalate concentration gradient among renal cortex, medulla, and papilla 15 . 16 as the human kidneys. I? Thus, urinary oxalate concentration is highest in the collecting ducts, and renal papilla has more oxalate than other parts of the kidneys. These similarities have allowed us to critically explore various aspects of the oxalate stone disease by experimental modeling in rats. The primary site for CaOx crystal retention in kidneys is the renal papilla, where retention is accomplished by the involvement of tubular epithelium and its basement membrane. Even in idiopathic urolithiasis, crystal deposition is generally restricted to the renal papillae and has been suggested to start as so-called Randall's plaques. 18 Randall's plaques are subepithelially retained crystals and are formed as mineral deposits in basement membrane of the papillary tubular epithelium. These retained crystals are incipient stones that accrete mass on exposure to a metastable urine. Our studies demonstrate that even mild hyperoxaluria causes injury to the renal tubular epithelial cells and that chronic and acute hyperoxaluria result in different crystallization patterns in the kidneys. Injury to the epithelial cells results in membrane damage and shedding, which can cause crystal formation, as well as their retention. The shed membranes can induce crystal nucleation in situ or move downstream and induce nucleation in the papillary collecting ducts where oxalate concentration is highest, or in the bladder where urine stays the longest. A transient increase in filtered oxalate may trigger crystal nucleation in the upper nephron. These nuclei may grow to form crystals and be retained in the upper nephron because of their aggregated size or may move downstream to grow into crystals and be retained at the papillary tip. Thus, depending on the intensity of hyperoxaluria, duration of the hyperoxaluric state, and crystallization inhibitory capacity of the urine,
401
HYPEROXALURIA IN CALCIUM OXALATE UROLITHIASIS
crystal nucleation, growth, and retention can be separate in time and space and can occur in upper nephron or papillary collecting ducts or in the bladder.
ACKNOWLEDGMENT I am indebted to Dr Raymond L. Hackett for his various suggestions and critique and to John D. Konicek and Paula N. Shevock for their diligent technical support.
REFERENCES 1. Robertson WG, Peacock M: Pathogenesis of urolithiasis, in Schneider H-J (ed): Urolithiasis: Etiology, Diagnosis. Berlin, Germany, Springer-Verlag, 1985, pp 185-334 2. Khan SR, Hackett RL: Calcium oxalate urolithiasis in rat: Is it a model for human stone disease? A review of recent literature. Scanning Microsc 2:759-774, 1985 3. Khan SR, Finlayson B, Hackett RL: Relationship between experimentally induced crystalluria and relative supersaturation of various stone salts in rats. Urol Res 12:271-273, 1984 4. Werness PG, Bergert JH, Smith LH: Crystalluria. J Crystal Growth 53:166-181,1981 5. Gregory JG, Park KY, Burns T: The interaction of urinary oxalate, calcium and pH in renal calculus formation as demonstrated in a rat model, in Ryall R, Brockis JG, Marshall V, et al (eds): Urinary Stone. Proceedings of Second International Symposium. London, England, Churchill Livingstone, 1984, pp 355-362 6. Khan SR, Shevock PN, Hackett RL: Urinary enzymes and calcium oxalate urolithiasis. J Urol 142:846-849, 1989 7. Khan SR, Finlayson B, Hackett RL: Histologic study of the early events in oxalate induced intranephronic calculosis. Invest Urol 17: 199-202, 1979 8. Finlayson B: Physicochemical aspects of urolithiasis. Kidney Int 13:334-360, 1978 9. Hackett RL, Shevock PN, Khan SR: Cell injury associated calcium oxalate crystalluria, J Urol 144:1535-1538, 1990
10. Khan SR, Shevock PN, Hackett RL: Membrane associated crystallization of calcium oxalate in vitro. Calcif Tissue Int 46: 116-120, 1990 11. Khan SR, Finlayson B, Hackett RL: Experimental calcium oxalate nephrolithiasis in the rat, role of renal papilla. Am J Pathol 107:59-69, 1982 12. Riese RJ, Riese J\v, Kleinman JG, et al: Specificity in calcium oxalate adherence to papillary epithelial cells in culture. Am J Physiol 255:FI025-FI032, 1988 13. Gill WB, Ruggiero KJ, Straus FH: Crystallization studies in a urothelial-lined living test tube (the catheterized female rat bladder). I. Calcium oxalate crystal adhesion to the chemically injured rat bladder. Invest Urol 17:257-261, 1979 14. Khan SR, Cockrell CA, Finlayson B, et al: Crystal retention by injured urothelium of the