Kidney stone growth through the lens of Raman mapping.

Bulk composition of kidney stones, often analyzed with infrared spectroscopy, plays an essential role in determining the course of treatment for kidney stone disease. Though bulk analysis of kidney stones can hint at the general causes of stone formation, it is necessary to understand kidney stone microstructure to further advance potential treatments that rely on in vivo dissolution of stones rather than surgery. The utility of Raman microscopy is demonstrated for the purpose of studying kidney stone microstructure with chemical maps at ≤ 1 µm scales collected for calcium oxalate, calcium phosphate, uric acid, and struvite stones. Observed microstructures are discussed with respect to kidney stone growth and dissolution with emphasis placed on < 5 µm features that would be difficult to identify using alternative techniques including micro computed tomography. These features include thin concentric rings of calcium oxalate monohydrate within uric acid stones and increased frequency of calcium oxalate crystals within regions of elongated crystal growth in a brushite stone. We relate these observations to potential concerns of clinical significance including dissolution of uric acid by raising urine pH and the higher rates of brushite stone recurrence compared to other non-infectious kidney stones.

The Rise in Tubular pH during Hypercalciuria Exacerbates Calcium Stone Formation.

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca2+ entry triggers a transepithelial Ca2+ flux to regulate proximal tubular (PT) luminal [Ca2+], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca2+ entry) inhibitors (Pyr10), Ca2+ entry by WTT cells was moderately inhibited, suggesting that the Ca2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Composition analysis of 1,495 cases of upper urinary tract calculi: the role of age and gender.

OBJECTIVE: This study aimed to quantitatively analyze the calculi components of upper urinary tract calculi and to explore the relationship between calculus components, demographic characteristics, and underlying diseases. PATIENTS AND METHODS: Clinical data of 1,495 patients with upper urinary tract calculi were retrospectively collected. The calculi were divided into simple calcium oxalate, calcium oxalate mixed, calcium phosphate mixed, uric acid, magnesium ammonium phosphate, and other components. Statistical software SPSS 22.0 was used to analyze the differences between the stone compositions and various factors. The influencing factors (p < 0.05) were analyzed using multiple logistic regression analysis. RESULTS: Among 1,495 patients with upper urinary tract calculi, simple calcium oxalate calculi were the most common component (39.7%), followed by calcium oxalate mixed calculi (30.4%), uric acid calculi (13.6%), calcium phosphate mixed calculi (10.4%), magnesium ammonium phosphate calculi (5.8%) and other component calculi (0.1%). Univariate analysis revealed statistically significant differences in stone composition according to gender, age, and hyperuricemia (p < 0.05). Multiple logistic regression analysis showed that compared to men, the odds ratio (OR) values of calcium oxalate mixed stones, calcium phosphate mixed stones, and magnesium ammonium phosphate stones in women were 1.61, 2.50, and 4.17, respectively (p < 0.001). Compared with elderly patients, the OR values of calcium phosphate mixed stones in young and middle-aged patients were 3.14 and 2.70, respectively (p < 0.05). CONCLUSIONS: Patients with different stone components had different demographic characteristics, and stone components were significantly different between gender and age. Calcium oxalate mixed stones were more common in females, and calcium phosphate mixed stones and magnesium ammonium phosphate stones were more common in females, young patients, and middle-aged patients.

FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury.

The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.

Antioxidant activity and Inhibitory effects of Cydonia oblonga Miller. leaves extracts against calcium oxalate stones.

Urinary stone disease is a widespread health problem in both adults and children, and its prevalence has been increasing worldwide. Various plants preparations have already been used since ancient times in order to treat urolithiasis. The aim of this study is to evaluate the antioxidant capacity and litholytic effect on kidney stones of Cydonia oblonga Miller. leaves. The infusion, methanol and acetone extracts were made from Cydonia oblonga Miller. leaf at different concentration. Estimation of mass fractions of total polyphenol, flavonoid, and flavonol contents, as well as the in vitro radical scavenging potential on 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH·) of the investigated extracts was carried out using colorimetric methods. The litholytic property of the extracts was performed by an in-vitro model using experimentally prepared kidney stones- calcium oxalate. As results, the quince leaf extracts revealed stronger antioxidant properties in the DPPH assay, which was proved by the semi-maximal inhibitory concentration values, being about 36.06 ± 3.55, 74.15 ± 6.29, and 142.35 ± 5.09 µg/ml for methanol, acetone and infusion extracts respectively. Furthermore, the tested extracts were found to be more effective in dissolving calcium oxalate stones compared to the control solutions, the mass loss is about 15.13 ± 1.10% with methanol extract, while it is 14.77 ± 1.74% and 11.14 ± 2.86% for acetone and infusion extracts respectively. These findings confirm the quince leaf's richness in phyto-components, offering anti-oxidant property and being able to be used as a remedy for the management of kidney stones by dissolving calcium oxalate stones in the kidneys.

