Search

Calcium oxalate (CaOx) nephrolithiasis constitutes approximately 75% of nephrolithiasis cases, resulting from the supersaturation and deposition of CaOx crystals in renal tissues. Despite their prevalence, precise biomarkers for CaOx nephrolithiasis are lacking. With advances in high-throughput sequencing, we aimed to identify biomarkers of CaOx nephrolithiasis by combining two CaOx nephrolithiasis datasets (GSE73680 and GSE117518). Utilizing weighted gene co-expression network analysis (WGCNA) and four machine learning, we identified six hub genes (DLK2, BHLHA15, C12orf5, ICMT, LOXHD1, and TP73) as potential biomarkers. Additionally, CIBERSORT immune infiltration analysis suggested that these core genes may influence immune cell recruitment and infiltration in CaOx nephrolithiasis. Then, TP73 emerged as a significant hub gene in CaOx nephrolithiasis via receiver operating characteristic (ROC) analysis (AUC = 0.885). Furthermore, the role of TP73 was validated in CaOx nephrolithiasis rat models induced by 1% ethylene glycol, as well as clinical samples and renal tubular epithelial cell models treated with 1 mM oxalate. Immunohistochemistry, RNA-Sequencing, and RT-qPCR experiments demonstrated an increased expression of TP73 in CaOx nephrolithiasis rat models and clinical samples. After transfection with TP73 lentivirus, CCK-8 assays suggested that TP73 could inhibit the proliferation of HK-2 and NRK-52E cells. In oxalate-induced cell models, dihydroethidium staining and flow cytometry apoptosis assays indicated that TP73 could enhance ROS levels and cell apoptosis. In summary, our study preliminarily identified TP73 as a diagnostic biomarker and elucidated the promoting role of TP73 in CaOx nephrolithiasis, providing a deeper understanding of the clinical diagnosis and pathogenesis., Competing Interests: Declarations Conflict of interests The authors declare no competing interests. Ethical approval This study was approved by the Fudan Laboratory Animal Ethics Board and the Huashan Institutional Review Board of Fudan University (HIRB). Consent for publication Not applicable., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Calcium oxalate (CaOx) kidney stones accumulate within the renal tubule due to high concentrations of insoluble deposits in the urine. Pb 2+ -induced Ca 2+ mobilization along with Pb 2+ -induced nephrotoxic effects within the proximal tubule have been well established; however, Pb 2+ mediated effects within the collecting duct remains insufficiently studied. Thus in vitro and ex vivo model systems were treated with increasing concentrations of lead (II) acetate (PbAc) ± sodium oxalate (Na 2 C 2 O 4 ) for 1 h, both individually and in combination. Pb 2+ -mediated solution turbidity increased 2 to 5 times greater post-exposure to 75, 100 and 200 μM Pb 2+ with the additional co-treatment of 10 mM oxalate in mouse inner medullary collecting duct (mIMCD-3) cells. Additionally, 100 μM and 200 μM Pb 2+ alone induced significant levels of intracellular Ca 2+ release. To validate Pb 2+ -mediated effects on the formation of CaOx crystals, alizarin red staining confirmed the presence of CaOx crystallization. Pb 2+ -induced intracellular Ca 2+ was also observed ex vivo in fly Malpighian tubules with significant increases in CaOx crystal formation via Pb 2+ -induced intracellular Ca 2+ release significantly increasing the average crystal number, size, and total area of crystal formation, which was ameliorated by tissue-specific SPoCk C transporter and Capa receptor knockdown. These studies demonstrate Pb 2+ -induced Ca 2+ release likely increases the formation of CaOx crystals, which is modulated by a G q -linked mechanism with concurrent Ca 2+ extracellular mobilization., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Greg M. Landry reports financial support was provided by American Association of Colleges of Pharmacy. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Kidney stone formation is a common disease that causes a significant threat to human health. The crystallization mechanism of calcium oxalate, the most common type of kidney stone, has been extensively researched, yet the damaging effects and mechanisms of calcium oxalate crystals on renal tubular epithelial cells remain incompletely elucidated. Regulated mitochondrial dynamics is essential for eukaryotic cells, but its role in the occurrence and progression of calcium oxalate (CaOx) nephrolithiasis is not yet understood., Methods: An animal model of calcium oxalate-related nephrolithiasis was established in adult male Sprague‒Dawley (SD) rats by continuously administering drinking water containing 1% ethylene glycol for 28 days. The impact of calcium oxalate crystals on mitochondrial dynamics and apoptosis in renal tubular epithelial cells was investigated using HK2 cells in vitro. Blood samples and bilateral kidney tissues were collected for histopathological evaluation and processed for tissue injury, inflammation, fibrosis, oxidative stress detection, and mitochondrial dynamics parameter analysis., Results: Calcium oxalate crystals caused higher levels of mitochondrial fission and apoptosis in renal tubular epithelial cells both in vivo and in vitro. Administration of a PPARγ agonist significantly alleviated mitochondrial fission and apoptosis in renal tubular epithelial cells, and improved renal function, accompanied by reduced levels of oxidative stress, increased antioxidant enzyme expression, alleviation of inflammation, and reduced fibrosis in vivo., Conclusion: Our results indicated that increased mitochondrial fission in renal tubular epithelial cells is a critical component of kidney injury caused by calcium oxalate stones, leading to the accumulation of reactive oxygen species within the tissue and the subsequent initiation of apoptosis. Regulating mitochondrial dynamics represents a promising approach for calcium oxalate nephrolithiasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Kidney Stone Disease (KSD) constitutes a multifaceted disorder, emerging from a confluence of environmental and genetic determinants, and is characterized by a high frequency of occurrence and recurrence. Our objective is to elucidate potential causative proteins and identify prospective pharmacological targets within the context of KSD. This investigation harnessed the unparalleled breadth of plasma protein and KSD pooled genome-wide association study (GWAS) data, sourced from the United Kingdom Biobank Pharma Proteomics Project (UKBPPP) and the FinnGen database version R10. Through Mendelian randomization analysis, proteins exhibiting a causal influence on KSD were pinpointed. Subsequent co-localization analyses affirmed the stability of these findings, while enrichment analyses evaluated their potential for pharmacological intervention. Culminating the study, a phenome-wide association study (PheWAS) was executed, encompassing all phenotypes (2408 phenotypes) catalogued in the FinnGen database version R10. Our MR analysis identified a significant association between elevated plasma levels of proteins FKBPL, ITIH3, and SERPINC1 and increased risk of KSD based on genetic predictors. Conversely, proteins CACYBP, DAG1, ITIH1, and SEMA6C showed a protective effect against KSD, documented with statistical significance (P FDR <0.05). Co-localization analysis confirmed these seven proteins share genetic variants with KSD, signaling a shared genetic basis (PPH3 + PPH4 > 0.8). Enrichment analysis revealed key pathways including hyaluronan metabolism, collagen-rich extracellular matrix, and serine-type endopeptidase inhibition. Additionally, our PheWAS connected the associated proteins with 356 distinct diseases (P FDR <0.05), highlighting intricate disease interrelations. In conclusion, our research elucidated a causal nexus between seven plasma proteins and KSD, enriching our grasp of prospective therapeutic targets., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oxalate-induced damage to renal tubular epithelial cells (RTECs) is an essential factor in the incident kidney stone, but the specific mechanism is unclear. Recent research has pinpointed interacting areas within the endoplasmic reticulum and mitochondria, called mitochondria-associated membranes (MAMs). These studies have linked endoplasmic reticulum stress (ERS) and oxidative imbalance to kidney disease development. The sigma-1 receptor (S1R), a specific protein found in MAMs, is involved in various physiological processes, but its role in oxalate-induced kidney stone formation remains unclear. In this study, we established cellular and rat models of oxalate-induced kidney stone formation to elucidate the S1R's effects against ERS and apoptosis and its mechanism in oxalate-induced RTEC injury. We found that oxalate downregulated S1R expression in RTECs and escalated oxidative stress and ERS, culminating in increased apoptosis. The S1R agonist dimemorfan up-regulated S1R expression and mitigated ERS and oxidative stress, thereby reducing apoptosis. This protective effect was mediated through S1R inhibition of the CHOP pathway. Animal experiments demonstrated that S1R's activation attenuated oxalate-induced kidney injury and alleviated kidney stone formation. This is the first study to establish the connection between S1R and kidney stones, suggesting S1R's protective role in inhibiting ERS-mediated apoptosis to ameliorate kidney stone formation.

