- Email: [email protected]
Shock Wave Induced Kidney Injury Promotes Calcium Oxalate Deposition
Shock Wave Induced Kidney Injury Promotes Calcium Oxalate Deposition Yu-Quan Xue, Da-Lin He,* Xing-Fa Chen, Xiang Li, Jin Zeng and Xin-Yang Wang From the First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an, Shaanxi, China
Abbreviations and Acronyms Cr ⫽ creatinine NAG ⫽ N-acetyl-beta-dglucosaminidase Ox ⫽ oxalate Submitted for publication October 19, 2008. Study received Xi’an Jiao Tong University institutional animal care committee approval. Supported by China National Natural Science Fund Grant 30700833 and Technology Key Project in Shaanxi Province Grant 2006k10-G-1. * Correspondence: First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an, Shaanxi, China.
Purpose: Extracorporeal shock wave lithotripsy is the preferred treatment for upper urinary tract renal calculi. However, this treatment is associated with a high rate of recurrent renal calculi. Shock wave therapy can result in renal epithelial cell injury, which in turn is a most important factor in calculus formation. We investigated the influence of kidney damage secondary to shock waves on Ca oxalate crystal retention in the kidney. Materials and Methods: A total of 32 rats were randomly divided into 4 groups, including group 1— controls, group 2—sham treated rats given 25 ml 0.75% ethylene glycol per day for 14 days, group 3—rats given 15 kV 1Hz shock waves 500 times to the left kidney, followed by 25 ml 0.75% ethylene glycol daily for 14 days, and group 4 —rats with the same treatment as group 3 except the number of impacts was increased to 1,000. The 2 kidneys were removed at the end of the experiment. Ca oxalate crystals were observed by surgical microscopy in kidney sections stained with hematoxylin and eosin. Crystal morphology was determined using polarizing microscopy. Acidified kidney tissue homogenate was examined for Ca and oxalate content by colorimetry (Sigma®). Results: Kidney sections showed that kidneys that did not receive shock waves had fewer crystals than kidneys with shock waves, which had crystals in major areas. In the left kidney in groups 2 to 4 the mean ⫾ SD quantity of Ca was 16.88 ⫾ 6.41, 28.58 ⫾ 7.54 and 40.81 ⫾ 15.29 mol/gm wet kidney and the mean quantity of oxalate was 8.44 ⫾ 6.80, 20.52 ⫾ 7.70, 31.76 ⫾ 14.14 mol/gm wet kidney, respectively. Ca oxalate density increased with the number of shock wave impacts. Conclusions: Kidney damage caused by shock wave treatment can increase Ca oxalate crystal retention in the kidneys of rats in this stone model. Key Words: kidney, lithotripsy, high-energy shock waves, calcium oxalate, iatrogenic disease
762
www.jurology.com
EXTRACORPOREAL shock wave lithotripsy is the preferred clinical treatment for upper urinary tract stones.1 However, it is associated with a high rate of stone recurrence.2– 4 Studies demonstrate that the shock waves used in ESWL® can lead to the release of free radicals, which in turn cause renal tubular epithelial cell injury.5,6 Further studies show that tu-
bular epithelial cell injury is a key factor in stone formation.7,8 Thus, we hypothesized that renal tubular epithelial cell injury secondary to shock wave therapy can increase CaOx crystal deposition, which in turn may lead to the high stone recurrence rate. Previous studies of the stone recurrence rate after shock wave lithotripsy are principally clini-
0022-5347/09/1822-0762/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 182, 762-765, August 2009 Printed in U.S.A. DOI:10.1016/j.juro.2009.03.080
SHOCK WAVE INDUCED KIDNEY INJURY PROMOTES CALCIUM OXALATE DEPOSITION
cal followup studies. These series are limited by patient variability in stone size, location and composition. In addition, the number and energy of shock waves that patients received varied. Therefore, definitive conclusions regarding the influence of ESWL on stone formation cannot be drawn from these studies. To address this hypothesis we performed animal experiments to study the influence of kidney injury caused by shock wave therapy on CaOx deposition.
MATERIALS AND METHODS Animals A total of 32 male Sprague Dawley rats weighing a mean ⫾ SD of 200 ⫾ 10 gm were obtained from the laboratory animal center at our institution. The experimental protocol was reviewed and approved by the Xi’an Jiao Tong University institutional animal care committee. All animals were housed in individual metabolic cages under a mean temperature of 22C ⫾ 1C, 50% relative humidity and a 12:12-hour light/dark cycle. Animals were acclimatized to these conditions for 7 days before experiments were started.
