Calcium Oxalate Crystal Deposition Research Articles

Bidirectional Impact of Varying Severity of Acute Kidney Injury on Calcium Oxalate Stone Formation

Introduction: Acute kidney injury (AKI) is a prevalent renal disorder. The occurrence of AKI may promote the formation of renal calcium oxalate stones by exerting continuous effects on renal tubular epithelial cells (TECs). We aimed to delineate the molecular interplay between AKI and nephrolithiasis. Methods: A mild (20 min) and severe (30 min) renal ischemia-reperfusion injury model was established in mice. Seven days after injury, calcium oxalate stones were induced using glyoxylate (Gly) to evaluate the impact of AKI on the formation of kidney stones. Transcriptome sequencing was performed on TECs to elucidate the relationship between AKI severity and kidney stones. Key transcription factors (TFs) regulating differential gene transcription levels were identified using motif analysis, and pioglitazone, ginkgetin, and fludarabine were used for targeted therapy to validate key TFs as potential targets for kidney stone treatment. Results: Severe AKI led to increased deposition of calcium oxalate crystals in renal, impaired kidney function, and upregulation of kidney stone-related gene expression. In contrast, mild AKI was associated with decreased crystal deposition, preserved kidney function, and downregulation of similar gene expression. Transcriptomic analysis revealed that genes associated with inflammation and cell adhesion pathways were significantly upregulated after severe AKI, while genes related to energy metabolism pathways were significantly upregulated after mild AKI. An integrative bioinformatic analysis uncovered a TF regulatory network within TECs, pinpointing that PKNOX1 was involved in the upregulation of inflammation-related genes after severe AKI, and inhibiting PKNOX1 function with pioglitazone could simultaneously reduce the increase of calcium oxalate crystals after severe AKI in kidney. On the other hand, motif analysis also revealed the protective role of STAT1 in the kidneys after mild AKI, enhancing the function of STAT1 with ginkgetin could reduce kidney stone formation, while the specific inhibitor of STAT1, fludarabine, could eliminate the therapeutic effects of mild AKI on kidney stones. Conclusion: Inadequate repair of TECs after severe AKI increases the risk of kidney stone formation, with the upregulation of inflammation-related genes regulated by PKNOX1 playing a role in this process. Inhibiting PKNOX1 function can reduce kidney stone formation. Conversely, after mild AKI, effective cell repair through upregulation of STAT1 expression can protect TEC function and reduce stone formation, and activating STAT1 function can also achieve the goal of treating kidney stones.

Multiorgan Crystal Deposition in Acute Ethylene Glycol Toxicity.

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

NINJ1-mediated Macrophage Plasma Membrane Rupture and Neutrophil Extracellular Trap Formation Contribute to Oxalate Nephropathy.

Oxalate nephropathy is characterized by calcium oxalate crystals deposition, which triggers necrosis of renal tubular epithelial cells, initiates an inflammatory cascade characterized by neutrophil and macrophage activation within the renal microenvironment. Despite the close association of immune cells with acute oxalate nephropathy, the underlying mechanisms still remain unclear. Nerve injury-induced protein 1 (NINJ1) plays an essential role in the induction of plasma membrane rupture (PMR), leading to damage-associated molecular patterns (DAMPs) release and triggering inflammation. We hypothesize that NINJ1-mediated high mobility group box 1 (HMGB1) release from macrophage PMR and neutrophil extracellular traps (NETs) formation synergistically contribute to the progression of acute oxalate nephropathy. Using a murine model of acute oxalate nephropathy, myeloid cell-specific deletion of Ninj1 mice (Ninj1fl/flvavcre) and their wild-type littermate control mice (Ninj1wt/wtvavcre) were administered intraperitoneal injection of 100 mg/kg sodium oxalate followed by drinking water with 3% sodium oxalate. Evaluation was conducted on tubular injury and inflammatory cell infiltration. In vitro studies involved isolation and culture of renal proximal tubular epithelial cells (RTECs), bone marrow-derived macrophages, and neutrophils to investigate NETs formation and HMGB1 release. Targeted deletion of Ninj1 in myeloid cells significantly mitigated oxalate-induced acute kidney injury by suppressing both HMGB1 release and NETs formation in vivo. In vitro investigations demonstrated that HMGB1 release from macrophage PMR and NETs formation in neutrophils mediated by NINJ1 oligomerization, which consequently coordinated to enhance renal tubular epithelial cell death. Our findings elucidate the pivotal role of NINJ1-dependent macrophage PMR and NETs formation in the progression of acute oxalate nephropathy, providing novel insights for its prevention and therapeutic targets.