Calcium-containing stones represent the most common form of kidney calculi, frequently linked to idiopathic hypercalciuria, though their precise pathogenesis remains elusive. This research aimed to elucidate the molecular mechanisms involved by employing urinary exosomal microRNAs as proxies for renal tissue analysis. Elevated miR-148b-5p levels were observed in exosomes derived from patients with kidney stones. Systemic administration of miR-148b-5p in rat models resulted in heightened urinary calcium excretion, whereas its inhibition reduced stone formation. RNA immunoprecipitation combined with deep sequencing identified miR-148b-5p as a suppressor of calcitonin receptor (Calcr) expression, thereby promoting urinary calcium excretion and stone formation. Mice deficient in Calcr in distal epithelial cells demonstrated elevated urinary calcium excretion and renal calcification. Mechanistically, miR-148b-5p regulated Calcr through the circRNA-83536/miR-24-3p signaling pathway. Human kidney tissue samples corroborated these results. In summary, miR-148b-5p regulates the formation of calcium-containing kidney stones via the circRNA-83536/miR-24-3p/Calcr axis, presenting a potential target for novel therapeutic interventions to prevent calcium nephrolithiasis., (© 2024. The Author(s).)

Urolithiasis or nephrolithiasis is a condition of kidney stone formation and is considered a painful disease of the urinary tract system. In this work, we planned to discover the therapeutic roles of myricetin on the ethylene glycol (EG)-induced nephrolithiasis in rats. The experimental rats were treated with 0.75% of EG through drinking water for 4 weeks to initiate the nephrolithiasis and subsequently treated with 25 and 50 mg/kg of myricetin. The body weight and urine volume were measured regularly. After the sacrification of rats, the samples were collected, and serum and urinary biomarkers such as creatinine, urea, Ca2 + ion, and BUN, OPN, oxalate, and citrate levels were determined using assay kits. These biomarkers, the MDA level and CAT, SOD, and GPx activities, were assessed in the kidney tissue homogenates. The IL-6, IL-1β, and TNF-α levels were also quantified using respective kits. The histopathological analysis was done on the kidney tissues. Myricetin treatment did not show major changes in the body weight and kidney weight in the EG-induced rats. The treatment with 25 and 50 mg/kg of myricetin considerably reduced the urea, creatinine, BUN, Ca2 + ion, and oxalate and increased the citrate content in serum and urine samples of EG-induced rats. Further, myricetin depleted the inflammatory cytokines and MDA levels and elevated the CAT, SOD, and GPx activities in the renal tissues. The activities of ALT, AST, ALP, GGT, and LDH were also reduced by the myricetin. Furthermore, the myricetin upheld the histoarchitecture of the kidneys. The outcomes of this investigation propose that myricetin is effective in EG-induced urolithiasis probably because of its antioxidant, anti-inflammatory, and renoprotective activities. In addition, further studies are still required to verify the precise therapeutic mechanism of myricetin., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Calcium-sensing receptor (CASR) influences the expression pattern of multiple genes in renal tubular epithelial cells. The objective of this inquiry was to explore the molecular mechanisms of CASR in renal tubular epithelial cells and nephrolithiasis., Methods: HK-2 cells were transfected with lentiviruses carrying either CASR (named CASR) or an empty vector negative control (named NC), as well as shRNA intended to target CASR (named shCASR) or its corresponding negative control (named shNC). CCK-8 assay was used to detect the effect of CASR on the proliferation of HK-2 cells. RNA-Sequencing was applied to explore potential pathways regulated by CASR in HK-2 cells., Results: PCR and western blot results showed that CASR expression was significantly increased in CASR cells and was decreased in shCASR cells when compared to their corresponding negative control, respectively. CCK-8 assay revealed that CASR inhibited the proliferation of HK-2 cells. RNA-Sequencing results suggested that the shCASR HK-2 cells exhibited a significant up-regulation of 345 genes and a down-regulation of 366 genes. These differentially expressed genes (DEGs) were related to cell apoptosis and cell development. In CASR HK-2 cells, 1103 DEGs primarily functioned in mitochondrial energy metabolism, and amino acid metabolism. With the Venn diagram, 4 DEGs (Clorf116, ENPP3, IL20RB, and CLDN2) were selected as the hub genes regulated by CASR. Enrichment analysis revealed that these hub genes were involved in cell-cell junction, and epithelial cell development., Conclusions: In summary, our investigation has the potential to offer novel perspectives on CASR regulating cell-cell junction in HK-2 cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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 Ca 2+ entry triggers a transepithelial Ca 2+ flux to regulate proximal tubular (PT) luminal [Ca 2+ ], 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 Ca 2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca 2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca 2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca 2+ entry) inhibitors (Pyr10), Ca 2+ entry by WTT cells was moderately inhibited, suggesting that the Ca 2+ 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.

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.

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.

Ferroptosis is a specific type of programmed cell death characterized by iron-dependent lipid peroxidation. Understanding the involvement of ferroptosis in calcium oxalate (CaOx) stone formation may reveal potential targets for this condition. The publicly available dataset GSE73680 was used to identify 61 differentially expressed ferroptosis-related genes (DEFERGs) between normal kidney tissues and Randall's plaques (RPs) from patients with nephrolithiasis through employing weighted gene co-expression network analysis (WGCNA). The findings were validated through in vitro and in vivo experiments using CaOx nephrolithiasis rat models induced by 1% ethylene glycol administration and HK-2 cell models treated with 1 mM oxalate. Through WGCNA and the machine learning algorithm, we identified LAMP2 and MDM4 as the hub DEFERGs. Subsequently, nephrolithiasis samples were classified into cluster 1 and cluster 2 based on the expression of the hub DEFERGs. Validation experiments demonstrated decreased expression of LAMP2 and MDM4 in CaOx nephrolithiasis animal models and cells. Treatment with ferrostatin-1 (Fer-1), a ferroptosis inhibitor, partially reversed oxidative stress and lipid peroxidation in CaOx nephrolithiasis models. Moreover, Fer-1 also reversed the expression changes of LAMP2 and MDM4 in CaOx nephrolithiasis models. Our findings suggest that ferroptosis may be involved in the formation of CaOx kidney stones through the regulation of LAMP2 and MDM4.

Nephrolithiasis is highly prevalent and associated with the increased risk of kidney cancer. The tumor suppressor von Hippel-Lindau (VHL) is critical for renal cancer development, however, its role in kidney stone disease has not been fully elucidated until now. Here we reported VHL expression was upregulated in renal epithelial cells upon exposure to crystal. Utilizing Vhl +/mu mouse model, depletion of VHL exacerbated kidney inflammatory injury during nephrolithiasis. Conversely, overexpression of VHL limited crystal-induced lipid peroxidation and ferroptosis in a BICD2-depdendent manner. Mechanistically, VHL interacted with the cargo adaptor BICD2 and promoted itsd K48-linked poly-ubiquitination, consequently resulting in the proteasomal degradation of BICD2. Through promoting STAT1 nuclear translocation, BICD2 facilitated IFNγ signaling transduction and enhanced IFNγ-mediated suppression of cystine/glutamate antiporter system X c - , eventually increasing cell sensitivity to ferroptosis. Moreover, we found that the BRAF inhibitor impaired the association of VHL with BICD2 through triggering BICD2 phosphorylation, ultimately causing severe ferroptosis and nephrotoxicity. Collectively, our results uncover the important role of VHL/BICD2/STAT1 axis in crystal kidney injury and provide a potential therapeutic target for treatment and prevention of renal inflammation and drug-induced nephrotoxicity., (© 2023. The Author(s).)

