Calcium Oxalate Kidney Stones Research Articles

Effects of oxalate-degrading enzyme on the calcium oxalate kidney stones formation in intestinal hy-peroxaluria rats

Objective To observe the effects of oxalate-degrading enzyme on the formation of calcium oxalate kidney stones in intestinal hyperoxaluria rats. Methods From May to June, 2014, 8 healthy rats were selected as the negative control group named Group A. Intestinal hyperoxaluria rats were randomly divided into three groups (n=8): positive control group (Group B), low-dose enzyme gavage group (Group C) and high-dose enzyme gavage group (Group D). Group A were treated with normal saline by gavage (3 ml/d). Group B were treated with 4% ammonium oxalate solution by gavage (3 ml/d). Group C and Group D were treated with 20 U/d or 40 U/d enzyme on the basis of Group B. The intervention lasted for 4 weeks. The oxalate concentrations of 24 hours urine in each group were detected in 0, 7th, 14th, 21th, 28th day. The renal function, the serum and urine Ca2+ in each group were measured in 0 and 28th day. In 28th day, the kidneys of rats in Group A, B, D were examined by spectral CT. The kidneys of 4 groups were examined microscopically for possible calcium oxalate crystals. Results In 0 day, the urinary oxalate concentrations of Group A, B, C and D were (0.61±0.06), (1.58±0.15), (1.59±0.10) and (1.64±0.20) mmol/L. There were no significant differences among B, C and D(P>0.05). Compared with A, the urinary oxalate concentrations of B, C and D were significantly higher (P<0.001). After the enzyme gavage, the urinary oxalate concentrations of Group C and D were significantly decreased, and Group D decreased more significantly. In 28th day, the urinary oxalate concentrations of Group A, B, C and D were (0.61±0.11), (1.64±0.12), (1.17±0.12) and (0.87±0.11) mmol/L. After statistical analysis, the urinary oxalate concentration of Group C and D were significantly lower than Group B (P<0.01), and Group D decreased more significantly than that in Group C (P<0.01). The CT post-processing workstation data analysis suggested that the average CT value of Group B were significantly higher than D (P<0.01), and both of the two groups were higher than Group A (P<0.01); The differences were more obvious in conditions of low-kilovolt. Renal tubular calcium oxalate crystal deposition were seen the most in Group B, less in Group C and lowest in Group D. Whereas, there was no crystallization in Group A. Conclusion Oxalate-degrading enzymes could inhibit the formation of calcium oxalate kidney stones in intestinal hyperoxaluria rats. Key words: Kidney calculi; Oxalate-degrading enzymes; Hyperoxaluria; Calcium oxalate stones; Rats

Oxalate-degrading microorganisms or oxalate-degrading enzymes: which is the future therapy for enzymatic dissolution of calcium-oxalate uroliths in recurrent stone disease?

Renal urolithiasis is a pathological condition common to a multitude of genetic, physiological and nutritional disorders, ranging from general hyperoxaluria to obesity. The concept of quickly dissolving renal uroliths via chemolysis, especially calcium-oxalate kidney stones, has long been a clinical goal, but yet to be achieved. Over the past 25 years, there has been a serious effort to examine the prospects of using plant and microbial oxalate-degrading enzymes known to catabolize oxalic acid and oxalate salts. While evidence is emerging that bacterial probiotics can reduce recurrent calcium-oxalate kidney stone disease by lowering systemic hyperoxaluria, the possible use of free oxalate-degrading enzyme therapy remains a challenge with several hurdles to overcome before reaching clinical practice.

