Calcium Oxalate Crystal Formation Research Articles

Multiorgan Crystal Deposition in Acute Ethylene Glycol Toxicity.

Ethylene glycol (EG) is a widely available chemical and a well-known toxin in forensic pathology, which can lead to severe metabolic acidosis and death. Its metabolism in the body leads to formation of calcium oxalate (CO) crystals, which can precipitate in the kidney leading to renal failure. Although the identification of CO crystals in the kidney is widely known, the deposition of CO crystals in other organs has only occasionally been reported and seldom studied. This report examines 18 deaths due to EG toxicity for the presence of CO crystals in various organs. CO crystals were identified most often in the kidneys, followed by the brain and lungs. It is hypothesized that crystal deposition in these other organs may contribute to the toxicity of EG.

Antiurolithic activity of Zaleya pentandra (L.) C Jeffrey in ethylene glycol-induced calcium oxalate crystal rat model; A scientific validation of traditional use for kidney stone prevention

Ethnopharmacological relevanceTraditional herbal remedies have been used for treating nephrolithiasis, but the relevant scientific evidence is limited. Zaleya pentandra (L.) C. Jeffrey is traditionally used for the prevention of kidney stones in various cultures. However, its efficacy has not been scientifically studied. Aim of the studyThis study aimed to investigate the antiurolithic activity of Zaleya pentandra, and validate its traditional used for preventing kidney stones. Materials and methodsThe crude ethanolic extract of Z. pentandra (Zp.Crd) was evaluated through in vitro and in vivo studies. In vitro experiments assessed its impact on crystal count and morphology in metastable calcium oxalate solutions. In vivo studies involved diuretic and ethylene glycol-induced calcium oxalate crystal formation in male Wistar rats. ResultsZp.Crd transforms calcium oxalate crystals from harmful calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD). In vivo, Zp.Crd exhibited dose-dependent (30–300 mg/kg) diuretic activity in rats by significantly increasing urinary sodium (Na+) and potassium (K+) excretion, similar to the standard diuretic hydrochlorothiazide (HCT). In the urolithiasis model, Zp.Crd exhibited dose-dependent antiurolithic effects by reducing kidney crystals and significantly altering lithogenic factors induced by ethylene glycol, including crystalluria, oxaluria, calcium, creatinine, and urea, in the urine and serum of treated rats. Zp.Crd also exhibited antioxidant effects, effectively combating oxidative lithogenic stress in rats. ConclusionZp.Crd has been shown to demonstrate antiurolithic activity against CaOx stones through CaOx crystal inhibition, diuretic activity, antioxidant properties, hypocalciuric effects, and hypercitrauric actions. The findings underscore Zp.Crd's potential as a viable alternative or supplemental therapy to current urolithiasis treatments, paving the door for further clinical trials and its inclusion into modern medical practices.

Crystallization ripening and erosion of calcium oxalate under the effect of bacteria and a polymer materials surface.

As typical examples of pathological biomineralization, urinary stones and stent encrustation have been associated with bacteria, yet the underlying mechanisms remain unclear. In this study, the effect of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus on the nucleation and growth of calcium oxalate crystals both in solution and on material surfaces in vitro was investigated. Both bacteria can promote calcium oxalate crystallization, and E. coli shows a prominent ability to boost the nucleation and growth rate. Interestingly, we discovered an Ostwald ripening phenomenon after the initial nucleation on the material surfaces, where larger particles emerge upon the disappearance of small nuclei particles, evident in the case of S. aureas. Over an extended period of time, erosion and disintegration of the crystals was observed when bacteria were involved. Based on these understandings, we developed a new functional surface by synthesizing an antibacterial polypeptoid in-house and utilizing polyurethane as the substrate material. This surface exhibits a synergistic effect that inhibits the formation of calcium oxalate crystals. This study helps to elucidate the role of bacteria in calcium oxalate biomineralization and supports further development of treatment approaches such as anti-encrustation polymer materials.