Intervention The left kidney of each rat was exposed through an incision under the ribs. A silver medical folder was clamped in the perirenal fascia for x-ray positioning. All animals were divided into 4 groups of 8 each. Group 1 served as controls that received normal drinking water during the whole study. Sham treated group 2 was given drinking water containing 25 ml 0.75% ethylene glycol per day per rat. Group 3 animals were anesthetized by 40 mg/kg pentobarbital sodium and received 500 shock wave impacts of 15 kV and 1 Hz. From the day of shock wave treatment the animals were given drinking water containing 25 ml 0.75% ethylene glycol per day. Group 4 animals received an increased shock wave impact of 15 kV and 1 Hz for 1,000 times and drinking water containing 25 ml 0.75% ethylene glycol per day. There were no other differences among the high urinary Ox groups except the number of shock waves. All rats were fed standard chow. After 14 days of treatment all rats received fresh drinking water for 2 days to wash out free intratubular crystals from the kidneys. All animals were then sacrificed.
Laboratory Analysis Urine collection and analysis. Urine was collected for 24 hours before and after shock wave impact. A drop of concentrated HCl was added to urine before storage at 4C. Urine was analyzed for NAG and Cr by spectrophotometry.
763
using the scale, 0 points—no deposit, 1 point— crystal deposit at the papillary tip, 2 points— crystal deposit at the cortical medullary junction and 3 points— crystal deposit in the cortex.9 When crystal deposition was observed at multiple areas, the points were combined. Kidney homogenate analysis. The second half of each kidney was milled into homogenate, which was acidified with 10 mmol/ml concentrated HCl. This was centrifuged for 10 minutes at 1,000 ⫻ gravity and the supernatant was separated. The quantity of Ca and Ox in supernatant was determined by colorimetry.
Statistical Analysis Results are expressed as the mean ⫾ SD. Differences among data were determined using 1-way ANOVA, followed by the Student-Newman-Keul test with differences considered significant at p ⬍0.05.
RESULTS Shock Wave Therapy and Urinary NAG-to-Cr Ratio Table 1 shows urinary NAG-to-Cr ratios before and after impact. Groups 1 and 2 had no significant changes in the urinary NAG-to-Cr ratio. In groups 3 and 4 the ratio was increased significantly after shock wave therapy (p ⬍0.01). The change in the urinary NAG-to-Cr ratio in group 4 animals was greater than that in group 3 animals. At 24 hours after impact NAG-to-Cr values in the rats increased significantly (p ⬍0.001). Surgical Microscopy of Kidney Cross Sections The right kidneys of group 3 and 4 rats, and each kidney in group 1 rats had some CaOx crystals at the cortex-medulla borderline in 26 of 32 cross sections (fig. 1, A). The left kidneys of group 3 rats had crystal clumping deposited in the medulla in 5 of 8 cross sections (fig. 1, B). In 3 of 8 group 4 rats cross sections showed massive crystal sedimentation in the renal papillae and even small stones attached to the papillary tip were macroscopically visible (fig. 1, C). Renal Histopathology Examination of paraffin kidney sections revealed that kidneys without shock wave impact, including the left and right kidneys in group 2, and the right kidney in groups 3 and 4, had only few crys-
Table 1. Urinary NAG-to-Cr ratios before and after impact
Microscopy of kidney cross sections. The 2 kidneys were removed and transversely sectioned into 2 parts parallel to the corticomedullary axis. Transverse kidney sections were examined using a surgical microscope to determine the presence of CaOx crystals. Half of each kidney was fixed with 4% polyoxymethylene (pH 7.2) and samples were stored for 24 hours at 4C. Hematoxylin and eosin staining was done to observe the microdeposition of CaOx crystals in renal tissue. Crystal deposit was evaluated
Mean ⫾ SD NAG/Cr (IU/gm) Group No.
Before Impact
After Impact
1 2 3* 4*
12.81 ⫾ 3.60 11.97 ⫾ 3.77 13.18 ⫾ 3.72 14.00 ⫾ 3.41
12.96 ⫾ 3.23 12.34 ⫾ 2.17 28.37 ⫾ 3.71 46.50 ⫾ 5.82
* Significantly different at 24 hours (p ⬍0.001).