Carboxymethylated Rhizoma alismatis polysaccharides reduces the risk of calcium oxalate stone formation by reducing cellular inflammation and oxidative stress.

This study aims to elucidate the mechanism and potential of Rhizoma alismatis polysaccharides (RAPs) in preventing oxidative damage to human renal proximal tubule epithelial cells. The experimental approach involved incubating HK-2 cells with 100 nm calcium oxalate monohydrate for 24 h to establish a cellular injury model. Protection was provided by RAPs with varying carboxyl group contents: 3.57%, 7.79%, 10.84%, and 15.33%. The safeguarding effect of RAPs was evaluated by analyzing relevant cellular biochemical indicators. Findings demonstrate that RAPs exhibit notable antioxidative properties. They effectively diminish the release of reactive oxygen species, lactate dehydrogenase, and malondialdehyde, a lipid oxidation byproduct. Moreover, RAPs enhance superoxide dismutase activity and mitochondrial membrane potential while attenuating the permeability of the mitochondrial permeability transition pore. Additionally, RAPs significantly reduce levels of inflammatory factors, including NLRP3, TNF-α, IL-6, and NO. This reduction corresponds to the inhibition of overproduced pro-inflammatory mediator nitric oxide and the caspase 3 enzyme, leading to a reduction in cellular apoptosis. RAPs also display the ability to suppress the expression of the HK-2 cell surface adhesion molecule CD44. The observed results collectively underscore the substantial anti-inflammatory and anti-apoptotic potential of all four RAPs. Moreover, their capacity to modulate the expression of cell surface adhesion molecules highlights their potential in inhibiting the formation of kidney stones. Notably, RAP3, boasting the highest carboxyl group content, emerges as the most potent agent in this regard.

Determinants and impact of calcium oxalate crystal deposition on renal outcomes in acute kidney injury patients.

OBJECTIVES: Calcium oxalate (CaOx) crystal deposition in acute kidney injury (AKI) patients is under recognized but impacts renal outcomes. This study investigates its determinants and effects. METHODS: We studied 814 AKI patients with native kidney biopsies from 2011 to 2020, identifying CaOx crystal deposition severity (mild: <5, moderate: 5-10, severe: >10 crystals per section). We assessed factors like urinary oxalate, citrate, urate, electrolytes, pH, tubular calcification index, and SLC26A6 expression, comparing them with creatinine-matched AKI controls without oxalosis. We analyzed how these factors relate to CaOx severity and their impact on renal recovery (eGFR < 15 mL/min/1.73 m2 at 3-month follow-up). RESULTS: CaOx crystal deposition was found in 3.9% of the AKI cohort (32 cases), with 72% due to nephrotoxic medication-induced tubulointerstitial nephritis. Diuretic use, higher urinary oxalate-to-citrate ratio induced by hypocitraturia, and tubular calcification index were significant contributors to moderate and/or severe CaOx deposition. Poor baseline renal function, low urinary chloride, high uric acid and urea nitrogen, tubular SLC26A6 overexpression, and glomerular sclerosis were also associated with moderate-to-severe CaOx deposition. Kidney recovery was delayed, with 43.8%, 31.2%, and 18.8% of patients having eGFR < 15 mL/min/1.73 m2 at 4, 12, and 24-week post-injury. Poor outcomes were linked to high urinary α1-microglobulin-to-creatinine (α1-MG/C) ratios and active tubular injury scores. Univariate analysis showed a strong link between this ratio and poor renal outcomes, independent of oxalosis severity. CONCLUSIONS: In AKI, CaOx deposition is common despite declining GFR. Factors worsening tubular injury, not just oxalate-to-citrate ratios, are key to understanding impaired renal recovery.

The impact of crystal phase transition on the hardness and structure of kidney stones.