Efficient and safe in vivo treatment of primary hyperoxaluria type 1 via LNP-CRISPR-Cas9-mediated glycolate oxidase disruption

Primary hyperoxaluria type 1 (PH1) is a severe genetic metabolic disorder caused by mutations in the AGXT gene, leading to defects in enzymes crucial for glyoxylate metabolism. PH1 is characterized by severe, potentially life-threatening manifestations due to excessive oxalate accumulation, which leads to calcium oxalate crystal deposits in the kidneys and, ultimately, renal failure and systemic oxalosis. Existing substrate reduction therapies, such as inhibition of liver-specific glycolate oxidase (GO) encoded by HAO1 using siRNA or CRISPR/Cas9 delivered by adeno-associated virus (AAV), either require repeated dosing or have raised safety concerns. To address these limitations, our study employed lipid nanoparticles (LNPs) for CRISPR/Cas9 delivery to rapidly generate a PH1 mouse model and validate the therapeutic efficacy of LNP-CRISPR/Cas9 targeting the Hao1 gene. The LNP-CRISPR/Cas9 system exhibited efficient editing of the Hao1 gene, significantly reducing GO expression and lowering urinary oxalate levels in treated PH1 mice. Notably, these effects persisted for 12 months with no significant off-target effects, liver-induced toxicity, or substantial immune responses, highlighting the approach's safety and specificity. Furthermore, the developed humanized mouse model validated the efficacy of our therapeutic strategy. These findings support LNP-CRISPR/Cas9 targeting HAO1 as a promising and safer alternative for PH1 treatment with a single administration.

Puerarin alleviates apoptosis and inflammation in kidney stone cells via the PI3K/AKT pathway: Network pharmacology and experimental verification.

Puerarin(PUE), an isoflavonoid extracted from Pueraria root, has anti-apoptotic effects. The objective of this research is to examine the impact of PUE on renal apoptosis and inflammation resulting from renal calculi and to elucidate its mechanism. The approach of network pharmacology and molecular docking was employed to discover potential targets and pathways of PUE. An animal model of calcium oxalate crystal deposition by intraperitoneal injection of glyoxylate and a model of COM-induced human renal tubular epithelial cells (HK2) were used to investigate the pharmacological mechanisms of PUE against apoptosis and inflammation. We used haematoxylin-eosin (H&E) and Periodic Acid-Schiff staining (PAS) to assess the effect of PUE on crystal deposition and damage. The mechanism of PUE was elucidated and validated using Western blotting, histology and immunohistochemical staining. Network pharmacology findings indicated that the PI3K/AKT pathway plays a crucial role in PUE. We experimentally demonstrate that PUE alleviated COM-induced changes in apoptotic proteins, increased inflammatory indicators and changes in oxidative stress-related indicators in HK2 cells by activating the PI3K/AKT pathway, reduced serum creatinine and urea nitrogen levels in mice caused by CaOx, alleviated crystal deposition and damage, and alleviated apoptosis, oxidative stress and inflammation. Puerarin attenuates renal apoptosis and inflammation caused by kidney stones through the PI3K/AKT pathway.

Isovaleramide attenuates ethylene glycol poisoning-induced acute kidney injury and reduces mortality by inhibiting alcohol dehydrogenase activity in rats.

We explored the potential value of the alcohol dehydrogenase (ADH) inhibitor isovaleramide (ISO) in the treatment of acute ethylene glycol (EG) poisoning-induced acute kidney injury. Sprague-Dawley rats were divided into the control, EG, EG + ISO (10mg/kg) and EG + ISO (20 mg/kg) groups. It is found that ISO intervention significantly reduced the ADH activity in liver tissue by using visible spectrophotometry, inhibited the in vivo metabolism of EG by using gas chromatography, lowered the levels of toxic metabolites glycolic acid and oxalic acid by using high-performance liquid chromatography and decreased the expression of kidney injury markers serum creatinine (sCr), KIM-1, neutrophil gelatinase-associated lipocalin (NGAL) and liver fatty acid-binding protein (L-FABP) by ELISA. Additionally, Western blotting results showed that ISO down-regulated the expression of apoptotic factors Bax and cleaved caspase-3 in the kidneys and upregulated the expression of antiapoptotic factor Bcl-2. Pizzolato staining and polarized light microscopy results revealed the reduced deposition of calcium oxalate crystals in the kidney tubules. Using haematoxylin and eosin (H&E), periodic acid-Schiff (PAS) and Masson staining, we found attenuated kidney tissue pathological injury. Finally, ISO significantly reduced the mortality rate. In conclusion, ISO has the potential to be a valuable drug for the treatment of EG poisoning-induced acute kidney injury.

