Proposal for pathogenesis-based treatment options to reduce calcium oxalate stone recurrence

Objective:Prevalence of kidney stone disease continues to increase globally with recurrence rates between 30%and 50%despite technological and scientific advances.Reduction in recurrence would improve patient outcomes and reduce cost and stone morbidities.Our objective was to review results of experimental studies performed to determine the efficacy of readily available compounds that can be used to prevent recurrence.Methods: All relevant literature up to October 2020,listed in PubMed is reviewed.Results: Clinical guidelines endorse the use of evidence-based medications,such as alkaline agents and thiazides,to reduce urinary mineral supersaturation and recurrence.However,there may be additional steps during stone pathogenesis where medications could moderate stone risk.Idiopathic calcium oxalate stones grow attached to Randall’s plaques or plugs.Results of clinical and experimental studies suggest involvement of reactive oxygen species and oxidative stress in the formation of both the plaques and plugs.The renin-angiotensin-aldosterone system(RAAS),nicotinamide adenine dinucleotide phosphate(NADPH)oxidase,mitochondria,and NOD-like receptor pyrin domain containing-3(NLRP3)inflammasome have all been implicated at specific steps during stone pathogenesis in animal models.Conclusion: In addition to supersaturation-reducing therapies,the use of anti-oxidants,free radical scavengers,and inhibitors of NADPH oxidase,NLRP3 inflammasome,and RAAS may prove beneficial for stone prevention.Compounds such as statins and angiotensin converting enzyme inhibitors are already in use as therapeutics for hypertension and cardio-vascular disease and have previously shown to reduce calcium oxalate nephrolithiasis in rats.Although clinical evidence for their use in stone prevention in humans is limited,experimental data support they be considered along with standard evidence-based medications and clinical expertise when patients are being counselled for stone prevention.

Modulation of Tartrates with Various Counterions on the Phases of Calcium Oxalate in Gelatinous Systems

Effect of various counterions of tartrate on the crystallization of calcium oxalate in gel system was investigated using scanning electron microscopy and X-ray diffraction. Various tartrates with hydrogen (H2tart), sodium (Na2tart), potassium (K2tart), ammonium ((NH4)2tart), and a mixture of sodium and potassium cations (NaKtart) were considered. For H2tart, Na2tart, and (NH4)2tart, calcium oxalate dihydrate (COD) was induced. However, for K2tart and NaKtart, calcium oxalate trihydrate (COT) was obtained.

A Comparative Study on Several Models of Experimental Renal Calcium Oxalate Stones Formation in Rats

In order to compare the effects of several experimental renal calcium oxalate stones formation models in rats and to find a simple and convenient model with significant effect of calcium oxalate crystals deposition in the kidney, several rat models of renal calcium oxalate stones formation were induced by some crystal-inducing drugs （CID） including ethylene glycol （EG）, ammonium chloride （AC）, vitamin D3 [ 1 α（OH）VitD3, alfacalcidol], calcium gluconate, ammonium oxalate, gentamicin sulfate, L-hydroxyproline. The rats were fed with drugs given singly or unitedly. At the end of experiment, 24-h urines were collected and the serum creatinine （Cr）, blood urea nitrogen （BUN）, the extents of calcium oxalate crystal deposition in the renal tissue, urinary calcium and oxalate excretion were measured. The serum Cr levels in the stone-forming groups were significantly higher than those in the control group except for the group EG＋L-hydroxyproline, group calcium gluconate and group oxalate. Blood BUN concentration was significantly higher in rats fed with CID than that in control group except for group EG＋L-hydroxyproline and group ammonium oxalate plus calcium gluconate. In the group of rats administered with EG plus Vitamin D3, the deposition of calcium oxalate crystal in the renal tissue and urinary calcium excretion were significantly greater than other model groups. The effect of the model induced by EG plus AC was similar to that in the group induced by EG plus Vitamin D3. EG plus Vitamin D3 or EG plus AC could stably and significantly induced the rat model of renal calcium oxalate stones formation .