Calcium oxalate kidney stones, the most prevalent type of kidney stones, undergo a multi-step process of crystal nucleation, growth, aggregation, and secondary transition. The secondary transition has been rather overlooked, and thus, the effects on the disease and the underlying mechanism remain unclear. Here, we show, by periodic micro-CT images of human kidney stones in an ex vivo incubation experiment, that the growth of porous aggregates of calcium oxalate dihydrate (COD) crystals triggers the hardening of the kidney stones that causes difficulty in lithotripsy of kidney stone disease in the secondary transition. This hardening was caused by the internal nucleation and growth of precise calcium oxalate monohydrate (COM) crystals from isolated urine in which the calcium oxalate concentrations decreased by the growth of COD in closed grain boundaries of COD aggregate kidney stones. Reducing the calcium oxalate concentrations in urine is regarded as a typical approach for avoiding the recurrence. However, our results revealed that the decrease of the concentrations in closed microenvironments conversely promotes the transition of the COD aggregates into hard COM aggregates. We anticipate that the suppression of the secondary transition has the potential to manage the deterioration of kidney stone disease.

Proof-of-concept for a novel nanotechnology-based treatment for urolithiasis.

Proof-of-concept of photonic lithotripsy in an in vitro setting and its ability to fragment the most common stone types is demonstrated. Effectiveness of different classes of photonic nanoparticles in fragmenting human stones is assessed. De-identified human stones were collected after institutional approval. Stones of a size range between 2-4 mm were rehydrated in simulated urine for 24 h. Stones were then coated with a solution of nanoparticles prior to activation with either a 785 nm or 1320 nm near-infrared energy source. Photonic lithotripsy achieved greater than 70% success rate in fragmentating calcium oxalate monohydrate stones using carbon-based nanoparticles for both near-infrared wavelengths. For gold-based nanoparticles, there was a similar success rate with the 785 nm wavelength but a significant decrease when using the 1320 nm wavelength energy source. All stones fragmented with the energy source at a distance ≥ 20 mm from the stone's surface. Limitations include the use of mixed-composition stones, a lack of complete stone immersion in liquid during treatment, and smaller stone size. Different classes of nanoparticles when excited with a near-infrared energy source can fragment common stone types in vitro. This technology has the potential to change the way we approach and treat patients with urolithiasis in a clinical setting.

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.

Lycopene from tomatoes and tomato products exerts renoprotective effects by ameliorating oxidative stress, apoptosis, pyroptosis, fibrosis, and inflammatory injury in calcium oxalate nephrolithiasis: the underlying mechanisms.

Several mechanisms underlying nephrolithiasis, one of the most common urological diseases, involve calcium oxalate formation, including oxidative stress, inflammatory reactions, fibrosis, pyroptosis, and apoptosis. Although lycopene has strong antioxidant activity, its protective effects against CaOx-induced injury have not yet been reported. This study aimed to systematically investigate the protective effects of lycopene and explore its mechanisms and molecular targets. Crystal deposition, renal function, oxidative stress, inflammatory response, fibrosis, pyroptosis, and apoptosis were assessed to evaluate the renoprotective effects of lycopene against crystal formation in a CaOx rat model and oxalate-stimulated NRK-52E and HK-2 cells. Lycopene markedly ameliorated crystal deposition, restored renal function, and suppressed kidney injury by reducing oxidative stress, apoptosis, inflammation, fibrosis, and pyroptosis in the rats. In cell models, lycopene pretreatment reversed reactive oxygen species increase, apoptotic damage, intracellular lactate dehydrogenase release, cytotoxicity, pyroptosis, and extracellular matrix deposition. Network pharmacology and proteomic analyses were performed to identify lycopene target proteins under CaOx-exposed conditions, and the results showed that Trappc4 might be a pivotal target gene for lycopene, as identified by cellular thermal shift assay and surface plasmon resonance analyses. Based on molecular docking, molecular dynamics simulations, alanine scanning mutagenesis, and saturation mutagenesis, we observed that lycopene directly interacts with Trappc4 via hydrophobic bonds, which may be attributed to the PHE4 and PHE142 residues, preventing ERK1/2 or elevating AMPK signaling pathway phosphorylation events. In conclusion, lycopene might ameliorate oxalate-induced renal tubular epithelial cell injury via the Trappc4/ERK1/2/AMPK pathway, indicating its potential for the treatment of nephrolithiasis.

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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Taken together, Lithom could be a potential clinical-therapeutic alternative for management of nephrolithiasis.

Cell death­related molecules and targets in the progression of urolithiasis (Review).