Metabolism of 13C5-hydroxyproline in mouse models of Primary Hyperoxaluria and its inhibition by RNAi therapeutics targeting liver glycolate oxidase and hydroxyproline dehydrogenase

Excessive endogenous oxalate synthesis can result in calcium oxalate kidney stone formation and renal failure. Hydroxyproline catabolism in the liver and kidney contributes to endogenous oxalate production in mammals. To quantify this contribution we have infused Wt mice, Agxt KO mice deficient in liver alanine:glyoxylate aminotransferase, and Grhpr KO mice deficient in glyoxylate reductase, with 13C5-hydroxyproline. The contribution of hydroxyproline metabolism to urinary oxalate excretion in Wt mice was 22±2%, 42±8% in Agxt KO mice, and 36%±9% in Grhpr KO mice. To determine if blocking steps in hydroxyproline and glycolate metabolism would decrease urinary oxalate excretion, mice were injected with siRNA targeting the liver enzymes glycolate oxidase and hydroxyproline dehydrogenase. These siRNAs decreased the expression of both enzymes and reduced urinary oxalate excretion in Agxt KO mice, when compared to mice infused with a luciferase control preparation. These results suggest that siRNA approaches could be useful for decreasing the oxalate burden on the kidney in individuals with Primary Hyperoxaluria.

Assessment of Probe-to-Specimen Distance Effect in Kidney Stone Treatment With Hydrodynamic Cavitation

The aim of this study is to focus on the effect of probe-to-specimen distance in kidney stone treatment with hydrodynamic bubbly cavitation. Cavitating bubbles were generated by running phosphate buffered saline (PBS) through stainless steel tubing of inner diameter of 1.56 mm at an inlet pressure of ∼10,000 kPa, which was connected to a 0.75 mm long probe with an inner diameter of 147 μm at the exit providing a sudden contraction and thus low local pressures. The bubbles were targeted on the surface of nine calcium oxalate kidney stones (submerged in a water pool at room temperature and atmospheric pressure) from three different distances, namely, 0.5 mm, 2.75 mm, and 7.75 mm. The experiments were repeated for three different time durations (5 min, 10 min, and 20 min). The experimental data show that amongst the three distances considered, the distance of 2.75 mm results in the highest erosion amount and highest erosion rate (up to 0.94 mg/min), which suggests that a closer distance does not necessarily lead to a higher erosion rate and that the probe-to-specimen distance is a factor of great importance, which needs to be optimized. In order to be able to explain the experimental results, a visualization study was also conducted with a high speed CMOS camera. A new correlation was developed to predict the erosion rates on kidney stones exposed to hydrodynamic cavitation as a function of material properties, time, and distance.

The Presence of Oxalobacter formigenes in the Microbiome of Healthy Young Adults

Oxalobacter formigenes, a member of the human colonic microbiota with a major role in net colonic oxalate transport and secretion, is protective against the formation of calcium oxalate kidney stones. We describe the prevalence, relative abundance and stability of O. formigenes in healthy young adults in the United States. We used HMP (Human Microbiome Project) data on fecal samples from 242 healthy young adults who had 1 to 3 study visits. Samples underwent whole genomic shotgun sequencing and/or 16S rRNA sequencing. Three data sets available from the processed sequence data were studied, including whole genomic shotgun metagenomic analysis by alignment to reference genomes using shotgun community profiling, or MetaPhlAn (http://huttenhower.sph.harvard.edu/metaphlan) or QIIME (http://qiime.org/) analysis of the V1-3 or V3-5 16S sequences. O. formigenes was detected in fecal samples using whole genomic shotgun and 16S rRNA data. Analysis of the whole genomic shotgun data set using shotgun community profiling showed that 29 of 94 subjects (31%) were O. formigenes positive. V1-3 and V3-5 analyses were less sensitive for O. formigenes detection. When present, O. formigenes relative abundance varied over 3 log10 and was normally distributed. All assays agreed in 58 of 66 samples (88%) studied by all 3 methods. Of 14 subjects who were O. formigenes positive at baseline 13 (93%) were positive at the followup visit, indicating the stability of colonization. O. formigenes appears to be stably present in fewer than half of healthy young adults in the United States. It is most sensitively detected by whole genomic shotgun.