764
SHOCK WAVE INDUCED KIDNEY INJURY PROMOTES CALCIUM OXALATE DEPOSITION
Figure 1. Macroscopic examination of renal tissue. A, tiny CaOx crystals deposits at cortex-medulla border. B, clumping CaOx crystal deposit in renal medulla. C, large CaOx crystals deposit sheet in renal papilla. Reduced from ⫻400.
tals scattered in some parts of the kidney (fig. 2, A). The mean value of crystal deposit formation in these sections was 3.5, 3.5, 3.3 and 3.6 points, respectively. There were no significant differences in these groups (p ⬎0.05). In contrast, the left kidney in group 3 and 4 rats, which underwent shock wave impact, had crystals in major kidney areas (fig. 2, B). In many tubules crystals were agglomerated in large aggregates (fig. 2, C). The mean value of crystal deposit formation in the left kidney in group 3 rats was 4.9 points and in group 4 rats it was 5.4 points. There was no significant difference in crystal distribution between groups 3 and 4 (p ⫽ 0.47). However, the quantity of crystals in group 4 was greater than in group 3. In group 3 and 4 rats kidneys that underwent impact therapy had significantly greater crystal deposition than kidneys that did not undergo impact therapy (p ⬍0.05). Ca and Ox in Kidney Homogenate Ca and Ox deposition was significantly greater in rats receiving ethylene glycol (group 2) than in control rats (group 1) (p ⬍0.001, table 2). In groups 3 and 4 the kidney that underwent shock wave impact had higher Ca and Ox concentrations than the contralateral kidney (p ⬍0.01). The group 4 left kidney with 1,000 shocks had more Ca and Ox than the group 3 left kidney with 500.
DISCUSSION Kidney stone disease is common and ESWL remains the principal treatment option in China. However, many studies indicate that shock wave treatment can cause damage to patient kidneys. Our first aim was to evaluate urinary NAG concentrations as an early indicator of renal tubular injury in relation to shock wave therapy. Shock wave therapy increased the urinary NAG concentration, confirming the presence of renal tubular injury after treatment. The severity of renal damage increased with the treatment dose. Studies to date suggest that the kidney injury caused by shock wave treatment is temporary.10 –13 Therefore, little exploration has been done of the possible long-term consequences of injury caused by ESWL. Our second purpose was to determine the possible relevance of renal injury induced by ESWL in terms of involvement in recurrent stone formation. The experiments show that the kidney damage caused by shock wave therapy was sufficient to trigger new stone formation in the presence of urinary CaOx supersaturation and the quantity of stone formation was related to kidney injury severity. This supports previous studies showing that renal tubular epithelial cell injury is a key factor in the stone formation process.7,8,14,15
Figure 2. Microscopic examination for CaOx crystals. A, kidneys without shock wave therapy had only minimal crystal deposition. B and C, kidneys with shock wave therapy had major crystal deposition. Reduced from ⫻400.
SHOCK WAVE INDUCED KIDNEY INJURY PROMOTES CALCIUM OXALATE DEPOSITION
Table 2. Kidney homogenate Ca and Ox Mean ⫾ SD Concentration (mol/gm wet kidney)* Kidney Ca: Lt Rt Ox: Lt Rt
Group 1
Group 2†
Group 3
Group 4
9.64 ⫾ 2.01 9.53 ⫾ 3.05
16.88 ⫾ 6.41 17.34 ⫾ 6.89
28.58 ⫾ 7.54‡ 16.15 ⫾ 4.47†
40.81 ⫾ 15.29§ 15.51 ⫾ 5.87†
1.33 ⫾ 0.38 1.51 ⫾ 0.53
8.44 ⫾ 6.80 9.25 ⫾ 5.45
20.52 ⫾ 7.70‡ 8.19 ⫾ 4.15†
31.76 ⫾ 14.14§ 11.21 ⫾ 6.25†
* In 8 rats per group. † Vs control p ⬍0.001. ‡ Vs group 2 p ⬍0.001. § Vs group 3 left kidney p ⬍0.001.
The stone recurrence rate after ESWL is greater than that after other treatments.2,3 Kamihira et al followed 903 patients who were stone-free after ESWL.4 At a mean followup of 25 months stones had recurred in 183 of 903 patients (20.3%). The KaplanMeier recurrence rate was 6.7%, 28.0% and 41.8% after 1, 3 and 5 years, respectively. A separate study revealed that stones recurred in 51% of 436 patients followed to a mean of 7.1 years.16 Late recurrence was noted in as many as 70% of patients after 9 years. The origin of recurrent stone formation may be microscopic sand particles migrating to dependent calices that act as a nidus for new stone formation.3 However, our study shows that ESWL induced renal tubular injury increased CaOx crystal retention in the kidney even without microscopic sand particle migration.
