High-Throughput in vitro Gel-Based Plate Assay to Screen for Calcium Oxalate Stone Inhibitors

<b><i>Objective:</i></b> Kidney stones are a common medical condition that is increasing in prevalence worldwide. Approximately, ∼80% of urinary calculi are composed of calcium oxalate (CaOx). There is a growing interest toward identifying therapeutic compounds that can inhibit the formation of CaOx crystals. However, some chemicals (e.g., antibiotics and bacterial metabolites) may directly promote crystallization. Current knowledge is limited regarding crystal promoters and inhibitors. Thus, we have developed an in vitro gel-based diffusion model to screen for substances that directly influence CaOx crystal formation. <b><i>Materials and Methods:</i></b> We used double diffusion of sodium oxalate and calcium chloride-loaded paper disks along an agar medium to facilitate the controlled formation of monohydrate and dihydrate CaOx crystals. A third disk was used for the perpendicular diffusion of a test substance to assess its influence on CaOx crystal formation. <b><i>Results:</i></b> We confirmed that citrates and magnesium are effective inhibitors of CaOx crystals. We also demonstrated that 2 strains of uropathogenic <i>Escherichia coli</i> are able to promote crystal formation. While the other tested uropathogens and most antibiotics did not change crystal formation, ampicillin was able to reduce crystallization. <b><i>Conclusion:</i></b> We have developed an inexpensive and high-throughput model to evaluate substances that influence CaOx crystallization.

Lactiplantibacillus plantarum Reduced Renal Calcium Oxalate Stones by Regulating Arginine Metabolism in Gut Microbiota

Renal calcium oxalate (CaOx) stones are a common kidney disease. There are few methods for reducing the formation of these stones. However, the potential of probiotics for reducing renal stones has received increasing interest. We previously isolated a strain of Lactiplantibacillus plantarum N-1 from traditional cheese in China. This study aimed to investigate the effects of N-1 on renal CaOx crystal deposition. Thirty rats were randomly allocated to three groups: control group (ddH2O by gavage), model group [ddH2O by gavage and 1% ethylene glycol (EG) in drinking water], and Lactiplantibacillus group (N-1 by gavage and 1% EG in drinking water). After 4 weeks, compared with the model group, the group treated with N-1 exhibited significantly reduced renal crystals (P &lt; 0.05). In the ileum and caecum, the relative abundances of Lactobacillus and Eubacterium ventriosum were higher in the control group, and those of Ruminococcaceae UCG 007 and Rikenellaceae RC9 were higher in the N-1-supplemented group. In contrast, the relative abundances of Staphylococcus, Corynebacterium 1, Jeotgalicoccus, Psychrobacter, and Aerococcus were higher in the model group. We also predicted that the arginase level would be higher in the ileal microbiota of the model group than in the N-1-supplemented group with PICRUSt2. The arginase activity was higher, while the level of arginine was lower in the ileal contents of the model group than in the N-1-supplemented group. The arginine level in the blood was also higher in the N-1-supplemented group than in the model group. In vitro studies showed that exposure to arginine could reduce CaOx crystal adhesion to renal epithelial HK-2 cells. Our findings highlighted the important role of N-1 in reducing renal CaOx crystals by regulating arginine metabolism in the gut microbiota. Probiotics containing L. plantarum N-1 may be potential therapies for preventing renal CaOx stones.

Calcium Oxalate Crystals in Leaves of the Extremophile Plant Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae)

The presence of calcium oxalate (CaOx) crystals has been widely reported in the plant kingdom. These structures play a central role in various physiological functions, including calcium regulation, metal detoxification, and photosynthesis. However, precise knowledge about their possible roles and functions in plants is still limited. Therefore, the present work aims to study the ecotypic variability of Colobanthus quitensis, an extremophile species, concerning CaOx crystal accumulation. The CaOx crystals were studied in leaves of C. quitensis collected from different provenances within a latitudinal gradient (From Andes mountains in central Chile to Antarctica) and grown under common garden conditions. Polarized light microscopy, digital image analysis, and electron microscopy were used to characterize CaOx crystals. The presence of CaOx crystals was confirmed in the four provenances of C. quitensis, with significant differences in the accumulation among them. The Andean populations presented the highest accumulation of crystals and the Antarctic population the lowest. Electron microscopy showed that CaOx crystals in C. quitensis are classified as druses based on their morphology. The differences found could be linked to processes of ecotypic differentiation and plant adaptation to harsh environments.