Osteogenic changes in kidneys of hyperoxaluric rats

Many calcium oxalate (CaOx) kidney stones develop attached to renal papillary sub-epithelial deposits of calcium phosphate (CaP), called Randall's plaque (RP). Pathogenesis of the plaques is not fully understood. We hypothesize that abnormal urinary environment in stone forming kidneys leads to epithelial cells losing their identity and becoming osteogenic. To test our hypothesis male rats were made hyperoxaluric by administration of hydroxy-l-proline (HLP). After 28days, rat kidneys were extracted. We performed genome wide analyses of differentially expressed genes and determined changes consistent with dedifferentiation of epithelial cells into osteogenic phenotype. Selected molecules were further analyzed using quantitative-PCR and immunohistochemistry. Genes for runt related transcription factors (RUNX1 and 2), zinc finger protein Osterix, bone morphogenetic proteins (BMP2 and 7), bone morphogenetic protein receptor (BMPR2), collagen, osteocalcin, osteonectin, osteopontin (OPN), matrix-gla-protein (MGP), osteoprotegrin (OPG), cadherins, fibronectin (FN) and vimentin (VIM) were upregulated while those for alkaline phosphatase (ALP) and cytokeratins 10 and 18 were downregulated. In conclusion, epithelial cells of hyperoxaluric kidneys acquire a number of osteoblastic features but without CaP deposition, perhaps a result of downregulation of ALP and upregulation of OPN and MGP. Plaque formation may additionally require localized increases in calcium and phosphate and decrease in mineralization inhibitory potential.

Oxalobacter formigenes Colonization and Oxalate Dynamics in a Mouse Model.

Animal and human studies have provided compelling evidence that colonization of the intestine with Oxalobacter formigenes reduces urinary oxalate excretion and lowers the risk of forming calcium oxalate kidney stones. The mechanism providing protection appears to be related to the unique ability of O. formigenes to rely on oxalate as a major source of carbon and energy for growth. However, much is not known about the factors that influence colonization and host-bacterium interactions. We have colonized mice with O. formigenes OxCC13 and systematically investigated the impacts of diets with different levels of calcium and oxalate on O. formigenes intestinal densities and urinary and intestinal oxalate levels. Measurement of intestinal oxalate levels in mice colonized or not colonized with O. formigenes demonstrated the highly efficient degradation of soluble oxalate by O. formigenes relative to other microbiota. The ratio of calcium to oxalate in diets was important in determining colonization densities and conditions where urinary oxalate and fecal oxalate excretion were modified, and the results were consistent with those from studies we have performed with colonized and noncolonized humans. The use of low-oxalate purified diets showed that 80% of animals retained O. formigenes colonization after a 1-week dietary oxalate deprivation. Animals not colonized with O. formigenes excreted two times more oxalate in feces than they had ingested. This nondietary source of oxalate may play an important role in the survival of O. formigenes during periods of dietary oxalate deprivation. These studies suggest that the mouse will be a useful model to further characterize interactions between O. formigenes and the host and factors that impact colonization.

MP33-18 “THE ROLE OF RENAL TUBULAR CELL INJURY IN THE EARLY PERIOD OF RENAL CRYSTAL FORMATION” IDENTIFIED FROM THE CELL INJURY-INHIBITING EFFECT OF GREEN TEA

RESULTS: The examinations at every weekend showed that Ox of 24 hours urine did not change significantly in both Group A and B£P>0.05£ , Group C and D were significantly lower than those in group B (P 0.05£ £Group C and D were significantly increased compared with those in Group B(P<0.05), Group D increased more significantly than that Group C (P<0.05). There were no significant changes of the renal function and serum Ca2þ of rats in all groups. The CT post-processing workstation data analysis suggested that the average CT value of Group B were significantly higher than Group D, and both of the two groups were higher than Group A; The differences were more obvious in conditions of low-kilovolt (P<0.05). The calcium oxalate crystal of tubules in Group B wer more serious than in Group C and D, and Group C were more serious than Group D. There were no significent calcium oxalate crystal deposition and pathological changes in renal biopsy specimens of Group A. CONCLUSIONS: Oxalate-degrading enzymes can effectively reduce the formation of calcium oxalate kidney stones in intestinal hyperoxaluria rat.