The Effect of Lidan Huashi Pills on the Glutathione Peroxidase Activity and Growth of Calcium Oxalate Crystals in the Kidneys of Rats

Objective: To explore the effects of Lidan Huashi Pills on the glutathione peroxidase (GSH-Px) activity and growth of calcium oxalate crystals in the kidneys of rats. Methods: 30 rats were randomly divided into a blank group, a model group, and an experimental group, with 10 rats in each group. The blank group was free to eat and drink water for 8 weeks; the ethylene glycol method was used for the standard calcium oxalate crystal modeling in the control group and experimental group for 4 weeks. The model group was given free feeding and physiological saline (2 ml/d) by continuous gavage for 4 weeks; the experimental group was given free feeding and Lidan Huashi Pills (450 mg/kg, 2 ml/d) by continuous gavage for 4 weeks. After 4 weeks, all rats were euthanized, and the left kidney was taken for GSH-Px level detection. The right kidney was stained with hematoxylin and eosin (H&E) to observe the formation of calcium oxalate crystals. Results: After 4 weeks of modeling, the urinary calcium levels in the model group and experimental group significantly increased compared to the blank group (868.00 ± 39.29 vs 929.40 ± 33.61, P < 0.05), indicating successful modeling. The urine calcium ion concentration in the experimental group after modeling was significantly lower than that in the model group (929.40 ± 33.61 vs 888.60 ± 25.92, P < 0.05). The grading score of calcium oxalate crystals in the model group was significantly higher than that in the blank group (P < 0.05); the grading score of calcium oxalate crystals in the experimental group was lower than that in the model group, and the difference was not statistically significant (P > 0.05). The GSH-Px activity in the left kidney of the model group was significantly lower than that of the blank group [203.49 (208.21, 144.22) vs 494.91 (431.32, 538.18), P < 0.05); the GSH-Px activity in the left kidney of the experimental group was significantly higher than that of the model group [433.60 (383.86, 504.49) vs 203.49 (208.21, 144.22), P < 0.05). Morphological observation and H&E staining suggest that the formation of right kidney crystals and inflammation in the experimental group are between the blank group and the model group. Conclusion: Lidan Huashi Pills can enhance the serum GSH-Px activity in rats and inhibit the growth of calcium oxalate crystals and inflammatory response in the kidneys, thus playing a role in preventing and treating urinary tract stones.

Role of urinary tract microbiota and urine proteome in urolithiasis pathogenesis

The pathogenetic approach to the metaphylaxis of urolithiasis is universally a socially significant health problem due to the increase in morbidity and, especially, its recurrent course, despite the achievements of pharmacotherapy and the use of lithotripsy. Pathological crystallogenesis is considered as a consequence of exceeding the solubility threshold of a number of mineral components of urine, on the one hand, and as a consequence of posttranslational defects of the main proteome of urination - uromodulin (UMOD), demonstrating in healthy people a sanogenetic system for ensuring the stability of biophysical homeostasis: colloidal properties of urine. However, in addition, UMOD, by binding mannose-dependent pills of infectious agents, blocks the contact of bacteria with urotheliocytes, despite the fact that infection of the urinary tract is a known factor of urolithiasis. By modeling the urine of healthy individuals by coincubation with E. coli bacteria, a decrease in the concentration of polymerized uromodulin in urine was demonstrated as a factor in increasing the risk of formation of calcium oxalate crystals

Protective Role of Rosmarinic Acid in Experimental Urolithiasis: Understanding Its Impact on Renal Parameters.