Urolithiasis is a high­incidence disease caused by calcium oxalate (mainly), uric acid, calcium phosphate, struvite, apatite, cystine and other stones. The development of kidney stones is closely related to renal tubule cell damage and crystal adhesion and aggregation. Cell death, comprising the core steps of cell damage, can be classified into various types (i.e., apoptosis, ferroptosis, necroptosis and pyroptosis). Different crystal types, concentrations, morphologies and sizes cause tubular cell damage via the regulation of different forms of cell death. Oxidative stress caused by high oxalate or crystal concentrations is considered to be a precursor to a variety of types of cell death. In addition, complex crosstalk exists among numerous signaling pathways and their key molecules in various types of cell death. Urolithiasis is considered a metabolic disorder, and tricarboxylic acid cycle­related molecules, such as citrate and succinate, are closely related to cell death and the inhibition of stone development. However, a literature review of the associations between kidney stone development, metabolism and various types of cell death is currently lacking, at least to the best of our knowledge. Thus, the present review summarizes the major advances in the understanding of regulated cell death and urolithiasis progression.

Comparison of metabolic parameters between pure-uric acid and mixed-uric acid kidney stone formers.

PURPOSE: We seek to compare clinical and 24-h urine parameters between pure-uric acid (UA) and UA-CaOx stone formers in our practice and explore how any differences in metabolic profiles could suggest different prevention strategies between the two groups. METHODS: We retrospectively reviewed patients with either pure- or mixed-UA nephrolithiasis from 2020 to 2023 at a tertiary care center. We included patients with a 24-h urine collection and a stone analysis detecting any amount of UA. Patients were organized into two cohorts: (1) those with 100% UA stones and (2) < 100% UA stones. Differences in demographic characteristics were compared between pure-UA and UA-CaOx stone formers. Twenty-four hour urine metabolic parameters as well as metabolic abnormalities were compared between the pure-uric acid and mixed-uric acid groups. RESULTS: We identified 33 pure-UA patients and 33 mixed-UA patients. Patient demographics were similar between the groups (Table 1). Pure- and mixed-UA patients had a similar incidence of metabolic syndrome, diabetes, history of stones, and stone burden. Table 1 Demographic and baseline characteristics among pure- and mixed-uric acid stone formers Pure-uric acid stones (n = 33) Mixed-uric acid stones (n = 33) p-value Median age [IQR] 63.00 [58.00-72.50] 63.00 [53.50-68.00] 0.339 Median BMI [IQR] 28.79 [25.81-33.07] 27.96 [25.81-29.55] 0.534 Gender, n (%) 1.000  Male 21 (63.6) 21 (63.6)  Female 12 (36.4) 12 (36.4) Metabolic syndrome, n (%) 17 (51.5) 16 (48.5) 0.806 Diabetes, n (%) 13 (39.4) 12 (36.4) 0.800 History of stones, n (%) 23 (69.7) 22 (66.7) 0.792 Median total stone burden, mm [IQR] 12.00 [6.00-26.50] 13.00 [7.05-20.00] 0.995 Median serum uric acid, mg/dL [IQR] 6.20 [4.80-7.15] 5.90 [4.98-6.89] 0.582 IQR Interquartile range BMI Body Mass Index n number We found the pure-UA cohort to have 24-h lower urine volume (1.53 vs. 1.96 L/day, p = 0.045) and citrate levels (286 vs. 457 mg/day, p = 0.036). UA-CaOx stone formers had higher urinary calcium levels (144 vs. 68 mg/day, p = 0.003), higher urinary oxalate levels (38 vs. 30 mg/day, p = 0.017), and higher median urinary calcium oxalate super-saturation (3.97 vs. 3.06, p = 0.047). CONCLUSIONS: Pure-UA kidney stone formers have different urinary metabolic parameters when compared with UA-CaOx stone formers, thus requiring different and tailored medical management.

N-acetylcysteine regulates oxalate induced injury of renal tubular epithelial cells through CDKN2B/TGF-ß/SMAD axis.