rat urinary bladder. J Urol 132: 153-157, 1984 15. Wright RJ, Hodgkinson A: Oxalic acid, calcium and phosphorus in renal papilla of normal and stone forming rats. Invest Urol 9:369-375, 1972 16. Hodgkinson A: Oxalic acid in biology and medicine. San Diego, CA, Academic, 1977, pp 159-192 17. Hautmann RE, Lehman A, Osswald H: Intrarenal calcium and oxalate concentration gradients in healthy and stone forming kidneys, the renal papilla as the primary nucleation site, in Smith LH, Robertson WG, Finlayson B (eds): Urolithiasis, Clinical and Basic Research. New York, NY, Plenum, 1980, pp 509-515 18. Randall A: The etiology of primary renal calculus. Int Abstr Surg 71 :209-236, 1940
C
ALCIUM OXALATE (CaOx) urolithiasis is the most common human urinary stone disease in the western hemisphere. To understand the pathobiology of this disease, the rat has generally been used as the in vivo model, although rats seldom form urinary stones spontaneously. CaOx urolithiasis in rat is therefore experimentally induced by causing hyperoxaluria either by administration of excess oxalate or precursors of oxalate such as ethylene glycol or hydroxY-L-proline, or by nutritional manipulations as exemplified by pyridoxine deficiency. HYPEROXALURIA
Hyperoxaluria is one of the main risk factors of human idiopathic CaOx stone disease' and its experimental induction is necessary for the production of CaOx urolithiasis in rat. 2 An increase in the urinary oxalate results in a proportional increase in the urinary CaOx supersaturation, which shows a positive correlation with the number of crystals in the urine. 3 ,4 Various methods of induction have been used and have resulted in two basic types of hyperoxaluria, (1) chronic, when a rat is challenged with multiple, generally small doses of lithogen for a long period of time, and (2) acute, when a single, generally large dose of lithogen is administered. Different types of hyperoxaluria From the Department of Pathology, College of Medicine, University of Florida, Gainesville, FL. Supported by National Institutes of Health Grant No.5 POl DK20586. Address reprint requests to Saeed R. Khan, PhD, J-275, JHMHC, College of Medicine, University of Florida, Gainesvlle, FL 32610. © 1991 by the National Kidney Foundation, Inc. 0272-6386/9111704-0010$3.00/0
398
produce different crystallization patterns in the rats. In the absence of other mitigating factors, urinary CaOx crystals are not encountered in lowgrade chronic hyperoxaluria. In one experiment in which 0.25%, 0.5%, or 0.75% ethylene glycol (EG) was administered to rats through drinking water for 24 days, there was an increase in urinary oxalate of all animals, but consistent crystalluria was present only in the rats receiving 0.75% EG. On day 10 of the experiment (Table 1), urinary oxalate was 62 % higher in rats on 0.25 % EG than in normal controls, 105 % higher on 0.5 % EG, and 206% higher on 0.75%. Rats on 0.25% EG had no crystalluria and no renal crystal deposition for up to 24 days, whereas two of six rats on 0.5% EG showed transient crystalluria and one rat had crystal deposits in the renal papilla after 24 days. All rats on 0.75% EG developed crystalluria by day 12, and three of six rats had crystals in the renal papillae by day 24. Apparently crystalluria and renal crystal deposition start after urinary oxalate has reached a threshold level, which in case of male Sprague-Dawley rats appears to be at least 100 % over the normal control for induction of crystalluria and over at least 200 % of the normal control for induction of crystal deposition in the kidneys. In some of our experiments, similar administration of 0.75 % ethylene glycol did not result in consistent crystalluria or nephrolithiasis, because urinary oxalate did not cross this threshold. Renal papillary tip and calyces are the preferential sites for early crystal deposition in chronic hyperoxaluria. 2 ,5 Chances of crystal deposition in the upper nephron increase with increasing urinary oxalate and length of hyperoxaluric state. Chronic hyperoxaluria even at the low level
American Journal of Kidney Diseases, Vol XVII, No 4 (April), 1991: pp 398-401
399
HYPEROXALURIA IN CALCIUM OXALATE UROLITHIASIS
Table 1. Day 10 of Chronic Hyperoxaluria Induced by EG Administration Dose
OX INCR
Crystalluria (Total No. of Rats)
Nephrolithiasis (Total No. of Rats)
0.25% 0.5% 0.75%
63% 106% 206%
No 2 (3) 3 (3)
No No 2 (3) PAP
NOTE. N = 3 rats per group. Abbreviations: OX INCR, oxalate increase; PAp, papilla.
