No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Calcium oxalate stone formation in genetic hypercalciuric stone-forming rats
Abstract
Over 54 generations, we have successfully bred a strain of rats that maximizes urinary calcium excretion. The rats now consistently excrete 8 to 10 times as much calcium as controls, uniformly form poorly crystalline calcium phosphate kidney stones, and are termed genetic hypercalciuric stone-forming (GHS) rats. These rats were used to test the hypothesis that increasing urinary oxalate excretion would not only increase the supersaturation with respect to the calcium oxalate solid phase, but also would increase the ratio of calcium oxalate-to-calcium phosphate supersaturation and result in calcium oxalate stone formation.
To increase urine oxalate excretion an oxalate precursor, hydroxyproline, was added to the diet of male GHS rats. The GHS rats were fed a standard 1.2% calcium diet alone or with 1%, 3% or 5% trans-4-hydroxy-l-proline (hydroxyproline).
The addition of 1% hydroxyproline to the diet of GHS rats led to an increase in urinary oxalate excretion, which did not increase further with the provision of additional hydroxyproline. The addition of 1% and 3% hydroxyproline did not alter calcium excretion while the provision of 5% hydroxyproline led to a decrease in urine calcium excretion. The addition of 1% hydroxyproline led to an increase in urinary calcium oxalate supersaturation, which did not further increase with additional hydroxyproline. The addition of 1% and 3% hydroxyproline did not alter urinary supersaturation with respect to calcium hydrogen phosphate while the addition of 5% hydroxyproline tended to lower this supersaturation. Compared to rats fed the control and the 3% hydroxyproline diet the addition of 5% hydroxyproline increased the ratio of calcium oxalate supersaturation to calcium phosphate supersaturation. Virtually all rats formed stones. In the control and 1% hydroxyproline group, all of the stones were composed of calcium and phosphate (apatite), in the 3% hydroxyproline group the stones were a mixture of apatite and calcium oxalate, while in the 5% hydroxyproline group all of the stones were calcium oxalate.
The provision of additional dietary hydroxyproline to GHS rats increases urinary oxalate excretion, calcium oxalate supersaturation and the ratio of calcium oxalate-to-calcium phosphate supersaturation, resulting in the formation of calcium oxalate kidney stones. Thus, with the addition of a common amino acid, the GHS rats now not only model the most common metabolic abnormality found in patients with nephrolithiasis, hypercalciuria, but form the most common type of kidney stone, calcium oxalate.
... Although several studies have previously investigated the physiological effects of gelatin or pure hydroxyproline in animals [13][14][15][16] and humans [1,2,17,18], urinary pH was found in their rat study that urinary pH increased significantly relative to controls after 6-week ingestion of a diet supplemented with hydroxyproline but there were no further increases after 12 weeks and 18 weeks on the same diet [13]. Khan et al. found that urinary pH was generally lower in rats which had been fed hydroxyproline compared to controls, but this was not statistically significant [15]. ...
... Although several studies have previously investigated the physiological effects of gelatin or pure hydroxyproline in animals [13][14][15][16] and humans [1,2,17,18], urinary pH was found in their rat study that urinary pH increased significantly relative to controls after 6-week ingestion of a diet supplemented with hydroxyproline but there were no further increases after 12 weeks and 18 weeks on the same diet [13]. Khan et al. found that urinary pH was generally lower in rats which had been fed hydroxyproline compared to controls, but this was not statistically significant [15]. ...
... Calcium speciation calculated by JESS at baseline and after low and high doses of hydroxyproline reported in only three of them[13,15,16]. Buchinsky et al. ...
Purpose
Dietary hydroxyproline may be involved in the endogenous synthesis of oxalate. Glycolate, produced during the metabolism of hydroxyproline, may exert physicochemical effects on urinary calcium by virtue of its dihydroxycarboxylic acid structure. The aim of this study was to investigate these possible stone-risk scenarios.
Methods
We modelled the effect of different glycolic acid concentrations on ionized calcium (iCa²⁺) and relative supersaturation (RSS) of calcium oxalate (CaOx) using the program JESS. Thereafter, three healthy white males and two healthy black males ingested 30 g gelatin for 3 days. 24-h urines were collected at baseline and after completion of the protocol. Urines were analysed for physicochemical risk factors and for iCa²⁺ and glycolic acid. Speciation concentrations and RSS values were calculated.
Results
Theoretical modelling showed that binding between calcium and glycolate does not occur and that iCa²⁺ and RSS CaOx are unaffected. However, after ingestion of hydroxyproline, iCa²⁺ decreased significantly. Urinary pH and glycolate increased significantly. Oxalate excretion and RSS CaOx did not change
Conclusions
We attribute the decrease in iCa²⁺ to increases in the concentrations of several Ca–phosphate species, the formation of which is due to the increase in pH. We speculate that the absence of an increase in oxalate excretion despite an increase in glycolate excretion may be due to the mixed racial composition of our test group in which some pathways may be preferred to others. Our findings alert stone researchers to the importance of measuring urinary pH in their workup of subjects and to select racially homogenous groups for investigation.
... When fed a standard Ca diet, all GHS rats develop kidney stones [34,35] composed of calcium phosphate (CaP) [34,[36][37][38]. The addition of hydroxyproline to the diet of GHS rats results in CaOx stone formation [39][40][41]. In this study, GHS rats were fed either a normal Na diet (NNaD, 0.4%) or a low Na diet (LNaD, 0.05%). ...
... All of these methods have been used previously [31,36,[42][43][44]. Urine SS With the measured solute excretion, the urinary SS with respect to CaOx, CaP, and UA solid phases were calculated using the computer program EQUIL2 [45] as we have done previously [37,39,44,46,47]. Ratios of 1 denote a urine at equilibrium, those > 1 denote SS and those < 1 denote undersaturation. ...
... GHS rats have alterations in Ca homeostasis including increased intestinal absorption, reduced renal tubular reabsorption, and increased bone resorption, similar to those of many patients with idiopathic IH [24]. Hypercalciuria in the GHS rats leads directly to stone formation as it does in humans [1,37,39]. GHS rats also have reduced BMD, as do humans with nephrolithiasis, as well as lower trabecular volume and thickness compared to control Sprague-Dawley rats [32,39]. Previous studies on the effect of dietary Na intake, performed on humans with IH, as well as normal rats [48,49], demonstrate that increased dietary Na leads to increased hypercalciuria, as well as an increase in osteoporotic risk factors such as increased bone resorption and decreased BMD [48,49]. ...
Background:
Urine (u) calcium (Ca) excretion is directly dependent on dietary sodium (Na) intake leading to the recommendation for Na restriction in hypercalciuric kidney stone formers. However, there is no direct evidence that limiting Na intake will reduce recurrent stone formation.
Materials and methods:
We used genetic hypercalciuric stone-forming (GHS) rats, which universally form Ca phosphate (P) kidney stones, fed either a low Na (LNa, 0.05%) or normal Na (NNa, 0.4%) Na diet (D) for 18 weeks. Urine was collected at 6-week intervals. Radiographic analysis for stone formation and bone analyses were done at the conclusion of the study.
Results:
Mean uCa was lower with LNaD than NNaD as was uP and LNaD decreased mean uNa and uChloride. There were no differences in urine supersaturation (SS) with respect to calcium phosphate (CaP) or Ca oxalate (CaOx). However, stone formation was markedly decreased with LNaD by radiographic analysis. The LNaD group had significantly lower femoral anterior-posterior diameter and volumetric bone mineral density (vBMD), but no change in vertebral trabecular vBMD. There were no differences in the bone formation rate or osteoclastic bone resorption between groups. The LNaD group had significantly lower femoral stiffness; however, the ultimate load and energy to fail was not different.
Conclusion:
Thus, a low Na diet reduced uCa and stone formation in GHS rats, even though SS with respect to CaP and CaOx was unchanged and effects on bone were modest. These data, if confirmed in humans, support dietary Na restriction to prevent recurrent Ca nephrolithiasis.
... [4][5][6] When fed a standard, ample calcium diet, all GHS rats develop kidney stones, 27,28,31,32 which are composed of CaP. 27, 31,33,34 Addition of hydroxyproline to the diet of GHS rats results in CaOx stone formation. 37,38,40 To determine the effects of K-cit on urine solute excretion, supersaturation with respect to the common stone solid phases, and CaP stone formation, GHS rats were fed a normal calcium diet without hydroxyproline with K-cit or potassium chloride (KCl), as control. ...
... The GHS rats were derived from Sprague-Dawley rats (Charles River Laboratories, Kingston, NY) by successively inbreeding the most hypercalciuric progeny of each generation. [24][25][26][27][36][37][38][39]49 Eight-weekold male GHS rats from the 95th generation were used in this study. ...
... With the measured solute excretion, the urinary supersaturation with respect to CaOx, CaP, and uric acid solid phases were calculated using the computer program EQUIL 2 70 , as we have done previously. 28,29,33,34,37,40,43,48,[67][68][69] Ratios of 1 denote a urine at equilibrium, those .1 denote supersaturation, and those ,1 denote undersaturation. We have found excellent correspondence between calculated and experimentally measured saturation in urine and blood and in bone culture medium. ...
Potassium citrate is prescribed to decrease stone recurrence in patients with calcium nephrolithiasis. Citrate binds intestinal and urine calcium and increases urine pH. Citrate, metabolized to bicarbonate, should decrease calcium excretion by reducing bone resorption and increasing renal calcium reabsorption. However, citrate binding to intestinal calcium may increase absorption and renal excretion of both phosphate and oxalate. Thus, the effect of potassium citrate on urine calcium oxalate and calcium phosphate supersaturation and stone formation is complex and difficult to predict. To study the effects of potassium citrate on urine supersaturation and stone formation, we utilized 95th-generation inbred genetic hypercalciuric stone-forming rats. Rats were fed a fixed amount of a normal calcium (1.2%) diet supplemented with potassium citrate or potassium chloride (each 4 mmol/d) for 18 weeks. Urine was collected at 6, 12, and 18 weeks. At 18 weeks, stone formation was visualized by radiography. Urine citrate, phosphate, oxalate, and pH levels were higher and urine calcium level was lower in rats fed potassium citrate. Furthermore, calcium oxalate and calcium phosphate supersaturation were higher with potassium citrate; however, uric acid supersaturation was lower. Both groups had similar numbers of exclusively calcium phosphate stones. Thus, potassium citrate effectively raises urine citrate levels and lowers urine calcium levels; however, the increases in urine pH, oxalate, and phosphate levels lead to increased calcium oxalate and calcium phosphate supersaturation. Potassium citrate induces complex changes in urine chemistries and resultant supersaturation, which may not be beneficial in preventing calcium phosphate stone formation.
Copyright © 2015 by the American Society of Nephrology.
... Studies in humans (Knight et al., 2006), rats Gershoff, 1981, 1982;Bushinsky et al., 2002;Takayama et al., 2003;Ogawa et al., 2007), and mice (Jiang et al., 2012) have shown that substantial amounts of the glyoxylate and oxalate are synthesized endogenously through the metabolism of the AA hydroxyproline (hyp). In a randomized controlled trial, cats fed diets containing collagen (rich in hyp) had a 2-to 3-fold greater Uox excretion compared with diets containing soy isolate and horse meat as the protein source (Zentek and Schulz, 2004). ...
... The additional Uox excretion with increasing hyp intake (Lhyp-Lox, Mhyp-Lox, and Hhyp-Lox) was of endogenous origin because the fecal oxalate output was similar among these diets, and the greater negative oxalate balance indicated that endogenous synthesis of oxalate occurred. The l-hydroxyproline can be metabolized to glyoxylate and then to oxalate in the hepatocyte Gershoff, 1981, 1982;Bushinsky et al., 2002;Takayama et al., 2003;Ogawa et al., 2007;Jiang et al., 2012) and subsequently excreted in the urine. In the present study, 0.32% (on a molar basis) of the supplemented hyp was recovered as oxalate in the urine. ...
... This result is consisting with the results obtained by other researchers [21]. The high level of 24-h urinary oxalate causes an increase in the supersaturation of the CaOx solid phase, which leads to the production of CaOx stones [23]. ...
Contradictory results are existed in the literature regarding the impact of trace elements on the pathogenesis of calcium oxalate (CaOx) stone patients. Therefore, the aim of our study was to investigate the effect of Cu and Zn on biochemical and molecular characteristics of CaOx stones. Plasma and urine concentrations of Cu and Zn in 30 CaOx stones patients and 20 controls were determined by flame atomic absorption spectrometry (FAAS). Urinary levels of citric acid and oxalate were measured by commercial spectrophotometric kits. Blood levels of glutathione reduced (GSH) and catalase (CAT) were determined as markers of antioxidant activity, while blood malondialdehyde (MDA) and urine level of nitric oxide (NO) were used to assess oxidative stress. Gene expression of MAPk pathway (ERK, P38, and JNK) were estimated. The plasma and urine levels of Cu were significantly increased in the patient group compared to those of controls, while the levels of Zn were decreased. Excessive urinary excretion of citric acid and oxalate were found among CaOx stone patients. The GSH and CAT concentration were significantly reduced in CaOx stones patients compared to healthy group. The plasma MDA and urine NO concentration were significantly increased in CaOx stones patients compared to control group. The expressions of the studied genes were significantly increased in CaOx stones patients. These findings suggest that alteration in Cu and Zn might contribute to pathogenesis of CaOx patients through oxidative stress and MAPK pathway genes (ERK, P38 and JNK).