This study aimed to assess the ability of rosmarinic acid (RA) to prevent kidney stone formation in an ethylene glycol and ammonium chloride (EG/AC) model. There was an increase in diuresis in the normotensive (NTRs) and hypertensive rats (SHRs) treated with hydrochlorothiazide (HCTZ) and exposed to EG/AC, while RA restored urine volume in NTRs. The EG/AC groups exhibited lower urine pH and electrolyte imbalance; these parameters were not affected by any of the treatments. Both HCTZ+EG/AC and RA+EG/AC reduced calcium oxalate crystal formation in NTR and SHR urine. Kidney tissue analysis revealed alterations in oxidative stress and inflammation parameters in all EG/AC-receiving groups, with RA enhancing antioxidant defenses in SHRs. Additionally, crystals were found in the kidney histology of all EG/AC-exposed groups, with reduced Bowman's capsule areas in NTRs and SHRs. The NTR VEH+EG/AC group showed intense renal damage, while the others maintained their structures, where treatments with HCTZ and RA were fundamental for kidney protection in the NTRs. Docking analysis showed that RA exhibited good binding affinity with matrix metalloproteinase-9, phosphoethanolamine cytidylyltransferase, and human glycolate oxidase enzymes. The data disclosed herein underscore the importance of further research to understand the underlying mechanisms better and validate the potential of RA for clinical use.

STUB1 exacerbates calcium oxalate-induced kidney injury by modulating reactive oxygen species-mediated cellular autophagy via regulating CFTR ubiquitination.

The formation of calcium oxalate (CaOx) crystals in the kidneys leads to renal epithelial damage and the progression of crystalline nephropathy. This study investigated the role of STIP1 homology and U-box protein 1 (STUB1), an E3 ubiquitin ligase, and cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel, in CaOx-related renal damage and autophagy regulation.HK-2 cells were treated with various doses of CaOx monohydrate (COM) to simulate kidney injury in vitro. Cell viability, reactive oxygen species (ROS) production, and apoptosis were assessed. The regulation of CFTR ubiquitination by STUB1 was confirmed by immunoprecipitation. An in vivo model was established by injecting mice with glyoxylate.COM treatment dose-dependently decreased cell viability, increased TNF-α and ROS production, and induced apoptotic cell death in HK-2 cells. COM-treated cells also showed decreased CFTR protein expression. CFTR overexpression improved cell viability and reduced ROS production in COM-stimulated HK-2 cells. Bioinformatics analysis predicted CFTR's ubiquitination binding site for STUB1. Further analysis confirmed the role of STUB1 as a ubiquitin ligase in CFTR degradation. Knockdown of STUB1 upregulated CFTR expression, while STUB1 overexpression had the opposite effect. Knockdown of CFTR reversed the impact of STUB1 deficiency on autophagy. The in vivo experiments showed that CFTR overexpression attenuated kidney tissue damage and CaOx deposition in mice.STUB1-mediated CFTR ubiquitination plays a crucial role in mitigating calcium oxalate-related renal damage by regulating autophagy. Targeting the STUB1/CFTR axis may hold therapeutic potential for treating kidney injury associated with calcium oxalate deposition.

Long-Term Sodium Deficiency Reduces Sodium Excretion but Impairs Renal Function and Increases Stone Formation in Hyperoxaluric Calcium Oxalate Rats.

Excessive sodium intake is associated with nephrolithiasis, but the impact of sodium-deficient (SD) diets is unknown. Hence, we investigated the effects of short- and long-term SD diets on the expression of renal aquaporins and sodium transporters, and thus calcium oxalate (CaOx) crystal formation in hyperoxaluria rats. In a short-term sodium balance study, six male rats received drinking water and six received 0.75% ethylene glycol (EG) to induce hyperoxaluria. After a 30-day period of feeding on normal chow, both groups were treated with a normal-sodium diet for 5 days, followed by a sodium-free diet for the next 5 days. In a long-term SD study (42 days), four groups, induced with EG or not, were treated with normal-sodium water and sodium-free drinking water, alternately. Short-term sodium restriction in EG rats reversed the daily positive sodium balance, but progressively caused a negative cumulative water balance. In the long-term study, the abundant levels of of Na/H exchanger, thiazide-sensitive Na-Cl cotransporter, Na-K-ATPase, and aquaporins-1 from SD + EG rats were markedly reduced, corresponding to a decrease in Uosm, as compared to SD rats. Increased urine calcium, AP(CaOx)index, and renal CaOx deposition were also noted in SD + EG rats. Although the SD treatment reduced sodium excretion, it also increased urinary calcium and impaired renal function, ultimately causing the formation of more CaOx crystals.