Antilithic Effects of Extracts from Urtica dentata Hand on Calcium Oxalate Urinary Stones in Rats

This study examined the potential antilithic effects of a traditional Chinese medicine Urtica dentata Hand （UDH） in experimental rats and screened the optimal extract of UDH as a possible therapeutic agent for kidney stones. The rat model of urinary calcium oxalate stones was induced by intragastric （i.g.） administration of 2 mL of 1.25% ethylene glycol （EG） and 1% ammonium chloride （AC） for 28 days and was confirmed by Color Doppler ultrasound imaging. The rats in different experimental groups were then intragastrically given petroleum ether extract （PEE）, N-butanol extract （NBE）, aqueous extract （AqE） of UDH, Jieshitong （positive control drug）, and saline, respectively. Treatment with NBE significantly reduced the elevated levels of urinary calcium, uric acid, phosphate, as well as increased urinary output. Accordingly, the increased calcium, oxalate levels and the number of calcium oxalate crystals deposits were remarkably reverted in the renal tissue of NBE-treated rats. In addition, NBE also prevented the impairment of renal function to decrease the contents of blood urea nitrogen （BUN） and creatinine. Taken together, these data suggest that NBE of UDH has a beneficial effect on calcium oxalate urinary stones in rats by flushing the stones out and protecting renal function.

Decreased Expression of Vitamin K Epoxide Reductase Complex Subunit 1 in Kidney of Patients with Calcium Oxalate Urolithiasis

Urinary prothrombin fragment 1 （UPTFl） is a potent inhibitor of urinary stone formation. UPTF1 exerts such inhibitory effect by effective 7-carboxylation in which vitamin K epoxide reductase complex subunit 1 （VKORC1）, the rate-limiting enzyme, is involved. This study examined the correlation between VKORC1 expression and calcium oxalate urolithiasis. The renal cortex samples were obtained from patients undergoing nephrectomy and then divided into 3 groups： urolithiasis group, control group A [hydronephrosis-without-stone （HWS） group], control group B （normal control group）, The localization and expression of VKORC1 in renal tissues were determined by using immunohistochemistry, immunofluorescence microscopy, Western blotting and SYBR Green I real-time reverse-transcription PCR. The rapid amplification of cDNA ends （RACE） were conducted to obtain the 3＇- and 5＇-untranslated region （UTR） of VKORC1. The results showed that VKORC1 was located in the cytoplasm of renal tubular epithelial cells. The expression of VKORC1 in the uro- lithiasis group was significantly lower than that in the other two control groups （P〈0.05）. Moreover, the 3＇- and 5＇-UTR sequence of the VKORC1 gene was successfully cloned. No insertion or deletion was found in the 3＇- and 5＇-UTR. However, a 171-bp new base sequence was discovered in the up- stream of 5＇-UTR end in the urolithiasis group. It was concluded that the decreased expression of VKORC 1 may contribute to the development of calcium oxalate urolithiasis in the kidney.

Inhibition of the Crystal Growth and Aggregation of Calcium Oxalate by Algae Sulfated Polysaccharide In-vitro

The influence of sulfated polysaccharide （SPS） isolated from marine algae Sargassum fusiforme on the morphology and phase compositions of urinary crystal calcium oxalate was investigated in vitro by means of scanning electron microscopy and X-ray diffraction. SPS maybe is a potential inhibitor to CaOxa urinary stones by inhibiting the growth of calcium oxalate monohydrate （COM）, preventing the aggregation of COM, and inducing the formation of calcium oxalate dihydrate （COD） crystals.

Genetic Mutation of Vitamin K-dependent Gamma-glutamyl Carboxylase Domain in Patients with Calcium Oxalate Urolithiasis

To investigate the exon mutation of vitamin K-dependent gamma-glutamyl carboxylase （GGCX or VKDC） in patients with calcium oxalate urolithasis, renal cortex and peripheral blood samples were obtained from severe hydronephrosis patients （with or without calculi）, and renal tumor patients undergoing nephrectomy. GGCX mutations in all 15 exons were examined in 44 patients with calcium oxalate urolithiasis （COU） by polymerase chain reaction （PCR） and denatured high pressure liquid chromatography （DHPLC）, and confirmed by sequencing. Mutation was not found in all COU samples compared to the controls. These data demonstrated that functional GGCX mutations in all 15 exons do not occur in most COU patients. It was suggested that there may be no significant association between the low activity and mutation of GGCX in COU.