Analysis of Commercial Kidney Stone Probiotic Supplements

To examine the levels of Oxalobacter formigenes in probiotic supplements marketed by PRO-LAB, Ltd, Toronto, Canada, and capsules of Oxalo purchased from Sanzyme Ltd, Hyderabad, India, and to measure the ability of these preparations to degrade oxalate invitro. Probiotic supplements and pure cultures of O. formigenes were cultured in a number of media containing oxalate. Optical density at 595 nm (OD595) was used to measure bacterial growth, and ion chromatography was used to measure loss of oxalate in culture media. O. formigenes-specific and degenerate Lactobacillus primers to the oxalate decarboxylase gene (oxc) were used in polymerase chain reaction (PCR). Incubating probiotic supplements in different media did not result in the growth of oxalate-degrading organisms. PCR indicated the absence of organisms harboring the oxc gene. Culture and 16S ribosomal ribonucleic acid gene sequencing indicated the PRO-LAB supplement contained viable Lactococcus lactis subsp. lactis (GenBank accession no. KJ095656.1), whereas Oxalo contained several Bacillus species and Lactobacillus plantarum. The probiotic supplement sold over the Internet by PRO-LAB Ltd and Sanzyme Ltd did not contain identifiable O. formigenes or viable oxalate-degrading organisms, and they are unlikely tobe of benefit to calcium oxalate kidney stone patients.

Effects of acid-base variables and the role of carbonic anhydrase on oxalate secretion by the mouse intestine in vitro.

Hyperoxaluria is a major risk factor for calcium oxalate kidney stones and the intestine is recognized as an important extra-renal pathway for eliminating oxalate. The membrane-bound chloride/bicarbonate (Cl−/) exchangers are involved in the transcellular movement of oxalate, but little is understood about how they might be regulated. , CO2, and pH are established modulators of intestinal NaCl cotransport, involving Na+/H+ and Cl−/ exchange, but their influence on oxalate transport is unknown. Measuring 14C-oxalate and 36Cl fluxes across isolated, short-circuited segments of the mouse distal ileum and distal colon we examined the role of these acid-base variables and carbonic anhydrase (CA) in oxalate and Cl− transport. In standard buffer both segments performed net oxalate secretion (and Cl− absorption), but only the colon, and the secretory pathway were responsive to and CO2. Ethoxzolamide abolished net oxalate secretion by the distal colon, and when used in tandem with an impermeant CA inhibitor, signaled an intracellular CA isozyme was required for secretion. There was a clear dependence on as their removal eliminated secretion, while at 42 mmol/L was also decreased and eradicated. Independent of pH, raising Pco2 from 28 to 64 mmHg acutely stimulated net oxalate secretion 41%. In summary, oxalate secretion by the distal colon was dependent on , CA and specifically modulated by CO2, whereas the ileum was remarkably unresponsive. These findings highlight the distinct segmental heterogeneity along the intestine, providing new insights into the oxalate transport mechanism and how it might be regulated.

Randall's plaque as the origin of calcium oxalate kidney stones.

Eight decades ago, Alexander Randall identified calcium phosphate deposits at the tip of renal papillae as the origin of renal calculi. The awareness that these "Randall's plaque" promote renal stone formation has been amplified during the past years by the development of endoscopic procedures allowing the in situ visualization of these plaques. Recent studies based upon kidney biopsies evidenced that apatite deposits at the origin of these plaque originate from the basement membranes of thin loops of Henle and then spread in the surrounding interstitium. In addition, scanning electron microscopy examination of calcium oxalate stones developed on Randall's plaque evidenced that plaque may also be made of tubules obstructed by calcium phosphate plugs. Hypercalciuria has been associated to Randall's plaque formation. However, several additional mechanisms may be involved resulting in increased tissular calcium phosphate supersaturation and the role of macromolecules in plaque formation remains elusive. At last, apatite crystals are the main mineral phase identified in plaques, but other calcium phosphates and various chemical species such as purines have been evidenced, revealing thereby that several mechanisms may be responsible for plaque formation.

