- Email: [email protected]
Endourology
UROLITHIASIS/ENDOUROLOGY
1219
suppressor cascade that promotes prostate cancer growth and metastasis by coordinately activating the small GTPase Ras and nuclear factor-kappaB (NF-kappaB). Specifically, we show that loss of the Ras GTPase-activating protein (RasGAP) gene DAB2IP induces metastatic prostate cancer in an orthotopic mouse tumor model. Notably, DAB2IP functions as a signaling scaffold that coordinately regulates Ras and NF-kappaB through distinct domains to promote tumor growth and metastasis, respectively. DAB2IP is suppressed in human prostate cancer, where its expression inversely correlates with tumor grade and predicts prognosis. Moreover, we report that epigenetic silencing of DAB2IP is a key mechanism by which the polycomb-group protein histone-lysine N-methyltransferase EZH2 activates Ras and NF-kappaB and triggers metastasis. These studies define the mechanism by which two major pathways can be simultaneously activated in metastatic prostate cancer and establish EZH2 as a driver of metastasis. Editorial Comment: Ras is regulated positively by guanine nucleotide exchange factors and negatively by GAPs. It is unknown whether guanine nucleotide exchange factors have a role in cancer. However, one RasGAP, neurofibromin (encoded by NF1), is a known tumor suppressor. There are 14 human RasGAPs, few of which have been studied in detail. The authors investigated whether other RasGAP genes might also function as tumor suppressors. This study identifies a new tumor and metastasis suppressor within this gene family, defines the mechanism by which its loss promotes metastatic prostate cancer and demonstrates that its epigenetic suppression is a key mechanism by which the polycomb-group protein EZH2 triggers metastasis. Anthony Atala, M.D.
Urolithiasis/Endourology The Effect of Dietary Protein on Intestinal Calcium Absorption in Rats E. Gaffney-Stomberg, B. H. Sun, C. E. Cucchi, C. A. Simpson, C. Gundberg, J. E. Kerstetter and K. L. Insogna Department of Allied Health Sciences, University of Connecticut, Storrs, Connecticut Endocrinology 2010; 151: 1071–1078.
Increasing dietary protein intake in humans acutely increases urinary calcium. Isotopic absorption studies have indicated that, at least in the short term, this is primarily due to increased intestinal Ca absorption. To explore the mechanisms underlying dietary protein’s effect on intestinal Ca absorption, female Sprague Dawley rats were fed a control (20%), low (5%), or high (40%) protein diet for 7 d, and Ca balance was measured during d 4 –7. On d 7, duodenal mucosa was harvested and brush border membrane vesicles (BBMVs) were prepared to evaluate Ca uptake. By d 7, urinary calcium was more than 2-fold higher in the 40% protein group compared with control (4.2 mg/d vs. 1.7 mg/d; P ⬍ 0.05). Rats consuming the 40% protein diet both absorbed and retained more Ca compared with the 5% protein group (absorption: 48.5% vs. 34.1% and retention: 45.8% vs. 33.7%, respectively; P ⬍ 0.01). Ca uptake was increased in BBMVs prepared from rats consuming the high-protein diet. Maximum velocity (V(max)) was higher in the BBMVs prepared from the high-protein group compared with those from the low-protein group (90 vs. 36 nmol Ca/mg protein x min, P ⬍ 0.001; 95% CI: 46 –2486 and 14 –55, respectively). The Michaelis Menten constant (K(m)) was unchanged (2.2 mm vs. 1.8 mm, respectively; P ⫽ 0.19). We conclude that in rats, as in humans, acute increases in protein intake result in hypercalciuria due to augmented intestinal Ca absorption. BBMV Ca uptake studies suggest that higher protein intake improves Ca absorption, at least in part, by increasing transcellular Ca uptake. Editorial Comment: The increase in calcium excretion associated with protein consumption has been attributed to release of calcium from bone as a buffering mechanism for the
1220
UROLITHIASIS/ENDOUROLOGY
associated acid load. These investigators demonstrate that another mechanism is increased calcium absorption via transcellular pathways. The mechanisms for this response need to be defined. Dean Assimos, M.D.
Hydroxyproline-Induced Hyperoxaluria Using Acidified and Traditional Diets in the Porcine Model D. M. Kaplon, K. L. Penniston, C. Darriet, T. D. Crenshaw and S. Y. Nakada Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin J Endourol 2010; 24: 355–359.