Herbo-Mineral Medicine, Lithom Exhibits Anti-Nephrolithiasis Activity in Rat Model of Hyperoxaluria by Attenuating Calcium Oxalate Crystal Formation and Oxidative Stress.

Calcium oxalate monohydrate (COM) forms the most common type of kidney stones observed in clinics, elevated levels of urinary oxalate being the principal risk factor for such an etiology. The objective of the present study was to evaluate the anti-nephrolithiatic effect of herbo-mineral formulation, Lithom. The in vitro biochemical synthesis of COM crystals in the presence of Lithom was performed and observations were made by microscopy and Scanning Electron Microscope (SEM) based analysis for the detection of crystal size and morphology. The phytochemical composition of Lithom was evaluated by Ultra-High-Performance Liquid Chromatography (UHPLC). The in vivo model of Ethylene glycol-induced hyperoxaluria in Sprague-Dawley rats was used for the evaluation of Lithom. The animals were randomly allocated to 5 different groups namely Normal control, Disease control (ethylene glycol (EG), 0.75%, 28 days), Allopurinol (50 mg/kg, q.d.), Lithom (43 mg/kg, b.i.d.), and Lithom (129 mg/kg, b.i.d.). Analysis of crystalluria, oxalate, and citrate levels, oxidative stress parameters (malondialdehyde (MDA), catalase, myeloperoxidase (MPO)), and histopathology by hematoxylin and eosin (H&E) and Von Kossa staining was performed for evaluation of Lithom. The presence of Lithom during COM crystals synthesis significantly reduced the average crystal area, feret's diameter, and area-perimeter ratio, in a dose-dependent manner. SEM analysis revealed that COM crystals synthesized in the presence of 100 and 300 μg/mL of Lithom exhibited a veritable morphological transition from irregular polygons with sharp edges to smoothened smaller cuboid polygons. UHPLC analysis of Lithom revealed the presence of Trigonelline, Bergenin, Xanthosine, Adenosine, Bohoervinone B, Vanillic acid, and Ellagic acid as key phytoconstituents. In EG-induced SD rats, the Lithom-treated group showed a decrease in elevated urinary oxalate levels, oxidative stress, and renal inflammation. Von Kossa staining of kidney tissue also exhibited a marked reduction in crystal depositions in Lithom-treated groups. Taken together, Lithom could be a potential clinical-therapeutic alternative for management of nephrolithiasis.

Naringen's Effects on Diuresis and Prevention of Urolithiasis in Hypertensive Rats.

This research demonstrates the diuretic effect of naringenin, a flavanone aglycone found in citrus, on spontaneously hypertensive female and male rats (SHR). The data reinforces existing literature findings that male SHR exhibits higher systolic blood pressure than age-matched females. Urine volume assessed over 8 hours was lower when obtained from SHR males than females. When these animals were orally treated with different doses of naringenin (0.1-1 mg/kg), this increased urinary volume in both genders at the highest dose tested. In contrast, the lowest dose promoted a significant natriuretic effect. The other electrolytes analyzed in urine were not significantly altered, except potassium excretion, which was shown to be increased in the urine of SHR males. Furthermore, naringenin showed promise in reducing calcium oxalate (CaOx) crystal formation in an in vitro model, presenting potential advantages in lithiasis prevention.

Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis ‘Hongyang’ and A.eriantha ‘White’). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.Graphical abstract

3-Demethyl-2-geranyl-4-prenylbellidifoline, a natural xanthone with diuretic and kidney protective properties.