... Urine measurements included volume, calcium, magnesium, oxalate, phosphate, pH, uric acid (UA), sodium, potassium, chloride, creatinine, citrate, and ammonia; serum measurements included calcium, phosphate, UA, creatinine, magnesium, sodium, potassium, chloride and bicarbonate, using previously published methods [11]. Gastrointestinal (GI) anion was calculated as previously described [12]. ...
We examined how physicians made therapeutic choices to decrease stone risk in patients with bowel disease without colon resection, many of whom have enteric hyperoxaluria (EH), at a single clinic. We analyzed clinic records and 24-h urine collections before and after the first clinic visit, among 100 stone formers with bowel disease. We used multivariate linear regression and t tests to compare effects of fluid intake, alkali supplementation, and oxalate-focused interventions on urine characteristics. Patients advised to increase fluid intake had lower initial urine volumes (L/day; 1.3 ± 0.5 vs. 1.7 ± 0.7) and increased volume more than those not so advised (0.7 ± 0.6 vs. 0.3 ± 0.6 p = 0.03; intervention vs. non-intervention). Calcium oxalate supersaturation (CaOx SS) fell (95% CI −4.3 to −0.8). Alkali supplementation increased urine pH (0.34 ± 0.53 vs. 0.22 ± 0.55, p = 0.26) and urine citrate (mg/d; 83 ± 256 vs. 98 ± 166, p = 0.74). Patients advised to reduce oxalate (mg/day) absorption had higher urine oxalate at baseline (88 ± 44 vs. 50 ± 26) which was unchanged on follow-up (88 (baseline) vs. 91 (follow-up), p = 0.90). Neither alkali (95% CI −1.4 to 2.1) nor oxalate-focused advice (95% CI −1.2 to 2.3) lowered CaOx SS. Physicians chose treatments based on baseline urine characteristics. Advice to increase fluid intake increased urine volume and decreased CaOx SS. Alkali and oxalate interventions were ineffective.
... Nephrolithiasis is mainly associated with various systemic comorbidities, primarily affecting calcium metabolism, lipid metabolism, and diabetes mellitus. Calcium-containing stones are most prevalent among the patients, mainly composed of calcium phosphate and calcium oxalate (CaOx) [2]. ...
Objective: The search for anti-calculi drugs from natural sources has been believed of greater importance. Hence, the present study explored the effectiveness of lycopene against experimentally induced nephrolithiasis. Methods: The experimental study lasted for 28 days. Adult male Wistar rats were divided into six groups. Group I (Normal control) received drinking water. Group II (Disease control) received 0.75% ethylene glycol and 1% ammonium chloride in drinking water to induce nephrolithiasis. Group III–V was treated with lycopene (50 mg/kg, 100 mg/kg, and 200 mg/kg, p.o.) along with 0.75% ethylene glycol and 1% ammonium chloride. Group VI treated standard (750 mg/kg, p.o.) along with 0.75% ethylene glycol and 1% ammonium chloride. Results: The study results showed significantly high levels of urinary and serum creatinine, urea, calcium, and uric acid levels and a decrease in magnesium levels in Group II (Disease control) compared with Group I (Normal control). Treatment with lycopene (50 mg/kg, 100 mg/kg, and 200 mg/kg) restored the elevated urinary and serum parameters in Group III–VI compared with Group II. Ethylene glycol administrations lead to the production of oxidative stress and decrease superoxide dismutase, reduced glutathione, and catalase activity. Lycopene treatment restored the elevated oxidative stress parameters to normal. Histologically, lycopene has alleviated the damaged integrity of the renal structure. Conclusion: Supplementation with lycopene (100 mg/kg and 200 mg/kg) reduces and prevents the toxicity caused by ethylene glycol administration and protects the renal cells from damage.
... A patient is considered to exhibit hypercalciuria when the amount of calcium in urine reaches values greater than 300 mg/24 h for men and 250 mg/24 h for women. 2,7,23,24 The daily oxalate excretion can vary between 10 and 40 mg per 24 h. The amount of oxalate exceeding 40− 45 mg per 24 h is considered as hyperoxaluria. ...
The increasing prevalence of urolithiasis in industrialized societies triggered considerable interest in how various species found in urine regulate the nucleation and growth of common kidney stone constituents such as calcium oxalate (CaOx). Yet, the role macromolecules play in kidney stone formation is often overlooked due to their low concentration in urine. In this study, we investigate the nucleation kinetics of CaOx in artificial urine with droplet-based microfluidic induction time measurements at varying concentrations of oxalate and hyaluronic acid (HA), a polysaccharide commonly found in urine. The formation of two pseudo-polymorphic forms of calcium oxalate crystals, calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD), are carefully monitored using polarized light microscopy in induction time experiments. COM and COD nucleated concomitantly in artificial urine yet with distinct kinetics. Our results indicate that higher oxalate concentrations favor the formation of COD, the metastable form, over COM, the most stable form. Moreover, COD is also the fastest nucleating form in droplets under studied conditions. Furthermore, increasing the concentration of HA at fixed calcium and oxalate concentrations favored the nucleation of COM. We observed that in droplets where COM nucleated first, COD was not formed within the experimental time scale. However, in the droplets where COD appeared first, COM crystals were also observed later. We hope our findings shed light on the role macromolecules such as HA plays in dictating the pseudo-polymorphic form of CaOx and guide next generation treatments.
... This condition is referred to as hypercalciuria, i.e., high Ca levels in the urine, and the amount of calcium can reach values greater than 300 mg/24 h for men and 250 mg/24 h for women. 2,5,18,19 In the human body, there are numerous organic and inorganic compounds that might either facilitate (promoters) or prevent (inhibitors) stone formation. Low urine volume and high calcium, sodium, oxalate, and urate concentrations in the urine are known to promote the formation of kidney stones. ...
A droplet-based microfluidic platform is presented to study the nucleation kinetics of calcium oxalate monohydrate (COM), the most
common constituent of kidney stones, while carefully monitoring the pseudo-polymorphic transitions. The precipitation kinetics of
COM is studied as a function of supersaturation and pH as well as in the presence of inhibitors of stone formation, magnesium ions
(Mg2+), and osteopontin (OPN). We rationalize the trends observed in the measured nucleation rates leveraging a solution chemistry
model validated using isothermal solubility measurements. In equimolar calcium and oxalate ion concentrations with different buffer solutions,
dramatically slower kinetics is observed at pH 6.0 compared to pHs 3.6 and 8.6. The addition of both Mg2+ and OPN to the solution
slows down kinetics appreciably. Interestingly, complete nucleation inhibition is observed at significantly lower OPN, namely,
3.2 × 10−8 M, than Mg2+ concentrations, 0.875 × 10−4 M. The observed inhibition effect of OPN emphasizes the often-overlooked role of
macromolecules on COM nucleation due to their low concentration presence in urine. Moreover, analysis of growth rates calculated
from observed lag times suggests that inhibition in the presence of Mg2+ cannot be explained solely on altered supersaturation. The presented
study highlights the potential of microfluidics in overcoming a major challenge in nephrolithiasis research, the overwhelming
physiochemical complexity of urine.
... Many studies show that calcium oxalate can be inherited. (12,13) Lifestyle and dietary habits were also important factors for stone formation (14) . Due to the instructions of stone prevention, such as "Drinking more than 8 glasses of water per day can protect against stone formation" (15) , our study found that most of the urolithiasis patients drank more than 8 glasses per day. ...
Objective: To study and analyze stones in urolithiasis patients in Loei province. Material and Method: A case-control study that included 161 urolithiasis patients who underwent any treatment at Loei Hospital and received stone infrared spectroscopy and 170 patients who did not have urolithiasis or a history of urolithiasis with investigations that did not show urinary tract stones between July 2015 and January 2016. The parameters of the demographic data, urolithiasis data, and stone analysis data were collected and analyzed using the descriptive statistic method. Result: The percentage of urolithiasis patients in Loei province who had a family history of urolithiasis was 24.8%. The most common occupation was farmer at 80.1%. Boiled water and bottled water were statistically significant for stone prevention. Papaya salad with pickled fish and Laab/Koi eaten by patients more than 3 times per day were statistically significant for stone formation. The percentage of urolithiasis patients in Loei province who had pure stones was 56%, and with more than mixed stones 44%. Stone types, regarding the major components found: the most common group was oxalate, followed by the phosphate group and the uric group. Conclusion: Urolithiasis in Loei province comprised pure stones more than mixed stones. Stone type with regard to major components found: the most common group was oxalate. Risk factors for stone formation were papaya salad with pickled fish and Laab/Koi. Boiled water and bottled water were protective urinary tract stone formation factors.
... Twenty-four-hour urine data included volume, creatinine, calcium, oxalate, citrate, potassium, uric acid, pH, chloride, phosphorus, magnesium, ammonium, and sulfate. Measurements were made using methods detailed elsewhere (Bushinsky et al., 2002). ...
Background:
Animal models have demonstrated an interactive relationship between the epithelial anion exchanger SLC26A6 and transporter NaDC-1 that regulates citrate and oxalate homeostasis. This relationship is a potential mechanism to protect against kidney stones as higher urine oxalate is accompanied by higher urine citrate but it has not been explored in humans.
Methods:
We examined 24-h urine data on 13,155 kidney stone forming patients (SF) from separate datasets at the University of Chicago and Litholink, a national laboratory, and 143 non-kidney stone forming participants (NSF) to examine this relationship in humans. We used multivariate linear regression models to examine the association between oxalate and citrate in all study participants and separately in SF and NSF.
Results:
Higher urinary oxalate was associated with higher urinary citrate in both SF and NSF. In NSF, the multivariate adjusted urine citrate excretion was 3.0 (1.5-4.6) (mmol)/creatinine (mmol) per oxalate (mmol)/creatinine (mmol). In SF, the multivariate adjusted urine citrate excretion was 0.3 (0.2-0.4) (mmol)/creatinine (mmol) per oxalate (mmol)/creatinine (mmol).
Conclusions:
Higher urinary oxalate excretion was associated with higher urinary citrate excretion and this effect was larger in non-kidney stone forming participants compared with those who form kidney stones.
... Hydroxy-L-proline (HLP), a physiologic precursor of oxalate is also used for induction of hyperoxaluria in the rats by oral administration or by intraperitoneal injection [54,143]. Other hyperoxaluric rat models are produced by inbreeding of hyperoxaluric progeny [144,145], the administration of sodium oxalate [146], or glycolic acid [147]. In addition, several other models of nephrolithiasis have been developed such as transgenic mouse with selective knockout (KO) of osteopontin (OPN) [148] and Tamm-Horsefall protein (THP) [149], oxalate, sodium phosphate [150], and cysteine transporter [151]. ...
Urolithiasis is one of the oldest diseases affecting humans, while plants are one of our oldest companions providing food, shelter, and medicine. In spite of substantial progress in understanding the pathophysiological mechanisms, treatment options are still limited, often expensive for common people in most parts of the world. As a result, there is a great interest in herbal remedies for the treatment of urinary stone disease as an alternative or adjunct therapy. Numerous in vivo and in vitro studies have been carried out to understand the efficacy of herbs in reducing stone formation. We adopted PRISMA guidelines and systematically reviewed PubMed/Medline for the literature, reporting results of various herbal products on in vivo models of nephrolithiasis/urolithiasis. The Medical Subject Heading Terms (Mesh term) “Urolithiasis” was used with Boolean operator “AND” and other related Mesh Unique terms to search all the available records (July 2019). A total of 163 original articles on in vivo experiments were retrieved from PubMed indexed with the (MeshTerm) “Urolithiasis” AND “Complementary Therapies/Alternative Medicine, “Urolithiasis” AND “Plant Extracts” and “Urolithiasis” AND “Traditional Medicine”. Most of the studies used ethylene glycol (EG) to induce hyperoxaluria and nephrolithiasis in rats. A variety of extraction methods including aqueous, alcoholic, hydro-alcoholic of various plant parts ranging from root bark to fruits and seeds, or a combination thereof, were utilized. All the investigations did not study all aspects of nephrolithiasis making it difficult to compare the efficacy of various treatments. Changes in the lithogenic factors and a reduction in calcium oxalate (CaOx) crystal deposition in the kidneys were, however, considered favorable outcomes of the various treatments. Less than 10% of the studies examined antioxidant and diuretic activities of the herbal treatments and concluded that their antiurolithic activities were a result of antioxidant, anti-inflammatory, and/or diuretic effects of the treatments.
... A similar protocol was used for screening and inbreeding of subsequent generations as described previously. (23,24,26,29,31,34,36,(38)(39)(40)(41)(42)(43) Beyond the 30th generation, GHS rats have consistently excreted 8 to 10 times more urine Ca than of WT SD rats. Urine Ca excretion in SD rats has not varied with time. ...
To study human idiopathic hypercalciuria (IH) we developed an animal model, genetic hypercalciuric stone‐forming (GHS) rats, whose pathophysiology parallels that in IH. All GHS rats form kidney stones and have decreased bone mineral density (BMD) and bone quality compared to the founder Sprague‐Dawley (SD) rats. To understand the bone defect we characterized osteoclast and osteoblast activity in the GHS compared to SD rats. Bone marrow cells were isolated from femurs of GHS and SD rats and cultured to optimize differentiation into osteoclasts or osteoblasts. Osteoclasts were stained for TRAcP, cultured to assess resorptive activity and analyzed for specific gene expression. Marrow stromal cells or primary neonatal calvarial cells were differentiated to osteoblasts and osteoblastic gene expression as well as mineralization was analyzed. There was increased osteoclastogenesis and increased resorption pit formation in GHS compared to SD cultures. Osteoclasts had increased expression of cathepsin K, Tracp and MMP9 in cells from GHS compared to SD rats. Osteoblastic gene expression and mineralization was significantly decreased. Thus alterations in baseline activity of both osteoclasts and osteoblasts in GHS rats, perhaps due to their known increase in vitamin D receptors, lead to decreased BMD and bone quality, perhaps due to their known increase in vitamin D receptors. Better understanding of the role of GHS bone cells in decreased BMD and quality may provide new strategies to mitigate the low BMD and increased fracture risk found in patients with IH. This article is protected by copyright. All rights reserved.