This study was aimed to investigate the preventive effects of N-acetyl-L-cysteine (NAC) against renal tubular cell injury induced by oxalate and stone formation and further explore the related mechanism. Transcriptome sequencing combined with bioinformatics analysis were performed to identify differentially expressed gene (DEG) and related pathways. HK-2 cells were pretreated with or without antioxidant NAC/with or silencing DEG before exposed to sodium oxalate. Then, the cell viability, oxidative biomarkers of superoxidase dismutase (SOD) and malondialdehyde (MDA), apoptosis and cell cycle were measured through CCK8, ELISA and flow cytometry assay, respectively. Male SD rats were separated into control group, hyperoxaluria (HOx) group, NAC intervention group, and TGF-ß/SMAD pathway inhibitor group. After treatment, the structure changes and oxidative stress and CaOx crystals deposition were evaluated in renal tissues by H&E staining, immunohistochemical and Pizzolato method. The expression of TGF-ß/SMAD pathway related proteins (TGF-ß1, SMAD3 and SMAD7) were determined by Western blot in vivo and in vitro. CDKN2B is a DEG screened by transcriptome sequencing combined with bioinformatics analysis, and verified by qRT-PCR. Sodium oxalate induced declined HK-2 cell viability, in parallel with inhibited cellular oxidative stress and apoptosis. The changes induced by oxalate in HK-2 cells were significantly reversed by NAC treatment or the silencing of CDKN2B. The cell structure damage and CaOx crystals deposition were observed in kidney tissues of HOx group. Meanwhile, the expression levels of SOD and 8-OHdG were detected in kidney tissues of HOx group. The changes induced by oxalate in kidney tissues were significantly reversed by NAC treatment. Besides, expression of SMAD7 was significantly down-regulated, while TGF-ß1 and SMAD3 were accumulated induced by oxalate in vitro and in vivo. The expression levels of TGF-ß/SMAD pathway related proteins induced by oxalate were reversed by NAC. In conclusion, we found that NAC could play an anti-calculus role by mediating CDKN2B/TGF-ß/SMAD axis.

Pathophysiology and Main Molecular Mechanisms of Urinary Stone Formation and Recurrence.

Kidney stone disease (KSD) is one of the most common urological diseases. The incidence of kidney stones has increased dramatically in the last few decades. Kidney stones are mineral deposits in the calyces or the pelvis, free or attached to the renal papillae. They contain crystals and organic components, and they are made when urine is supersaturated with minerals. Calcium-containing stones are the most common, with calcium oxalate as the main component of most stones. However, many of these form on a calcium phosphate matrix called Randall's plaque, which is found on the surface of the kidney papilla. The etiology is multifactorial, and the recurrence rate is as high as 50% within 5 years after the first stone onset. There is a great need for recurrence prevention that requires a better understanding of the mechanisms involved in stone formation to facilitate the development of more effective drugs. This review aims to understand the pathophysiology and the main molecular mechanisms known to date to prevent recurrences, which requires behavioral and nutritional interventions, as well as pharmacological treatments that are specific to the type of stone.

Correlation research demonstrates that an inflammatory diet is a risk factor for calcium oxalate renal stone formation.

BACKGROUND AND AIMS: Previous studies have demonstrated associations between the Dietary Inflammatory Index (DII®), an analytical tool which evaluates the inflammatory potential of the diet according to the pro- and anti-inflammatory properties of its components, and renal stone formation. However, these have not comprehensively addressed important parameters such as stone type, gender, DII scores in stone formers (SFs) and healthy controls (Cs) and associations of DII with urine and blood chemistries. These were adopted as the survey parameters for the present study, the purpose of which was to test whether the contributory role of an inflammatory diet on stone formation could be further confirmed. METHODS: 97 calcium oxalate (CaOx) SFs and 63 Cs, matched for age and gender each completed a semi-quantitative food frequency questionnaire from which nutrient composition was computed. These data were used to calculate the DII® score. To control the effect of energy intake, energy-adjusted DII scores were calculated per 1000 kcal consumed (E-DII™). A single blood sample and two consecutive overnight (8h) urine samples were collected from a subset (n = 59 SFs and n = 54 Cs) of the overall number of particpants (n = 160). These were analysed for renal stone risk factors. Data were analysed using regression models fit in R software. RESULTS: E-DII scores were found to fit the data better than DII, so they were used throughout. E-DII scores were significantly more positive (more pro-inflammatory) in SFs than in controls in the combined gender group (-0.34 vs. -1.73, p < 0.0001) and separately in males (-0.43 vs. -1.78, p = 0.01) and females (-0.26 vs. - 1.61, p = 0.05). In blood, a significant negative correlation was seen between E-DII and HDL cholesterol. In urine significant positive correlations were seen between E-DII and each of calcium (ρ = 0.25, p = 0.02), phosphate (ρ = 0.48, p < 0.001), magnesium (ρ = 0.33, p < 0.0001) and uric acid (ρ = 0.27, p = 0.004) concentrations. A significant negative correlation was seen between E-DII and urinary volume ρ = -0.27, p = 0.003). There was no correlation between E-DII scores and the relative supersaturations of urinary CaOx, calcium phosphate (brushite) and uric acid. CONCLUSIONS: Our findings provide hitherto unreported quantitative evidence in support of the notion that the diet of calcium oxalate renal stone patients is significantly more pro-inflammatory than that of healthy controls.

