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.

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.

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 .

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.

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.

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.

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.

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

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.

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

A new method to prepare calcium oxalate films for marble protection

Artificial calcium oxalate films have been applied to protect sculptures,monuments,and buildings made of marble due to their potential protective capacities.Unfortunately,artificially prepared calcium oxalate films are usually loose,and easy to peel off in contrast to the natural ones.In this paper,a new method was developed for the preparation of a compact and cohesive calcium oxalate film,using ethylene glycol as an additive.The morphology,elemental composition,structure,and crystal phase of the film were analyzed by SEM,EDX,IR,and XRD,and the protective effects were investigated by acid resistance test,while its adhesion to the underlying marble by the Scotch tape test.The porosity,water absorption,and color of treated marble were also assessed.The results showed that a calcium oxalate film with a compact structure,good adhesion,and a protective effect against acid rain can be successfully obtained by including ethylene glycol as an additive.With the new preparation method,a better bond between the stone and the film is obtained which contributes to solving the spalling problem of the artificial calcium oxalate films.

Decreased renal vitamin K-dependent γ-glutamyl carboxylase activity in calcium oxalate calculi patients

To study the activity of vitamin K-dependent γ-glutamyl carboxylase in patients with calcium oxalate (CaOx) urolithiasis compared with healthy individuals and to assess its relationship to the renal calcium oxalate urolithiasis Methods Renal parenchymas were harvested from urolithic patients and renal tumor patients undergoing nephrectomy The renal carboxylase activity was evaluated as the radioactivity of [ 14 C] labeled sodium bicarbonate in carboxylic reactions in vitro using β-liquid scintillation counting Results Significantly reduced activity of renal vitamin K-dependent γ-glutamyl carboxylase was observed in the urolithic group as compared with normal controls (P<0 01) Conclusion It suggests that the reduced carboxylase activity observed in the urolithic patients may play an important role in the course of renal calcium oxalate urolithiasis

Differences in Adsorption of Anionic Surfactant AOT by Calcium Oxalate： Effect of Crystal Size and Crystalline Phase

The adsorption of anionic surfactant sodium diisooctyl sulfosuccinate（AOT） onto calcium oxalate mono- hydrate（COM） and dihydrate（COD） with sizes of 50, 100 nm, 1, 3 and 10 μm was comparatively studied to simulate the interaction between urinary crystallites and urine components. The adsorption quantity of different concentrations of AOT onto COD and COM with different sizes was detected using a UV-Vis spectrophotometer. The crystalline phase transition of COM and COD before and after adsorption was analyzed by X-ray powder diffraction and Fourier transform infrared spectrometry. The zeta potential of the crystal surface after adsorption of different concentrations of AOT was measured using a zeta potential analyzer. The adsorption quantity of AOT on COM and COD with different sizes was ranked in the following order： 50 nm〉100 nm〉1μm〉3 μm〉10 μm. The adsorption quantity of COM was greater than that of COD with the same size because the density of the positive charges on the COM surface was higher than that on COD surface. With the increase of AOT concentration, the adsorption curves of the large-sized COM and COD（3 and 10μm） were S-type, whereas the adsorption curves of the small-sized COM and COD（50 nm, 100 nm and 1μm） were linear. The adsorption capacities of small-sized COM and COD were much greater than those of the 3 and 10μm crystals. On the basis of the above results, we proposed a molecular model to summarize the absorption of AOT onto COM and COD crystals. Small crystals exhibit a large specific surface area and high surface energy. Thus, the adsorption capacities of them are stronger than those of large crystals. Overall, this study implies that small crystals can easily absorb anionic molecules in urine and may easily adhere to a negatively charged cell surface, thereby increasing the severity of cell injury.

Effect of Concentration of Structurally-Different Carboxylic Acids on Growth and Aggregation of Calcium Oxalate in Gel Systems

The effect of concentration of structurally-different carboxylic acids such as ethylene diamine tetraacetic acid （H4edta）, citric acid （H3cit）, tartaric acid （H2tart）, and acetic acid （HOAc） on growth and aggregation of calcium oxalate （CaOxa） in gel systems was comparatively investigated. H2tart and H3cit could change the morphology of cal- cium oxalate monohydrate （COM） and induce the formation of calcium oxalate dihydrate （COD）. H4edta could induce the formation of COD at a lower concentration of 0.33 mmol/L and have the strongest ability to inhibit aggregation of COM. HOAc inhibited COM aggregation only at a higher concentration than 500 mmol/L. With increasing the number of carboxylic groups in an acid or increasing the concentration of carboxylic acid, the capacity of this acid to induce COD formation and to inhibit growth and aggregation of COM crystals increased. That is, this capacity followed the order： H4edta〉H3cit〉H2tart〉 〉HOAc. The result in this work suggested that the presence of H3cit and H2tart in urine played a role in the natural defense against stone formation.

Promotion on Nucleation and Aggregation of Calcium Oxalate Crystals by Injured African Green Monkey Renal Epithelial Cells

The purpose of this work was to detect the properties of African green monkey renal epithelial cells （Vero） after oxidative injury and to study the mediation of the injured Vero on aggregation and formation of calcium oxalate crystals. This injury model was induced by 0.15 mmol/L H2O2 according to the pretest evaluation. The results suggested that H2O2 could injure Vero significantly and decrease cell viability in a time-dependent manner for exposure time of 0.5--2 h. After cell injury, the indexes connected with oxidative injury changed. The malondialdehyde （MDA） content and osteopontin （OPN） expression increased, while superoxide dismutase （SOD） level decreased. It resulted in the increase of both the amount of CaOxa crystals and the degree of crystal aggregation on the injured cells. This work indicated that injured cells promoted the formation of calcium oxalate monohydrate （COM） crystals, thus increased the risk of formation of urinary stone.

Effectiveness of Using Renalof®in the Elimination of Kidney Stones under 10 mm Located in the Renal-Ureteral Tract

Background: In view of the high recurrence of kidney stones in patients, the search for less aggressive, preventive treatments has become increasingly essential. Renalof® offers a phytotherapy alternative. Due to its diuretic and kidney stone demineralisation properties, it has been widely used in this patient population, disintegrating and eliminating calcium oxalate and struvite kidney stones painlessly in the genitourinary system. Methods: A Phase II, randomised, prospective, observational, single-blind study with two treatment arms was conducted in order to determine the efficacy of this alternative therapy: a total of 155 patients were enrolled, 120 were assigned to a Renalof® treatment group and 35 to the placebo group. All were over 18 years of age, of both genders, diagnosed with kidney stones of under 10 mm in diameter, present along the entire renal-ureteral-vesicular tract, diagnosed by ultrasound and renal CAT scan. Divided into two study arms, 120 were administered a dosage of a single 325 mg capsule of the Renalof® product half an hour before the two main meals for 3 months. The presence of kidney stones in any part of the renal-ureteral tract was assessed at monthly consultations using one of the above-mentioned diagnostic tools. Results: Study results show a high rate of effectiveness with Renalof®, finding up to a 65% expulsion rate (78 patients) in the first 8 weeks of treatment, compared to 11.4% (4 patients) in the placebo control group, P < 0.001. It is likely a longer follow-up period would be necessary in patients with kidney stones of 10 mm in diameter or larger than the period applied in the study. Discussion: We strongly recommend the inclusion of this product in kidney stone disease management protocols, especially for patients with kidney stones under 10 mm in diameter, where high response and effectiveness have been observed. Thus, it should be evaluated to reduce surgical treatment costs, as well as those for possible colic episodes and other associated complications.