765
While ESWL remains a principal treatment for renal calculi, strategies to decrease renal tubular injury may be appropriate in an attempt to decrease stone recurrence. Many substances can inhibit renal tubular cell injury and as a result inhibit renal stone formation.17 In particular free radical scavengers can reduce the kidney damage caused by ESWL.18,19 Further research is ongoing to determine whether using these substances can actually decrease CaOx deposition after ESWL. Although this study demonstrates that shock wave therapy increases CaOx deposition, there remains a great difference between crystal deposition and stone formation. Further studies are needed to confirm the relationship between crystal deposition and subsequent stone formation. Although stone recurrence includes stone regrowth and regeneration, the main objective of our study was stone regeneration, and so the applicability of our findings is limited. Further limitations of this study relate to the limits of a rat model to accurately simulate the human renal injury caused by ESWL. Thus, further studies would be appropriate to evaluate the effects of renal injury caused by ESWL in stone recurrence in humans.
CONCLUSIONS In a rat model we noted that shock wave therapy results in proximal tubular injury in a dose dependent manner. In turn this was associated with a markedly increased deposition of CaOx stones in kidney tissue.
REFERENCES 1. Argyropoulos AN and Tolley DA: Optimizing shock wave lithotripsy in the 21st century. Eur Urol 2007; 52: 344. 2. Krambeck AE, LeRoy AJ, Patterson DE et al: Long-term outcomes of percutaneous nephrolithotomy compared to shock wave lithotripsy and conservative management. J Urol 2008; 17: 2233. 3. Carr LK, D’A Honey JR, Jewett MAS et al: New stone formation: a comparison of extracorporeal shock wave lithotripsy and percutaneous nephrolithotomy. J Urol 1996; 155: 1565. 4. Kamihira O, Ono Y, Katoh N et al: Long-term stone recurrence rate after extracorporeal shock wave lithotripsy. J Urol 1996; 156: 1267. 5. Weinberg K and Ortiz M: Shock wave induced damage in kidney tissue. Comp Mat Sci 2005; 32: 588. 6. Delvecchio FC, Auge BK, Munver R et al: Shock wave lithotripsy causes ipsilateral renal injury remote from the focal point: the role of regional vasoconstriction. J Urol 2003; 169: 1526. 7. Rabinovich YI, Esayanur M, Daosukho S et al: Adhesion force between calcium oxalate monohydrate crystal and kidney epithelial cells and
possible relevance for kidney stone formation. J Coll I Sci 2006; 300: 131.
rics a safe procedure? J Pediatr Surg 2008; 43: 591.
8. Khan SJ: Role of renal epithelial cells in the initiation of calcium oxalate stones. Nephron Exp Nephrol 2004; 98: e55.
14. Escobar C, Byer KJ, Khaskheli H et al: Apatite induced renal epithelial injury: insight into the pathogenesis of kidney stones. J Urol 2008; 180: 379.
9. Yamaguchi S, Yachiku S, Okuyama M et al: Early stage of urolithiasis formation in experimental hyperparathyroidism. J Urol 2001; 165: 1268. 10. Strohmaier WL, Carl AM, Wilbert DM et al: Effects of extracorporeal shock wave lithotripsy on plasma concentrations of endothelin and renin in humans. J Urol 1996; 155: 48.
15. Tsujihata M: Mechanism of calcium oxalate renal stone formation and renal tubular cell injury. Int J Urol 2008; 15: 115. 16. Sun BYC, Lee YH, Jiaan BP et al: Recurrence rate and risk factors for urinary calculi after extracorporeal shock wave lithotripsy. J Urol 1996; 156: 903.
11. Villanyi KK, Szekely JG, Farkas LM et al: Shortterm changes in renal function after extracorporeal shock wave lithotripsy in children. J Urol 2001; 166: 222.
17. Park HK, Jeong BC, Sung MK et al: Reduction of oxidative stress in cultured renal tubular cells and preventive effects on renal stone formation by the bioflavonoid quercetin. J Urol 2008; 179: 1620.
12. El-Assmy A, El-Nahas AR, Hekal IA et al: Longterm effects of extracorporeal shock wave lithotripsy on renal function: our experience with 156 patients with solitary kidney. J Urol 2008; 179: 2229.
18. Serel TA, Ozguner F and Soyupek S: Prevention of shock wave-induced renal oxidative stress by melatonin: an experimental study. Urol Res 2004; 32: 69.