... 16,[26][27][28][29][30] Urine Supersaturation Urine supersaturation with respect to CaOx and CaP solid phases were calculated from solute measurements using the computer program EQUIL2, 31 as we have done previously. 10,27,30,[32][33][34] Kidney Stone Formation Kidneys and ureters were removed from each rat en bloc, frozen, and imaged in a Faxitron radiography device (Tucson, AZ) to determine extent of kidney stone formation. Three observers blinded to treatment scored all radiographs on a scale ranging from 0 (no stones) to 4 (extensive stones). ...
Background:
The pathophysiology of genetic hypercalciuric stone-forming rats parallels that of human idiopathic hypercalciuria. In this model, all animals form calcium phosphate stones. We previously found that chlorthalidone, but not potassium citrate, decreased stone formation in these rats.
Methods:
To test whether chlorthalidone and potassium citrate combined would reduce calcium phosphate stone formation more than either medication alone, four groups of rats were fed a fixed amount of a normal calcium and phosphorus diet, supplemented with potassium chloride (as control), potassium citrate, chlorthalidone (with potassium chloride to equalize potassium intake), or potassium citrate plus chlorthalidone. We measured urine every 6 weeks and assessed stone formation and bone quality at 18 weeks.
Results:
Potassium citrate reduced urine calcium compared with controls, chlorthalidone reduced it further, and potassium citrate plus chlorthalidone reduced it even more. Chlorthalidone increased urine citrate and potassium citrate increased it even more; the combination did not increase it further. Potassium citrate, alone or with chlorthalidone, increased urine calcium phosphate supersaturation, but chlorthalidone did not. All control rats formed stones. Potassium citrate did not alter stone formation. No stones formed with chlorthalidone, and rats given potassium citrate plus chlorthalidone had some stones but fewer than controls. Rats given chlorthalidone with or without potassium citrate had higher bone mineral density and better mechanical properties than controls, whereas those given potassium citrate did not.
Conclusions:
In genetic hypercalciuric stone-forming rats, chlorthalidone is superior to potassium citrate alone or combined with chlorthalidone in reducing calcium phosphate stone formation and improving bone quality.
... In our second study we will use the genetic hypercalciuric stone-forming (GHS) rat to determine if calcium stones can be prevented in vivo. The GHS rats spontaneously form CaP stones when fed standard rat chow [57], but will form CaOx stones when fed a diet enriched with hydroxyproline [58]. The rat studies will allow us to address both types of calcium stones and use kidney stone formation as an end point in a time frame much shorter than feasible in a human study. ...
Alkali supplements are used to treat calcium kidney stones owing to their ability to increase urine citrate excretion which lowers stone risk by inhibiting crystallization and complexing calcium. However, alkali increases urine pH, which may reduce effectiveness for patients with calcium phosphate stones and alkaline urine. Hydroxycitrate is a structural analog of citrate, widely available as an over-the-counter supplement for weight reduction. In vitro studies show hydroxycitrate has the capacity to complex calcium equivalent to that of citrate and that it is an effective inhibitor of calcium oxalate monohydrate crystallization. In fact, hydroxycitrate was shown to dissolve calcium oxalate crystals in supersaturated solution in vitro. Hydroxycitrate is not known to be metabolized by humans, so it would not be expected to alter urine pH, as opposed to citrate therapy. Preliminary studies have shown orally ingested hydroxycitrate is excreted in urine, making it an excellent candidate as a stone therapeutic. In this article, we detail the crystal inhibition activity of hydroxycitrate, review the current knowledge of hydroxycitrate use in humans, and identify gaps in knowledge that require appropriate research studies before hydroxycitrate can be recommended as a therapy for kidney stones.
... 4 Patients with hypercalciuria often excrete more calcium than they absorb, reflecting a net loss of total body calcium. 5 In the present study, an excessive urinary calcium excretion was associated with osteoporosis in women, but not to osteopenia. In the multiple logistical regression analysis, the effect of an excessive urinary calcium excretion on osteoporosis, independent of the gender or BMI, was substantial (OR= 3.26 and p<0.05). ...
... Modification of the diet by high dosing of HP or ethylene glycol may lead to appetite loss, lowering or gaining weight and may interfere the water intake and urine excretion. 10,11 Moreover, some data suggests that it may also cause metabolic acidosis and may lead to renal dysfunction. 10 All of the above mentioned discrepancies may have an 727 none none minimal none none minimal 747 none none minimal none none minimal 757 none none none none none minimal 760 none none minimal none none minimal 765 minimal minimal minimal none minimal moderate 777 minimal minimal moderate none minimal moderate 724 none none minimal none none minimal 730 none minimal minimal none none minimal 735 none minimal minimal none minimal minimal 751 none none none none none none 759 none none minimal none none minimal 775 minimal minimal moderate minimal minimal moderate 731 minimal minimal moderate minimal minimal moderate 749 none none none none none none 754 none none none none none none 761 none minimal minimal none minimal moderate 763 minimal minimal minimal minimal minimal moderate 783 none none minimal none none impact on the results of the studies with enzymes, drugs, etc., for which, in fact, animal kidney stone models are designed for, and, which is obviously of great importance for final outcome of studies. ...
Introduction. Kidney stone formation may be a result of increased urinary oxalate supersaturation.
Material and Methods. Eighteen pigs were randomly divided into: Control group, where standard cereal-based feed was supplemented
with 4% HP only, Prevention group, where treatment with OxDc slurry started at the end of the adaptation period
when pigs were switched to 4% HP diet, Reduction group, where the treatment with OxDc lyo powder started after pigs were
already on a 4% HP diet for 6 days.
Results. OxDc slurry prevented oxalate excretion in urine. The reduction effect of OxDc lyo feed addition was generally visible
during the first two days of the therapy (p<0.05). Both dietary intake of 4% HP and OxDc preparations did not influence weight
gain, water or feed intake, urine excretion and creatinine clearance.
Conclusions. The capacity of OxDc in preventing induced hyperoxaluria was moderate. Most probably, this is due to the incoherent
response of animals to the HP enriched diet dependent on their gut pH, since optimum pH for OxDc is around 5-6.
A higher pH essentially reduces the activity of OxDc. The capacity of OxDc in reversing the hyperoxaluria induced by a HP enriched
diet was significant during the first 2 days after introducing OxDc to the diet.
Keywords. Nephrocalcinosis, Oxalate decarboxylase, Hydroxyproline, Pig model
... In young pigs fed sow's milk that naturally contains a large amount of Hyp, this AA is a major substrate for endogenous synthesis of glycine. This recent finding challenges the traditional view that Hyp in its free or small-peptide form is merely a metabolic waste in animals, including pigs (Bushinsky et al. 2002;Khan et al. 2006;Mandel et al. 2004 ...
Glycine, proline, and hydroxyproline (Hyp) contribute to 57% of total amino acids (AAs) in collagen, which accounts for one-third of proteins in animals. As the most abundant protein in the body, collagen is essential to maintain the normal structure and strength of connective tissue, such as bones, skin, cartilage, and blood vessels. Mammals, birds, and fish can synthesize: (1) glycine from threonine, serine, choline, and Hyp; (2) proline from arginine; and (3) Hyp from proline residues in collagen, in a cell- and tissue-specific manner. In addition, livestock (e.g., pigs, cattle, and sheep) produces proline from glutamine and glutamate in the small intestine, but this pathway is absent from birds and possibly most fish species. Results of the recent studies indicate that endogenous synthesis of glycine, proline, and Hyp is inadequate for maximal growth, collagen production, or feed efficiency in pigs, chickens, and fish. Although glycine, proline and Hyp, and gelatin can be used as feed additives in animal diets, these ingredients except for glycine are relatively expensive, which precludes their inclusion in practical rations. Alternatively, hydrolyzed feather meal (HFM), which contains 9% glycine, 5% Hyp, and 12% proline, holds great promise as a low cost but abundant dietary source of glycine, Hyp, and proline for ruminants and nonruminants. Because HFM is deficient in most AAs, future research efforts should be directed at improving the bioavailability of its AAs and the balance of AAs in HFM-supplemented diets. Finally, HFM may be used as a feed additive to prevent or ameliorate connective tissue disorders in domestic and aquatic animals.
... Physiological experiments on animals in the 17th century are documented [4]. Ogawa et al. [50] Rats Glycolic acid in diet X X X Khan et al. [51] Rats Ethylene glycol in drinking water for 8 weeks X X X X Bushinsky et al. [52] Genetic hypercalciuric rats 1, 3 or 5% HP in drinking water for 18 weeks X X X Khan et al. [53] Rats 5% HP diet for 63 days X X X X Canales et al. [54] Rats Diet induced obese rats s/p Roux-en-Y gastric bypass surgery X Mandel et al. [55] Young pigs 10% HP added to standard diet for 20 days X X X X Content courtesy of Springer Nature, terms of use apply. Rights reserved. ...
Animal models are useful in the study of many human diseases. Our current understanding of the biological, physiological, and biochemical aspects of hyperoxaluria and calcium oxalate urolithiasis has been greatly informed by studies using animals. Recently, limitations in the extrapolation to humans of research results derived from laboratory rodents have been identified. The use in biomedical research of a variety of organisms, including large animals, is increasingly encouraged. The purpose of this article is to review the use of pigs in biomedical and stone research, to provide a rationale for using pigs in metabolic stone research, and to describe our 8-year experience in developing a porcine platform for studying hyperoxaluria and calcium oxalate urolithiasis. In this article, we share and review some of the highlights of our findings. We also report results from a recent feeding swine study that demonstrated oxalate-induced renal nephropathy. Finally, we offer ideas for future directions in urolithiasis research using swine.
... Consumption of AIN-76 diet by female rats results in the intratubular deposition of CaP crystals, mostly at the cortico-medullary junction [79], where proximal tubules and loops of Henle meet, somewhat similar to what happens in rats with hyperoxaluria [28]. Increased urinary excretion of calcium in a genetic model of hypercalciuria produces aggregates of free floating CaP crystals in the urinary space [80]. However, sometimes, particularly in renal fornix, crystals were found attached to the papillary surface epithelium. ...
Crystallization by itself is not harmful as long as the crystals are not retained in the kidneys and are allowed to pass freely down the renal tubules to be excreted in the urine. A number of theories have been proposed, and studies performed, to determine the mechanisms involved in crystal retention within the kidneys. It has been suggested that urinary transit through the nephron is too fast for crystals to grow large enough to be retained. Thus, free particle mechanism alone cannot lead to stone formation, and there must be a mechanism for crystal fixation within the kidneys. Animal model studies suggest that crystal retention is possible through both the free- and fixed-particle mechanisms. Crystal–cell interaction leads to pathological changes which promote crystal attachment to either epithelial cells or their basement membrane. Alternatively, crystals aggregate and produce large enough particles to block the tubules particularly at sites, where urinary flow is affected because of changes in the luminal diameter of the tubule. Crystal deposits plugging the openings of the ducts of Bellini may be the result of such a phenomenon. Intratubular crystals translocating to renal interstitium may produce osteogenic changes in the epithelial or endothelial cells resulting in the formation of the Randall’s plaques. Thus, fixation appears to be either through the formation of Randall’s plugs, crystal plugs clogging the openings of the ducts of Bellini or sub-epithelial crystal deposits, and the Randall’s plaques.
... A C C E P T E D ACCEPTED MANUSCRIPT Bushinsky et al. found that the addition of 1%, 3%, and 5% trans-4-HLP to GHS rats altered urine calcium and stone type, with rats receiving 5% HLP having lower urine calcium excretion and consistent calcium oxalate calculi composition. 33 ...
... A C C E P T E D ACCEPTED MANUSCRIPT Bushinsky et al. found that the addition of 1%, 3%, and 5% trans-4-HLP to GHS rats altered urine calcium and stone type, with rats receiving 5% HLP having lower urine calcium excretion and consistent calcium oxalate calculi composition. 33 ...
The etiology of stone disease remains unknown despite the major technological advances in the treatment of urinary calculi. Clinically, urologists have relied on 24-h urine collections for the last 30–40 years to help direct medical therapy in hopes of reducing stone recurrence; yet little progress has been made in preventing stone disease. As such, there is an urgent need to develop reliable animal models to study the pathogenesis of stone formation and to assess novel interventions. A variety of vertebrate and invertebrate models have been used to help understand stone pathogenesis. Genetic knockout and exogenous induction models are described. Surrogates for an endpoint of stone formation have been urinary crystals on histologic examination and/or urinalyses. Other models are able to actually develop true stones. It is through these animal models that real breakthroughs in the management of urinary stone disease will become a reality.
... Dietary calcium has a major influence on kidney stone formation in GHS rats (30). GHS rats also form calcium phosphate stones under standard diet (31). The model described herein might also be useful to analyze the impact of vitamin D with or without calcium enriched-diet on calcium intestinal net flux and on bone turn-over in further studies. ...