Introduction: Swine models have proven useful for many different disease processes, especially for urologic research. In this study, we sought to create a model of hyperoxaluria in the adult sow by feeding hydroxyproline (HP). The development of an adult porcine model for calcium oxalate stone disease would represent a significant contribution to stone research as previous animal models have been developed only for rats and baby pigs. Methods: The experiment included a total of 12 multiparous, gestating sows (Large White x Landrace). Sows were randomly allotted to one of the two treatment groups. Treatments involved basal diets that were either control diet (CD) or acidogenic diet (AD). Urine was collected for 6 consecutive days. On days 1 and 2, each sow was fed 2 kg of the assigned basal diet (CD or AD). On days 3, 4, and 5, 200 g of L-hydroxyproline (Wilshire Technologies, Princeton, NJ) was added to each basal diet for all the 12 sows. The HP was evenly mixed with the basal diets before feeding. On day 6, each sow was fed the basal diet originally assigned without HP (Fig. 1). Urine was collected for each entire 24-hour period to control for differences in the diurnal and postprandial variations in the renal handling of oxalate and glycolate. Results: The addition of HP to the diet increased urinary oxalate excretion. Overall, there was a 192% (CD) and 187% (AD) increase in urinary oxalate between days 1 and 3. The increase peaked on day 3 and gradually returned to baseline by day 6. Student’s paired t-test was performed and it confirmed that oxalate on days 3 and 5 was significantly different than baseline (p ⫽ 0.009 and p ⫽ 0.03, respectively). Urinary glycolate also increased as a result of adding HP to the diet. Overall, there was a 12,340% (CD) and 14,400% (AD) increase in urinary glycolate between days 1 and 3. The increase peaked on day 3 and then declined, although remained more than 10 x greater than baseline at day 6. Student’s paired t-test confirmed that glycolate levels on days 3, 5, and 6 were significantly different than baseline (p ⬍ 0.001, p ⫽ 0.01, and p ⫽ 0.03, respectively). Conclusion: The role of oxalate in the formation of kidney stones cannot be understated. Medical prevention and management of calcium oxalate nephrolithiasis will require a comprehensive understanding of oxalate metabolism in humans. A model for human hyperoxaluria can be reliably created in the adult sow. Such a model is necessary to further our understanding of oxalate metabolism and ultimately aid in the prevention of calcium oxalate calculi. Editorial Comment: Hydroxyproline is metabolized in the mitochondrial compartment to glyoxylate, the immediate precursor to oxalate. The porcine kidney is structurally and functionally similar to that of humans. The authors showed that administration of hydroxyproline in this animal model significantly increased oxalate excretion. Further development of this model may provide insights into endogenous oxalate synthesis, something thought to contribute to 40% to 50% of the urinary oxalate pool. Dean Assimos, M.D.
1219
suppressor cascade that promotes prostate cancer growth and metastasis by coordinately activating the small GTPase Ras and nuclear factor-kappaB (NF-kappaB). Specifically, we show that loss of the Ras GTPase-activating protein (RasGAP) gene DAB2IP induces metastatic prostate cancer in an orthotopic mouse tumor model. Notably, DAB2IP functions as a signaling scaffold that coordinately regulates Ras and NF-kappaB through distinct domains to promote tumor growth and metastasis, respectively. DAB2IP is suppressed in human prostate cancer, where its expression inversely correlates with tumor grade and predicts prognosis. Moreover, we report that epigenetic silencing of DAB2IP is a key mechanism by which the polycomb-group protein histone-lysine N-methyltransferase EZH2 activates Ras and NF-kappaB and triggers metastasis. These studies define the mechanism by which two major pathways can be simultaneously activated in metastatic prostate cancer and establish EZH2 as a driver of metastasis. Editorial Comment: Ras is regulated positively by guanine nucleotide exchange factors and negatively by GAPs. It is unknown whether guanine nucleotide exchange factors have a role in cancer. However, one RasGAP, neurofibromin (encoded by NF1), is a known tumor suppressor. There are 14 human RasGAPs, few of which have been studied in detail. The authors investigated whether other RasGAP genes might also function as tumor suppressors. This study identifies a new tumor and metastasis suppressor within this gene family, defines the mechanism by which its loss promotes metastatic prostate cancer and demonstrates that its epigenetic suppression is a key mechanism by which the polycomb-group protein EZH2 triggers metastasis. Anthony Atala, M.D.
Urolithiasis/Endourology The Effect of Dietary Protein on Intestinal Calcium Absorption in Rats E. Gaffney-Stomberg, B. H. Sun, C. E. Cucchi, C. A. Simpson, C. Gundberg, J. E. Kerstetter and K. L. Insogna Department of Allied Health Sciences, University of Connecticut, Storrs, Connecticut Endocrinology 2010; 151: 1071–1078.