13. D’Addessi A, Bongiovanni L, Racioppi M et al: Is extracorporeal shock wave lithotripsy in pediat-
19. Sarica K, Ozer G, Soygür T et al: Preservation of shock-wave-induced renal histologic changes by dermatan sulfate. Urology 1997; 49: 145.
Abbreviations and Acronyms Cr ⫽ creatinine NAG ⫽ N-acetyl-beta-dglucosaminidase Ox ⫽ oxalate Submitted for publication October 19, 2008. Study received Xi’an Jiao Tong University institutional animal care committee approval. Supported by China National Natural Science Fund Grant 30700833 and Technology Key Project in Shaanxi Province Grant 2006k10-G-1. * Correspondence: First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an, Shaanxi, China.
Purpose: Extracorporeal shock wave lithotripsy is the preferred treatment for upper urinary tract renal calculi. However, this treatment is associated with a high rate of recurrent renal calculi. Shock wave therapy can result in renal epithelial cell injury, which in turn is a most important factor in calculus formation. We investigated the influence of kidney damage secondary to shock waves on Ca oxalate crystal retention in the kidney. Materials and Methods: A total of 32 rats were randomly divided into 4 groups, including group 1— controls, group 2—sham treated rats given 25 ml 0.75% ethylene glycol per day for 14 days, group 3—rats given 15 kV 1Hz shock waves 500 times to the left kidney, followed by 25 ml 0.75% ethylene glycol daily for 14 days, and group 4 —rats with the same treatment as group 3 except the number of impacts was increased to 1,000. The 2 kidneys were removed at the end of the experiment. Ca oxalate crystals were observed by surgical microscopy in kidney sections stained with hematoxylin and eosin. Crystal morphology was determined using polarizing microscopy. Acidified kidney tissue homogenate was examined for Ca and oxalate content by colorimetry (Sigma®). Results: Kidney sections showed that kidneys that did not receive shock waves had fewer crystals than kidneys with shock waves, which had crystals in major areas. In the left kidney in groups 2 to 4 the mean ⫾ SD quantity of Ca was 16.88 ⫾ 6.41, 28.58 ⫾ 7.54 and 40.81 ⫾ 15.29 mol/gm wet kidney and the mean quantity of oxalate was 8.44 ⫾ 6.80, 20.52 ⫾ 7.70, 31.76 ⫾ 14.14 mol/gm wet kidney, respectively. Ca oxalate density increased with the number of shock wave impacts. Conclusions: Kidney damage caused by shock wave treatment can increase Ca oxalate crystal retention in the kidneys of rats in this stone model. Key Words: kidney, lithotripsy, high-energy shock waves, calcium oxalate, iatrogenic disease
762
www.jurology.com
EXTRACORPOREAL shock wave lithotripsy is the preferred clinical treatment for upper urinary tract stones.1 However, it is associated with a high rate of stone recurrence.2– 4 Studies demonstrate that the shock waves used in ESWL® can lead to the release of free radicals, which in turn cause renal tubular epithelial cell injury.5,6 Further studies show that tu-
bular epithelial cell injury is a key factor in stone formation.7,8 Thus, we hypothesized that renal tubular epithelial cell injury secondary to shock wave therapy can increase CaOx crystal deposition, which in turn may lead to the high stone recurrence rate. Previous studies of the stone recurrence rate after shock wave lithotripsy are principally clini-
0022-5347/09/1822-0762/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 182, 762-765, August 2009 Printed in U.S.A. DOI:10.1016/j.juro.2009.03.080
SHOCK WAVE INDUCED KIDNEY INJURY PROMOTES CALCIUM OXALATE DEPOSITION
cal followup studies. These series are limited by patient variability in stone size, location and composition. In addition, the number and energy of shock waves that patients received varied. Therefore, definitive conclusions regarding the influence of ESWL on stone formation cannot be drawn from these studies. To address this hypothesis we performed animal experiments to study the influence of kidney injury caused by shock wave therapy on CaOx deposition.
MATERIALS AND METHODS Animals A total of 32 male Sprague Dawley rats weighing a mean ⫾ SD of 200 ⫾ 10 gm were obtained from the laboratory animal center at our institution. The experimental protocol was reviewed and approved by the Xi’an Jiao Tong University institutional animal care committee. All animals were housed in individual metabolic cages under a mean temperature of 22C ⫾ 1C, 50% relative humidity and a 12:12-hour light/dark cycle. Animals were acclimatized to these conditions for 7 days before experiments were started.