and in the absence of crystalluria and nephrolithiasis can result in renal tubular injury. 6 Acute hyperoxaluria induced by a single intraperitoneal injection of sodium oxalate resulted in CaOx crystalluria and CaOx crystal deposition in the kidneys within 15 minutes. 7 The degree and the length of both hyperoxaluria (Table 2) and crystalluria depended on the severity of the challenge. In acute hyperoxaluria, crystals appeared first in the proximal tubules and then in the rest of nephron. At the low dose of 3 mg sodium oxalate per 100 g of rat body weight, crystals appeared to lie free in the tubular lumina, and tubular epithelial cells appeared normal. After 3 hours of the challenge, no crystals were observed anywhere in the kidneys. However, at higher doses of 5, 7, or 9 mg sodium oxalate per 100 g body weight, crystals were found associated with flocculent eosinophilic cellular debris. Renal tubules were dilated and demonstrated distinct cellular necrosis. There were numerous cells with dividing nuclei. Epitheliallining of the loop of Henle was often denuded, exposing the basement membrane. Crystals were retained in the renal papilla and were there even after 3 days. These observations indicate that at low doses of acute lithogenic challenge, crystals form in the renal tubules and quickly move down the nephron with the urinary flow, without causing detectable changes in the kidneys. But, crystals formed as a result of the higher dose lithogenic challenge are retained in the tubules. Apparently this retention causes renal tubular injury resulting in epithelial necrosis and release of cellular degradation products, including membranes. During early stages of the experimental CaOx nephrolithiasis, crystals are almost always found in the lumina of renal tubules. It is much later, in some cases even after the lithogenic challenge is removed and the urinary oxalate has reached a pla-
teau, that some crystals are found to be interstitially located. These crystals are mostly cortical. It appears that crystals retained in the tubules secondarily become interstitial. CRYSTAL NUCLEATION
In a low- to medium-grade chronic hyperoxaluria, nucleation of CaOx crystals appears to be heterogeneous, because in these situations, both urinary oxalate and CaOx supersaturation are not high enough for the homogeneous nucleation. For homogeneous nucleation to occur, urinary CaOx relative supersaturation should exceed 80,8 and that would require more than 20 mmol/L of oxalate in the urine of male Sprague-Dawley rats. Normal urinary oxalate is less than 1 mmollL. In experimentally induced high-grade chronic hyperoxaluria, where urinary oxalate may reach more than 20 mmollL, homogeneous nucleation is a distinct possibility. Similarly, in acute hyperoxaluria, where urinary oxalate can increase manyfold within a few minutes, initial crystal nucleation can be homogeneous. Even though urine is not a clean system and virtually all nucleation is claimed to be heterogeneous, conditions favoring homogeneous nucleation of both CaOx and calcium phosphate can exist in the urine.' A low-grade chronic hyperoxaluria on its own does not result in urinary crystal formation in the rats. However, additional membrane shedding from the renal tubular epithelium can induce crystallization. Subcutaneous injections of gentamicin sulfate result in membrane shedding from the proximal tubules. These membranes move downstream with the urine and have been detected in the bladder urine. When rats on 0.25% EG with lowgrade hyperoxaluria were exposed to subcutaneous administration of gentamicin sulfate, they developed CaOx crystalluria. 9 It appears that similar to calcium phosphate nucleation, CaOx nucleation is also aided by cellular membranes and their Table 2. Urinary Oxalate (mmollL) After Intraperitoneal Administration of Sodium Oxalate at 3 mg/100 9 or 7 mg/100 9 Rat Body Weight Time
3 mg/100 9
Control
2.66 ± 1.12 0.3 1st 6 hours 1.02 ± 0.48 0.49 2nd 6 hours Next 12 hours 0.6 ± 0.11 0.48 Next 24 hours 0.65 ± 0.1 0.62
± ± ± ±
0.15 0.0 0.09 0.12
7 mg/100 9
4.87 2.48 1.37 1.48
± ± ± ±
1.36 0.49 0.33 0.41
400
SAEED R. KHAN
lipids. Recent in vitro studies lO have also shown that renal proximal tubular brush border membrane and their lipids, as well as the urinary stone matrix and their lipids, are capable of nucleating CaOx crystals from a metastable CaOx solution that would otherwise not support it. CRYSTAL RETENTION AND STONE FORMATION