Oxidative stress is a major risk factor for calcium oxalate nephrolithiasis. Reports suggest that oxidative stress response is induced in animals and humans with kidney stones. Keap1, Nrf2, and HO-1 are known as oxidative stress mediators. However, the association between oxidative stress response and stone formation is unclear. In this study, we analyzed oxidative stress response from the acute to the crystal formation phase when crystal formation was applied to renal crystal mice model and bioimaging mice and investigated the effect on crystal formation. In renal tissues, after glyoxylate administration, HO-1 increased for up to 6 h and returned to baseline at 24 h. This was observed following each daily dose until five days after the crystallization phase; however, the range of increase was attenuated. The possibility that Nrf2 activity influenced the number of crystals was considered in the experiment. Crystal formation increased in Nrf2-deficient mice and could be reduced by Nrf2 activators. In conclusion, the oxidative stress response via the Keap1-Nrf2 pathway may contribute to crystal formation. Particularly, this pathway may be a prospective target for drug development to prevent and cure nephrolithiasis., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Nephrolithiasis is a common and frequently-occurring disease in the urinary system with high recurrence. The present study aimed to explore the protective effect and underlying mechanism of hydroxycitric acid (HCA) in hyperoxaluria-induced nephrolithiasis in vitro and in vivo . Crystal deposition and pathophysiological injury in rat models of glyoxylate-induced nephrolithiasis were examined using H&E staining. Cell models of nephrolithiasis were established by oxalate-treated renal tubular epithelial cells. The levels of oxidative stress indexes were determined by ELISA kits. Cell proliferation in vivo and in vitro was evaluated using a cell counting kit-8 (CCK-8) assay and Ki-67 cell proliferation detection kit. Cell apoptosis was measured by flow cytometry and TUNEL staining. The protein levels were examined by western blotting. Our results showed that HCA administration significantly reduced crystal deposition and kidney injury induced by glyoxylate. HCA also alleviated oxidative stress via upregulating the antioxidant enzyme activities of superoxide dismutase (SOD) and catalase (CAT) and reducing the malondialdehyde (MDA) content. Moreover, HCA treatment promoted cell proliferation and inhibited apoptosis of renal tubular epithelial cells exposed to hyperoxaluria. Of note, Nrf2 activator dimethyl fumarate (DMF) exerted the same beneficial effects as HCA in nephrolithiasis. Mechanistically, HCA prevented crystal deposition and oxidative stress induced by hyperoxaluria through targeting the Nrf2/Keap1 antioxidant defense pathway, while knockdown of Nrf2 significantly abrogated these effects. Taken together, HCA exhibited antioxidation and anti-apoptosis activities in nephrolithiasis induced by hyperoxaluria via activating Nrf2/Keap1 pathway, suggesting that it may be an effective therapeutic agent for the prevention and treatment of nephrolithiasis.

Purpose: Oxidative damage is important in calcium oxalate (CaOx) stone development but occurs via multiple pathways. Studies have shown that klotho plays an essential role in ameliorating oxidative damage. This study aims to explore the role of klotho in CaOx stones and whether the underlying mechanism is related to the regulation of Keap1-Nrf2-ARE signaling., Methods: The levels of GSH, SOD, CAT, MDA, and ROS were examined by ELISA. The klotho, Bcl-2, caspase-3, Keap1, Nrf2, HO-1, and NQO1 mRNA levels were measured by qRT‒PCR, and their protein levels were detected by Western blotting. Renal tissue apoptosis was examined by TUNEL staining, and crystal cell adherence and apoptosis in HKC cells were assessed based on the Ca 2+ concentrations and by flow cytometry. The renal pathological changes and the adhesion of CaOx crystals in the kidneys were examined by hematoxylin-eosin and von Kossa staining, respectively., Results: We constructed a CaOx kidney stone model in vitro. By regulating the klotho gene, klotho overexpression inhibited the CaOx-induced promotion of crystal cell adherence and apoptosis in HKC cells, and these effects were reversed by klotho knockdown. Moreover, our in vivo assay demonstrated that klotho overexpression alleviated glyoxylate administration-induced renal oxidative damage, renal apoptosis, and crystal deposition in the kidneys of mice, and these effects were also associated with activation of the Keap1-Nrf2-ARE pathway., Conclusion: Klotho protein inhibits the oxidative stress response of HKC cells through the Keap1-Nrf2-ARE signaling pathway, reduces the apoptosis of and adhesion of CaOx crystals to HKC cells, and decreases the occurrence of CaOx kidney stones., Clinical Trial Registration: 20220304., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Nephrolithiasis (NL) is a complex multifactorial disease affecting up to 10%-20% of the human population and causing a significant burden on public health systems worldwide. It results from a combination of environmental and genetic factors. Hyperoxaluria is a major risk factor for NL., Methods: We used a whole exome-based approach in a patient with calcium oxalate NL. The effects of the mutation were characterised using cell culture and in silico analyses., Results: We identified a rare heterozygous missense mutation (c.1519C>T/p.R507W) in the SLC26A6 gene that encodes a secretory oxalate transporter. This mutation cosegregated with hyperoxaluria in the family. In vitro characterisation of mutant SLC26A6 demonstrated that Cl - -dependent oxalate transport was dramatically reduced because the mutation affects both SLC26A6 transport activity and membrane surface expression. Cotransfection studies demonstrated strong dominant-negative effects of the mutant on the wild-type protein indicating that the phenotype of patients heterozygous for this mutation may be more severe than predicted by haploinsufficiency alone., Conclusion: Our study is in line with previous observations made in the mouse showing that SLC26A6 inactivation can cause inherited enteric hyperoxaluria with calcium oxalate NL. Consistent with an enteric form of hyperoxaluria, we observed a beneficial effect of increasing calcium in the patient's diet to reduce urinary oxalate excretion., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Nephrolithiasis is a highly prevalent urological disease and results in a correspondingly heavy socioeconomic and healthcare burden. Calcium oxalate (CaOx) stones are among the most common types of kidney stones. They are associated with renal tubular damage, interstitial fibrosis and chronic kidney disease (CKD). However, the molecular mechanisms in CaOx crystal deposition-induced renal fibrosis remain unclear. Chemokines and their receptors act a crucial role in the progression of renal fibrosis through inflammatory cell infiltration, autophagy activation, and epithelial-mesenchymal transition (EMT). The current work aims to study the action and mechanism of the C-X-C motif chemokine receptor 4 (CXCR4) in CaOx crystal deposition-induced renal fibrosis. Transcriptome RNA sequencing, qPCR, and immunohistochemistry revealed that the expression of CXCR4 was significantly upregulated in patients with nephrolithiasis and hyperoxaluric mice. Renal injury and fibrosis were significantly suppressed by inhibiting CXCR4 with AMD3100 or siRNA in hyperoxaluric mice and oxalate-stimulated HK-2 cells; EMT, reactive oxygen species (ROS) levels, and autophagy were also suppressed. Bioinformatic analysis revealed that the NF-κB pathway was activated in hyperoxaluric mice. Mechanistically, activation of the NF-κB pathway was suppressed by CXCR4 inhibition in CaOx crystal-induced renal fibrosis; this suppression was significantly aggravated by the NF-κB inhibitor BAY-11-7085. Moreover, inhibition of autophagy attenuated EMT progression in vitro. Our results suggest that CXCR4 inhibition attenuates CaOx crystal deposition-induced renal fibrosis by suppressing autophagy and EMT through the NF-κB pathway. Therefore, CXCR4 is a potential target for preventing renal fibrosis in patients with nephrolithiasis., (Copyright © 2022. Published by Elsevier B.V.)