Reactive oxygen species, inflammation and calcium oxalate nephrolithiasis.

Calcium oxalate (CaOx) kidney stones are formed attached to Randall’s plaques (RPs) or Randall’s plugs. Mechanisms involved in the formation and growth are poorly understood. It is our hypothesis that stone formation is a form of pathological biomineralization or ectopic calcification. Pathological calcification and plaque formation in the body is triggered by reactive oxygen species (ROS) and the development of oxidative stress (OS). This review explores clinical and experimental data in support of ROS involvement in the formation of CaOx kidney stones. Under normal conditions the production of ROS is tightly controlled, increasing when and where needed. Results of clinical and experimental studies show that renal epithelial exposure to high oxalate and crystals of CaOx/calcium phosphate (CaP) generates excess ROS, causing injury and inflammation. Major markers of OS and inflammation are detectable in urine of stone patients as well as rats with experimentally induced CaOx nephrolithiasis. Antioxidant treatments reduce crystal and oxalate induced injury in tissue culture and animal models. Significantly lower serum levels of antioxidants, alpha-carotene, beta-carotene and beta-cryptoxanthine have been found in individuals with a history of kidney stones. A diet rich in antioxidants has been shown to reduce stone episodes. ROS regulate crystal formation, growth and retention through the timely production of crystallization modulators. In the presence of abnormal calcium, citrate, oxalate, and/or phosphate, however, there is an overproduction of ROS and a decrease in the antioxidant capacity resulting in OS, renal injury and inflammation. Cellular degradation products in the urine promote crystallization in the tubular lumen at a faster rate thus blocking the tubule and plugging the tubular openings at the papillary tips forming Randall’s plugs. Renal epithelial cells lining the loops of Henle/collecting ducts may become osteogenic, producing membrane vesicles at the basal side. In addition endothelial cells lining the blood vessels may also become osteogenic producing membrane vesicles. Calcification of the vesicles gives rise to RPs. The growth of the RP’s is sustained by mineralization of collagen laid down as result of inflammation and fibrosis.

How much calcium supplement is recommended in a patient with osteoporosis and a history of calcium oxalate kidney stones?

Evidence-Based Answer The answer is unclear. When extrapolating from trials with postmenopausal women or patients with history of kidney stones, calcium intake alone does not appear to greatly increase the risk of recurrent calcium oxalate stones (SOR: C, extrapolation from RCTs).

Cloning, localization and characterization of a Zn transporter (Zip10) from fly, dog, and human (893.39)

Background: Zn is implicated as having a role in the formation of calcium oxalate (CaOx) kidney stones. Our studies indicate that Slc39a10 (Zip10) may be important in this process. Zip10 has identifiable sequences in human, dog and fly (Drosophila). As we are interested in a translational model of kidney stones, we examined Zip10 from all 3 species.Methods: Zip10 cDNAs were cloned from human &amp; dog (kidney) and fly (whole fly) using gene specific primers and RT‐PCR. Clones were put into a Xenopus oocyte expression vector so that Zip10 function could be measured. Three days post‐injection, oocytes were pre‐incubated in ND96, followed by 30 min in ND96 + 10 μM ZnCl + the PET isotope 63Zn, with varying pHs and ion concentrations. 63Zn uptake was measured in each oocyte using a gamma counter. Zip10 oocytes were also checked using electrophysiology (ion selective electrodes &amp; voltage clamping).Results: Zip10 mRNA &amp; protein are found in gut and kidney. In mouse kidney, Zip10 antibodies stained the apical membrane of proximal tubules. All clones transported 63Zn (nmol/oocyte/hr): hZip10 = 70±8; cZip10= 44 ±7; dZip10= 64 ±8; water = 0.4 ±0.1. These clones seem to be most active at pH 7.5 as uptakes decreased at 5.5 and 8.5. Replacement of either all Cl‐ (with HCO3舉) or all Na+ (with choline) reduced Zn‐uptake. Electrophysiology revealed no evidence of I or V changes with addition of Zn (electroneutral).Conclusions: Our results indicated that all three Zip10 clones transport Zn2+. Understanding the detailed mechanisms of these Zn transporters in oocytes presents the opportunity to better use our Drosophila model in the study of CaOx stone formation and absorption.Grant Funding Source: Supported by DK83007, DK92408