Influence of Konjac Glucomannan on the Crystallization Morphology and Structure of Calcium Oxalate

The influence of additive Konjac Glucomannan （KGM） in a variety of con- centrations on the crystallization morphology and structure of calcium oxalate （CaOxa） has been investigated by infrared spectroscopy, scanning electron microscope, X-ray diffraction and so on. The results showed KGM can complex with the Ca^2＋ ions; low concentration KGM prevents CaOxa from aggregating, raises the concentration of ions in the solution, reduces the quantity of crystals and inhibits their growth, and the crystals are round and blunt; while high concentration KGM promotes the growth of crystal, which appears in sheet-like or irregular shape. Only CaOxa monohydrate was observed in a certain system with or without the presence of KGM.

Formation of ring calcium oxalate patterns induced by domains in DPPC Langmuir-Blodgett films

The ring patterns of calcium oxalate crystals were induced by domains in Langmuir-Blodgett （LB） films of dipalmitoylpho- sphatidylcboline （DPPC）. The result was explained by the defects at the ring boundaries of liquid condensed （LC） and liquid expanded （LE） phases of LB film. These boundaries could provide less free energy and much more nucleating sites for COM crystals.

The Effect of Potassium Citrate on Simultaneous Growth of Calcium Oxalate Mono-, Di-, and Trihydrate in Synthetic Urine

The inducing effect of potassium citrate （K3cit） on simultaneous growth of calcium oxalate mono-（COM）, di-（COD）, and trihydrate （COT） crystals in synthetic urine was observed with double diffusion gelatinous technique. K3cit can induce the formation of COD and COT, inhibit the aggregation and decrease the surface area of COM crystals. It supported the clinical use of K3cit and may provide important clues to this disease in cure and in search for new drugs.

Association of Vitamin D Receptor Gene Polymorphisms with Calcium Oxalate Calculus Disease

To study the relationship between polymorphism of vitamin D receptor (VDR) allele with formation of calcium oxalate calculus and find the predisposing genes of calcium oxalate calculus, we screened out 150 patients who suffered from calcium oxalate calculus. 36 of them had idiopathic hypercalciuria according to analysis of calculus component and assay of urine calcium. The polymorphisms of VDR gene Taq1, Apa1 and Fok1 were detected using PCR-RFLP technique and the correlation were analyzed between the polymorphism and urinary calculus or between the polymorphism and hypercalciuria. The difference in each genotypic frequency of the allele of promoter Fok1 between calculus group and healthy group or between idiopathic hypercalciuria calculus group and health group was significant. The content of 24-h urine calcium of those who had genotype ff was obviously higher than that of those who have other genotypes in the same group. There was no significant difference in the polymorphism of gene Apa1 and Taq1 between each two groups. It is concluded that hypercalciuria and calcium oxalate calculus were related to the polymorphism of VDR gene's promoter Fok1 allele, but it had nothing to do with the polymorphism of gene Apa1 and Taq1. The genotype ff was a candidate heredity marker of calcium calculus disease.

The Relationship of Oxalobacter Formigenes and Calcium Oxalate Calculi

The intestinal Oxalobacter Formigenes were isolated in 30 cases of urolithiasis and in 45 controls. The biologic characters and morphology of the bacteria were also observed. The results showed that the colony counts in urolith group 9 (mean 103/g. faeces) were significantly less than that of controls (mean 108/g. faeces) (P<0. 001). It is believed that the lesser amount of oxalobacter formigenes in urolith was the important factor of the calcium oxalate calculi formation.