It is now widely accepted that retention of incipient stones is essential for the development of stone disease. Studies of experimental CaOx urolithiasis have demonstrated that crystal retention in upper nephron is generally caused by high-grade chronic or acute hyperoxaluria, resulting in a large number of crystals being deposited in the tubules in a short period of time. Diameter of the proximal tubular lumen is approximately 25 p.m, that of thin loop even less. But crystal deposits of up to 30 J.tm in the proximal tubules of rat kidneys occur within 1 hour of sodium oxalate injection. These crystals are unable to pass through the lumen because of their aggregated mass. On the other hand, crystal retention in the papilla, particularly the papillary tip, appears to be accomplished by crystal fixation to the tubular epithelium. II Crystals are seen anchored to the tubular basement membrane. The mechanisms involved are not well understood, but in studies of crystal! epithelium interaction I2 where a monolayer of renal papillary epithelial cells was exposed to CaOx and apatite crystals, the crystals were found to preferentially adhere to clumps of rounded cells. It was suggested that these cells lose their attachment to the basement membrane, resulting in their rounding up and exposure of molecules that bind crystals. Thus, crystal binding molecules may be of basement membrane or basolateral membrane origin. Gill et al demonstrated increased retention of CaOx crystals in rat urinary bladder I3 after its urothelium was experimentally injured by treatment with 0.1 N HCI or 5% Triton X 100. It was suggested that the retention was effected by removal of normal protective coating of the urothelium or by release of a gluing substance by the injured cells. Electron microscopic examination of similarly injured urothelium l4 demonstrated that such chemical treatments denuded the urothelium to its basement membrane. Crystal retention and aggregation was facilitated by being trapped in the cellular degradation products. Exposed basement
membrane also appeared to play a role in this phenomenon. DISCUSSION AND CONCLUSIONS
Even though concentration and rate of excretion is higher in rats than in man, IS. 16 CaOx urolithiasis is not a spontaneous phenomenon in rats.2 However, the physiology and metabolism of oxalate in rats and humans are similar. 15 . 16 In addition, rat kidneys exhibit a similar oxalate concentration gradient among renal cortex, medulla, and papilla 15 . 16 as the human kidneys. I? Thus, urinary oxalate concentration is highest in the collecting ducts, and renal papilla has more oxalate than other parts of the kidneys. These similarities have allowed us to critically explore various aspects of the oxalate stone disease by experimental modeling in rats. The primary site for CaOx crystal retention in kidneys is the renal papilla, where retention is accomplished by the involvement of tubular epithelium and its basement membrane. Even in idiopathic urolithiasis, crystal deposition is generally restricted to the renal papillae and has been suggested to start as so-called Randall's plaques. 18 Randall's plaques are subepithelially retained crystals and are formed as mineral deposits in basement membrane of the papillary tubular epithelium. These retained crystals are incipient stones that accrete mass on exposure to a metastable urine. Our studies demonstrate that even mild hyperoxaluria causes injury to the renal tubular epithelial cells and that chronic and acute hyperoxaluria result in different crystallization patterns in the kidneys. Injury to the epithelial cells results in membrane damage and shedding, which can cause crystal formation, as well as their retention. The shed membranes can induce crystal nucleation in situ or move downstream and induce nucleation in the papillary collecting ducts where oxalate concentration is highest, or in the bladder where urine stays the longest. A transient increase in filtered oxalate may trigger crystal nucleation in the upper nephron. These nuclei may grow to form crystals and be retained in the upper nephron because of their aggregated size or may move downstream to grow into crystals and be retained at the papillary tip. Thus, depending on the intensity of hyperoxaluria, duration of the hyperoxaluric state, and crystallization inhibitory capacity of the urine,
401
HYPEROXALURIA IN CALCIUM OXALATE UROLITHIASIS
crystal nucleation, growth, and retention can be separate in time and space and can occur in upper nephron or papillary collecting ducts or in the bladder.