Glycyrrhizae radix et rhizoma processing ameliorates adverse reactions of polygalae radix in zebra fish and rabbit models.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygala tenuifilia Willd (Polygalaceae), a traditional Chinese medicine, has been used for a long time to treat various illnesses with serious adverse reactions. Glycyrrhizae radix et rhizoma processing is generally used to reduce the adverse reactions. AIM OF THE STUDY: The aim of this study was to validate the irritation caused by raw Polygalaceae (RPA), to investigate whether processed Polygalaceae (PGA) was less irritating, and to screen and validate irritant properties of virgaureagenin G (polygala acid, PA), 3,6'-disinapoylsucrose (DSS), Tenuifolia (TEN) and polygalaxanthone III (POL), which had pharmacologically active in Polygalaceae. Zebrafish model, Draize test and High-Performance Liquid Chromatography (HPLC) were utilized to achieve the aim. MATERIALS AND METHODS: Scanning Electron Microscopy (SEM) and optical microscope were used to determine the presence of calcium oxalate needle crystal in RPA and PGA. Zebrafish egg spinning changes and zebrafish embryo behavior were used for irritation validation, irritation comparison and irritant screening. For additional evidence, the Draize test, HE staining of rabbit eyes and ELISA kit were used. Finally, changes in the composition of RPA and PGA were investigated using HPLC. RESULTS: SEM and optical microscopy revealed no calcium oxalate needle crystals in Polygalaceae. RPA, PGA, PA and DSS were able to accelerate the spinning of zebrafish eggs and the movement of embryos, while TEN and POL were not. RPA, PGA, DSS and PA may cause rabbit eyes to become hyperemic and swollen, resulting in damage to the iris, cornea and conjunctiva and increased levels of interleukin-6 (IL-6) and interleukin-10 (IL-10). Comparatively, the effects caused by PGA were less severe than those caused by RPA. In addition, compared to RPA, PGA had lower levels of DSS and PA. CONCLUSIONS: RPA, PGA, DSS, and PA were irritating. However, processing and curing could reduce the irritation by reducing the levels of DSS and PA. DSS and PA could be two potential irritants of Polygalaceae.

Phagocytosis model of calcium oxalate monohydrate crystals generated using human induced pluripotent stem cell-derived macrophages.

Macrophages play a role in nephrolithiasis, offering the possibility of developing macrophage-mediated preventive therapies. To establish a system for screening drugs that could prevent the formation of kidney stones, we aimed to develop a model using human induced pluripotent stem cell (iPSC)-derived macrophages to study phagocytosis of calcium oxalate monohydrate (COM) crystals. Human iPSCs (201B7) were cultured. CD14+ monocytes were recovered using a stepwise process that involved the use of growth factors and cytokines. These cells were then allowed to differentiate into M1 and M2 macrophages. The macrophages were co-cultured with COM crystals and used in the phagocytosis experiments. Live cell imaging and polarized light observation via super-resolution microscopy were used to visualize phagocytosis. Localization of phagocytosed COM crystals was observed using transmission electron microscopy. Intracellular fluorescence intensity was measured using imaging cytometry to quantify phagocytosis. Human iPSCs successfully differentiated into M1 and M2 macrophages. M1 macrophages adhered to the culture plate and moved COM crystals from the periphery to cell center over time, whereas M2 macrophages did not adhere to the culture plate and actively phagocytosed the surrounding COM crystals. Fluorescence assessment over a 24-h period showed that M2 macrophages exhibited higher intracellular fluorescence intensity (5.65-times higher than that of M1 macrophages at 4.5 h) and maintained this advantage for 18 h. This study revealed that human iPSC-derived macrophages have the ability to phagocytose COM crystals, presenting a new approach for studying urinary stone formation and highlighting the potential of iPSC-derived macrophages as a tool to screen nephrolithiasis-related drugs.