Nephrolithiasis is the most common type of urinary system disease in developed countries, with high morbidity and recurrence rates. Nephrolithiasis is a serious health problem, which eventually leads to the loss of renal function and is closely related to hypertension. Modern medicine has adopted minimally invasive surgery for the management of kidney stones, but this does not resolve the root of the problem. Thus, nephrolithiasis remains a major public health issue, the causes of which remain largely unknown. Researchers have attempted to determine the causes and therapeutic targets of kidney stones and calculus‑related hypertension. Solute carrier family 26 member 6 (SLC26A6), a member of the well‑conserved solute carrier family 26, is highly expressed in the kidney and intestines, and it primarily mediates the transport of various anions, including OXa 2‑ , HCO 3 ‑ , Cl ‑ and SO 4 2‑ , amongst others. Na + ‑dependent dicarboxylate‑1 (NADC‑1) is the Na + ‑carboxylate co‑transporter of the SLC13 gene family, which primarily mediates the co‑transport of Na + and tricarboxylic acid cycle intermediates, such as citrate and succinate, amongst others. Studies have shown that Ca 2+ oxalate kidney stones are the most prevalent type of kidney stones. Hyperoxaluria and hypocitraturia notably increase the risk of forming Ca 2+ oxalate kidney stones, and the increase in succinate in the juxtaglomerular device can stimulate renin secretion and lead to hypertension. Whilst it is known that it is important to maintain the dynamic equilibrium of oxalate and citrate in the kidney, the synergistic molecular mechanisms underlying the transport of oxalate and citrate across kidney epithelial cells have undergone limited investigations. The present review examines the results from early reports studying oxalate transport and citrate transport in the kidney, describing the synergistic molecular mechanisms of SLC26A6 and NADC‑1 in the process of nephrolithiasis formation. A growing body of research has shown that nephrolithiasis is intricately associated with hypertension. Additionally, the recent investigations into the mediation of succinate via regulation of the synergistic molecular mechanism between the SLC26A6 and NADC‑1 transporters is summarized, revealing their functional role and their close association with the inositol triphosphate receptor‑binding protein to regulate blood pressure.

Nephrolithiasis causes severe pain and is a highly recurrent pathophysiological state. Calcium-containing stones, specifically calcium oxalate (CaOx), is the most common type accounting for approximately 75 % of stone composition. Genetic predisposition, gender, geographic region, diet, and low fluid intake all contribute to disease pathogenesis. However, exposure to environmental pollutants as a contribution to kidney stone formation remains insufficiently studied. Lead (Pb2+) is of particular interest as epidemiological data indicate that low-level exposure (BLL = 0.48-3.85 μM) confers a 35 % increased risk of developing CaOx nephrolithiasis. However, mechanisms underlying this association have yet to be elucidated. Drosophila melanogaster provide a useful genetic model where major molecular pathophysiological pathways can be efficiently studied. Malpighian tubules (MT) were isolated from either Wild-Type or InsP 3 R knockdown flies and treated with oxalate (5 mM) ± Pb 2+ (2μM) for 1 h. Following exposure, MTs were imaged and crystals quantified. CaOx crystal number and total area were significantly increased (˜5-fold) in Pb 2+ (pre-treatment) + oxalate-exposed MTs when compared to oxalate alone controls. However, CaOx crystal number and total crystal area in Pb 2+ + oxalate-exposed InsP 3 R knockdown MTs were significantly decreased (˜3-fold) indicating the role for principal cell-specific InsP 3 R-mediated Ca 2+ mobilization as a mechanism for Pb 2+ -induced increases in CaOx crystallization inset model of nephrolithiasis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Urolithiasis is the third most common urological disease after urinary tract infections and prostate diseases, and it is characterised by an occurrence rate of about 15%, which continues to rise. The increase in the incidence of kidney stones observed in recent decades, is most likely caused by modifications in dietary habits (high content of protein, sodium and sugar diet) and lifestyle (reduced physical activity) in all industrialised countries. Moreover, men are more likely than women to be diagnosed with kidney stones. A growing body of evidence suggests that inflammation, oxidant-antioxidant imbalance, angiogenesis, purine metabolism and urea cycle disorders may play a crucial role in nephrolithiasis development. Patients with urolithiasis were characterised by an increased level of reactive oxygen species (ROS), the products of lipid peroxidation, proinflammatory cytokines as well as proangiogenic factors, compared to controls. Furthermore, it has been shown that deficiency and disorders of enzymes involved in purine metabolism and the urea cycle might be causes of deposit formation. ROS generation suggests that the course of kidney stones might be additionally potentiated by inflammation, purine metabolism and the urea cycle. On the other hand, ROS overproduction may induce activation of angiogenesis, and thus, allows deposit aggregation.

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl-/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype-phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell-migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology., (© The Author(s) 2021. Published by Oxford University Press.)

GeoBioMed - a new transdisciplinary approach that integrates the fields of geology, biology and medicine - reveals that kidney stones composed of calcium-rich minerals precipitate from a continuum of repeated events of crystallization, dissolution and recrystallization that result from the same fundamental natural processes that have governed billions of years of biomineralization on Earth. This contextual change in our understanding of renal stone formation opens fundamentally new avenues of human kidney stone investigation that include analyses of crystalline structure and stratigraphy, diagenetic phase transitions, and paragenetic sequences across broad length scales from hundreds of nanometres to centimetres (five Powers of 10). This paradigm shift has also enabled the development of a new kidney stone classification scheme according to thermodynamic energetics and crystalline architecture. Evidence suggests that ≥50% of the total volume of individual stones have undergone repeated in vivo dissolution and recrystallization. Amorphous calcium phosphate and hydroxyapatite spherules coalesce to form planar concentric zoning and sector zones that indicate disequilibrium precipitation. In addition, calcium oxalate dihydrate and calcium oxalate monohydrate crystal aggregates exhibit high-frequency organic-matter-rich and mineral-rich nanolayering that is orders of magnitude higher than layering observed in analogous coral reef, Roman aqueduct, cave, deep subsurface and hot-spring deposits. This higher frequency nanolayering represents the unique microenvironment of the kidney in which potent crystallization promoters and inhibitors are working in opposition. These GeoBioMed insights identify previously unexplored strategies for development and testing of new clinical therapies for the prevention and treatment of kidney stones.

Lowe syndrome and Dent II disease are X-linked monogenetic diseases characterised by a renal reabsorption defect in the proximal tubules and caused by mutations in the OCRL gene, which codes for an inositol-5-phosphatase. The life expectancy of patients suffering from Lowe syndrome is largely reduced because of the development of chronic kidney disease and related complications. There is a need for physiological human in vitro models for Lowe syndrome/Dent II disease to study the underpinning disease mechanisms and to identify and characterise potential drugs and drug targets. Here, we describe a proximal tubule organ on chip model combining a 3D tubule architecture with fluid flow shear stress that phenocopies hallmarks of Lowe syndrome/Dent II disease. We demonstrate the high suitability of our in vitro model for drug target validation. Furthermore, using this model, we demonstrate that proximal tubule cells lacking OCRL expression upregulate markers typical for epithelial-mesenchymal transition (EMT), including the transcription factor SNAI2/Slug, and show increased collagen expression and deposition, which potentially contributes to interstitial fibrosis and disease progression as observed in Lowe syndrome and Dent II disease.

The prospect of using the antioxidant dipeptide carnosine for the treatment of urate nephrolithiasis was evaluated. Urate nephrolithiasis was modeled in rats by intragastric administration of a mixture of oxonic and uric acids. Carnosine was administered intragastrically through a tube in a dose of 15 mg/kg. In rats treated with carnosine, the concentration of TBA-reactive products decreased by 1.4 times, the total antioxidant activity increased by 1.4 times, and catalase activity increased by 1.3 times. By the end of the experiment, the lactate dehydrogenate level in experimental rats was 2-fold lower than in the control, and the number of urate deposits decreased by 1.6 times with a concomitant alleviation of the inflammatory processes. Thus, the use of direct peptide antioxidant carnosine attenuated the manifestations of urate nephrolithiasis.

Competing Interests: J. Agudelo reports other interests/relationships as a 2020 Urology Care Foundation Research Scholar Award Recipient. A.W. Miller reports research funding from the National Institutes of Health and honoraria from the Mayo Clinic.

Calcium (Ca 2+ ) is an important mediator of multicellular homeostasis and is involved in several diseases. The interplay among the kidney, bone, intestine, and parathyroid gland in Ca 2+ homeostasis is strictly modulated by numerous hormones and signaling pathways. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor, that is expressed in calcitropic tissues such as the parathyroid gland and the kidney, plays a pivotal role in Ca 2+ regulation. CaSR is important for renal Ca 2+ , as a mutation in this receptor leads to hypercalciuria and calcium nephrolithiasis. In addition, CaSR is also widely expressed in the vascular system, including vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) and participates in the process of vascular calcification. Aberrant Ca 2+ sensing by the kidney and VSMCs, owing to altered CaSR expression or function, is associated with the formation of nephrolithiasis and vascular calcification. Based on emerging epidemiological evidence, patients with nephrolithiasis have a higher risk of vascular calcification, but the exact mechanism linking the two conditions is unclear. However, a dysregulation in Ca 2+ homeostasis and dysfunction in CaSR might be the connection between the two. This review summarizes renal calcium handling and calcium signaling in the vascular system, with a special focus on the link between nephrolithiasis and vascular calcification.