Intervention The left kidney of each rat was exposed through an incision under the ribs. A silver medical folder was clamped in the perirenal fascia for x-ray positioning. All animals were divided into 4 groups of 8 each. Group 1 served as controls that received normal drinking water during the whole study. Sham treated group 2 was given drinking water containing 25 ml 0.75% ethylene glycol per day per rat. Group 3 animals were anesthetized by 40 mg/kg pentobarbital sodium and received 500 shock wave impacts of 15 kV and 1 Hz. From the day of shock wave treatment the animals were given drinking water containing 25 ml 0.75% ethylene glycol per day. Group 4 animals received an increased shock wave impact of 15 kV and 1 Hz for 1,000 times and drinking water containing 25 ml 0.75% ethylene glycol per day. There were no other differences among the high urinary Ox groups except the number of shock waves. All rats were fed standard chow. After 14 days of treatment all rats received fresh drinking water for 2 days to wash out free intratubular crystals from the kidneys. All animals were then sacrificed.
Laboratory Analysis Urine collection and analysis. Urine was collected for 24 hours before and after shock wave impact. A drop of concentrated HCl was added to urine before storage at 4C. Urine was analyzed for NAG and Cr by spectrophotometry.
763
using the scale, 0 points—no deposit, 1 point— crystal deposit at the papillary tip, 2 points— crystal deposit at the cortical medullary junction and 3 points— crystal deposit in the cortex.9 When crystal deposition was observed at multiple areas, the points were combined. Kidney homogenate analysis. The second half of each kidney was milled into homogenate, which was acidified with 10 mmol/ml concentrated HCl. This was centrifuged for 10 minutes at 1,000 ⫻ gravity and the supernatant was separated. The quantity of Ca and Ox in supernatant was determined by colorimetry.
Statistical Analysis Results are expressed as the mean ⫾ SD. Differences among data were determined using 1-way ANOVA, followed by the Student-Newman-Keul test with differences considered significant at p ⬍0.05.
RESULTS Shock Wave Therapy and Urinary NAG-to-Cr Ratio Table 1 shows urinary NAG-to-Cr ratios before and after impact. Groups 1 and 2 had no significant changes in the urinary NAG-to-Cr ratio. In groups 3 and 4 the ratio was increased significantly after shock wave therapy (p ⬍0.01). The change in the urinary NAG-to-Cr ratio in group 4 animals was greater than that in group 3 animals. At 24 hours after impact NAG-to-Cr values in the rats increased significantly (p ⬍0.001). Surgical Microscopy of Kidney Cross Sections The right kidneys of group 3 and 4 rats, and each kidney in group 1 rats had some CaOx crystals at the cortex-medulla borderline in 26 of 32 cross sections (fig. 1, A). The left kidneys of group 3 rats had crystal clumping deposited in the medulla in 5 of 8 cross sections (fig. 1, B). In 3 of 8 group 4 rats cross sections showed massive crystal sedimentation in the renal papillae and even small stones attached to the papillary tip were macroscopically visible (fig. 1, C). Renal Histopathology Examination of paraffin kidney sections revealed that kidneys without shock wave impact, including the left and right kidneys in group 2, and the right kidney in groups 3 and 4, had only few crys-
Table 1. Urinary NAG-to-Cr ratios before and after impact
Microscopy of kidney cross sections. The 2 kidneys were removed and transversely sectioned into 2 parts parallel to the corticomedullary axis. Transverse kidney sections were examined using a surgical microscope to determine the presence of CaOx crystals. Half of each kidney was fixed with 4% polyoxymethylene (pH 7.2) and samples were stored for 24 hours at 4C. Hematoxylin and eosin staining was done to observe the microdeposition of CaOx crystals in renal tissue. Crystal deposit was evaluated
Mean ⫾ SD NAG/Cr (IU/gm) Group No.
Before Impact
After Impact
1 2 3* 4*
12.81 ⫾ 3.60 11.97 ⫾ 3.77 13.18 ⫾ 3.72 14.00 ⫾ 3.41
12.96 ⫾ 3.23 12.34 ⫾ 2.17 28.37 ⫾ 3.71 46.50 ⫾ 5.82
* Significantly different at 24 hours (p ⬍0.001).