Vitamin D supplementation in humans should be accompanied by calcium administration to avoid bone demineralization through vitamin D receptor signaling. Here we analyzed whether long-term exposure of rats to vitamin D supplementation, with or without a calcium-rich diet, would promote kidney stone formation. Four groups of rats received vitamin D alone (100,000 UI/kg/3 weeks), a calcium-enriched diet alone, both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Serum and urine parameters and crystalluria were monitored. Kidney stones were assessed by 3-dimensional micro-computed tomography, infrared spectroscopy, von Kossa/Yasue staining, and field emission scanning electron microscopy. Although serum calcium levels were similar in the 4 groups, rats receiving vitamin D had a progressive increase in urinary calcium excretion over time, especially those receiving both calcium and vitamin D. However, oral calcium supplementation alone did not increase urinary calcium excretion. At 6 months, rats exposed to both calcium and vitamin D, but not rats exposed to calcium or vitamin D alone, developed significant apatite kidney calcifications (mean volume, 0.121 mm3). Thus, coadministration of vitamin D and increased calcium intake had a synergistic role in tubular calcifications or kidney stone formation in this rat model. Hence, one should be cautious about the cumulative risk of kidney stone formation in humans when exposed to both vitamin D supplementation and high calcium intake.
... Noteworthy, stone composition is mostly apatite (CaHPO4) when animals are fed with a standard 1.2% calcium diet, probably explained by urine CaHPO4 supersaturation which increases faster than CaOx supersaturation [26] . Conversely, an additional diet supplement of HyP 5% induces CaOx stones formation [27] with crystal deposits mainly in contact with urothelial cells lining the papilla and in the fornix areas. Interestingly, similarly to tubular cells surrounding crystals, some urothelial cells in contact with crystals are indeed proliferating and also expressing high levels of OPN [28] . ...
In human nephrolithiasis, most stones are containing calcium and are located within urinary cavities; they may contain monohydrate calcium oxalate, dihydrate calcium oxalate and/or calcium phosphates in various proportion. Nephrolithiasis may also be associated with nephrocalcinosis, i.e., crystal depositions in tubular lumen and/or interstitium, an entity which suggests specific pathological processes. Several rodents models have been developed in order to study the pathophysiology of intrarenal crystal formation. We review here calcium rodent models classified upon the presence of nephrolithiasis and/or nephrocalcinosis. As rodents are not prone to nephrolithiasis, models require the induction of a long standing hypercalciuria or hyperoxaluria (thus explaining the very few studies reported), conversely to nephrocalcinosis which may occur within hours or days. Whereas a nephrotoxicity leading to tubular injury and regeneration appears as a critical event for crystal retention in nephrocalcinosis models, surprisingly very little is known about the physiopathology of crystal attachment to urothelium in nephrolithiasis. Creating new models of nephrolithiasis especially in different genetic mice strains appears an important challenge in order to unravel the early mechanisms of urinary stone formation in papilla and fornices.
... Calculi resulting from the precipitation of stone constituents might be due to super saturation in urine also. Previous studies found that the incidence of upper urinary tract stones was many fold greater in men than in women, and stones were composed mainly of calcium oxalate mostly in males than in females (Bushinsky et al. 2002;Trinchieri 2008). Although the pathogenesis of urolithiasis appears to be multi-factorial and intricate, sex hormone-derived gender differences are thought to influence the process. ...
Context There have not been any conclusive studies of the effects of diosmin, a modified flavanone glycoside obtained from Teucrium gnaphalodes L’Her (Lamiaceae), on urolithiasis.
Objective To evaluate anti-urolithiatic effects of diosmin in ammonium chloride and ethylene glycol-induced renal stone in experimental animals.
Materials and methods Thirty Sprague-Dawley were divided into five groups (n=6) receiving the following treatments, respectively, p.o. for 15 consecutive days: distilled water, 0.75% v/v ethylene glycol + 2% w/v ammonium chloride, 0.75% v/v ethylene glycol + 2% w/v ammonium chloride + cystone® 750 mg/kg, 0.75% v/v ethylene glycol + 2% w/v ammonium chloride + diosmin 10 mg/kg or 0.75% v/v ethylene glycol + 2% w/v ammonium chloride + diosmin 20 mg/kg. Different biomarkers of urolithiasis in urine and serum were evaluated and histopathological examination of kidney was done.
Results Animals treated with diosmin (both 10 and 20 mg/kg) had significantly (p < 0.005) decreased in kidney weight, urinary pH, total urinary protein, urinary calcium, phosphorus, serum potassium, sodium, magnesium, creatinine, uric acid and blood urea nitrogen levels and significantly (p < 0.005) increased in urinary volume, urinary magnesium, potassium, sodium, creatinine, uric acid and serum calcium levels in comparison to animals treated with ethylene glycol and ammonium chloride. However, results were better with diosmin 20 mg/kg in comparison to the control group.
Conclusion Diosmin (10 and 20 mg/kg) has very good anti-urolithiatic activity similar to the standard drug cystone®.
... We recognize that the absence of documented stone formation in our models is another limitation of the current study. However, most models of nephrolithiasis in rodents represent in fact nephrocalcinosis [31], with the rat and mouse exceptions being anchored on specific genetic abnormalities [32,33]. nevertheless, to the best of our knowledge, this is the first experimental study addressing the potential effects of Pkd1 haploinsufficiency and of the development of renal cysts secondary to Pkd1 deficiency on potential lithogenic metabolic factors associated with nephrolithiasis in aDPKD. ...
... HS intake increases the urinary excretion of calcium, in part via reduced passive reabsorption of calcium following that of sodium and water in tubules [1,3]. Excessive urinary calcium excretion is part of the etiology of calcium-containing stone formation and contributes to the supersaturation of calcium oxalate (CaOx) crystals [4]. Moreover, HS diet may also produce an undesirable reduction in urinary citrate excretion via an uncertain mechanism [5]. ...
Enhanced sodium excretion is associated with intrarenal oxidative stress. The present study evaluated whether oxidative stress caused by high sodium (HS) may be involved in calcium oxalate crystal formation. Male rats were fed a sodium-depleted diet. Normal-sodium and HS diets were achieved by providing drinking water containing 0.3% and 3% NaCl, respectively. Rats were fed a sodium-depleted diet with 5% hydroxyl-L-proline (HP) for 7 and 42 days to induce hyperoxaluria and/or calcium oxalate deposition. Compared to normal sodium, HS slightly increased calcium excretion despite diuresis; however, the result did not reach statistical significance. HS did not affect the hyperoxaluria, hypocalciuria or supersaturation caused by HP; however, it increased calcium oxalate crystal deposition soon after 7 days of co-treatment. Massive calcium oxalate formation and calcium crystal excretion in HS+HP rats were seen after 42 days of treatment. HP-mediated hypocitraturia was further exacerbated by HS. Moreover, HS aggravated HP-induced renal injury and tubular damage via increased apoptosis and oxidative stress. Increased urinary malondialdehyde excretion, in situ superoxide production, NAD(P)H oxidase and xanthine oxidase expression and activity, and decreased antioxidant enzyme expression or activity in the HS+HP kidney indicated exaggerated oxidative stress. Interestingly, this redox imbalance was associated with reduced renal osteopontin and Tamm-Horsfall protein expression (via increased excretion) and sodium-dependent dicarboxylate cotransporter NaDC-1 upregulation. Collectively, our results demonstrate that a HS diet induces massive crystal formation in the hyperoxaluric kidney; this is not due to increased urinary calcium excretion but is related to oxidative injury and loss of anticrystallization defense.
... The possible stone forming effect of VDR polymorphisms may be mediated by intestinal calcium absorption, although it may also be attributable to decreased citrate excretion, possibly caused by vitamin D. 41 The lithogenic activity of VDR is confirmed by its greater intestinal expression in the genetically hypercalciuric and stone forming rats in the study by Bushinsky, representing a model of human hypercalciuria in stone formers. 42 The 3=-UTR polymorphisms of VDR may also influence BMD values. The b allele was found to be associated with higher BMD 43 and calcium absorption in women. ...
The paper is an update on genes involved in primary hypercalciuria
... When fed a standard, ample Ca diet, all GHS rats develop kidney stones [33,52,54,55] which are composed of calcium phosphate (CaP) [33,49,50,54]. Addition of the amino acid hydroxyproline to the diet of GHS rats results in calcium oxalate (CaOx) stone formation [31,34,47]. The GHS rats have been maintained with continuous selection for hypercalciuria for over 100 generations (Fig. 1) [31-36,39,40,42,44,47, 49-52,54- ...
In this review, we discuss how the genetic hypercalciuric stone-forming (GHS) rats, which closely model idiopathic hypercalciuria and stone formation in humans, provide insights into the pathophysiology and consequences of clinical hypercalciuria.
Hypercalciuria in the GHS rats is due to a systemic dysregulation of calcium transport, as manifest by increased intestinal calcium absorption, increased bone resorption and decreased renal tubule calcium reabsorption. Increased levels of vitamin D receptor in intestine, bone and kidney appear to mediate these changes. The excess receptors are biologically active and increase tissue sensitivity to exogenous vitamin D. Bones of GHS rats have decreased bone mineral density (BMD) as compared with Sprague-Dawley rats, and exogenous 1,25(OH)2D3 exacerbates the loss of BMD. Thiazide diuretics improve the BMD in GHS rats.
Studying GHS rats allows direct investigation of the effects of alterations in diet and utilization of pharmacologic therapy on hypercalciuria, urine supersaturation, stone formation and bone quality in ways that are not possible in humans.
Background/aims:
Hypercalciuria is the most common identifiable risk factor predisposing to CaOx stone formation. Increased oral magnesium intake may lead to decreased CaOx stone formation by binding intestinal Ox leading to decreased absorption and/or binding urinary Ox to decrease urinary supersaturation. This study assessed the effect of oral magnesium on 24-hour urine ion excretion, supersaturation, and kidney stone formation in a genetic hypercalciuric stone-forming (GHS) rat model of human idiopathic hypercalciuria.
Methods:
When fed the oxalate precursor, hydroxyproline, every GHS rat develops CaOx stones. The GHS rats were fed a normal calcium and phosphorus diet with hydroxyproline to induce CaOx , were divided into three groups of ten rats per group: control diet with 4.0 g/kg MgO, low MgO diet (0.5 g/kg), and high MgO diet (8 g/kg). At 6 weeks, twenty-four-hour urines were collected, and urine chemistry and supersaturation were determined. Stone formation was quantified.
Results:
The GHS rats fed the low and high Mg diets had a significant reduction and increase, respectively, in urinary Mg compared to those fed the control diet. Dietary Mg did not alter urine Ca excretion while the low Mg diet led to a significant fall in urinary Ox. Urine supersaturation with respect to CaOx was significantly increased with low Mg, whereas urine supersaturation was significantly decreased with high Mg. There was no effect of dietary Mg on stone formation within 6 weeks of treatment.
Conclusion:
Dietary magnesium decreases urine supersaturation but not CaOx stone formation in GHS rats.
Introduction:
Lowering kidney stone risk and urine calcium oxalate supersaturation is a primary clinical focus for kidney stone prevention and can be achieved with multiple strategies. Common strategies include advice to increase fluid intake, restrict dietary sodium, or prescribing a thiazide-type diuretic. We investigated how physicians make these decisions in real-world practice and evaluate their efficacy based on 24-h urine collections.
Methods:
We reviewed medical charts for 203 kidney stone formers with idiopathic calcium stones from University of Chicago Kidney Stone Clinic between 2005 and 2020. Patients had three 24-h urines before an initial pre-treatment clinic visit and one follow-up 24-h urine. We analyzed changes in urine composition based on treatment advice using t tests and ANOVA.
Results:
Patients who received advice to increase fluid intake had lower urine volume at baseline (1.5 vs. 2.5 L/day, p < 0.001) and larger increase in urine volume at follow-up (0.6 vs. 0.1 L/day, p < 0.001) compared to those who did not receive the advice. Patients who were advised to restrict dietary sodium had a higher urine sodium at baseline (208 vs. 139 mEq/day, p < 0.001), a larger reduction in urine sodium (-28 vs. 13 mEq/day, p = 0.002), and larger reduction in urine calcium (-74 vs. -28 mg/day, p = 0.005) compared with those not advised to restrict dietary sodium. Patients started on a thiazide had a higher baseline urine calcium (281 vs. 213 mg/day) and larger reduction in urine calcium (-83 vs. -9 mg/day, p < 0.001) compared with patients not started on a thiazide. In combination, thiazide prescriptions with dietary sodium restriction reduced urine calcium by 99 mg/day and reduced calcium oxalate supersaturation from 8.0 to 5.5 and calcium phosphate supersaturation from 1.4 to 1.0.
Conclusion:
Providers use 24-h urine data to guide treatment strategy decisions. These strategies achieved the intended effects on urine composition and lowered kidney stone risk.
Introduction About 1-in-11 Americans will experience a kidney stone, but underlying causes remain obscure. The objective of the present study was to separate idiopathic calcium oxalate stone formers by whether or not they showed positive evidence of forming a stone on Randall's plaque (RP). Materials and Methods In patients undergoing either percutaneous or ureteroscopic procedures for kidney stone removal, all stone material was extracted, and analyzed using micro computed tomographic imaging (micro CT), in order to identify those attached to RP. 24-hour urines were collected weeks after the stone removal procedure and off of medications that would affect urine composition. Endoscopic video was analyzed for papillary pathology (RP, pitting, plugging, dilated ducts, loss of papillary shape) by an observer blinded to the data on stone type. Percent papillary area occupied by RP and ductal plugging was quantified using image analytic software. Results Patients having even 1 stone on RP (N=36) did not differ from Non-RP patients (N=37) in age, sex, BMI, or other clinical characteristics. Compared to the Non-RP group, RP stone formers had more numerous but smaller stones, more abundant papillary RP, and fewer ductal plugs, both by quantitative measurement of surface area (on average, 3 times more plaque area, but only 41% as much plug area as Non-RP) and by semi-quantitative visual grading. Serum and blood values did not differ between RP and Non-RP stone formers by any measure. Conclusions Growth of many small stones on plaque seems the pathogenetic scheme for the RP stone forming phenotype, whereas the Non-RP phenotype stone pathogenesis pathway is less obvious. Higher papillary plugging in Non-RP suggests that plugs play a role in stone formation, and that these patients have a greater degree of papillary damage. Underlying mechanisms that create these distinctive phenotypes are presently unknown.