Proximal tubular (PT) acidosis, which alkalinizes the urinary filtrate, together with Ca 2+ supersaturation in PT can induce luminal calcium phosphate (CaP) crystal formation. While such CaP crystals are known to act as a nidus for CaP/calcium oxalate (CaOx) mixed stone formation, the regulation of PT luminal Ca 2+ concentration ([Ca 2+ ]) under elevated pH and/or high [Ca 2+ ] conditions are unknown. Since we found that transient receptor potential canonical 3 (TRPC3) knockout (KO; -/-) mice could produce mild hypercalciuria with CaP urine crystals, we alkalinized the tubular pH in TRPC3-/- mice by oral acetazolamide (0.08%) to develop mixed urinary crystals akin to clinical signs of calcium nephrolithiasis (CaNL). Our ratiometric (λ340/380) intracellular [Ca 2+ ] measurements reveal that such alkalization not only upsurges Ca 2+ influx into PT cells, but the mode of Ca 2+ entry switches from receptor-operated to store-operated pathway. Electrophysiological experiments show enhanced bicarbonate related current activity in treated PT cells which may determine the stone-forming phenotypes (CaP or CaP/CaOx). Moreover, such alkalization promotes reactive oxygen species generation, and upregulation of calcification, inflammation, fibrosis, and apoptosis in PT cells, which were exacerbated in absence of TRPC3. Altogether, the pH-induced alteration of the Ca 2+ signaling signature in PT cells from TRPC3 ablated mice exacerbated the pathophysiology of mixed urinary stone formation, which may aid in uncovering the downstream mechanism of CaNL.

Renal tubular cell injury induced by calcium oxalate (CaOx) is a critical initial stage of kidney stone formation. Theaflavin (TF) has been known for its strong antioxidative capacity; however, the effect and molecular mechanism of TF against oxidative stress and injury caused by CaOx crystal exposure in kidneys remains unknown. To explore the potential function of TF on renal crystal deposition and its underlying mechanisms, experiments were conducted using a CaOx nephrocalcinosis mouse model established by glyoxylate intraperitoneal injection, and HK-2 cells were subjected to calcium oxalate monohydrate (COM) crystals, with or without the treatment of TF. We discovered that TF treatment remarkably protected against CaOx-induced kidney oxidative stress injury and reduced crystal deposition. Additionally, miR-128-3p expression was decreased and negatively correlated with SIRT1 level in mouse CaOx nephrocalcinosis model following TF treatment. Moreover, TF suppressed miR-128-3p expression and further abolished its inhibition on SIRT1 to attenuate oxidative stress in vitro . Mechanistically, TF interacted with miR-128-3p and suppressed its expression. In addition, miR-128-3p inhibited SIRT1 expression by directly binding its 3'-untranslated region (UTR). Furthermore, miR-128-3p activation partially reversed the acceerative effect of TF on SIRT1 expression. Taken together, TF exhibits a strong nephroprotective ability to suppress CaOx-induced kidney damage through the recovery of the antioxidant defense system regulated by miR-128-3p/SIRT1 axis. These findings provide novel insights for the prevention and treatment of renal calculus., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Dietary modifications and patient-tailored medical management are significant in controlling renal stone disease. Nevertheless, the literature regarding effectiveness is sparse., Objectives: To explore the impact of dietary modifications and medical management on 24-hour urinary metabolic profiles (UMP) and renal stone status in recurrent kidney stone formers., Methods: We reviewed our prospective registry database of patients treated for nephrolithiasis. Data included age, sex, 24-hour UMP, and stone burden before treatment. Under individual treatment, patients were followed at 6-8 month intervals with repeat 24-hour UMP and radiographic images. Nephrolithiasis-related events (e.g., surgery, renal colic) were also recorded. We included patients with established long-term follow-up prior to the initiation of designated treatment, comparing individual nephrolithiasis status before and after treatment initiation., Results: Inclusion criteria were met by 44 patients. Median age at treatment start was 60.5 (50.2-70.2) years. Male:Female ratio was 3.9:1. Median follow-up was 10 (6-25) years and 5 (3-6) years before and after initiation of medical and dietary treatment, respectively. Metabolic abnormalities detected included: hypocitraturia (95.5%), low urine volume (56.8%), hypercalciuria (45.5%), hyperoxaluria (40.9%), and hyperuricosuria (13.6%). Repeat 24-hour UMP under appropriate diet and medical treatment revealed a progressive increase in citrate levels compared to baseline and significantly decreased calcium levels (P = 0.001 and 0.03, respectively). A significant decrease was observed in stone burden (P = 0.001) and overall nephrolithiasis-related events., Conclusions: Dietary modifications and medical management significantly aid in correcting urinary metabolic abnormalities. Consequently, reduced nehprolithiasis-related events and better stone burden control is expected.

Context: Nephrolithiasis (NL) and primary hyperparathyroidism (HPTH) are metabolic complications of Paget disease of bone (PDB), but recent data regarding their prevalence in PDB patients are lacking., Objectives: Study 1: To compare the prevalence of primary HPTH and NL in 708 patients with PDB and in 1803 controls. Study 2: To evaluate the prevalence of NL-metabolic risk factors in 97 patients with PDB and NL, 219 PDB patients without NL, 364 NL patients without PDB, and 219 controls, all of them without HPTH., Design: Cross-sectional multicentric study., Setting: Italian referral centers for metabolic bone disorders., Participants: Patients with PDB from the Associazione Italiana malati di osteodistrofia di Paget registry. Participants in the Olivetti Heart and the Siena Osteoporosis studies., Main Outcome Measures: HPTH; NL; NL-metabolic risk factors., Results: Patients with PDB showed higher prevalence of primary HPTH and NL compared with controls (P < 0.01). The NL recurrence occurs more frequently in patients with polyostotic PDB. About one-half of patients with PDB but without NL showed 1 or more NL-related metabolic risk factors. The hyperoxaluria (HyperOx) prevalence was higher in patients with PDB and NL compared with patients with NL but without PDB and in patients with PDB without NL compared with controls (P = 0.01). Patients with PDB and HyperOx showed a longer lapse of time from the last aminobisphosphonate treatment., Conclusions: NL and HPTH are frequent metabolic complication of PDB. The NL occurrence should be evaluated in patients with PDB, particularly in those with polyostotic disease and/or after aminobisphosphonate treatment to apply an adequate prevention strategy., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The relationship of gut microbiota and calcium oxalate stone has been limited investigated, especially with no study of gut microbiota and short chain fatty acids (SCFAs) in nephrolithiasis. We provided Sprague Dawley rats of renal calcium oxalate stones with antibiotics and examined the renal crystals deposition. We also performed a case-control study by analyzing 16S rRNA microbial profiling, shotgun metagenomics and SCFAs in 153 fecal samples from non-kidney stone (NS) controls, patients with occasional renal calcium oxalate stones (OS) and patients with recurrent stones (RS). Antibiotics reduced bacterial load in feces and could promote the formation of renal calcium crystals in model rats. In addition, both OS and RS patients exhibited higher fecal microbial diversity than NS controls. Several SCFAs-producing gut bacteria, as well as metabolic pathways associated with SCFAs production, were considerably lower in the gut microbiota among the kidney stone patients compared with the NS controls. Representation of genes involved in oxalate degradation showed no significance difference among groups. However, fecal acetic acid concentration was the highest in RS patients with high level of urinary oxalate, which was positively correlated with genes involvement in oxalate synthesis. Administration of SCFAs reduced renal crystals. These results shed new light on bacteria and SCFAs, which may promote the development of treatment strategy in nephrolithiasis., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: The relationship between the composition and function of gut microbial communities and early-onset calcium oxalate kidney stone disease is unknown., Methods: We conducted a case-control study of 88 individuals aged 4-18 years, which included 44 individuals with kidney stones containing ≥50% calcium oxalate and 44 controls matched for age, sex, and race. Shotgun metagenomic sequencing and untargeted metabolomics were performed on stool samples., Results: Participants who were kidney stone formers had a significantly less diverse gut microbiome compared with controls. Among bacterial taxa with a prevalence >0.1%, 31 taxa were less abundant among individuals with nephrolithiasis. These included seven taxa that produce butyrate and three taxa that degrade oxalate. The lower abundance of these bacteria was reflected in decreased abundance of the gene encoding butyryl-coA dehydrogenase ( P =0.02). The relative abundance of these bacteria was correlated with the levels of 18 fecal metabolites, and levels of these metabolites differed in individuals with kidney stones compared with controls. The oxalate-degrading bacterial taxa identified as decreased in those who were kidney stone formers were components of a larger abundance correlation network that included Eggerthella lenta and several Lactobacillus species. The microbial ( α ) diversity was associated with age of stone onset, first decreasing and then increasing with age. For the individuals who were stone formers, we found the lowest α diversity among individuals who first formed stones at age 9-14 years, whereas controls displayed no age-related differences in diversity., Conclusions: Loss of gut bacteria, particularly loss of those that produce butyrate and degrade oxalate, associates with perturbations of the metabolome that may be upstream determinants of early-onset calcium oxalate kidney stone disease., (Copyright © 2020 by the American Society of Nephrology.)