ACKNOWLEDGMENT I am indebted to Dr Raymond L. Hackett for his various suggestions and critique and to John D. Konicek and Paula N. Shevock for their diligent technical support.
REFERENCES 1. Robertson WG, Peacock M: Pathogenesis of urolithiasis, in Schneider H-J (ed): Urolithiasis: Etiology, Diagnosis. Berlin, Germany, Springer-Verlag, 1985, pp 185-334 2. Khan SR, Hackett RL: Calcium oxalate urolithiasis in rat: Is it a model for human stone disease? A review of recent literature. Scanning Microsc 2:759-774, 1985 3. Khan SR, Finlayson B, Hackett RL: Relationship between experimentally induced crystalluria and relative supersaturation of various stone salts in rats. Urol Res 12:271-273, 1984 4. Werness PG, Bergert JH, Smith LH: Crystalluria. J Crystal Growth 53:166-181,1981 5. Gregory JG, Park KY, Burns T: The interaction of urinary oxalate, calcium and pH in renal calculus formation as demonstrated in a rat model, in Ryall R, Brockis JG, Marshall V, et al (eds): Urinary Stone. Proceedings of Second International Symposium. London, England, Churchill Livingstone, 1984, pp 355-362 6. Khan SR, Shevock PN, Hackett RL: Urinary enzymes and calcium oxalate urolithiasis. J Urol 142:846-849, 1989 7. Khan SR, Finlayson B, Hackett RL: Histologic study of the early events in oxalate induced intranephronic calculosis. Invest Urol 17: 199-202, 1979 8. Finlayson B: Physicochemical aspects of urolithiasis. Kidney Int 13:334-360, 1978 9. Hackett RL, Shevock PN, Khan SR: Cell injury associated calcium oxalate crystalluria, J Urol 144:1535-1538, 1990
10. Khan SR, Shevock PN, Hackett RL: Membrane associated crystallization of calcium oxalate in vitro. Calcif Tissue Int 46: 116-120, 1990 11. Khan SR, Finlayson B, Hackett RL: Experimental calcium oxalate nephrolithiasis in the rat, role of renal papilla. Am J Pathol 107:59-69, 1982 12. Riese RJ, Riese J\v, Kleinman JG, et al: Specificity in calcium oxalate adherence to papillary epithelial cells in culture. Am J Physiol 255:FI025-FI032, 1988 13. Gill WB, Ruggiero KJ, Straus FH: Crystallization studies in a urothelial-lined living test tube (the catheterized female rat bladder). I. Calcium oxalate crystal adhesion to the chemically injured rat bladder. Invest Urol 17:257-261, 1979 14. Khan SR, Cockrell CA, Finlayson B, et al: Crystal retention by injured urothelium of the rat urinary bladder. J Urol 132: 153-157, 1984 15. Wright RJ, Hodgkinson A: Oxalic acid, calcium and phosphorus in renal papilla of normal and stone forming rats. Invest Urol 9:369-375, 1972 16. Hodgkinson A: Oxalic acid in biology and medicine. San Diego, CA, Academic, 1977, pp 159-192 17. Hautmann RE, Lehman A, Osswald H: Intrarenal calcium and oxalate concentration gradients in healthy and stone forming kidneys, the renal papilla as the primary nucleation site, in Smith LH, Robertson WG, Finlayson B (eds): Urolithiasis, Clinical and Basic Research. New York, NY, Plenum, 1980, pp 509-515 18. Randall A: The etiology of primary renal calculus. Int Abstr Surg 71 :209-236, 1940