Regulation of Laminaria Polysaccharides with Different Degrees of Sulfation during the Growth of Calcium Oxalate Crystals and their Protective Effects on Renal Epithelial Cells

The original Laminaria polysaccharide (LP0) was sulfated using the sulfur trioxide-pyridine method, and four sulfated Laminaria polysaccharides (SLPs) were obtained, namely, SLP1, SLP2, SLP3, and SLP4. The sulfated (–OSO3–) contents were 8.58%, 15.1%, 22.8%, and 31.3%, respectively. The structures of the polysaccharides were characterized using a Fourier transform infrared (FT-IR) spectrometer and nuclear magnetic resonance (NMR) techniques. SLPs showed better antioxidant activity than LP0, increased the concentration of soluble Ca2+ in the solution, reduced the amount of CaOx precipitation and degree of CaOx crystal aggregation, induced COD crystal formation, and protected HK-2 cells from damage caused by nanometer calcium oxalate crystals. These effects can inhibit the formation of CaOx kidney stones. The biological activity of the polysaccharides increased with the content of –OSO3−, that is, the biological activities of the polysaccharides had the following order: LP0 < SLP1 < SLP2 < SLP3 < SLP4. These results reveal that SLPs with high –OSO3− contents are potential drugs for effectively inhibiting the formation of CaOx stones.

Evaluations of the curative efficacy of G. fruticosus solvent extracts in experimentally induced nephrolithiatic Wistar male rats

Background In Ethiopian folk medicine, there is a claim that medicinal plants can treat urolithiasis although there is insufficient scientific evidence. The objective of this study was to evaluate the curative efficacy of Gomphocarpus fruticosus extracts in experimentally induced nephrolithiatic rats. Methods Urolithiasis was induced in male Wistar rats by feeding ethylene glycol in drinking water for 28 days. The curative effects were evaluated after oral administrations of 200 mg/kg of the extracts from 15 to 28 days. Urine samples were collected 1 day before sacrificing the rats. Blood, liver and kidney samples were gathered under anaesthetic condition at day 28. Crystals in the urine were also analyzed by light microscopy. Results G. fruticosus EtOAc extract reduced significantly the level of sodium (P < 0.001), whereas it was significantly elevated the levels of magnesium and citrate (P < 0.01) compared to lithiatic control. G. fruticosus BuOH extract lowered the levels of potassium (P < 0.01), calcium and phosphate in urolithiatic rats. It was also observed that G. fruticosus EtOAc extract decreased the level of oxalate in the urine (P < 0.001), whereas it was increased the levels of magnesium (P < 0.05) and citrate (P < 0.01) in serum analysis after exposure to BuOH extract. In the kidneys, CaOx crystal deposits were reduced significantly by G. fruticosus EtOAc extract (P < 0.01). Conclusion It has been noted that G. fruticosus EtOAc extract was potent in treating urolithiasis. However, further study is required to assess the efficacy of the active compounds against urolithiasis.