Dent disease 1 (DD1) is caused by mutations in the CLCN5 gene encoding a voltage-gated electrogenic nCl - /H + exchanger ClC-5. Using ion-selective microelectrodes and Xenopus oocytes, here we studied Cl - /H + coupling properties of WT ClC-5 and four DD1-associated variants (S244L, R345W, Q629*, and T657S), along with trafficking and localization of ClC-5. WT ClC-5 had a 2Cl - /H + exchange ratio at a V h of +40 mV with a [Cl - ] out of 104 mm, but the transport direction did not reverse with a [Cl - ] out of 5 mm, indicating that ClC-5-mediated exchange of two Cl - out for one H + in is not permissible. We hypothesized that ClC-5 and H + -ATPase are functionally coupled during H + -ATPase-mediated endosomal acidification, crucial for ClC-5 activation by depolarizing endosomes. ClC-5 transport that provides three net negative charges appeared self-inhibitory because of ClC-5's voltage-gated properties, but shunt conductance facilitated further H + -ATPase-mediated endosomal acidification. Thus, an on-and-off "burst" of ClC-5 activity was crucial for preventing Cl - exit from endosomes. The subcellular distribution of the ClC-5:S244L variant was comparable with that of WT ClC-5, but the variant had a much slower Cl - and H + transport and displayed an altered stoichiometry of 1.6:1. The ClC-5:R345W variant exhibited slightly higher Cl - /H + transport than ClC-5:S244L, but co-localized with early endosomes, suggesting decreased ClC-5:R345W membrane trafficking is perhaps in a fully functional form. The truncated ClC-5:Q629* variant displayed the lowest Cl - /H + exchange and was retained in the endoplasmic reticulum and cis -Golgi, but not in early endosomes, suggesting the nonsense mutation affects ClC-5 maturation and trafficking., (© 2020 Chang et al.)

Background: Nephrolithiasis is a common disease in urology, and its pathogenesis is associated with various factors. Recent studies have shown that reactive oxygen species (ROS) can promote autophagy in the formation of kidney stones and exacerbate kidney injury. Endoplasmic reticulum stress (ERS), a key factor in regulating intracellular environmental homeostasis, is also directly related to ROS production. Therefore, this study aimed to investigate the regulatory effect of superoxide dismutase (SOD) on autophagy-ERS response during the formation of calcium oxalate (CaOx) kidney stones in rats., Methods: Thirty-two rats were randomly divided into four groups (n = 8): normal control group, stone model group, stone model with atorvastatin group, and stone model with diethyldithiocarbamic acid (DETC) group. Rat models of CaOx kidney stones were established by intragastric administration of 0.75 % ethylene glycol for 4 weeks. Kidney/body weight was used to assess renal enlargement. Renal function was assessed by measuring serum SOD, creatinine (CRE), and blood urea nitrogen (BUN) levels. The expression of autophagy-related proteins LC3B and BECN1 was detected through immunohistochemical staining. Meanwhile, the expression of autophagy-ERS response-related proteins LC3B, BECN1, p62, GRP78, and CHOP was detected using Western blot and RT-PCR. Renal tubular injury markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (Kim-1) were determined through enzyme-linked immunosorbent assay. The apoptosis of renal tubular cells and the expression of their signature proteins cleaved Caspase-3, Bax and Bcl-2 were detected using Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Western blot assays, respectively. Crystal deposition and histological tissue injury were assessed through Von Kossa staining., Results: Compared with the control group, the stone model group showed higher kidney/body weight ratio; evidently higher expression of autophagy-ERS response- and apoptosis-related proteins LC3B, BECN1, GRP78, CHOP, Bax and cleaved Caspase-3; and lower levels of p62, bcl-2 protein, and SOD. The stone model group also showed higher levels of apoptosis, serum CRE, BUN, NGAL, and Kim-1, as well as considerably greater crystal deposition and renal injury, than the control group. Atorvastatin reduced the levels of autophagy-ERS response, kidney injury, and crystal deposition, but they were increased by DETC., Conclusion: Enhanced SOD activity can protect the kidneys by reducing autophagy-ERS response and CaOx kidney stone formation. Atorvastatin may be a new option for the prevention and treatment of nephrolithiasis., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Nephrolithiasis or renal stone disease is an increasingly common problem, and its relatively high recurrence rate demands better treatment options. The majority of patients with nephrolithiasis have stones that contain calcium (Ca 2+ ), which develop upon "supersaturation" of the urine with insoluble Ca 2+ salts; hence processes that influence the delivery and renal handling of Ca 2+ may influence stone formation. Idiopathic hypercalciuria is indeed frequently observed in patients with kidney stones that contain Ca 2+ . Genetic screens of nephrolithiasis determinants have identified an increasing number of gene candidates, most of which are involved in renal Ca 2+ handling. This review provides an outline of the current knowledge regarding genetics of nephrolithiasis and will mainly focus on the epithelial Ca 2+ channel transient receptor potential vanilloid 5 (TRPV5), an important player in Ca 2+ homeostasis. Being a member of the TRP family of ion channels, TRPV5 is currently part of a revolution in structural biology. Recent technological breakthroughs in the cryo-electron microscopy field, combined with improvements in biochemical sample preparation, have resulted in high-resolution 3-dimensional structural models of integral membrane proteins, including TRPV5. These models currently are being used to explore the proteins' structure-function relationship, elucidate the molecular mechanisms of channel regulation, and study the putative effects of disease variants. Combined with other multidisciplinary approaches, this approach may open an avenue toward better understanding of the pathophysiological mechanisms involved in hypercalciuria and stone formation, and ultimately it may facilitate prevention of stone recurrence through the development of effective drugs., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Peroxisome proliferator-activated receptor- (PPAR-) γ is a ligand-dependent transcription factor, and it has become evident that PPAR- γ agonists have renoprotective effects, but their influence and mechanism during the development of calcium oxalate (CaOx) nephrolithiasis remain unknown. Rosiglitazone (RSG) was used as a representative PPAR- γ agonist in our experiments. The expression of transforming growth factor- β 1 (TGF- β 1), hepatocyte growth factor (HGF), c-Met, p-Met, PPAR- γ , p-PPAR- γ (Ser112), Smad2, Smad3, pSmad2/3, and Smad7 was examined in oxalate-treated Madin-Darby canine kidney (MDCK) cells and a stone-forming rat model. A CCK-8 assay was used to evaluate the effects of RSG on cell viability. In addition, intracellular reactive oxygen species (ROS) levels were monitored, and lipid peroxidation in renal tissue was detected according to superoxide dismutase and malondialdehyde levels. Moreover, the location and extent of CaOx crystal deposition were evaluated by Pizzolato staining. Our results showed that, both in vitro and in vivo , oxalate impaired PPAR- γ expression and phosphorylation, and then accumulative ROS production was observed, accompanied by enhanced TGF- β 1 and reduced HGF. These phenomena could be reversed by the addition of RSG. RSG also promoted cell viability and proliferation and decreased oxidative stress damage and CaOx crystal deposition. However, these protective effects of RSG were abrogated by the PPAR- γ -specific inhibitor GW9662. Our results revealed that the reduction of PPAR- γ activity played a critical role in oxalate-induced ROS damage and CaOx stone formation. RSG can regulate TGF- β 1 and HGF/c-Met through PPAR- γ to exert antioxidant effects against hyperoxaluria and alleviate crystal deposition. Therefore, PPAR- γ agonists may be expected to be a novel therapy for nephrolithiasis, and this effect is related to PPAR- γ -dependent suppression of oxidative stress., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Ya-Dong Liu et al.)