To study human idiopathic hypercalciuria we developed an animal model, genetic hypercalciuric stone-forming rats, whose pathophysiology parallels that of human idiopathic hypercalciuria. Fed the oxalate precursor, hydroxyproline, every rat in this model develops calcium oxalate stones. Using this rat model, we tested whether chlorthalidone and potassium citrate combined would reduce calcium oxalate stone formation and improve bone quality more than either agent alone. These rats (113 generation) were fed a normal calcium and phosphorus diet with hydroxyproline and divided into four groups: diets plus potassium chloride as control, potassium citrate, chlorthalidone plus potassium chloride, or potassium citrate plus chlorthalidone. Urine was collected at six, 12, and 18 weeks and kidney stone formation and bone parameters were determined. Compared to potassium chloride, potassium citrate reduced urinary calcium, chlorthalidone reduced it further and potassium citrate plus chlorthalidone even further. Potassium citrate plus chlorthalidone decreased urine oxalate compared to all other groups. There were no significant differences in calcium oxalate supersaturation in any group. Neither potassium citrate nor chlorthalidone altered stone formation. However, potassium citrate plus chlorthalidone significantly reduced stone formation. Vertebral trabecular bone increased with chlorthalidone and potassium citrate plus chlorthalidone. Cortical bone area increased with chlorthalidone but not potassium citrate or potassium citrate plus chlorthalidone. Mechanical properties of trabecular bone improved with chlorthalidone, but not with potassium citrate plus chlorthalidone. Thus in genetic hypercalciuric stone-forming rats fed a diet resulting in calcium oxalate stone formation, potassium citrate plus chlorthalidone prevented stone formation better than either agent alone. Chlorthalidone alone improved bone quality, but adding potassium citrate provided no additional benefit.
Мочекаменная болезнь занимает одно из ведущих мест в структуре урологических заболеваний, что позволяет отнести данное заболевание к разряду социально значимых заболеваний. Причём на долю камней, представленных оксалатами кальция, приходится 72%, доля фосфатов кальция составляет 14,7% от общего количества камней почек. Целью настоящей работы является исследование экспериментальных моделей мочекаменной болезни для выявления сходства и различия подобных процессов, протекающих у экспериментальных животных и в организме человека. В данной обзорной статье рассматриваются патофизиологические модели развития мочекаменной болезни при введении оксалата, этиленгликоля, гидроксипролина и гликолевой кислоты. Рассматриваются модели формирования кальциевого почечного камня в зависимости от наличия нефрокальциноза, нефролитиаза или наличия одновременно этих двух метаболических состояний у грызунов.
Urolithiasis takes one of leading places among urological diseases, which allows attributing this disease to the category of socially significant diseases. Calcium oxalates account for 72% and calcium phosphates - 14.7% of all kidney stones. The aim of this study was to evaluate experimental models of urolithiasis to identify similarities and differences in similar processes occurring in experimental animals and humans. In this review, we focused on pathophysiological models for development of urolithiasis using administration of oxalate, ethylene glycol, hydroxyproline, and glycolic acid. We addressed modeling the formation of kidney calcium stones in rodents depending on the presence of nephrocalcinosis, nephrolithiasis or both.
The anti-urolithiatic effect of Diosgenin was determined on ethylene glycol induced urolithiasis in male wistar rats. The urolithiasis was induced in rats by administration of ethylene glycol in drinking water (0.75%v/v) for 28 days. Suspension of Diosgenin(50mg/kg and 100mg/kg) was administered orally from 1 st day for preventive treatment and from 15 th day for curative regimen. Ethylene glycol administration elevated the calcium, oxalate, urea and uric acid in urine. Treatment with Diosgenin significantly reduced these biochemical parameters in urine. Also treatment with Diosgenin significantly elevated the urinary concentration of citrate. The elevated serum creatinine and blood urea nitrogen levels of urolithiatic rats were reduced by prophylactic and curative treatment with Diosgenin. A considerable reduction in oxidative stress and histopathological parameters were also noted. In vitro and ex vivo antiurolithiatic activity of diosgenin were carried out using aggregation assay in synthetic urine and in rat plasma. The study revealed that diosgenin in plasma has significant inhibitory potential.
This review explores the relationship between vitamin D supplementation and lithogenesis. A causal relationship has been assumed despite myriad studies demonstrating that therapeutic doses of vitamin D do not increase lithogenic risk. Select stone formers may be at increased risk for recurrence with vitamin D supplementation, possibly from CYP24A1 gene mutations. Additionally, the evidence for who is vitamin D deficient, and the benefits of supplementation in those not at risk for rickets, is sparse. Concerns may be avoidable as vitamin D screening appears unnecessary in most patients, and superior pharmacology is available which increases bone density, while decreasing stone formation.
Kidney stones are prevalent, cause considerable morbidity though little mortality. The most common metabolic abnormality in patients with kidney stones is hypercalciuria, which is a complex metabolic trait that is dependent on three major organs: the amount of dietary calcium absorbed, any net calcium released from bone resorption in excess of formation, and the extent to which filtered calcium is reabsorbed in the renal tubule. Each of these calcium fluxes is under control of number of factors and hormones, including parathyroid hormone and 1,25-dihydroxy-vitamin D. Whether a patient forms a kidney stone is dependent not only on the magnitude of the hypercalciuria but also on urinary volume, excretion of other ions, including oxalate, citrate, and phosphate, and on local factors in the urinary tract. Hypercalciuria, and resultant stone formation, is a partially inherited trait. Given the many determinants of not only urine calcium excretion, but also the other factors that determine whether a patient will form a kidney stone, it is clear that multiple genetic loci are involved. In this chapter, we will describe the progress made in understanding the genetic basis for hypercalciuria and stone formation in both experimental models and in man.
The etiology of stone disease remains unknown. Clinically, urologists have relied on 24-h urine collections to help direct medical therapy in hopes of reducing stone recurrence. Despite these efforts, little progress has been made in preventing stone disease. As such, there is an urgent need to develop reliable animal models to study the pathogenesis of stone formation and to assess novel interventions. A variety of vertebrate and invertebrate models have been used to help understand stone pathogenesis. Genetic knockout and exogenous induction models are described. Surrogates for an endpoint of stone formation have been urinary crystals on histologic examination and/or urinalyses. Other models are able to actually develop true stones. It is only through these animal models that real breakthroughs in the management of urinary stone disease will become a reality.
Die konservative, instrumenteile oder operative Steinentfernung beseitigt zwar das Endprodukt eines Krankheitsgeschehens, das Symptom, aber nicht die Ursache des Leidens. Der Harnsteinbildner muss ohne Prophylaxe mit einer Wahrscheinlichkeit von ca. 50% mit einem Rezidiv rechnen.Steinauflösung und Steinprophylaxe bzw. Harnsteinmetaphylaxe sind die erwünschten Ziele der Harnsteinbehandlung.
Calcium is a major component of 85 % of renal stones. The incidence of renal stone diseases is increasing, possibly consequent to the widespread use of calcium supplementation. A genetic contribution with defects in the regulation of renal calcium excretion has been suggested as a cause of renal stone disease. The parathyroid hormone (PTH) and vitamin D axis plays a key role in the calcium and phosphate homeostasis. The parathyroid gland responds with rapid changes of PTH in response to fluctuations in the extracellular calcium concentration, thereby regulating minute-to-minute normalization of serum ionized calcium, through stimulation of renal tubular calcium reabsorption and bone resorption. On a more chronic basis, PTH also stimulates the conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D (calcitriol) in the proximal renal tubular cells, thereby stimulating intestinal calcium absorption. Hypersecretion of parathyroid hormone results in hypercalcemia thus predisposing the individual to development of nephrolithiasis.
Vitamin D is recognized as an important hormone in health and disease. There has been an increasing appreciation of the complexity and importance of its regulation, functions, and supplementation. The use of calcium and vitamin D supplementation, though safe, has been shown to be associated with an increase in the incidence of renal lithiasis.
Abnormalities related to vitamin D deficiency and parathyroid hormone disorders in the general population are reported globally with an increased incidence of cardiovascular disease, diabetes, metabolic syndrome, and cancer rates, which is linked to protracted exposure to abnormal internal milieu such as occurs with vitamin D deficiency. This chapter reviews the recent advances and interrelationship in the understanding of calcium, vitamin D, and PTH axis as they affect the process of stone formation in the kidney in health and diseases.
Primary Hypercalciuria (PH) is very often accompanied with some degrees of bone demineralization. The most frequent clinical condition in which this association has been observed is calcium nephrolithiasis. In patients affected by this disorder bone density is very frequently low and increased susceptibility to fragility fractures is reported. The very poor definition of this bone disease from a histomorphometric point of view is a crucial aspect. At present, the most common finding seems to be a low bone turnover condition. Many factors are involved in the complex relationships between bone loss and PH. Since bone loss was mainly reported in patients with fasting hypercalciuria, a primary alteration in bone metabolism was proposed as a cause of both hypercalciuria and bone demineralization. This hypothesis was strengthened by the observation that some bone resorbing-cytokines, such as IL-1, IL-6, and TNF-a are high in hypercalciuric patients. The effect of an excessive response to the acid load induced by dietary protein intake seems an additional factor explaining a primitive alteration of bone. The intestine plays a major role in the clinical course of bone disease in PH. Patients with absorptive hypercalciuria less frequently show bone disease and a reduction in dietary calcium greatly increases the probability of bone loss in PH subjects. It has recently been reported that greater bone loss is associated with a larger increase in intestinal calcium absorption in PH patients. Considering the absence of PTH alterations, it was proposed that this is not a compensatory phenomenon, but probably the marker of disturbed cell calcium transport, involving both intestinal and bone tissues. While renal hypercalciuria is rather uncommon, the kidney still seems to play a role in the pathogenesis of bone loss of PH patients, possibly via the effect of mild to moderate urinary phosphate loss with secondary hypophosphatemia. In conclusion, bone loss is very common in PH patients. Even if most of the factors involved in this process have been identified, many aspects of this intriguing clinical condition remain to be elucidated.
In men, low bone mass and fracture have several etiologies. Bone loss with hypercalciuria and normal serum calcium may result from primary and secondary hypogonadal states. This chapter focuses on idiopathic hypercalciuria (IH), a common cause of low bone mass and the most common cause of Ca oxalate nephrolithiasis. Hypercalciuria was implicated in the pathogenesis of kidney stones in the 1930s when Flocks reported a high frequency of hypercalciuria among stone formers. In the 1950s, Albright and colleagues first described IH as consisting of hypercalciuria, normal serum Ca and the absence of the known systemic hypercalciuric disorders. Five percent of men in the adult population have hypercalciuria and, of them, about 10% will form a kidney stone. IH accounts for 50% of all Ca oxalate nephrolithiasis and is thought to contribute to stone formation by creating a urine supersaturated with respect to Ca and either oxalate or phosphate. A variety of non-invasive techniques have been used to quantify bone density in IH patients, however dual energy x-ray absorptiometry (DXA), which measures bone mineral density (BMD) at the lumbar spine, proximal femur and forearm, has been used most extensively. DXA scans are highly sensitive to small changes in BMD and are sufficiently reproducible to track changes in bone mass over time.
Supersaturation is the driving force behind crystal formation in the kidneys. It can, however, result only in the formation of crystals that can often be harmlessly excreted. For stone formation, crystals must form, grow, and be retained in the kidneys, which is indeed a rare occurrence. Crystalluria is universal while stone formation is not. Only pathological changes in the kidneys and renal cell dysfunction and injury can accomplish crystal retention and formation of stone nidus. Cellular dysfunction can be intrinsic or provoked. Lethal epithelial cellular injury promotes crystal nucleation, aggregation, and retention. Sublethal injury or dysfunctional cells may produce ineffective crystallization modulators and localized areas of supersaturation in the interstitium. The former will affect crystallization in the urine while the latter may cause precipitation in the interstitium and development of Randall's plaques. In addition, dysfunctional cells affect supersaturation, by influencing the excretion of participating ions such as calcium, oxalate, and citrate, and modulating urinary pH and production and excretion of macromolecular promoters and inhibitors of crystallization.
Kidney stones are prevalent, and cause considerable morbidity though little mortality. The most common metabolic abnormality in patients with kidney stones is hypercalciuria, which is a complex metabolic trait that is dependent on the amount of dietary calcium absorbed, any calcium released from net bone resorption in excess of formation and the extent to which filtered calcium is reabsorbed in the renal tubule. Each of these calcium fluxes is under the control of a number of factors and hormones including parathyroid hormone and 1,25 dihydroxyvitamin D. Whether a patient forms a kidney stone is dependent not only on the magnitude of the hypercalciuria but on urinary volume and excretion of other ions including oxalate and phosphate. Hypercalciuria, and resultant stone formation, is an inherited trait. Given the many determinants of not only urine calcium excretion, but also the other factors that determine whether a patient will form a kidney stone, it is clear that multiple genetic loci are involved. In this chapter, we will describe the progress that has been made in understanding the genetic basis for hypercalciuria and stone formation in both experimental models and in humans.