The diuretic and kidney protective effect of the 3-demethyl-2-geranyl-4-prenylbellidifoline (DGP) were evaluated in rats. The normotensive (NTR) and spontaneously hypertensive rats (SHR) received, once a day for 7 days, oral treatment with DGP (0.1 mg/kg), hydrochlorothiazide (10 mg/kg), or vehicle (10 ml/kg). Urine, blood, and kidney samples were collected for further analysis. The urine and Na+ elimination content were significantly higher in the groups that received DGP. Furthermore, a Ca2+-sparing action was detected in the urine of DGP-treated groups, which was consistent with the reduction in calcium oxalate crystal formation. Relevantly, the treatment did not change the parameters examined in the blood. Concerning the renal analyses, DGP treatment recovered the morphological damages of the kidney corpuscle area of SHR. In addition to the differences observed between the NTR and SHR vehicle groups, DGP augmented the amount of reduced glutathione and the activity of glutathione S-transferase GST while reducing the catalase and N-acetyl-β-D-glucosaminidase activity and nitrite levels. Together, this study displayed the prolonged diuretic action of DGP and its natriuretic, Ca2+-sparing, and antiurolytic effects. The antioxidative and anti-inflammatory effects of DGP were evidenced in SHR kidneys, opening perspectives for further studies regarding the benefits of this xanthone.

Effect of croton Tonkinensis Gagnep extract on calcium oxalate crystal formation causing kidney stones under in vitro conditions

The study investigated the use of Croton tonkinensis Gagnep extract to evaluate its ability to inhibit Calcium oxalate crystal formation, including three main stages: nucleation, growth, and aggregation. Croton tonkinensis Gagnep samples were extracted using a soaking method with 96% ethanol to obtain the concentrated extract. The percentage of inhibition of Calcium oxalate crystal nucleation by the Croton tonkinensis Gagnep extract was determined using spectrophotometry at a wavelength of 620 nm. Meanwhile, the inhibition efficacy on the growth of Calcium oxalate crystals was evaluated by measuring the optical density of the test sample at a wavelength of 214 nm over a period of 780 seconds. The inhibition efficacy on the aggregation of Calcium oxalate crystals by the extract was determined by measuring the optical density at a wavelength of 620 nm at different time intervals of 30, 60, 90, 180, 360, and 720 minutes. The research results showed that the extract had a high efficiency of 1.36%. The Croton tonkinensis Gagnep extract contained various compounds such as flavonoids, alkaloids, saponins, terpenoids, tannins, and phenols. The extract exhibited the ability to inhibit nucleation with an IC50 value of 7.78 mg/mL, as well as inhibit the growth and aggregation of Calcium oxalate crystals with times of 13 minutes and 90 minutes, respectively.

Metabolomics analysis reveals a protective effect of hydroxycitric acid on calcium oxalate-induced kidney injury.

Prior research has indicated that hydroxycitric acid (HCA) can impede the formation of calcium oxalate (CaOx) crystals, yet the specific mechanisms underlying its therapeutic effects remain unclear. In this study, we delved into the protective effects of HCA against glyoxylate-induced renal stones in rats and sought to elucidate the underlying metabolic pathways. Forty rats were randomly assigned to five groups: control group, model group, L-HCA-treated group, M-HCA-treated group, and H-HCA-treated group. Von Kossa staining was conducted on renal sections, and blood urea nitrogen and serum creatinine were determined by biochemical analysis. Meanwhile, body weight and urine volume were also measured. We subjected urine samples from the rats to analysis using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Next, we employed a metabolomic approach to scrutinize the metabolic profiles of each group. HCA significantly reduced blood urea nitrogen and serum creatinine, and increased body weight and urine volume. It also reduced CaOx crystal deposition. A total of 24 metabolites, exhibiting a significant reversal pattern following HCA administration, were identified as urine biomarkers indicative of HCA's preventive effects against CaOx crystal-induced renal injury. These metabolites are primarily associated with glycine, serine, and threonine metabolism; phenylalanine metabolism; tricarboxylic acid cycle; taurine and hypotaurine metabolism; and tryptophan metabolism. It was demonstrated that HCA has a protective effect against CaOx crystal-induced kidney injury in rats by modulating various metabolic pathways. Additionally, results suggest that HCA holds promise as a potential clinical therapeutic drug for both the prevention and treatment of renal stones.