Nephrolithiasis is one of the most common kidney diseases, with poorly understood pathophysiology, but experimental study has been hindered by lack of experimentally tractable models. Drosophila melanogaster is a useful model organism for renal diseases because of genetic and functional similarities of Malpighian (renal) tubules with the human kidney. Here, we demonstrated function of the sex-determining region Y protein-interacting protein-1 ( Sip1 ) gene, an ortholog of human Na + /H + exchanger regulatory factor ( NHERF1 ), in Drosophila Malpighian tubules and its impact on nephrolithiasis. Abundant birefringent calculi were observed in Sip1 mutant flies, and the phenotype was also observed in renal stellate cell-specific RNA interference Sip1 knockdown in otherwise normal flies, confirming a renal etiology. This phenotype was abolished in rosy mutant flies (which model human xanthinuria) and by the xanthine oxidase inhibitor allopurinol, suggesting that the calculi were of uric acid. This was confirmed by direct biochemical assay for urate. Stones rapidly dissolved when the tubule was bathed in alkaline media, suggesting that Sip1 knockdown was acidifying the tubule. SIP1 was shown to collocate with Na + /H + exchanger isoform 2 (NHE2) and with moesin in stellate cells. Knockdown of NHE2 specifically to the stellate cells also increased renal uric acid stone formation, and so a model was developed in which SIP1 normally regulates NHE2 activity and luminal pH, ultimately leading to uric acid stone formation. Drosophila renal tubules may thus offer a useful model for urate nephrolithiasis.

Purpose: Dietary hydroxyproline may be involved in the endogenous synthesis of oxalate. Glycolate, produced during the metabolism of hydroxyproline, may exert physicochemical effects on urinary calcium by virtue of its dihydroxycarboxylic acid structure. The aim of this study was to investigate these possible stone-risk scenarios., Methods: We modelled the effect of different glycolic acid concentrations on ionized calcium (iCa 2+ ) and relative supersaturation (RSS) of calcium oxalate (CaOx) using the program JESS. Thereafter, three healthy white males and two healthy black males ingested 30 g gelatin for 3 days. 24-h urines were collected at baseline and after completion of the protocol. Urines were analysed for physicochemical risk factors and for iCa 2+ and glycolic acid. Speciation concentrations and RSS values were calculated., Results: Theoretical modelling showed that binding between calcium and glycolate does not occur and that iCa 2+ and RSS CaOx are unaffected. However, after ingestion of hydroxyproline, iCa 2+ decreased significantly. Urinary pH and glycolate increased significantly. Oxalate excretion and RSS CaOx did not change CONCLUSIONS: We attribute the decrease in iCa 2+ to increases in the concentrations of several Ca-phosphate species, the formation of which is due to the increase in pH. We speculate that the absence of an increase in oxalate excretion despite an increase in glycolate excretion may be due to the mixed racial composition of our test group in which some pathways may be preferred to others. Our findings alert stone researchers to the importance of measuring urinary pH in their workup of subjects and to select racially homogenous groups for investigation.

Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzymes deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome, possibly by inhibiting neuronal lactate dehydrogenase 5 isoenzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that Stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, Stiripentol decreased in a dose-dependent manner the synthesis of oxalate by hepatocytes. In vivo, Stiripentol oral administration reduced significantly urine oxalate excretion in rats. Stiripentol protected kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, Stiripentol improved significantly renal function. Patients affected by Dravet syndrome and treated with Stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received Stiripentol for several weeks: urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.

Proton pump inhibitors, PPIs, are widely prescribed and sold globally. Although initially intended for time-limited treatment of acute disorders, such as gastric ulcers and esophagitis, PPIs are now commonly used for prolonged durations and are considered safe for over the counter access. Recent studies have raised concern over associations between PPI use and acute kidney injury, chronic kidney disease, end-stage renal disease, and electrolyte abnormalities. The growing concern over potentially serious adverse drug reactions warrants an evaluation of post marketing surveillance data. In this study of over ten million FDA Adverse Event Reporting System records, we provided evidence of kidney injury and electrolyte imbalances in an alarming number of patients taking PPIs. Additionally, we assessed differences between specific PPIs and observed significant electrolyte and renal abnormalities for each individual drug with varying magnitudes.

Aims: Preventing kidney stone recurrence relies on detecting and modifying urine chemistry abnormalities. The assumption is that an abnormality is due to a global metabolic defect present in both kidneys. However, we hypothesize that clinically significant unilateral defects may exist. We aimed to identify these patients by sampling urine from each renal unit., Materials and Methods: Adults undergoing retrograde upper urinary tract surgery were eligible for inclusion. Excluded were patients with a solitary kidney, suspected urothelial malignancy, or urinary tract infection. Following informed consent, all patients proceeded to the operating room. After induction of anesthesia, cystoscopy with ureteral catheterization was performed with urine collected via gravity drainage for 10 minutes. Urine samples with adequate volume were analyzed for chemistry concentrations. A difference greater than the 75 th percentile between matched pairs was considered significant. For urine pH, a difference of 0.5 was considered significant., Results: A total of 47 patients were screened for eligibility with only 13 (28%) electing to enroll in the study (26 renal units). All subjects underwent successful bilateral ureteral catheterization with no adverse events observed or later reported. The mean (± SD) urine volume captured from the right and left renal units was 5.0 ± 7.4 cm 3 and 6.6 ± 6.4 cm 3 , respectively. Urine was only captured from paired renal units in 8 participants (8/13; 62%). Of these 8 participants, 5 (5/8; 63%) had at least 1 unilateral metabolic defect., Conclusion: Unilateral renal unit urine sampling is safe and feasible. However, captured urine volumes are small and variable, but chemical analysis can still be performed. Unilateral defects in renal electrolyte handling are relatively common, but the clinical implications of these differences are still yet to be determined.
.

SUMOylation of proteins is an important regulatory element in modulating protein function and has been implicated in the pathogenesis of numerous human diseases such as cancers, neurodegenerative diseases, brain injuries, diabetes, and familial dilated cardiomyopathy. Growing evidence has pointed to a significant role of SUMO in kidney diseases such as DN, RCC, nephritis, AKI, hypertonic stress and nephrolithiasis. Recently, emerging studies in podocytes demonstrated that SUMO might have a protective role against podocyte apoptosis. However, the SUMO code responsible for beneficial outcome in the kidney remains to be decrypted. Our recent experiments have revealed that the expression of both SUMO and SUMOylated proteins is appreciably elevated in hypoxia-induced tubular epithelial cells (TECs) as well as in the unilateral ureteric obstruction (UUO) mouse model, suggesting a role of SUMO in TECs injury and renal fibrosis. In this review, we attempt to decipher the SUMO code in the development of kidney diseases by summarizing the defined function of SUMO and looking forward to the potential role of SUMO in kidney diseases, especially in the pathology of renal fibrosis and CKD, with the goal of developing strategies that maximize correct interpretation in clinical therapy and prognosis., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Primary hyperoxalurias (PHs) are rare inherited disorders of liver glyoxylate metabolism, characterized by the abnormal production of endogenous oxalate, a metabolic end-product that is eliminated by urine. The main symptoms are related to the precipitation of calcium oxalate crystals in the urinary tract with progressive renal damage and, in the most severe form named Primary Hyperoxaluria Type I (PH1), to systemic oxalosis. The therapies currently available for PH are either poorly effective, because they address the symptoms and not the causes of the disease, or highly invasive. In the last years, advances in our understanding of the molecular bases of PH have paved the way for the development of new therapeutic strategies. They include (i) substrate-reduction therapies based on small-molecule inhibitors or the RNA interference technology, (ii) gene therapy, (iii) enzyme administration approaches, (iv) colonization with oxalate-degrading intestinal microorganisms, and, in PH1, (v) design of pharmacological chaperones. This paper reviews the basic principles of these new therapeutic strategies and what is currently known about their application to PH.