The kidneys are primarily responsible for control of extracellular calcium balance. Renal calcium absorption occurs by a series of sequential events as the urine passes through the nephron. Most of the filtered calcium is recovered by proximal tubules, with progressively smaller fractions retrieved as the insipient urine passes through consecutive tubule segments. Thin descending and ascending limbs of Henle's loop exhibit low calcium permeability and do not contribute meaningfully to calcium economy. Distal convoluted tubules reabsorb 5—10% of the filtered calcium. Thiazide diuretics and functionally related agents such as metolazone have the unique ability to decrease renal calcium excretion, especially upon chronic administration, while simultaneously increasing sodium excretion. The primary end point for successful treatment of patients with calcium-containing kidney stones is a decrease in the rate of stone recurrence. The majority of human kidney stone formers with calcium-containing kidney stones are hypercalciuric when compared to non-stone formers.
La découverte d’une hypercalciurie au décours d’un épisode lithiasique ou lors des explorations biologiques de l’ostéoporose, impose une enquête clinique et biologique, pour rechercher une étiologie et en particulier une hyperparathyroïdie primitive ou une consommation excessive de vitamine D, calcium, de sel, d’alcool ou de protéines d’origine animale. Au cours de l’ostéoporose, la découverte d’une hypercalciurie idiopathique (HCI) ne modifie pas la prise en charge thérapeutique habituelle, en assurant des apports calciques adaptés sans dépasser 800 mg/j. Après un épisode lithiasique, si les mesures de régime ne n’avèrent pas suffisantes, la prescription d’un diurétique thiazique est possible en vérifiant l’efficacité biologique et la tolérance.
Offers the personal interpretations of authors as participant-observers
together with a data-based analysis of the evolution of the services
marketing literature. Bibliographic analysis of more than 1,000 English
language, general services marketing publications, spanning four
decades, provides an additional resource. Using an evolutionary
metaphor as the framework, traces the literature through three stages:
Crawling Out (1953-79): Scurrying About (1980-85); and Walking Erect
(1986-present). Shows how the literature has evolved from the early
services-marketing-is-different debate to the maturation of specific
topics (e.g. service quality, service encounters) and the legitimization
of the services marketing literature by major journals. Presents a
classification and summary of publications and authors. Closes with
discussion and speculation on the future of the services marketing
literature.
We evaluated a colorimetric method for the assay of uric acid in serum or urine, which utilises a Trinder chromogenic system modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid for oxidative coupling to p-aminophenazone. Colour development (Amax: 512 nm) is complete within five minutes. Measurement of a sample blank is not needed. The procedure involves pre-incubation with ascorbic acid oxidase and detergent to eliminate interference by ascorbic acid and to abolish turbidity due to lipaemia; this pretreatment was effective up to 1.14 mmol/l ascorbate and up to at least 25 mmol/l triacylglycerol. Interference by icteric sera was insignificant up to about 170 mumol/l bilirubin. The method is linear up to at least 1428 mumol/l. In human serum and urine the procedure correlates well with HPLC and the uricase p-aminophenazone method on the SMAC analyser. Within-run and between-run imprecisions of the enzymic test were higher than for HPLC, but did not exceed 1.2% (CV) and 2.5% (CV), respectively.
We have established a colony of genetic hypercalciuric (IH) rats as a model of idiopathic hypercalciuria in humans. To test the hypothesis that hypercalciuria can cause crystallization in kidneys through increased supersaturation, in the absence of confounding effects of diet and whatever complex inhibitor disorders underlay stone disease, we fed males and females of the 21st generation of IH rats 13 g per day of a low calcium (LCD, 0.02% Ca), followed by a normal calcium (NCD, 0.6% Ca) and then a high calcium (HCD, 1.2% Ca) diet, each for seven days. During the last 24 hours of each period complete urine collections were obtained and analyzed for all substances known to affect urinary calcium oxalate (CaOx) and brushite (CaHPO4) supersaturation. Relative supersaturation with respect to the solid phases of CaOx and CaHPO4 were then calculated. Compared to same gender controls (Ctl) urine calcium excretion was higher in the female IH rats on all diets and in the male IH rats on NCD and HCD. The female and male IH rats on NCD and HCD were supersaturated with respect to CaOx; however, the male and female Ctl were supersaturated with respect CaOx only on HCD. The female IH rats on NCD and HCD and the male IH rats on NCD were supersaturated with respect to CaHPO4; however, neither the male nor female Ctl rats were supersaturated with respect to CaHPO4 on any diet. On NCD and HCD urine supersaturation with respect to CaHPO4 by females exceeded that of males.(ABSTRACT TRUNCATED AT 250 WORDS)
A fundamental mechanism for hypercalciuria in genetic hypercalciuric rats appears due to a primary increase in intestinal calcium absorption. However previous studies could not exclude additional mechanisms to account for the hypercalciuria. To determine if enhanced bone mineral dissolution either as a primary abnormality or secondary to a defect in renal tubule calcium reabsorption is responsible for a component of the augmented calcium excretion we studied rats continually inbred for hypercalciuria. Nineteenth generation adult female idiopathic hypercalciuric (IH) and non-inbred control (Ctl) rats were fed 13 g/day of a normal calcium diet (0.6% calcium, NCD) for 10 days. Urine calcium excretion over the last seven days was greater in IH (34 +/- 2 mg/7 day) than in Ctl (2.9 +/- 0.3, P < 0.01) rats. Some rats in each group were continued on the same diet while others were fed a low calcium diet (0.02% calcium, LCD) for an additional 10 days; balance measurements were made over the final seven days. With LCD, urine calcium excretion was approximately 8-fold higher in IH compared to Ctl (13 +/- 2 mg/7 day vs. 1.6 +/- 0.1, IH vs. Ctl, respectively, P < 0.01). In IH rats percent calcium absorption was greater (59 +/- 3% vs. 45 +/- 3, IH vs. Ctl, P < 0.01), however calcium retention was negative (-1.9 +/- 2.0 mg/7 day vs. 6.5 +/- 0.5, IH vs. Ctl, P < 0.01) compared to Ctl rats. The fall in urine calcium excretion when IH rats are fed LCD indicates that enhanced intestinal calcium absorption is a primary mechanism of the hypercalciuria.(ABSTRACT TRUNCATED AT 250 WORDS)
Our genetic hypercalciuric (GH) rats have been selected and inbred for 29 generations to maximize urine calcium (UCa) excretion compared to identical gender controls (Ctl). To determine the effect of the increased UCa on urinary supersaturation and stone formation, we pair fed 15 GH and 15 Ctl rats a standard 1.2% calcium diet for 18 weeks, measured urine supersaturation every two weeks, and examined the urinary tract of 1/3 of the rats for the presence of stones every six weeks. Any stones formed were studied by SEM, X-ray and electron diffraction and X-ray microanalysis. Over the entire study UCa was increased in the GH compared to Ctl, resulting in greater supersaturation with respect to calcium hydrogen phosphate (CaHPO4) at all times and calcium oxalate (CaOx) at most times. There was a progressive increase in the incidence of stone formation in GH rats with one of five rats having stones at six weeks, three of five with stones at 12 weeks and five of five with stones at 18 weeks. There were no stones formed in Ctl rats. SEM reveals discrete stones and not nephrocalcinosis. X-ray and electron diffraction and X-ray microanalysis reveal the stones to be poorly crystalline apatite which is a solid phase of calcium and phosphate. Compared to Ctl, in the GH rats the saturation ratio for CaHPO4 increased proportionally more than that for CaOx, perhaps explaining why the rats formed apatite and not oxalate stones. This is the first description of an animal model of spontaneous nephrolithiasis.
As a model of human hypercalciuria, we have selectively inbred genetic hypercalciuric stone-forming (GHS) Sprague-Dawley rats whose mean urine calcium excretion is eight to nine times greater than that of controls. A large component of this excess urine calcium excretion is secondary to increased intestinal calcium absorption, which is not due to an elevation in serum 1,25(OH)2D3, but appears to result from an increased number of intestinal 1,25(OH)2D3 receptors (VDR). When GHS rats are fed a low-calcium diet, the hypercalciuria is only partially decreased and urine calcium excretion exceeds intake, suggesting that an additional mechanism contributing to the hypercalciuria is enhanced bone demineralization. To determine if GHS rat bones are more sensitive to exogenous 1,25(OH)2D3, we cultured calvariae from neonatal (2- to 3-day-old) GHS and control rats with or without 1,25(OH)2D3 or parathyroid hormone (PTH) for 48 h at 37 degrees C. There was significant stimulation of calcium efflux from GHS calvariae at 1 and 10 nM 1,25(OH)2D3, whereas control calvariae showed no significant response to 1,25(OH)2D3 at any concentration tested. In contrast, PTH induced similar bone resorption in control and GHS calvariae. Immunoblot analysis demonstrated a fourfold increase in the level of VDR in GHS calvariae compared with control calvariae, similar to the increased intestinal receptors described previously. There was no comparable change in VDR RNA levels as measured by slot blot analysis, suggesting the altered regulation of the VDR occurs posttranscriptionally. That both bone and intestine display an increased amount of VDR suggests that this may be a systemic disorder in the GHS rat and that enhanced bone resorption may be responsible, in part, for the hypercalciuria in the GHS rat.
Idiopathic hypercalciuria is a frequent cause of calcium (Ca) containing kidney stones. We have previously shown that there is increased intestinal Ca absorption in selectively inbred genetic hypercalciuric stone forming (GHS) rats; however, excess Ca excretion persists when the rats are fed a low Ca diet indicating a defect in renal Ca reabsorption and/or increased bone resorption. To determine if GHS rats have a defect in renal Ca reabsorption we performed 14C-inulin clearance studies on parathyroidectomized female GHS and control (Ctl) rats. After three baseline collections, chlorothiazide (CTZ) or furosemide (FUR) was infused and three more collections were obtained. Both GFR and filtered load of Ca did not differ among the groups; however, fractional and absolute excretion (UcaV) of Ca was three times higher in GHS rats. The increased Ca excretion was not diminished by a low Ca diet. Urine flow rate nearly tripled in all rats after either FUR or CTZ. After CTZ, UcaV was decreased to a greater extent in GHS compared to Ctl rats. After FUR, UcaV was increased to a greater extent in Ctl rats compared to GHS rats. These data indicate that GHS rats have a defect in renal Ca reabsorption, in addition to increased intestinal Ca absorption. The effect of CTZ was greater, and that of FUR was smaller, in GHS compared with Ctl rats, suggesting that the defect in renal Ca handling might be at the level of the thick ascending limb.
A new direct colorimetric procedure for uric acid assay in serum or urine is described, utilizing a 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminophenazone chromogenic system in the presence of horseradish peroxidase and uricase from Aspergillus flavus. This chromogen system has a high absorptivity, affording useful results with sample/reagent volume ratios as low as 0.025. The procedure is applicable to serum, plasma, or diluted urine. A single working reagent is used; the reaction is complete in less than 15 min at room temperature. The red dye formed is measured at 520 nm; a blank sample measurement is not needed. The standard curve for the method is linear for uric acid concentrations up to 1500 mumol/L. Average analytical recovery of uric acid in human sera and urine exceeded 99%; within-run and between-run precision studies showed CV's of less than or equal to 1.2 and less than or equal to 2.2%, respectively. The new procedure correlated well with the uricase/catalase and uricase/ultraviolet methods. The method is suitable for automation.
Histochemistry of calcium oxalate has not been definitive. The present author has critically investigated various histochemical methods for calcium salts and finally found a new method advantageous to those hitherto reported.
Celloidin models of calcium salts, rat kidneys of oxalosis induced with ethylene glycol and human materials were used for study.
New method: Firstly calcium phosphate and carbonate are removed by immersing sections into 5% acetic acid for 30 minutes. Secondly the sections still containing calcium oxalate are made to react with 5% aqueous silver nitrate for 15 minutes, and thirdly the reaction product appears dark brown to black with the use of rubeanic acid (saturated rubeanic acid in 70% alcohol with 2 drops of strong ammonium for 1 minute) that is very sensitive to silver without loosing the definite localization of calcium oxalate. This method has a merit; it does not depend on weather or illumination and takes a shorter time than the Kóssa method.
A confident histochemical method for identifying calcium oxalate was established by combining this method with other characteristics of calcium oxalate such as the unstainability with hematoxylin, birefringency, solubility after incineration and gypsum formation.
In the present study calcium oxalate was also found to react with lead besides silver and to appear brownish-black when one uses lead nitrate and ammonium polysulphide.
In humans, idiopathic hypercalciuria is associated with stone formation. To study the mechanisms that are responsible for the excess urine calcium excretion in ways that are difficult to impossible in humans, we have developed a rat model of idiopathic hypercalciuria. Hypercalciuric rats were successively inbred for more than 40 generations to produce a strain in which urine calcium excretion is far greater than that of controls and all rats form kidney stones. Analysis of the model has revealed that the rats not only exhibit increased intestinal calcium absorption but an independent defect in renal tubular resorption and an increased tendency for bone demineralization. These findings closely parallel those in patients with idiopathic hypercalciuria. In the intestine, bone, and kidney there is an increased number of vitamin D receptors which appear to make the rats more sensitive to the effect of 1,25(OH)2D3. Whether the increased number of vitamin D receptors can be directly translated into hypercalciuria and whether the same abnormality is present in humans with idiopathic hypercalciuria remains to be determined.
ABSTRACT Fifty recurrent stone formers were included in a double-blind randomized study (median 3 years) performed in a Norwegian general practice to compare twice daily administration of 25 mg hydrochlorothiazide versus placebo. The number of patients with new stones was significantly higher in the placebo group than in the thiazide group (p=0.05, one-tailed test). If a new stone was formed, thiazide, but not placebo, had the effect of prolonging the stone-free interval (p≤0.01). The probability of not forming a new stone during the treatment period was 45% for the placebo group and 75% for the thiazide group. The thiazide effect seemed to be independent of urinary calcium, but was less beneficial in patients with hyperuricosuria. The placebo group also showed a substantial decrease in the expected number of new stones (p≤0.01), emphasizing the importance of an adequate control group.