Calcium and Oxalate Contents of Curly Leaf (<i>Petroselinum crispum</i>) and Flat Leaf (<i>P. crispum var. neapolitanum </i>) Parsley Cultivars

The total, soluble and insoluble oxalate contents of the leaves and stems of curly leaf (Petroselinum crispum) and flat leaf (P. crispum var. neapolitanum) parsley cultivars were extracted from fresh tissue and measured using HPLC chromatography. There were no significant differences between the total and insoluble oxalate contents of the leaves between the flat leaf and curly leaf cultivars. There was a small difference (P < 0.05) between the soluble oxalate contents of the leaves of the two cultivars. The mean total, soluble and insoluble oxalates of the leaves of the two cultivars were 1137.0, 177.9 and 959.3 mg/100 g dry matter (DM), respectively. The mean total, soluble and insoluble oxalate contents of the stems were 1680.7, 386.2 and 1294.5 mg/100 g DM, respectively, and these were significantly higher than the mean values for the leaves of the two cultivars. Insoluble oxalate made up a mean of 77.0% of the curly leaf stems and leaves compared to a mean of 84.4% found in the flat-leaved cultivar. Unavailable calcium, that is, calcium bound to oxalate as insoluble oxalate, made up a mean of 26.9% of the total calcium in the leaves of both cultivars while the unavailable calcium made up 45.0% of the total calcium in the stems of the two cultivars. Overall, the oxalate contents of both parsley cultivars are relatively high, on a dry matter basis, but their overall contribution to dietary intake is likely to be quite small as parsley is an herb that is only used in small amounts to garnish foods.

Manufacture of a Low Oxalate Mitsumame-Type Dessert Using Rhubarb Juice and Calcium Salts

Rhubarb (Rheum rhabarbarum) juice was used to make a Japanese soft mitsumame-type dessert sweet. The dessert was prepared from extracted rhubarb juice, which was cooked with sugar, agar and guar gum, then allowed to set in sweet moulds. The total, soluble and insoluble oxalates were determined in the ingredients and the final products using HPLC chromatography. To reduce the soluble oxalate content of the dessert while retaining the colour and taste of the final product, increments of CaCl2 and CaCO3 were added to the test dessert mixes. The addition of CaCl2 reduced the pH from 3.55 ± 0.03 to pH 3.09 ± 0.02 while addition of CaCO3 increased the pH from 3.55 ± 0.03 to 4.96 ± 0.01. In both cases, the incremental addition of calcium reduced the soluble oxalate content of the sweets by converting it to insoluble oxalate.

Ergastic Substances (Calcium Oxalate Crystals) in the Leaf of <i>Combretum</i>Loefl. (Combretaceae) Species in Nigeria

Leaves of twenty-two (22) species of Combretum in Nigeria were examined for occurrence and distribution of different types and sizes of ergastic substances (calcium oxalate crystals). Fresh and herbarium specimens were used for the study. These specimens were wax embedded, sectioned, mounted and micro-photographed using Leica WILD MPS 52 microscope camera on Leitz Diaplan microscope. Results revealed two types of calcium oxalate crystals—crystal sand and druses. Based on the observed differences in the size of the crystals, three groups of calcium oxalate crystals are reported [large crystals: 180.0 - 360.0 μm (241 ± 44.57 μm), moderate crystals: 90.0 - 144.0 μm (117.0 ± 20.60 μm), and small crystals: 18.0 - 72.0 μm (50.21 ± 20.42 μm)]. Crystal sand was found only in Combretum sp. 1 but druses of varying sizes predominated amongst species of the genus. Crystals were distributed within the spongy mesophyll, palisade mesophyll, sub-epidermis adaxial, sub-epidermis abaxial and between the palisade and spongy mesophyll. The findings of this work provide information on the occurrence and distribution of the crystals in the leaf epidermis of these taxa in Nigeria. The formation, occurrence and distribution of the crystal type in the Combretum species constitute dependable taxonomic character especially when combined with other characters.