Calcium-sensing receptor (CaSR) is a plasma-membrane G protein-coupled receptor activated by extracellular calcium and expressed in kidney tubular cells. It inhibits calcium reabsorption in the ascending limb and distal convoluted tubule when stimulated by the increase of serum calcium levels; therefore, these tubular segments are enabled by CaSR to play a substantial role in the regulation of serum calcium levels. In addition, CaSR increases water and proton excretion in the collecting duct and promotes phosphate reabsorption and citrate excretion in the proximal tubule. These CaSR activities form a network in which they are integrated to protect the kidney against the negative effects of high calcium concentrations and calcium precipitates in urine. Therefore, the CaSR gene has been considered as a candidate to explain calcium nephrolithiasis. Epidemiological studies observed that calcium nephrolithiasis was associated with polymorphisms of the CaSR gene regulatory region, rs6776158, located within the promoter-1, rs1501899 located in the intron 1, and rs7652589 in the 5'-untranslated region. These polymorphisms were found to reduce the transcriptional activity of promoter-1. Activating rs1042636 polymorphism located in exon 7 was associated with calcium nephrolithiasis and hypercalciuria. Genetic polymorphisms decreasing CaSR expression could predispose individuals to stones because they may impair CaSR protective effects against precipitation of calcium phosphate and oxalate. Activating polymorphisms rs1042636 could predispose to calcium stones by increasing calcium excretion. These findings suggest that CaSR may play a complex role in lithogenesis through different pathways having different relevance under different clinical conditions.

The anion exchanger SAT-1 [sulfate anion transporter 1 (Slc26a1)] is considered an important regulator of oxalate and sulfate homeostasis, but the mechanistic basis of these critical roles remain undetermined. Previously, characterization of the SAT-1-knockout (KO) mouse suggested that the loss of SAT-1-mediated oxalate secretion by the intestine was responsible for the hyperoxaluria, hyperoxalemia, and calcium oxalate urolithiasis reportedly displayed by this model. To test this hypothesis, we compared the transepithelial fluxes of 14 C-oxalate, 35 S O 4 2 - , and 36 Cl - across isolated, short-circuited segments of the distal ileum, cecum, and distal colon from wild-type (WT) and SAT-1-KO mice. The absence of SAT-1 did not impact the transport of these anions by any part of the intestine examined. Additionally, SAT-1-KO mice were neither hyperoxaluric nor hyperoxalemic. Instead, 24-h urinary oxalate excretion was almost 50% lower than in WT mice. With no contribution from the intestine, we suggest that this may reflect the loss of SAT-1-mediated oxalate efflux from the liver. SAT-1-KO mice were, however, profoundly hyposulfatemic, even though there were no changes to intestinal sulfate handling, and the renal clearances of sulfate and creatinine indicated diminished rates of sulfate reabsorption by the proximal tubule. Aside from this distinct sulfate phenotype, we were unable to reproduce the hyperoxaluria, hyperoxalemia, and urolithiasis of the original SAT-1-KO model. In conclusion, oxalate and sulfate transport by the intestine were not dependent on SAT-1, and we found no evidence supporting the long-standing hypothesis that intestinal SAT-1 contributes to oxalate and sulfate homeostasis. NEW & NOTEWORTHY SAT-1 is a membrane-bound transport protein expressed in the intestine, liver, and kidney, where it is widely considered essential for the excretion of oxalate, a potentially toxic waste metabolite. Previously, calcium oxalate kidney stone formation by the SAT-1-knockout mouse generated the hypothesis that SAT-1 has a major role in oxalate excretion via the intestine. We definitively tested this proposal and found no evidence for SAT-1 as an intestinal anion transporter contributing to oxalate homeostasis.

Objectives: The involvement of the gut microbiota in the pathogenesis of calcium nephrolithiasis has been hypothesised since the discovery of the oxalate-degrading activity of Oxalobacter formigenes , but never comprehensively studied with metagenomics. The aim of this case-control study was to compare the faecal microbiota composition and functionality between recurrent idiopathic calcium stone formers (SFs) and controls., Design: Faecal samples were collected from 52 SFs and 48 controls (mean age 48±11). The microbiota composition was analysed through 16S rRNA microbial profiling approach. Ten samples (five SFs, five controls) were also analysed with deep shotgun metagenomics sequencing, with focus on oxalate-degrading microbial metabolic pathways. Dietary habits, assessed through a food-frequency questionnaire, and 24-hour urinary excretion of prolithogenic and antilithogenic factors, including calcium and oxalate, were compared between SFs and controls, and considered as covariates in the comparison of microbiota profiles., Results: SFs exhibited lower faecal microbial diversity than controls (Chao1 index 1460±363vs 1658±297, fully adjusted p=0.02 with stepwise backward regression analysis). At multivariate analyses, three taxa ( Faecalibacterium , Enterobacter , Dorea ) were significantly less represented in faecal samples of SFs. The Oxalobacter abundance was not different between groups. Faecal samples from SFs exhibited a significantly lower bacterial representation of genes involved in oxalate degradation, with inverse correlation with 24-hour oxalate excretion (r=-0.87, p=0.002). The oxalate-degrading genes were represented in several bacterial species, whose cumulative abundance was inversely correlated with oxaluria (r=-0.85, p=0.02)., Conclusions: Idiopathic calcium SFs exhibited altered gut microbiota composition and functionality that could contribute to nephrolithiasis physiopathology., Competing Interests: Competing interests: TM received an unconditioned grant for research in the field of nephrolithiasis by Fiuggi Acqua & Terme S.p.A. (Fiuggi, Frosinone, Italy). All the other authors have no conflict of interest to declare., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Aim: To evaluate the level of lipid peroxidation in patients with nephrolithiasis before and after extracorporeal shock wave lithotripsy (ESWL)., Materials & Methods: Isoprostane concentration (8-isoPGF 2α ) was measured in urine, and thiobarbituric acid reactive substance production in serum and erythrocytes. In addition, the concentrations of selected compounds (uric acid, glucose and creatinine) were measured in serum., Results: The patients (before and after ESWL) demonstrated significantly higher levels of two different biomarkers of lipid peroxidation compared with the control group. A correlation was identified between increased amounts of uric acid and biomarkers of lipid peroxidation in patients with nephrolithiasis, both before and after ESWL., Conclusion: Uric acid may be associated with lipid peroxidation in patients with nephrolithiasis.

Objective: To evaluate patient compliance with treatment for urinary lithiasis and to detect differences in adherence, causes of this behavior and associated factors., Methods: We performed a retrospective study of 93 patients with positive urinary metabolic study (UMS) for lithogenic pathology, diagnosed between 2013 and 2015, gathering data from the digital medical records and a structured telephonic questionnaire in 75 of them. Results were analyzed using the X2 test., Results: 68% of the patients were males. Median age 42.92 (12.17) years. Mean follow up was 2.65 years. Most frequent metabolic alterations were: Hyperoxaluria (42.7%), Hypercalciuria (33.3%) and hipocitraturia (30.7%). Most frequently prescribed drugs: Potassium citrate (70.7%), Thiazide diuretics (26.7%) and calcium supplements (15.1). 84.2% of the patients did not know their UMS and 29.8% did not know the treatment prescribed. 41.9% followed the doses prescribed less than 50% of the times. Dietetic treatment was abandoned by 65% of the patients and pharmacological treatment by 43.5%, mainly due to laziness (62.9% vs 46.2%). 72.6% of the compliant patients experienced improvement. We find a significant relationship between academic level and diagnosis knowledge (p=0.022) and treatment (p=0.036). There were no differences in compliance depending on the number of drugs taken., Conclusions: Despite urine metabolic study being well valued and treatment well tolerated therapeutic compliance is very low. Most patients would repeat or restart the treatment prescribed in case of recurrence. Diagnostic and therapeutic information provided was not understood.