MP20-14 UNCOUPLED ENDOTHELIAL NITRIC OXIDE SYNTHASE CONTRIBUTES TO OXALATE-INDUCED OXIDATIVE CELL INJURY IN RENAL EPITHELIAL CELLS: EFFECT OF ANTIOXIDANTS

You have accessJournal of UrologyStone Disease: Basic Research I1 Apr 2014MP20-14 UNCOUPLED ENDOTHELIAL NITRIC OXIDE SYNTHASE CONTRIBUTES TO OXALATE-INDUCED OXIDATIVE CELL INJURY IN RENAL EPITHELIAL CELLS: EFFECT OF ANTIOXIDANTS Vijayalakshmi Thamilselvan, Mani Menon, and Sivagnanam Thamilselvan Vijayalakshmi ThamilselvanVijayalakshmi Thamilselvan More articles by this author , Mani MenonMani Menon More articles by this author , and Sivagnanam ThamilselvanSivagnanam Thamilselvan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.736AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hyperoxaluria is one of the major risk factors for calcium oxalate urolithiasis and studies support the concept that cellular injury is a critical factor in calcium oxalate crystal nucleation and development of kidney stones. We have previously shown that oxalate-induced reactive oxygen species (ROS) mediates oxidative renal injury via NADPH oxidase (NOX) activation and mitochondrial dysfunction in renal epithelial cells. Here we demonstrate that Uncoupling of Endothelial Nitric Oxide Synthase (eNOS) due to increased NOX and mitochondrial dependent ROS generation also mediates oxalate-induced oxidative cell injury in renal epithelial cells. METHODS LLC-PK1 cells were exposed to oxalate (0.5 mM-1.0 mM) for different time periods in the presence or absence of inhibitors to NOX (Diphenyleneiodonium), mitochondrial electron transport chain (ETC) (rotenone and antimycin A), Nitric oxide synthase (L-NAME) and antioxidants (vitamin E and ascorbic acid). The ROS production, LDH release, NOX activity (lucigenin chemiluminescence), and nitric oxide (NO) production were determined. Mitochondrial ROS was determined by MitoSox oxidation. Protein expression levels of eNOS and nitrotyrosine were determined by western analysis. RESULTS Treatment of LLC-PK1 cells with oxalate increased superoxide, H2O2 generation, LDH release, NOX activity and mitochondrial superoxide generation in a dose and time dependent manner. Pretreatment of LLC-PK1 cells with inhibitors of NOX, mitochondria, and nitric oxide synthase significantly decreased oxalate-induced ROS and LDH release. Oxalate-induced uncoupling of eNOS in LLC-PK1 cells was confirmed by decreased levels of nitric oxide production, eNOS protein expression and increased levels of nitrotyrosine. NOX and mitochondrial inhibitors significantly attenuated oxalate-induced eNOS uncoupling and NO-dependent damage. Vitamin E and ascorbic acid treatment effectively prevented oxalate-induced eNOS uncoupling mediated renal cell injury. CONCLUSIONS We demonstrate that oxalate-induced eNOS uncoupling contributes to additional formation of ROS and subsequent induction of oxidative renal cell injury. Combination of vitamin E and ascorbic acid is more efficient in preventing NOX/mitochondria/eNOS uncoupling mediated renal cell injury. These novel findings suggest that a combination of vitamin E and ascorbic acid should be considered when treating patients with recurrent calcium oxalate kidney stones. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e201 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Vijayalakshmi Thamilselvan More articles by this author Mani Menon More articles by this author Sivagnanam Thamilselvan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Astaxanthin modulates osteopontin and transforming growth factor β1 expression levels in a rat model of nephrolithiasis: a comparison with citrate administration