Inhibition of calcium oxalate nephrotoxicity with Zamzam water

Zamzam water is well known of its high conductivity. For this fact urologist and nephrologists recommend their patients who are suffering from kidney stones not to drink this water because it could worse their health status. This study was conducted to investigate the effect of Zamzam water on calcium oxalate nephrotoxicity in experimentally induced kidney stones in male Wistar albino rats. Calcium oxalate crystals were induced by orally administration of 200 mg of glycolic acid dissolved in the drinking water. The rats were divided into three groups;six rats each. These include positive control group (given glycolic acid), test group (given glycolic acid plus Zamzam water) and negative group (given drinking water only). After two weeks of treatment, blood analysis of blood urea nitrogen (BUN) and creatinine showed significant differences in positive control group compared to the negative control group, whereas no significant differences were noticed in the level of BUN and creatinine between both the negative control and the test group. Moreover, urine analysis showed a high density of calcium oxalate crystals in the positive control group, whereas no crystals were detected in the negative control and the test groups. Histopathological investigations showed damaging in kidneys of the positive control group with no tissue abnormalities in the negative control and the test group. I concluded from this study that Zamzam water prevents the formation calcium oxalate stone, which probably mean that it has no negative effect on patients suffering from kidney disorders due to crystals formation.

Involvement of VKORC1 in the Inhibition of Calcium Oxalate Crystal Formation in HK-2 Cells

Summary： The vitamin K epoxide reductase complex subunit 1 （VKORC1）, the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate （CaOx） crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORClshRNA-2 as a PGPU6/GFP/Neo-VKORClshRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC 1 transfection group than in the pFLAG-CMV-7.1 control group （P〈0.01）. The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate （COM） crystal medium for 48 h was 14±4 per field （100×） in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field （100×） in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC 1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group （P〈0.05）. The expression levels of VKORC 1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC 1 shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group （P〈0.05）. The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field （100x） in the PGPU6/GFP/Neo-VKORC 1 shRNA-2 transfection group and 24±6 per field （100×） in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORClshRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group （P〈0.05）. These findings suggested that the VKORC 1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx. Key words： calcium oxalate crystals; kidney stone; vitamin K epoxide reduetase complex subunit 1; laser-scanning confocal microscopy

Mode of interaction of calcium oxalate crystal with human phosphate cytidylyltransferase 1: a novel inhibitor purified from human renal stone matrix

Nephrolithiasis is a common clinical disorder, and calcium oxalate (CaOx) is the principal crystalline component in approximately 75% of all renal stones. It is widely believed that proteins act as inhibitors of crystal growth and aggregation. Acidic amino acids present in these proteins play a significant role in the inhibition process. In this study, interaction of cal-cium oxalate with human phosphate cytidylyltrans-ferase 1(CCT), a novel calcium oxalate crystal growth inhibitor purified from human renal stone matrix has been elucidated in silico and involvement of acidic amino acids in the same. As only sequence of CCT is available, henceforth its 3-D structure was modeled via Homology modeling using Prime module of Schrodinger package. Molecular dynamic simulation of modeled protein with solvation was done by mac-romodel (Schrodinger). The quality of modeled pro-tein was validated by JCSG protein structure valida-tion (PROCHECK & ERRAT) server. To analyze the interaction of modeled protein CCT with calcium oxalate along with role played by acidic amino acids, ‘Docking simulation’ was done using MOE–Dock. Interaction between calcium oxalate and CCT was also studied by substituting acidic amino acid in the active sites of the protein with neutral and positively charged amino acids. The in silico analysis showed the bond formation between the acidic amino acids and calcium atom, which was further substantiated when substitution of these acidic amino acids with alanine, glycine, lysine, arginine and histidine com-pletely diminished the interaction with calcium ox-alate.

Comparative proteomic analysis of renal tubular epithelial cell injury caused by oxalic acid and calcium oxalate monohydrate

Objective To analyze and identify the differentially expressed proteins in human renal tubular epithelial ceils ( HK-2) after injury caused by oxalic acid and calcium oxalate monohydrate ( COM ) crystal,and to explore the potential role of renal tubular cell injury in kidney stone formation. Methods Normal HK-2 cells

Preparation of CaZrO_3 Powder ThroughOxalate Precipitation Route

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