MOF-818 Nanozyme Suppresses Calcium Oxalate Kidney Stones by Alleviating Oxidative Stress and Inflammatory Injury.

There remains a lack of effective drugs to alleviate the kidney stones caused by oxidative stress and inflammatory damage. The MOF-818 nanozyme is utilized to lessen the generation of reactive oxygen species (ROS) effectively, restore the membrane potential of mitochondria, regulate the cell cycle, decrease cell death, hinder the recruitment of macrophages, and mitigate the release of inflammatory factors in macrophages. These effects are attributed to the nanozyme's ability to mimic the enzyme properties of catalase (CAT) and superoxide dismutase (SOD). It is demonstrated that this nanozyme can reduce kidney calcium oxalate crystal deposition by reducing the renal injury caused by high concentration oxalate, upregulate the expression levels of SOD and CAT in tissues, downregulate adhesion proteins and inflammatory factor IL-6 and TNF-α, and promote the polarization of macrophages from M1 to M2 phenotype in the rat model induced by ethylene glycol. Overall, MOF-818 has the potential to effectively suppress oxidative stress and inflammatory harm caused by high levels of oxalate, hence lowering the likelihood of stone formation. MOF-818 nanozyme is also expected to be used as an alternative drug for the treatment of calcium oxalate kidney stones and provide an experimental theoretical basis for the development of new nanomedicines.

Mitochondrial dysfunction in kidney stones and relief of kidney stones after reducing mtROS.

Mitochondria are essential organelles because they generate the energy required for cellular functions. Kidney stones, as one of the most common urological diseases, have garnered significant attention. In this study, we first collected peripheral venous blood from patients with kidney stones and used qRT-PCR to detect mitochondrial DNA (mtDNA) copy number as a means of assessing mitochondrial function in these patients. Subsequently, through Western blotting, qPCR, immunofluorescence, immunohistochemistry, and transmission electron microscopy, we examined whether calcium oxalate crystals could cause mitochondrial dysfunction in the kidney in both in vitro and in vivo. We then examined the intersection of the DEGs obtained by transcriptome sequencing of the mouse kidney stone model with mitochondria-related genes, and performed KEGG and GO analyses on the intersecting genes. Finally, we administered the mitochondrial ROS scavenger Mito-Tempo in vivo and observed its effects. Our findings revealed that patients with kidney stones had a reduced mtDNA copy number in their peripheral venous blood compared to the control group, suggesting mitochondrial dysfunction in this population. This conclusion was further validated through in vitro and in vivo experiments. Enrichment analyses revealed that the intersecting genes were closely related to metabolism. We observed that after mitochondrial function was preserved, the deposition of calcium oxalate crystals decreased, and the kidney damage and inflammation caused by them were also alleviated. Our research indicates that kidney stones can cause mitochondrial dysfunction. After clearing mtROS, the damage and inflammation caused by kidney stones are reversed, providing new insights into the prevention and treatment of kidney stones.

Clinical manifestations and renal pathology of ethylene glycol.

Ethylene glycol (EG) poisoning is a critical medical emergency often associated with suicide attempts in adults. EG is metabolized by alcohol dehydrogenase, leading to the formation of toxic metabolites that cause metabolic acidosis, renal failure, hypocalcemia, aciduria, and disorders of the central nervous and cardiovascular systems. Calcium oxalate, a metabolite of EG, contributes to acute tubular necrosis. Despite limited reports on human renal pathology, we present a case detailing renal pathology following EG ingestion. A 44-year-old male, admitted due to loss of consciousness, had ingested a lethal dose of EG. Blood tests indicated metabolic acidosis, while urinary examination revealed calcium oxalate crystals. Continuous renal replacement therapy corrected the acidosis; however, nephrogenic diabetes insipidus subsequently developed. A renal biopsy on day 31 revealed calcium oxalate crystal deposition and tubulointerstitial damage. Notably, various stages of crystal deposition, adherence, and degradation were observed. This case underscores the importance of considering EG poisoning in cases of unexplained metabolic acidosis and renal dysfunction, with renal biopsy serving as a valuable diagnostic tool. Understanding the renal effects of EG is essential for timely intervention and effective management of poisoning cases.

#1846 Oxalate nephropathy in diabetic kidney disease: another factor to take into account?