To evaluate the effect of astaxanthin on renal angiotensin-I converting enzyme (ACE) levels, osteopontin (OPN) and transforming growth factor β1 (TGF-β1) expressions and the extent of crystal deposition in experimentally induced calcium oxalate kidney stone disease in a male Wistar rat model. To compare the efficacy of astaxanthin treatment with a currently used treatment strategy (citrate administration) for kidney stones. The expression of OPN was assessed by immunohistochemistry. One step reverse transcriptase polymerase chain reaction followed by densitometry was used to assess renal OPN and TGF-β1 levels. Renal ACE levels were quantified by an enzyme-linked immunosorbent assay method. Crystal deposition in kidney was analysed by scanning electron microscopic (SEM)-energy-dispersive X-ray (EDX). The renal ACE levels and the expression of OPN and TGF-β1 were upregulated in the nephrolithiasis-induced rats. Astaxanthin treatment reduced renal ACE levels and the expression OPN and TGF-β1. SEM-EDX analysis showed that crystal deposition was reduced in the astaxanthin-treated nephrolithiatic group. Astaxanthin treatment was more effective than citrate administration in the regulation of renal ACE levels, OPN and TGF-β1 expressions. Astaxanthin administration reduced renal calcium oxalate crystal deposition possibly by modulating the renal renin-angiotensin system (RAS), which reduced the expression of OPN and TGF-β1 levels. Astaxanthin administration was more effective than citrate treatment in reducing crystal deposition and down-regulating the expression of OPN and TGF-β1.

Variability in Kidney Stone Incidence Between Black and White South Africans: AGT Pro11Leu Polymorphism Is Not a Factor

Kidney stone disease is rare in the South African black (B) population and more prevalent in the white (W) population. Genetic studies have not previously examined this anomaly. The AGT Pro11Leu polymorphism in the alanine:glyoxylate aminotransferase (AGT) enzyme has been suggested as possibly playing a role in the pathogenesis of idiopathic calcium oxalate kidney stones. The present study was undertaken to investigate whether differences occur in the frequency of this polymorphism in subjects of both race groups. Healthy B (n=60) and W (n=60) male subjects each provided early morning spot urine, blood, and buccal cell samples. The AGT Pro11Leu locus was amplified using the polymerase chain reaction and polymorphism was genotyped using a restriction fragment length polymorphism. There was no difference in the frequency of the AGT Pro11Leu polymorphism, and the major allele (C) was present at a frequency of 0.82 in B and 0.83 in W. Thus, the most common genotype homozygous normal CC genotype was observed at similar frequencies in both groups (0.68 and 0.65 in B and W, respectively), as were the heterozygous CT genotype (CT) and the homozygous variant TT genotype (TT) genotypes (0.33 & 0.02 and 0.28 & 0.03 in B and W, respectively). Neither urinary oxalate nor any other component in the two groups was correlated with the frequency of the AGT Pro11Leu polymorphism. Our data imply that the AGT Pro11Leu polymorphism is not directly responsible for the low incidence of stone formation in B. We conclude that other factors must be instrumental in protecting the B population from urolithiasis.