764
SHOCK WAVE INDUCED KIDNEY INJURY PROMOTES CALCIUM OXALATE DEPOSITION
Figure 1. Macroscopic examination of renal tissue. A, tiny CaOx crystals deposits at cortex-medulla border. B, clumping CaOx crystal deposit in renal medulla. C, large CaOx crystals deposit sheet in renal papilla. Reduced from ⫻400.
tals scattered in some parts of the kidney (fig. 2, A). The mean value of crystal deposit formation in these sections was 3.5, 3.5, 3.3 and 3.6 points, respectively. There were no significant differences in these groups (p ⬎0.05). In contrast, the left kidney in group 3 and 4 rats, which underwent shock wave impact, had crystals in major kidney areas (fig. 2, B). In many tubules crystals were agglomerated in large aggregates (fig. 2, C). The mean value of crystal deposit formation in the left kidney in group 3 rats was 4.9 points and in group 4 rats it was 5.4 points. There was no significant difference in crystal distribution between groups 3 and 4 (p ⫽ 0.47). However, the quantity of crystals in group 4 was greater than in group 3. In group 3 and 4 rats kidneys that underwent impact therapy had significantly greater crystal deposition than kidneys that did not undergo impact therapy (p ⬍0.05). Ca and Ox in Kidney Homogenate Ca and Ox deposition was significantly greater in rats receiving ethylene glycol (group 2) than in control rats (group 1) (p ⬍0.001, table 2). In groups 3 and 4 the kidney that underwent shock wave impact had higher Ca and Ox concentrations than the contralateral kidney (p ⬍0.01). The group 4 left kidney with 1,000 shocks had more Ca and Ox than the group 3 left kidney with 500.
DISCUSSION Kidney stone disease is common and ESWL remains the principal treatment option in China. However, many studies indicate that shock wave treatment can cause damage to patient kidneys. Our first aim was to evaluate urinary NAG concentrations as an early indicator of renal tubular injury in relation to shock wave therapy. Shock wave therapy increased the urinary NAG concentration, confirming the presence of renal tubular injury after treatment. The severity of renal damage increased with the treatment dose. Studies to date suggest that the kidney injury caused by shock wave treatment is temporary.10 –13 Therefore, little exploration has been done of the possible long-term consequences of injury caused by ESWL. Our second purpose was to determine the possible relevance of renal injury induced by ESWL in terms of involvement in recurrent stone formation. The experiments show that the kidney damage caused by shock wave therapy was sufficient to trigger new stone formation in the presence of urinary CaOx supersaturation and the quantity of stone formation was related to kidney injury severity. This supports previous studies showing that renal tubular epithelial cell injury is a key factor in the stone formation process.7,8,14,15
Figure 2. Microscopic examination for CaOx crystals. A, kidneys without shock wave therapy had only minimal crystal deposition. B and C, kidneys with shock wave therapy had major crystal deposition. Reduced from ⫻400.
SHOCK WAVE INDUCED KIDNEY INJURY PROMOTES CALCIUM OXALATE DEPOSITION
Table 2. Kidney homogenate Ca and Ox Mean ⫾ SD Concentration (mol/gm wet kidney)* Kidney Ca: Lt Rt Ox: Lt Rt
Group 1
Group 2†
Group 3
Group 4
9.64 ⫾ 2.01 9.53 ⫾ 3.05
16.88 ⫾ 6.41 17.34 ⫾ 6.89
28.58 ⫾ 7.54‡ 16.15 ⫾ 4.47†
40.81 ⫾ 15.29§ 15.51 ⫾ 5.87†
1.33 ⫾ 0.38 1.51 ⫾ 0.53
8.44 ⫾ 6.80 9.25 ⫾ 5.45
20.52 ⫾ 7.70‡ 8.19 ⫾ 4.15†
31.76 ⫾ 14.14§ 11.21 ⫾ 6.25†
* In 8 rats per group. † Vs control p ⬍0.001. ‡ Vs group 2 p ⬍0.001. § Vs group 3 left kidney p ⬍0.001.
The stone recurrence rate after ESWL is greater than that after other treatments.2,3 Kamihira et al followed 903 patients who were stone-free after ESWL.4 At a mean followup of 25 months stones had recurred in 183 of 903 patients (20.3%). The KaplanMeier recurrence rate was 6.7%, 28.0% and 41.8% after 1, 3 and 5 years, respectively. A separate study revealed that stones recurred in 51% of 436 patients followed to a mean of 7.1 years.16 Late recurrence was noted in as many as 70% of patients after 9 years. The origin of recurrent stone formation may be microscopic sand particles migrating to dependent calices that act as a nidus for new stone formation.3 However, our study shows that ESWL induced renal tubular injury increased CaOx crystal retention in the kidney even without microscopic sand particle migration.