Potassium oxalate-based mouth rinse for rapid relief in dentinal hypersensitivity

Abstract Dentinal hypersensitivity (DHS) affects about 1.3%–92.1% of the population globally and has a significant impact on the quality of life of the affected individuals. Desensitizing agents that directly block the dentinal tubules or cause chemical blocking of the pulpal nerve endings are commonly recommended for the management of DHS. Potassium oxalate (KO) has been used as a desensitizing agent in the management of DHS. This narrative review aimed to assess the literature related to the efficacy of KO as a desensitizing agent, with a focus on mouth rinses containing 1.4% KO. A comprehensive search of the literature was carried out using PubMed and Google Scholar with relevant keywords. The results from the retrieved articles were assessed, and the findings regarding the efficacy of KO in managing DHS were analyzed. Studies suggest that the usage of 1.4% KO mouth rinses results in the occlusion of dentinal tubules by the formation of acid-resistant calcium oxalate crystals within dentinal tubules and on the dentine surface. These calcium oxalate crystals tend to form a strong bond with the inner surface of the dentinal tubules and lower the hydraulic conductivity of the dentine. These actions of KO can ensure both rapid and long-term relief from DHS.

PHYTOCONSTITUENTS AND IN VITRO ANTI-UROLITHIATIC ACTIVITY OF VARIOUS HYDROPHILIC EXTRACTS OF TERMINALIA CATAPPA LEAVES ON CALCIUM OXALATE CRYSTAL

Background: Medicinal plants play an important role in the alternative or complement therapy to manage of urinary stones at this time. Terminalia genus was proved anti-urolithiatic activity via in vitro inhibition of calcium oxalate formation. Among the samples, Terminalia catappa showed as a potential plants for this activity in India. Moreover, they were a common plant species in Vietnam and there was no research on this topic in our country. Objectives: To evaluate in vitro antiurolithiatic activity of Terminalia catappa leaves in Vietnam, through inhibition of calcium oxalate formation. In addition, moisture value, preliminary screening of the chemical composition and determination of tanninoid content of aqueous extract also were conducted. Materials and methods: Moisture content of herbs was conducted according to guidelines of 5th Vietnam pharmacopoeia, appendix 9.6. Qualitation of the phytochemical constituents of aqueous extract with appropriate reagents to confirm the presence of natural compounds via chemical reactions. Determination of tanninoid content of aqueous extract was evaluated by using oxidation-reduction titration method (Lowenthal assay) and skin powder method in instructing of appendix 12.6 from 5th Vietnam pharmacopoeia. Inhibitory effect of calcium oxalate formation of three hydrophilic extracts (45% ethanol, 96% ethanol and aqueous extracts) was confirmed by nucleation assay with cystone as a positive control. Results: Moisture content of medicinal plant was 11.625%. Terminalia catappa leaves contain the major phytochemical constituents such as flavonoids, tanninoids and saponins. The tanninoid content according to Lowenthal method and skin powder method were 10.88% and 10.70%, respectively. Therefore, the average tannin content was confirmed to be approximately 10.79%. Among the investigated samples, aqueous extract showed the best inhibitory activity of calcium oxalate crystal formation with an IC50 of 602.67 μg/mL when compared to cystone with IC50 423.05 μg/mL. Conclusions: The aqueous extract from Terminalia catappa leaves has been shown to be a promising source for anti-calcium oxalate crystals formation activity on experimental model. The anti-urolithic potential of Terminalia catappa leaves may be related to its major phytoconstituents.