Increasing dietary protein intake in humans acutely increases urinary calcium. Isotopic absorption studies have indicated that, at least in the short term, this is primarily due to increased intestinal Ca absorption. To explore the mechanisms underlying dietary protein’s effect on intestinal Ca absorption, female Sprague Dawley rats were fed a control (20%), low (5%), or high (40%) protein diet for 7 d, and Ca balance was measured during d 4 –7. On d 7, duodenal mucosa was harvested and brush border membrane vesicles (BBMVs) were prepared to evaluate Ca uptake. By d 7, urinary calcium was more than 2-fold higher in the 40% protein group compared with control (4.2 mg/d vs. 1.7 mg/d; P ⬍ 0.05). Rats consuming the 40% protein diet both absorbed and retained more Ca compared with the 5% protein group (absorption: 48.5% vs. 34.1% and retention: 45.8% vs. 33.7%, respectively; P ⬍ 0.01). Ca uptake was increased in BBMVs prepared from rats consuming the high-protein diet. Maximum velocity (V(max)) was higher in the BBMVs prepared from the high-protein group compared with those from the low-protein group (90 vs. 36 nmol Ca/mg protein x min, P ⬍ 0.001; 95% CI: 46 –2486 and 14 –55, respectively). The Michaelis Menten constant (K(m)) was unchanged (2.2 mm vs. 1.8 mm, respectively; P ⫽ 0.19). We conclude that in rats, as in humans, acute increases in protein intake result in hypercalciuria due to augmented intestinal Ca absorption. BBMV Ca uptake studies suggest that higher protein intake improves Ca absorption, at least in part, by increasing transcellular Ca uptake. Editorial Comment: The increase in calcium excretion associated with protein consumption has been attributed to release of calcium from bone as a buffering mechanism for the
1220
UROLITHIASIS/ENDOUROLOGY
associated acid load. These investigators demonstrate that another mechanism is increased calcium absorption via transcellular pathways. The mechanisms for this response need to be defined. Dean Assimos, M.D.
Hydroxyproline-Induced Hyperoxaluria Using Acidified and Traditional Diets in the Porcine Model D. M. Kaplon, K. L. Penniston, C. Darriet, T. D. Crenshaw and S. Y. Nakada Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin J Endourol 2010; 24: 355–359.
Introduction: Swine models have proven useful for many different disease processes, especially for urologic research. In this study, we sought to create a model of hyperoxaluria in the adult sow by feeding hydroxyproline (HP). The development of an adult porcine model for calcium oxalate stone disease would represent a significant contribution to stone research as previous animal models have been developed only for rats and baby pigs. Methods: The experiment included a total of 12 multiparous, gestating sows (Large White x Landrace). Sows were randomly allotted to one of the two treatment groups. Treatments involved basal diets that were either control diet (CD) or acidogenic diet (AD). Urine was collected for 6 consecutive days. On days 1 and 2, each sow was fed 2 kg of the assigned basal diet (CD or AD). On days 3, 4, and 5, 200 g of L-hydroxyproline (Wilshire Technologies, Princeton, NJ) was added to each basal diet for all the 12 sows. The HP was evenly mixed with the basal diets before feeding. On day 6, each sow was fed the basal diet originally assigned without HP (Fig. 1). Urine was collected for each entire 24-hour period to control for differences in the diurnal and postprandial variations in the renal handling of oxalate and glycolate. Results: The addition of HP to the diet increased urinary oxalate excretion. Overall, there was a 192% (CD) and 187% (AD) increase in urinary oxalate between days 1 and 3. The increase peaked on day 3 and gradually returned to baseline by day 6. Student’s paired t-test was performed and it confirmed that oxalate on days 3 and 5 was significantly different than baseline (p ⫽ 0.009 and p ⫽ 0.03, respectively). Urinary glycolate also increased as a result of adding HP to the diet. Overall, there was a 12,340% (CD) and 14,400% (AD) increase in urinary glycolate between days 1 and 3. The increase peaked on day 3 and then declined, although remained more than 10 x greater than baseline at day 6. Student’s paired t-test confirmed that glycolate levels on days 3, 5, and 6 were significantly different than baseline (p ⬍ 0.001, p ⫽ 0.01, and p ⫽ 0.03, respectively). Conclusion: The role of oxalate in the formation of kidney stones cannot be understated. Medical prevention and management of calcium oxalate nephrolithiasis will require a comprehensive understanding of oxalate metabolism in humans. A model for human hyperoxaluria can be reliably created in the adult sow. Such a model is necessary to further our understanding of oxalate metabolism and ultimately aid in the prevention of calcium oxalate calculi. Editorial Comment: Hydroxyproline is metabolized in the mitochondrial compartment to glyoxylate, the immediate precursor to oxalate. The porcine kidney is structurally and functionally similar to that of humans. The authors showed that administration of hydroxyproline in this animal model significantly increased oxalate excretion. Further development of this model may provide insights into endogenous oxalate synthesis, something thought to contribute to 40% to 50% of the urinary oxalate pool. Dean Assimos, M.D.