Editorial Comment

For decades, physicians caring for patients with nephrolithiasis have relied on abnormalities of 24-hour urine composition to explain recurrent stone disease and risk of future stone formation. 1 Levy F.L. Adams-Huet B. Pak C.Y. Ambulatory evaluation of nephrolithiasis: an update of a 1980 protocol. Am J Med. 1995; 98: 50-59 Abstract Full Text PDF PubMed Scopus (277) Google Scholar And yet, we are often puzzled to find either a patient with recurrent stone disease despite having a “normal” 24-hour urine, or a patient whose urine has been modified with drug therapy and nonetheless continues to form additional stones. In recent years, investigators have turned their attention to the immune system and its potential role in stone formation, and perhaps this work will result in future novel therapies to prevent recurrent nephrolithiasis. 2 Holoch P.A. Tracy C.R. Antioxidants and self-reported history of kidney stones: the National Health and Nutrition Examination Survey. J Endourol. 2011; 25: 1903-1908 Crossref PubMed Scopus (45) Google Scholar , 3 Khan S.R. Reactive oxygen species as the molecular modulators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations. J Urol. 2013; 189: 803-811 Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar Specifically, studies have demonstrated that oxidative stress might play an important role in the pathogenesis of calcium oxalate stones, 3 Khan S.R. Reactive oxygen species as the molecular modulators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations. J Urol. 2013; 189: 803-811 Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar and also that markers of oxidative stress and inflammation might be significantly elevated in stone-formers when compared with controls. 2 Holoch P.A. Tracy C.R. Antioxidants and self-reported history of kidney stones: the National Health and Nutrition Examination Survey. J Endourol. 2011; 25: 1903-1908 Crossref PubMed Scopus (45) Google Scholar , 4 Shoag J, Eisner BH. Relationship between C-reactive protein and kidney stone prevalence [e-pub ahead of print]. J Urol. doi: 10.1016/j.juro.2013.09.033, accessed October 4, 2013. Google Scholar Erythrocyte Oxidative Stress in Patients With Calcium Oxalate Stones Correlates With Stone Size and Renal Tubular DamageUrologyVol. 83Issue 2PreviewTo investigate how erythrocyte oxidative stress relates to renal tubular damage and calcium oxalate stone size in patients as oxidative stress has been demonstrated to be associated with stone formation in disease progression. Full-Text PDF ReplyUrologyVol. 83Issue 2PreviewUrinalysis of 24-hour urine sample reflects bona fide microenvironmental changes in kidneys. On the basis of such analysis, physicians prescribe medication to patients with stone disease, but poor outcomes in the form of stone recurrence and renal insufficiency are frequently observed. This clearly indicates a mechanism other than uncovered by urinalysis involved in stone formation contributes to disease progression not only in the kidney but also in association with blood circulation because the kidney has a higher rate of perfusion than any other organ. Full-Text PDF

Peeping into Human Renal Calcium Oxalate Stone Matrix: Characterization of Novel Proteins Involved in the Intricate Mechanism of Urolithiasis

BackgroundThe increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal–membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure.MethodsProteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin–Darby Canine Kidney (MDCK) renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS) followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated.ResultsFive proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level.ConclusionsWe identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone formation. Thus, these proteins having potential to modulate calcium oxalate crystallization will throw light on understanding and controlling urolithiasis in humans.

Diversity in protein profiles of individual calcium oxalate kidney stones.

Calcium oxalate kidney stones contain low amounts of proteins, some of which have been implicated in progression or prevention of kidney stone formation. To gain insights into the pathophysiology of urolithiasis, we have characterized protein components of calcium oxalate kidney stones by proteomic approaches. Proteins extracted from kidney stones showed highly heterogeneous migration patterns in gel electrophoresis as reported. This was likely to be mainly due to proteolytic degradation and protein-protein crosslinking of Tamm-Horsfall protein and prothrombin. Protein profiles of calcium oxalate kidney stones were obtained by in-solution protease digestion followed by nanoLC-MALDI-tandem mass spectrometry, which resulted in identification of a total of 92 proteins in stones from 9 urolithiasis patients. Further analysis showed that protein species and their relative amounts were highly variable among individual stones. Although proteins such as prothrombin, osteopontin, calgranulin A and calgranulin B were found in most stones tested, some samples had high contents of prothrombin and osteopontin, while others had high contents of calgranulins. In addition, calgranulin-rich stones had various neutrophil-enriched proteins such as myeloperoxidase and lactotransferrin. These proteomic profiles of individual kidney stones suggest that multiple systems composed of different groups of proteins including leucocyte-derived ones are differently involved in pathogenesis of individual kidney stones depending on situations.