Urinary endogenous oxalate was increased by feeding vitamin B6-deficient or control rats with 5.2% hydroxyproline, or 3% glycine plus 5.2% hydroxyproline. The activities of liver lactic dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD) , malic enzyme (ME), and ATP citrate lyase were decreased in vitamin B6-DEFICIENT RATS, AND THEIR LIVEr G6PD was further decreased by the addition of glycine and hydroxyproline to their diets. Supplementing control diets with the two amino acids decreased the activities of rat liver LDH, G6PD, and ATP citrate lyase. The effects of glycine and hydroxyproline feeding on the enzymes studied did not appear related to alterations in insulin availability. Since in vitamin B6-deficient rats, there are increases in urinary levels of oxalic and glycolic acids, and glycine, and increases in tissue levels of glyoxylic acid and glycine, the effects of these metabolites on the activities of the above mentioned enzymes were measured. Oxalic acid inhibited the activities of LDH, G6PD, and ME. Glyoxylic acid inhibited LDH and ME, but not G6PD. Glycolic acid inhibited G6PD and ME, but not LDH. ATP citrate lyase was not affected by these substances. Glycine had no effect on the enzymes studied. Diets which increased oxalate excretion generally reduced or did not alter liver and kidney levels of oxalate, glycolate, and glyoxylate. However, the feeding of glycine and hydroxyproline increased kidney oxalate, and liver and kidney glyoxylate in vitamin B6-deficient rats.
To determine whether Ca incorporation from medium into cultured bone represents normal mineralization, we labeled some neonatal mouse calvariae in vitro and others in vivo with the stable isotope 44Ca and compared surface label localization with a scanning ion microprobe utilizing secondary ion mass spectrometry. To label in vitro, we incubated live calvariae in medium containing 40Ca or 44Ca for 3 h. Compared with a 44Ca/40Ca ratio of 0.020 with 1 mM 40Ca, the ratio with 1 mM 44Ca was 0.135 and with 2 mM 44Ca was 0.556. Erosion revealed a marked decrease in 44Ca/40Ca with depth. To label in vivo, we subcutaneously injected 40Ca or 44Ca into mice equal to a percentage of their total body weight and dissected the calvariae 24 h later. Compared with a 44Ca/40Ca ratio of 0.021 with 2% 40Ca, the ratio with 2% 44Ca was 0.120 and with 6% 44Ca was 0.205. Erosion revealed only a slight decrease in 44Ca/40Ca with depth. Elemental distribution maps of in vivo labeled samples show broad deposition of 44Ca, whereas maps of in vitro labeled bones show 44Ca preferentially localized at the surface in contact with the medium. Thus calvariae can be labeled with 44Ca both in vitro and in vivo. However, the differing patterns of isotope localization under the conditions of this study indicate that in vitro Ca deposition differs from normal in vivo bone mineralization.
We extended the study of oxalate bioavailability by testing 7 additional food items: brewed tea, tea with milk, turnip greens, okra, peanuts and almonds. Nine normal subjects ingested a large serving of each of these items. The bioavailable oxalate was calculated from the increment in urinary oxalate during 8 hours after ingestion and bioavailability was determined as the percentage of total oxalate content in a given food item represented by bioavailable oxalate. Brewed tea and tea with milk, with a high oxalate content, had a low bioavailable oxalate level (1.17 and 0.44 mg. per load) because of the low oxalate availability (bioavailability of 0.08 and 0.03%). Turnip greens, with a satisfactory oxalate bioavailability (5.8%), had a negligible effect on urinary oxalate excretion, since oxalate content was relatively low (12 mg. per load). Okra, with a moderate oxalate content (264 mg. per load) had a negligible bioavailable oxalate (0.28 mg. per load). Only peanuts and almonds provided a moderate increase in oxalate excretion (3 to 5 mg. per load) due to the modest oxalate content (116 and 131 mg. per load) and oxalate bioavailability (3.8 and 2.8%). Thus, the ability of various oxalate-rich foods to augment urinary oxalate excretion depends not only on oxalate content but on the bioavailability.
Protons are buffered and calcium is released by bone during metabolic acidosis. Incubation of neonatal mouse calvariae in acid medium causes net calcium efflux from bone and net proton influx into bone, just as metabolic acidosis does in vivo. To determine whether the calcium carbonate phase of bone mineral is solubilized with increasing proton concentrations, we cultured calvariae for 3 h in medium in which the saturation was varied by changing pH or calcium and phosphate concentrations. We determined the driving force for crystallization by calculating the Gibbs free energy of formation (DG). With alteration of the medium pH, calcium carbonate entry or loss from bone varied linearly with the initial DG for medium calcium carbonate (r = -0.745, n = 41, P less than 0.001) as it did with alteration of the medium calcium and phosphate (r = -0.665, n = 118, P less than 0.001). There was dissolution of calcium carbonate into medium that was unsaturated with respect to calcium carbonate, net flux ceased at saturation, and calcium carbonate entered bone from supersaturated medium, indicating that the medium is in equilibrium with the calcium carbonate phase of bone mineral. Neither the mineral phase brushite nor apatite was in equilibrium with the medium. These observations indicate that in vitro, acute proton-induced calcium efflux is due to dissolution of bone calcium carbonate.
Excessive urine calcium excretion in patients with idiopathic hypercalciuria may involve a primary increase in intestinal calcium absorption, overproduction of 1,25-dihydroxyvitamin D3 or a defect in renal tubular calcium reabsorption. To determine the mechanism of hypercalciuria in an animal model, hypercalciuria was selected for in rats and the most hypercalciuric animals inbred. Animals from the fourth generation were utilized to study mineral balance and intestinal transport in relation to levels of serum 1,25(OH)2D3. Both urine calcium excretion and net intestinal calcium absorption were greater in hypercalciuric males (HM) than in normocalciuric males (NM) and in hypercalciuric females (HF) than in normocalciuric females (NF). However, serum 1,25(OH)2D3 was lower in HM than in NM and not different in HF than in NF. Net calcium balance was more positive in HM than in NM and in HF than in NF. In vitro duodenal calcium net flux was correlated with serum 1,25(OH)2D3 in HM and HF and in NM and NF. However, with increasing serum 1,25(OH)2D3 there was greater calcium net flux in hypercalciuric rats than in normocalciuric controls. Hypercalciuria in this colony of hypercalciuric rats is due to a primary intestinal overabsorption of dietary calcium and not an overproduction of 1,25(OH)2D3 or a defect in the renal tubular reabsorption of calcium.
We examined the effectiveness of chlorthalidone or magnesium hydroxide in the prevention of recurrent calcium oxalate kidney calculi. In a double-blind random allocation design daily dosages of 25 or 50 mg. chlorthalidone, 650 or 1,300 mg. magnesium hydroxide, or an identical placebo were administered. All groups showed significantly decreased calculous events compared to the pretreatment rates. During the trial 56.1 per cent fewer calculi than predicted developed in the placebo group (p less than 0.01), whereas the groups receiving low and high dosage magnesium hydroxide showed 73.9 and 62.3 per cent fewer calculi, respectively (p less than 0.001 and less than 0.01, respectively). Chlorthalidone treatment resulted in a 90.1 per cent decrease from predicted rates and both dosages yielded similar results. When the treatments were compared chlorthalidone was significantly better than the placebo or magnesium hydroxide (p less than 0.01). The large decreases in calculous events seen when placebo or ineffective therapy was given underscore the positive treatment bias that occurs when historical controls are used and they demonstrate the need for proper experimental design.
A BASIC computer program for the calculation of urinary supersaturation with respect to the common kidney stone components is described. In vitro and in vivo tests show that the program described accurately calculates supersaturation. The application of this computer program to urolithiasis research is discussed.
The Jaffe reaction has been modified by the addition of sodium-dodecyl-sulfate (SDS) and borate to the reaction mixture. A method has been elaborated permitting serum creatinine determination without prior deproteinisation.On direct serum creatinine determination using this reagent, values are obtained that correlate well to those obtained with a method involving isolation of creatinine with a chromatographic procedure.
1.Measurement of urinary sulphate is of clinical significance because the amount of endogenous acid production from sulphur-containing amino acid-metabolism can be back extrapolated from the total amount of urinary sulphate anions.2.A rapid and accurate method for determination of urinary sulphate concentration based on the precipitation of sulphate with barium is presented.3.Freezing (−20°C) and refrigeration (4°C) are shown to be satisfactory for storage up to at least 30 days.
The toxic effects of ethylene glycol in several macaque species are reported. The compound was administered in the drinking water at concentrations of 0.25 to 10%, and histologic studies were made after acute and chronic administration. With the exception of a few animals (which had calcium oxalate crystals in the brain) significant pathological alterations were limited to the kidneys. Animals receiving 15 ml/kg or more of ethylene glycol had calcium oxalate crystals within proximal renal tubules and associated tubular degeneration. Despite the absence of crystals in animals receiving less than 15 ml/kg, mild glomerular damage was found, and azotemia occurred in some animals, suggesting a toxic effect of ethylene glycol apart from its conversion to oxalic acid.
Fifty recurrent stone formers were included in a double-blind randomized study (median 3 years) performed in a Norwegian general practice to compare twice daily administration of 25 mg hydrochlorothiazide versus placebo. The number of patients with new stones was significantly higher in the placebo group than in the thiazide group (p = 0.05, one-tailed test). If a new stone was formed, thiazide, but not placebo, had the effect of prolonging the stone-free interval (p less than or equal to 0.01). The probability of not forming a new stone during the treatment period was 45% for the placebo group and 75% for the thiazide group. The thiazide effect seemed to be independent of urinary calcium, but was less beneficial in patients with hyperuricosuria. The placebo group also showed a substantial decrease in the expected number of new stones (p less than or equal to 0.01), emphasizing the importance of an adequate control group.
We studied the physiological importance of elevated serum 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] in male rats. Male and female rats of equal weight consumed either a high calcium diet (1.20% calcium; HCD), a low calcium diet (0.02% calcium; LCD), or a normal calcium diet (0.60% calcium; NCD). With all three diets, serum 1,25(OH)2D3 was higher in males than females and rose in both sexes with decreasing diet calcium. There was a direct correlation between serum 1,25(OH)2D3 and proximal duodenal 45Ca uptake (r = 0.667; P less than 0.001). With NCD, we performed a balance study and found a direct correlation between serum 1,25(OH)2D3 and both 5-day fractional calcium absorption (r = 0.703; P less than 0.01) and fractional phosphorus absorption (r = 0.679; P less than 0.01). In addition, serum 1,25(OH)2D3 was directly correlated with 5-day calcium retention (r = 0.772; P less than 0.001) and phosphorus retention (r = 0.830; P less than 0.001). Males grew faster than females (F ratio = 1,194; P less than 0.001). Elevated 1,25(OH)2D3 in males appears to be of biological significance and may help satisfy a greater, growth-related, need for calcium.
Magnesium in plasma is determined by diluting a 50-microL sample with 5.0 mL of stable calmagite reagent containing the amphoteric detergent Empigen BB, and buffered with 2-amino-2-methyl-1-propanol at pH 11.5. The calcium response is masked with strontium-buffered [ethylene-bis(oxyethylenenitrilo)]tetraacetate (EGTA), while iron is masked with triethanolamine. The detergent causes an increased separation of the test and blank spectral absorbance bands. The test absorbance peaks, which may be measured immediately, are at 520 nm, while the blank peak shifts from 610 to 655 nm on addition of detergent. The blank absorbance at 520 nm is relatively decreased by the detergent. This allows the use of a more concentrated calmagite reagent, which in turn extends the linear relation of absorbance to magnesium concentration to 5 mmol/L in plasma. This method (y) has been compared with atomic absorption procedure (x) of McDonald and Watson (Clin, Chim. Acta 14: 233, 1966) and gives favorable regression statistics (y = 1.001X + 0.057 mmol/L).
It has been observed that the feces as well as urine of rats fed diets supplemented with 3% glycine and 5.2% hydroxyproline contain unexpectedly high amounts of endogenously formed oxalate. That intestinal microorganisms do not synthesize significant amounts of oxalate was indicated by the findings that oral tetracycline had no effect on oxalate excretion and that germ-free rats excreted more oxalate than conventional rats. Since little intraperitoneally injected [14C] oxalate appeared in the feces, and rat intestinal mucosa homogenates were found to produce oxalate from a variety of precursors of which glyoxylic acid was far the most important, it is probable that the intestinal mucosa may be an important source of fecal oxalate observed in these studies. Ninety percent of weanling rats fed complete diets supplemented with glycine and hydroxyproline developed urinary stones in 38 days. It has been concluded that in the treatment of patients with histories of calcium oxalate urolithiasis, more concern than is commonly shown should be directed towards the feeding of diets high in precursors of endogenous oxalate synthesis.
The ability of 7 "oxalate-rich" foods to enhance urinary oxalate excretion was measured in 8 normal volunteers. The analyzed value for oxalate was high for spinach (1,236 mg.), moderate for chocolate (126 mg.) and tea (66 mg.), and for low vegetable juice, cranberry juice, pecans, and orange juice (2 to 26 mg.). The urinary oxalate increased by 29.3 mg. during eight hours after ingestion of spinach. However, it rose by less than 4.2 mg. from consumption of other food items. The bioavailable oxalate (per cent of total appearing in urine) was much less from food items of high or moderate oxalate content (spinach and chocolate) than from standard solutions of sodium oxalate (2.4 to 2.6 versus 6.5 to 7.3 per cent). Thus, only spinach among food items tested was capable of causing hyperoxaluria in normal subjects.