MO1156,7-DIHYDROXYCOUMARIN AMELIORATE EXPERIMENTAL NEPHROLITHIASIS BY INHIBITING MLKL PHOSPHORYLATION

Background and Aims Intrarenal deposition of organic and inorganic minerals attributes towards the pathogenesis of nephrolithiasis. Various current studies implicates crystal mineralization involves necroinflammation for the progression of crystal induced chronic kidney diseases (CKD). We hypothesized that 6,7-dihydroxycoumarin (6,7-DHC) inhibits calcium oxalate (CaOx) induced necroptosis and ameliorates nephrolithiasis. Method We used an in vitro unbiased high content screening for identifying natural compounds that inhibits CaOx induced necroptosis. Molecular docking and molecular dynamic simulations were done to study the interaction between 6,7-DHC - MLKL. Further, for in vivo studies mice and rats models of nephrolithiasis were used. Renal injuries, CaOx deposition and fibrosis were evaluated using histological analysis. Protein expressions were assessed using immunoblots. Data was analysed using one way ANOVA. Results An unbiased in vitro high content screening of a library of 24 natural compounds identified 6,7-DHC as a potential candidate. Further, pretreatment with 6,7-DHC protected human and mouse cells from CaOx crystal mediated necroptosis in vitro. Treatment with 6,7-DHC also protected both mice and rats from nephrolithiasis. Computational modelling have revealed 6,7-DHC interacts with MLKL and inhibits its phosphorylation by ATP which is a key event in necroptosis signaling cascade. Conclusion All together our studies indicates that 6,7-DHC owns a novel pharmacological inhibitory property towards MLKL and it could serve as a lead molecule for further development of novel coumarin based MLKL inhibitors. Moreover, our findings also suggests that 6,7-DHC could be used as a therapeutic strategy for combating nephrolithiasis.

LncRNA-ATB Participates in the Regulation of Calcium Oxalate Crystal-induced Renal Injury by Sponging the miR-200 Family

Background: LncRNA-ATB is a long noncoding RNAs (lncRNA) activated by transforming growth factor β (TGF-β) and it has important biological functions in tumours and nontumor diseases. Meanwhile, TGF-β is the most critical regulatory factor in the process of nephrotic fibrosis and calcium oxalate (CaOx) crystal-induced renal injury. The present study aimed to investigate the biological function and mechanism of lncRNA-ATB in CaOx crystal-induced renal injury. Methods: The expression level of lncRNA-ATB was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), the expression levels of epithelial-mesenchymal transition (EMT) markers and TGF-β1 were detected by qRT-PCR and western bloting, cell proliferation was measured with the CCK-8 kit, cell apoptosis was measured by flow cytometry, and cell injury was detected with the Cytotoxicity lactate dehydrogenase (LDH) Assay kit.Results: The expression levels of lncRNA-ATB and TGF-β1 significantly increased in HK-2 cells after coincubation with calcium oxalate monohydrate (COM). COM stimulation caused significant injury in HK-2 cells, induced cell apoptosis, inhibited cell proliferation, and induced EMT changes . After COM stimulation, the expression levels of epithelial cell markers E-cadherin and zonula occludens (ZO)-1 in HK-2 cells significantly decreased, whereas the levels of mesenchymal cell markers N-cadherin and vimentin significantly increased. Interference with lncRNA-ATB expression significantly relieved COM-induced cell injury, cell apoptosis, proliferation inhibition, and EMT changes. The expression levels of the microRNA-200 (miR-200) family in HK-2 cells after coincubation with COM significantly decreased. MiR-200a mimics relieved the COM-induced cell injury, apoptosis, proliferation inhibition, and EMT changes, whereas miR-200a inhibitors abolished the lncRNA-ATB interference-induced relief of COM-induced cell injury, apoptosis, proliferation inhibition, and EMT. Conclusion: lncRNA-ATB promote d COM-induced cell injury, cell apoptosis, proliferation inhibition, and EMT to participate in the process of CaOx crystal-induced renal injury by sponging miR-200s.

Genetic Background but Not Intestinal Microbiota After Co-Housing Determines Hyperoxaluria-Related Nephrocalcinosis in Common Inbred Mouse Strains