The Effect of Alpha Lipoic Acid on Pathogenesis of Experimental Nephrolithiasis and Epithelial Mesenchymal Transition

In this study, the effects of calcium oxalate (CaOx) crystal formation on the kidneys and the effect of alpha-lipoic acid (ALA) on this process were investigated in rats. The study consists of 6 groups with 10 rats in each group. Ethylene glycol (EG) and ammonium chloride (AC) (0.75% EG + 0.75% AC) were added to the drinking water of different groups for 7 or 14 days to induce nephrolithiasis. The effects of the CaOx on the kidneys and ALA (100 mg/kg/day/orally) on this process were investigated via histopathological, immunohistochemical, and biochemical methods. In the histopathological examination, EG+AC application for both 7 and 14 days caused crystal accumulation in the tubule lumens, cystically dilated tubules, and hydropic degeneration in the tubular epithelium. However, inflammatory cell infiltration was observed merely in 14 days. In the immunohistochemical examination, when EG+AC administration was applied for 14 days only, it caused expression of ED1, alpha smooth muscle actin (α-SMA), and vimentin in the tubulointerstitial areas. However, α-SMA and vimentin expression was not observed in tubular epithelial cells. Transforming growth factor beta-1 (TGF-β1) expression was also detected in the tubular epithelium and intertubular cells at 14 days. It was determined that ALA administration with EG+AC application reduced the crystal accumulation in the tubule lumens (p<0.001), the degeneration of the tubular epithelium (p<0.001), and the expression of TGF-β1. In addition, it was detected that ALA caused an increase in glutathione peroxidase (GPx) (p<0.001) and Catalase (CAT) (p>0.05) activities, which decreased with EG+AC application.

Assessment of Anti-urolithic effect of the Ocimum basilicum L. seed extract on Ethylene Glycolinduced urolithiasis in male rats

This research aimed to assess the effect of Ocimum basilicum L. seed extract on the urolithiasis. Rats were treated with Cystone (750mg/kg) and hydro-alcoholic extracts in 250 and 500mg/kg doses for 28days. On Day 29, Ethylene glycol induced urolithiasis was verified by evaluating urinary parameters, biochemical parameters, oxidative stress parameters and histology of kidneys. Extract treatment attenuated the urinary parameter elevation and biochemical serum parameters induced by the evidence. Extract treatment also reversed induced changes in urine volume, pH and Magnesium and creatinine clearance, oxidative and histological damage to the EG-caused kidneys. The histology of treated group showed small and low deposits of calculi in the kidney. Extract showed potential calcium oxalate crystal formation, diuretic, antioxidant activity and preserving the balance between stone promoters and inhibitors. This validated study rationalized its medicinal use in urolithiasis.