765
While ESWL remains a principal treatment for renal calculi, strategies to decrease renal tubular injury may be appropriate in an attempt to decrease stone recurrence. Many substances can inhibit renal tubular cell injury and as a result inhibit renal stone formation.17 In particular free radical scavengers can reduce the kidney damage caused by ESWL.18,19 Further research is ongoing to determine whether using these substances can actually decrease CaOx deposition after ESWL. Although this study demonstrates that shock wave therapy increases CaOx deposition, there remains a great difference between crystal deposition and stone formation. Further studies are needed to confirm the relationship between crystal deposition and subsequent stone formation. Although stone recurrence includes stone regrowth and regeneration, the main objective of our study was stone regeneration, and so the applicability of our findings is limited. Further limitations of this study relate to the limits of a rat model to accurately simulate the human renal injury caused by ESWL. Thus, further studies would be appropriate to evaluate the effects of renal injury caused by ESWL in stone recurrence in humans.
CONCLUSIONS In a rat model we noted that shock wave therapy results in proximal tubular injury in a dose dependent manner. In turn this was associated with a markedly increased deposition of CaOx stones in kidney tissue.
REFERENCES 1. Argyropoulos AN and Tolley DA: Optimizing shock wave lithotripsy in the 21st century. Eur Urol 2007; 52: 344. 2. Krambeck AE, LeRoy AJ, Patterson DE et al: Long-term outcomes of percutaneous nephrolithotomy compared to shock wave lithotripsy and conservative management. J Urol 2008; 17: 2233. 3. Carr LK, D’A Honey JR, Jewett MAS et al: New stone formation: a comparison of extracorporeal shock wave lithotripsy and percutaneous nephrolithotomy. J Urol 1996; 155: 1565. 4. Kamihira O, Ono Y, Katoh N et al: Long-term stone recurrence rate after extracorporeal shock wave lithotripsy. J Urol 1996; 156: 1267. 5. Weinberg K and Ortiz M: Shock wave induced damage in kidney tissue. Comp Mat Sci 2005; 32: 588. 6. Delvecchio FC, Auge BK, Munver R et al: Shock wave lithotripsy causes ipsilateral renal injury remote from the focal point: the role of regional vasoconstriction. J Urol 2003; 169: 1526. 7. Rabinovich YI, Esayanur M, Daosukho S et al: Adhesion force between calcium oxalate monohydrate crystal and kidney epithelial cells and
possible relevance for kidney stone formation. J Coll I Sci 2006; 300: 131.
rics a safe procedure? J Pediatr Surg 2008; 43: 591.
8. Khan SJ: Role of renal epithelial cells in the initiation of calcium oxalate stones. Nephron Exp Nephrol 2004; 98: e55.
14. Escobar C, Byer KJ, Khaskheli H et al: Apatite induced renal epithelial injury: insight into the pathogenesis of kidney stones. J Urol 2008; 180: 379.
9. Yamaguchi S, Yachiku S, Okuyama M et al: Early stage of urolithiasis formation in experimental hyperparathyroidism. J Urol 2001; 165: 1268. 10. Strohmaier WL, Carl AM, Wilbert DM et al: Effects of extracorporeal shock wave lithotripsy on plasma concentrations of endothelin and renin in humans. J Urol 1996; 155: 48.
15. Tsujihata M: Mechanism of calcium oxalate renal stone formation and renal tubular cell injury. Int J Urol 2008; 15: 115. 16. Sun BYC, Lee YH, Jiaan BP et al: Recurrence rate and risk factors for urinary calculi after extracorporeal shock wave lithotripsy. J Urol 1996; 156: 903.
11. Villanyi KK, Szekely JG, Farkas LM et al: Shortterm changes in renal function after extracorporeal shock wave lithotripsy in children. J Urol 2001; 166: 222.
17. Park HK, Jeong BC, Sung MK et al: Reduction of oxidative stress in cultured renal tubular cells and preventive effects on renal stone formation by the bioflavonoid quercetin. J Urol 2008; 179: 1620.
12. El-Assmy A, El-Nahas AR, Hekal IA et al: Longterm effects of extracorporeal shock wave lithotripsy on renal function: our experience with 156 patients with solitary kidney. J Urol 2008; 179: 2229.
18. Serel TA, Ozguner F and Soyupek S: Prevention of shock wave-induced renal oxidative stress by melatonin: an experimental study. Urol Res 2004; 32: 69.
13. D’Addessi A, Bongiovanni L, Racioppi M et al: Is extracorporeal shock wave lithotripsy in pediat-
19. Sarica K, Ozer G, Soygür T et al: Preservation of shock-wave-induced renal histologic changes by dermatan sulfate. Urology 1997; 49: 145.