1. Urinary composition was studied in nine healthy adults on unrestricted diet and low-oxalate diet with and without individual oxalate-rich foods.
2. Urine oxalate was constant on the low-oxalate and constant high-oxalate diets and only fluctuated greatly on unrestricted diet.
3. Urine oxalate was mainly due to dietary oxalate which accounts for up to two-thirds of urinary oxalate.
4. Urine oxalate was unaffected by urine volume.
5. Varying percentages of dietary oxalate were absorbed depending on the nature of the foodstuff.
6. Although tea was the main source of dietary oxalate in some people it, like strawberries, did not represent a real risk factor. Chocolates, peanuts, beetroot, rhubarb and spinach were considered as high-risk foods.
7. Calcium oxalate crystalluria at 4°C was increased significantly when the oxalate-rich foods were taken. When urine was examined at 37°C no increase in crystalluria was found.
The optimal reaction conditions and kinetic properties of glucose dehydrogenase were studied in order to develop a method for serum glucose determination. The Km value for glucose of this enzyme was influenced by the medium pH and ionic strength. Suitable conditions for the use of glucose dehydrogenase in rate assay and end point assay were identified. These methods showed very good reproducibility and were essentially unaffected by other reducing agents in the serum. The values obtained by these methods showed excellent correlations with those obtained with the hexokinase method. The present methods are rapid, simple and accurate.
A new direct colorimetric procedure for uric acid assay in serum or urine is described, utilizing a 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminophenazone chromogenic system in the presence of horseradish peroxidase and uricase from Aspergillus flavus. This chromogen system has a high absorptivity, affording useful results with sample/reagent volume ratios as low as 0.025. The procedure is applicable to serum, plasma, or diluted urine. A single working reagent is used; the reaction is complete in less than 15 min at room temperature. The red dye formed is measured at 520 nm; a blank sample measurement is not needed. The standard curve for the method is linear for uric acid concentrations up to 1500 mumol/L. Average analytical recovery of uric acid in human sera and urine exceeded 99%; within-run and between-run precision studies showed CV's of less than or equal to 1.2 and less than or equal to 2.2%, respectively. The new procedure correlated well with the uricase/catalase and uricase/ultraviolet methods. The method is suitable for automation.
A new colorimetric, enzymic method for determination of serum oxalate is described using oxalate oxidase from barley seedlings. This method, based on a specific reaction of oxalate with oxidase is rapid, simple and precise. The percentage recoveries of oxalate added to serum ranged from 91.5 to 103. The oxalate content in the serum obtained from 50 healthy non-fasting donors varied from 0.06 mg/dl to 0.41 mg/dl. The proposed method showed a good correlation with a chemical method.
An extensively updated version of the EQUIL software is described. The former version, designated EQUIL2, is widely used to study urolithiasis and related areas of biomineralization. In this report, we discuss recent enhancements which give EQUIL93 an expanded scope of application. This program has been frequently used in studies of the physicochemical processes underlying stone salt crystallization, especially crystal growth and nucleation, but it has also been employed as an aid for in vivo research and as an evaluator of therapeutic measures. We illustrate several new applications, including some outside the urologic realm, and we discuss how the enhanced software can be helpful in stone risk assessments.
In humans, familial or idiopathic hypercalciuria (IH) is a common cause of hypercalciuria and predisposes to calcium oxalate nephrolithiasis. Intestinal calcium hyperabsorption is a constant feature of IH and may be due to either a vitamin D-independent process in the intestine, a primary overproduction of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or a defect in renal tubular calcium reabsorption. Selective breeding of spontaneously hypercalciuric male and female Sprague-Dawley rats resulted in offspring with hypercalciuria, increased intestinal calcium absorption, and normal serum 1,25(OH)2D3 levels. The role of the vitamin D receptor (VDR) in the regulation of intestinal calcium absorption was explored in 10th generation male genetic IH rats and normocalciuric controls. Urine calcium excretion was greater in IH rats than controls (2.9 +/- 0.3 vs. 0.7 +/- 0.2 mg/24 h, P < 0.001). IH rat intestine contained twice the abundance of VDR compared with normocalciuric controls (536 +/- 73 vs. 243 +/- 42 nmol/mg protein, P < 0.001), with no difference in the affinity of the receptor for its ligand. Comparable migration of IH and normal intestinal VDR on Western blots and of intestinal VDR mRNA by Northern analysis suggests that the VDR in IH rat intestine is not due to large deletion or addition mutations of the wild-type VDR. IH rat intestine contained greater concentrations of vitamin D-dependent calbindin 9-kD protein. The present studies strongly suggest that increased intestinal VDR number and normal levels of circulating 1,25(OH)2D3 result in increased functional VDR-1,25(OH)2D3 complexes, which exert biological actions in enterocytes to increase intestinal calcium transport. Intestinal calcium hyperabsorption in the IH rat may be the first example of a genetic disorder resulting from a pathologic increase in VDR.
Increasing dietary calcium intake decreases urinary oxalate excretion by increasing intestinal precipitation of dietary oxalate as calcium oxalate. This mechanism was speculated to account for the decreased prospective incidence of kidney stones as estimated dietary calcium intake, adjusted for caloric intake, increased among men in a recent large epidemiological study. To further assess the relationship between estimated diet calcium and urinary oxalate, we studied 94 health adults, 50 women and 44 men, ages 20 to 70 years with weights ranging form 47 to 104 kg while they ate their customary diets. Each subject completed a semiquantitative food frequency questionnaire and collected three 24-hour urines preserved with HCl. The urines were collected accurately as judged by a mean intrasubject CV for creatinine excretion of 9.8% and direct relations between urinary creatinine excretion and body wt (r = 0.62; P < 0.0001), or predicted urine creatinine content for sex, age and weight using the Cockcroft and Gault formulas (r = 0.76; P < 0.0001). Estimated diet calcium intake ranged from 6.8 to 68 mmol/day (272 to 2720 mg/day) and averaged 29.5 mmol/day (1180 mg/day). Individual mean urinary oxalate excretion ranged from 0.079 go 0.332 mmol/day (7 to 29 mg/day) and averaged 0.198 mmol/day (17 mg/day). Among all subjects, daily oxalate excretion was directly related to creatinine excretion as an estimate of lean body mass (r = 0.61; P < 0.0001). Thus, oxalate excretion among men averaged 0.228 +/- 0.051 SD mmol/day, a value significantly higher than the average among women of 0.173 +/- 0.045 mmol/day (P < 0.001). Daily urine oxalate excretion/creatinine decreased curvilinearly as estimated dietary Ca intake increased (r = -0.30; P = 0.0035) and as the ratio of estimated dietary calcium to dietary oxalate increased (r = -0.39; P = 0.0001). We conclude that body size is the major determinant of urinary oxalate excretion among healthy adults, presumably reflecting variations in endogenous oxalate synthesis with lean body mass. Increasing estimated diet calcium intake, especially up to the range of 15 to 20 mmol/day (600 to 800 mg/day) has an additional effect to decrease during oxalate excretion, presumably by limiting intestinal absorption of dietary oxalate.
Supersaturation (SS) with respect to calcium oxalate monohydrate (COM), brushite (Br) and uric acid (UA), obtained in three 24-hour pretreatment urine samples from patients with stone disease were compared to the mineral composition of stones passed by the same patients to determine whether sparse urine SS measurements accurately reflect the long-term average SS values in the kidney and final urine. Among males and females elevation of SS above same sex normals corresponded to composition. As well, treatments that reduced stone rates also reduced these SS values. The degree of calcium phosphate (CaP) admixture was accurately matched by shifting magnitudes of COM and Br SS. As well, increasing CaP content was associated with falling urine citrate and rising urine pH, suggesting renal tubular acidosis. We conclude that sparse urine SS measurements accurately track stone admixtures, and are a reliable index of average renal and urine SS.
Calcium oxalate (CaOx) and calcium phosphate (CaP) crystals do not precipitate in large amounts in normal urine despite considerable supersaturation (SS), partly because urine inhibits crystal nucleation, aggregation, and growth. In normal rats and rats bred for hypercalciuria (GHS), we varied SS by varying calcium intake to test the hypothesis that increased SS might deplete inhibitors and reduce inhibition of crystal formation. In normal rats when compared to a low calcium diet (0.02% Ca), a high calcium diet (1.2% Ca) raised the SS of CaOx from 0.8 to 8.2. The high calcium diet also raised the upper limit of metastability (ULM) of CaOx (the SS at which crystals form in urine) from 11.8 to 36. In GHS rats, diet change altered CaOx SS from 1.5 to 12, and ULM from 17 to 50 (all differences, P < 0.001). Because ULM rose with SS, the increased SS had little potential to increase CaOx stone risk. For CaP, however, SS rose from 0.6 to 2.4 and 1.1 to 8 in normal and GHS rats (P < 0.001 for both), respectively, whereas ULM for CaP did not increase significantly (8 vs. 7 and 7 vs. 11; P = NS, both changes). Therefore, CaP SS rose close to the ULM, posing a high stone risk. The stones formed by these rats are composed of CaP. Increasing CaOx SS by diet raises ULM for CaOx thereby offsetting the risk of CaOx stones in rats.
Metabolic acidosis induces resorption of cultured bone, resulting in a net efflux of calcium (Ca) from the bone and an apparent loss of mineral potassium (K). However, in these organ cultures, there is diffusion of K between the medium and the crystal lattice, causing difficulty in interpretation of the acid-induced changes in mineral ion composition. To determine the effects of acidosis on bone mineral K, we injected 4-day-old neonatal mice with pure stable isotope 41K, equal to ~5% of their total body K. Calvariae were dissected 24 h later and then cultured for 24 h in medium without added 41K, either at pH ~7.4 (Ctl) or at pH ~7.1 (Ac), with or without the osteoclastic inhibitor calcitonin (3 x 10-9 M, CT). The bone isotopic ion content was determined with a high-resolution scanning ion microprobe utilizing secondary ion mass spectrometry. 41K is present in nature at 6.7% of total K. The injected 41K raised the ratio of bone 41K/(39K+41K) to 9.8 ± 0.5% on the surface (ratios of counts per second of detected secondary ions, mean ±95% confidence interval) but did not alter the ratio in the interior (6.9 ± 0.4%), indicating biological incorporation of the 41K into the mineral surface. The ratios of 41K/40Ca on the surface of Ctl calvariae was 14.4 ± 1.2, indicating that bone mineral surface is rich in K compared with Ca. Compared with Ctl, Ac caused a marked increase in the net Ca efflux from bone that was blocked by CT. Ac also induced a marked fall in the ratio of 41K/40Ca on the surface of the calvariae (4.3 ± 0.5, p < 0.01 vs. Ctl), which was partially blocked by CT (8.2 ± 0.9, p < 0.01 vs. Ctl and vs. Ac), indicating that Ac causes a greater release of bone mineral K than Ca which is partially blocked by CT. Thus, bone mineral surface is rich in K relative to Ca, acidosis induces a greater release of surface mineral K than Ca, and osteoclastic function is necessary to support the enriched levels of surface mineral K in the presence of acidosis.
Hypercalciuria in genetic hypercalciuric stone-forming (GHS) rats is accompanied by intestinal Ca hyperabsorption with normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels, elevation of intestinal, kidney, and bone vitamin D receptor (VDR) content, and greater 1,25(OH)2D3-induced bone resorption in vitro. To test the hypothesis that hyperresponsiveness of VDR gene expression to 1,25(OH)2D3 may mediate these observations, male GHS and wild-type Sprague- Dawley normocalciuric control rats were fed a normal Ca diet (0.6% Ca) and received a single intraperitoneal injection of either 1,25(OH)2D3 (10-200 ng/100 g body wt) or vehicle. Total RNAs were isolated from both duodenum and kidney cortex, and the VDR and calbindin mRNA levels were determined by Northern blot hybridization using specific cDNA probes. Under basal conditions, VDR mRNA levels in GHS rats were lower in duodenum and higher in kidney compared with wild-type controls. Administration of 1,25(OH)2D3 increased VDR gene expression significantly in GHS but not normocalciuric animals, in a time- and dose-dependent manner. In vivo half-life of VDR mRNA was similar in GHS and control rats in both duodenum and kidney, and was prolonged significantly (from 4-5 to > 8 h) by 1,25(OH)2D3 administration. Neither inhibition of gene transcription by actinomycin D nor inhibition of de novo protein synthesis with cycloheximide blocked the upregulation of VDR gene expression stimulated by 1,25(OH)2D3 administration. No alteration or mutation was detected in the sequence of duodenal VDR mRNA from GHS rats compared with wild-type animals. Furthermore, 1,25(OH)2D3 administration also led to an increase in duodenal and renal calbindin mRNA levels in GHS rats, whereas they were either suppressed or unchanged in wild-type animals. The results suggest that GHS rats hyperrespond to minimal doses of 1,25(OH)2D3 by an upregulation of VDR gene expression. This hyperresponsiveness of GHS rats to 1,25(OH)2D3 (a) occurs through an increase in VDR mRNA stability without involving alteration in gene transcription, de novo protein synthesis, or mRNA sequence; and (b) is likely of functional significance, and affects VDR-responsive genes in 1, 25(OH)2D3 target tissues. This unique characteristic suggests that GHS rats may be susceptible to minimal fluctuations in serum 1, 25(OH)2D3, resulting in increased VDR and VDR-responsive events, which in turn may pathologically amplify the actions of 1,25(OH)2D3 on Ca metabolism that thus contribute to the hypercalciuria and stone formation.