#4359 LOSS OF GASDERMIN D LEADS TO AN EXACERBATION OF KIDNEY DAMAGE IN NEPHROCALCINOSIS-RELATED CHRONIC KIDNEY DISEASE

Abstract Background and Aims Gasdermin D (GSDMD) is a membrane-targeting and pore-forming protein that is primarily expressed in immune cells. In the proinflammatory cell death process known as pyroptosis, cleaved GSDMD plays a crucial role through cell membrane pore formation, leading to the release of proinflammatory cytokines and membrane rapture. Despite this, the majority of GSDMD's other physiological functions remain to be elucidated. Previous reports have implicated GSDMD in the pathogenesis of kidney disease, utilizing some acute kidney injury (AKI) animal models and a few chronic kidney disease (CKD) animal models. However, it remains uncertain if GSDMD contributes to the pathogenesis of nephrocalcinosis-related CKD. Therefore, this study aimed to investigate the role of GSDMD in in this context. Method In our study, 8-week-old GSDMD-knockout (KO) mice and wildtype (WT) mice received an oxalate-rich diet (50 μmol/g sodium oxalate) for three weeks. All mice were sacrificed on day 21 and kidneys were harvested after the sacrifice. Plasma and urine samples were collected at different time points before the sacrifice. The glomerular filtration rate (GFR) was measured on day 0 and day 20 to assess kidney excretion function. Histological analysis was performed using Periodic Acid-Schiff (PAS) staining to assess renal injury, Sirius Red staining to check renal fibrosis, Pizzolato staining to evaluate calcium crystal deposition, F4/80 staining to evaluate the infiltration of macrophages, TUNEL staining to check for cell death, RIPK3 and phosphorylated MLKL (pMLKL) staining to detecting necroptosis. And GSDMD staining was also performed to examine the expression of GSDMD in the kidney. Results Pizzolato staining showed no differences in calcium crystal deposition between WT mice and KO mice. GFR was decreased in both WT mice and KO mice after the oxalate-rich diet (day 0 WT: 199.5±40.1 μl/min vs KO 175.0±44.8 μl/min, day 20 WT: 156.5±37.9 μl/min vs KO: 73.0±48.1 μl/min), and ΔGFR was much greater in KO mice (WT: −22.4±26.5% vs KO: −59.4±19.3%). The level of blood urea nitrogen was significantly higher in KO mice (WT: 41.9±3.90 mg/dl vs KO: 58.7±22.5 mg/dl) in accordance with the result of the GFR. PAS staining revealed more severe kidney injury in KO mice, and Sirius Red staining indicated a higher degree of renal fibrosis in KO mice. Furthermore, F4/80 staining showed greater infiltration of macrophages in KO mice. Although TUNEL staining showed no significant differences in positive cells between WT and KO, KO mice showed greater positive areas implicating more necroptosis in RIPK3 and pMLKL staining. GSDMD staining revealed that injured tubular cells during the chronic phase express GSDMD (Fig. 1). In in vitro experiments, we evaluated the functionality of macrophages such as phagocytic ability and migratory ability using bone marrow-derived macrophages (BMDMs), but we didn't find any significant disparities between BMDMs from WT mice and those from KO mice. Conclusion Genetic deletion of GSDMD resulted in more severe kidney injury, macrophage infiltration and fibrosis in mice with nephrocalcinosis-related CKD. Our results suggest a potential role for pyroptosis, especially GSDMD, in contributing to renal tubular cell necroptosis, a process downstream of calcium-oxalate crystal formation and deposition, in the progression of nephrocalcinosis-related CKD, similar to previous reports in mouse models of AKI.

Novel Model of Oxalate Diet-Induced Chronic Kidney Disease in Dahl-Salt-Sensitive Rats.

Diet-induced models of chronic kidney disease (CKD) offer several advantages, including clinical relevance and animal welfare, compared with surgical models. Oxalate is a plant-based, terminal toxic metabolite that is eliminated by the kidneys through glomerular filtration and tubular secretion. An increased load of dietary oxalate leads to supersaturation, calcium oxalate crystal formation, renal tubular obstruction, and eventually CKD. Dahl-Salt-Sensitive (SS) rats are a common strain used to study hypertensive renal disease; however, the characterization of other diet-induced models on this background would allow for comparative studies of CKD within the same strain. In the present study, we hypothesized that SS rats on a low-salt, oxalate rich diet would have increased renal injury and serve as novel, clinically relevant and reproducible CKD rat models. Ten-week-old male SS rats were fed either 0.2% salt normal chow (SS-NC) or a 0.2% salt diet containing 0.67% sodium oxalate (SS-OX) for five weeks.Real-time PCR demonstrated an increased expression of inflammatory marker interleukin-6 (IL-6) (p < 0.0001) and fibrotic marker Timp-1 metalloproteinase (p < 0.0001) in the renal cortex of SS-OX rat kidneys compared with SS-NC. The immunohistochemistry of kidney tissue demonstrated an increase in CD-68 levels, a marker of macrophage infiltration in SS-OX rats (p < 0.001). In addition, SS-OX rats displayed increased 24 h urinary protein excretion (UPE) (p < 0.01) as well as significant elevations in plasma Cystatin C (p < 0.01). Furthermore, the oxalate diet induced hypertension (p < 0.05). A renin-angiotensin-aldosterone system (RAAS) profiling (via liquid chromatography-mass spectrometry; LC-MS) in the SS-OX plasma showed significant (p < 0.05) increases in multiple RAAS metabolites including angiotensin (1-5), angiotensin (1-7), and aldosterone. The oxalate diet induces significant renal inflammation, fibrosis, and renal dysfunction as well as RAAS activation and hypertension in SS rats compared with a normal chow diet. This study introduces a novel diet-induced model to study hypertension and CKD that is more clinically translatable and reproducible than the currently available models.