Calcium oxalate (CaOx) crystal formation, aggregation and growth is a common cause of kidney stone disease and nephrocalcinosis-related chronic kidney disease (CKD). Genetically modified mouse strains are frequently used as an experimental tool in this context but observed phenotypes may also relate to the genetic background or intestinal microbiota. We hypothesized that the genetic background or intestinal microbiota of mice determine CaOx crystal deposition and thus the outcome of nephrocalcinosis. Indeed, Casp1-/-, Cybb-/- or Casp1-/-/Cybb-/- knockout mice on a 129/C57BL/6J (B6J) background that were fed an oxalate-rich diet for 14 days did neither encounter intrarenal CaOx crystal deposits nor nephrocalcinosis-related CKD. To test our assumption, we fed C57BL/6N (B6N), 129, B6J and Balb/c mice an oxalate-rich diet for 14 days. Only B6N mice displayed CaOx crystal deposits and developed CKD associated with tubular injury, inflammation and interstitial fibrosis. Intrarenal mRNA expression profiling of 64 known nephrocalcinosis-related genes revealed that healthy B6N mice had lower mRNA levels of uromodulin (Umod) compared to the other three strains. Feeding an oxalate-rich diet caused an increase in uromodulin protein expression and CaOx crystal deposition in the kidney as well as in urinary uromodulin excretion in B6N mice but not 129, B6J and Balb/c mice. However, backcrossing 129 mice on a B6N background resulted in a gradual increase in CaOx crystal deposits from F2 to F7, of which all B6N/129 mice from the 7th generation developed CaOx-related nephropathy similar to B6N mice. Co-housing experiments tested for a putative role of the intestinal microbiota but B6N co-housed with 129 mice or B6N/129 (3rd and 6th generation) mice did not affect nephrocalcinosis. In summary, genetic background but not the intestinal microbiome account for strain-specific crystal formation and, the levels of uromodulin secretion may contribute to this phenomenon. Our results imply that only littermate controls of the identical genetic background strain are appropriate when performing knockout mouse studies in this context, while co-housing is optional.

Antilithiatic effect of C. dactylon, E. officinalis, K. pinnata, and B. nutans ethyl acetate fraction on glyoxylate-induced nephrolithiasis

Background The plants Cynodon dactylon (C. dactylon), Emblica officinalis (E. officinalis), Kalanchoe pinnata (K. pinnata), and Bambusa nutans (B. nutans) have been reported to possess diuretic and antiurolithiatic potential against ethylene glycol and ammonium chloride along with in vitro calcium oxalate (CaOx) crystal growth inhibition property. Our previous research publications reported a rich presence of antioxidative phytocompounds like polyphenols and flavonoids in ethyl acetate fractions of these plants. This present study aims to explore antiurolithiatic potential of C. dactylon, E. officinalis, K. pinnata, and B. nutans ethyl acetate fraction following 7 days of sodium glyoxalate treatment on mice. Results Sodium glyoxylate treatment caused significant (P < 0.01–0.001) reduction in the urine magnesium and creatinine and elevation in oxalate, citrate, calcium, and phosphate levels. Ethyl acetate fraction of K. pinnata and B. nutans showed a highly significant antilithiatic effect by increasing urine volume, normalizing disrupted urine parameters, increasing LDH level, and decreasing kidney tissue oxalate content. E. officinalis and K. pinnata ethyl acetate fraction treatment showed a pronounced reversal of tubular dilation and damage of epithelial cell in kidney tissue with very less inflammatory cell infiltration. Conclusion The results signify the protective effect of K. pinnata and B. nutans ethyl acetate fraction rich with polyphenol and flavonoid on glyoxylate induced oxidative cell damage and morphological changes in mouse kidneys.

Evaluation of Phyllanthus niruri L. from Malaysia for In-vitro Anti-Urolithiatic Properties by Different Solvent Extraction

The Phyllanthus niruri is traditionally used for curing of kidney disorders and urinary stones in Malaysia. Hence the current work was aimed to evaluate the effect of different solvents extract (n- hexane, ethyl acetate, methanol and water) of P. niruri for in vitro anti-urolithiatic properties in terms of inhibition activity on CaOx by using the rate of CaOx aggregation assay and dissolution of calcium oxalate (CaOx) crystal by using titrimetry method. Cystone was used as positive control. The effects of cystone on slope of nucleation and aggregation as well as growth of CaOx were evaluated spectrophotometrically. The highest yield percentage of P.niruri was occupied by methanol (5.74 %). The maximum inhibition against aggregation of CaOx crystals was also occupied by methanol (66.67 % ± 1.61) and was comprised with alkaloid, steroid, terpenoid and tannin. Dissolution effect on calcium oxalate crystals indicates that the aqueous extracts of P. niruri was found to be more effective in dissolution of CaOx with 63.33 % ± 1.44. P. niruri significantly (P < 0.05) inhibited the slope of nucleation and aggregation of CaOx crystallization, and reduced the crystal density. The results of the present study confirmed that P. niruri leaves can be used as remedial mediator for urolithiasis. However, further studies are required for isolation and identification of active constituents and their in-vivo confirmation.