Abstract Background and Aims Patients with diabetes mellitus (DM) have an increased urinary excretion of oxalate. Around 40% of patients with oxalate nephropathy have DM. The aim of this study was to describe the prevalence of tubular deposits of calcium oxalate (CaOx) crystals in patients with diabetic kidney disease (DKD) and investigate its association with renal survival. Method We performed an observational retrospective study that included patients with DKD in renal biopsies performed at our centre between January 1999 and December 2021. We revised the biopsy specimens for the presence of intratubular CaOx crystals. We excluded patients with a histological findings of non-diabetic kidney disease and those lost to follow-up. Renal survival was defined as a state free from developing end-stage kidney disease (ESKD) requiring renal replacement therapy. Results The study included 87 patients with a mean age of 63.1 ± 13 years, 73.5% were male, and 88.5% had type 2 DM (11.5% type 1 DM). Baseline GFR at the moment of kidney biopsy was 38.5 ± 23 ml/min, and baseline median proteinuria was 3.46 (1.35-5.70) g/day. We found intratubular CaOx deposits in 11 biopsies (12.6%). These findings were only found in patients with type 2 DM (14.3% vs 0%), and was more frequently present in patients with a history of renal stones (33.3% vs 11.1%). There were no differences in baseline GFR, proteinuria or vintage of DM between patients with intratubular CaOx deposits and those with no signs of oxalte nephropathy. CaOx deposition was not associated with other histological lesions such as percentage of glomerular sclerosis, grade of interstitial fibrosis and/or tubular atrophy, interstitial inflammation, arterial hyalinosis or arteriosclerosis. After a median follow-up of 41 months, 49.4% of patients developed ESKD while 26.4% died without progressing to ESKD. The patients who progressed to ESKD had a higher baseline serum creatinine (2.56 ± 1.45 vs 1.98 ± 1.07 ml/min, p = 0.037) and lower serum albumin (3.08 ± 0.70 vs 3.57 ± 0.74 g/dl, p = 0.003). In the regression analysis there was no association between intratubular CaOx deposits and progression to ESKD (OR = 2.96, 95% CI 0.73-12.03). In the adjusted model, no histological lesions were associated with renal survival. In the survival analysis, the presence of intratubular CaOx deposits was not associated with neither renal nor patient survival. Conclusion Oxalate nephropathy, defined as the presence of intratubular CaOx deposits in kidney biopsies, may be found in patients with DKD. In our cohort, 14.3% of type 2 DM patients with DKD had concomitant oxalate nephropathy. Its presence was not associated with a worse renal or patient survival.

Porous Se@SiO2 nanocomposites play a potential inhibition role in hyperoxaluria associated kidney stone by exerting antioxidant effects.

Nephrolithiasis seriously affects people's health with increasing prevalence and high recurrence rates. However, there is still a lack of effective interventions for the clinical prevention of kidney stones. Hyperoxaluria-induced renal tubular epithelial cell (TEC) injury is a known key factor in kidney stone formation. Thus, developing new drugs to inhibit the hyperoxaluria-induced TEC injury may be the best way. We synthesized the Se@SiO2 nanocomposites as described in Zhu's study. The size and morphology of the Se@SiO2 nanocomposites were captured by transmission electron microscopy. Cell viability was measured by a Cell Counting Kit-8 (CCK-8) assay. The mice were randomly divided into the following four groups: (I) the control group (n=6); (II) the Se@SiO2 group (n=6); (III) the glyoxylic acid monohydrate (GAM) group; and (IV) the GAM + Se@SiO2 group (n=6). The concentration of Se in the mice was quantified using inductively coupled plasma atomic emission spectroscopy. The CCK-8 assays showed that Se@SiO2 nanocomposites had almost no obvious cytotoxicity on the Transformed C3H Mouse Kidney-1 (TCMK-1) cell. The mice kidney Se concentration levels in the Se@SiO2 groups (Se@SiO2 6.905±0.074 mg/kg; GAM + Se@SiO2 7.673±2.85 mg/kg) (n=6) were significantly higher than those in the control group (Control 0.727±0.072 mg/kg; GAM 0.747±0.074 mg/kg) (n=6). The Se@SiO2 nanocomposites reduced kidney injury, calcium oxalate crystal deposition, and the osteoblastic-associated proteins in the hyperoxaluria mice models. Se@SiO2 nanocomposites appear to protect renal TECs from hyperoxaluria by reducing reactive oxygen species production, suggesting the potential role of preventing kidney stone formation and recurrence.

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.

In vivo base editing rescues primary hyperoxaluria type 1 in rats

Primary hyperoxaluria type 1 (PH1) is a childhood-onset autosomal recessive disease, characterized by nephrocalcinosis, multiple recurrent urinary calcium oxalate stones, and a high risk of progressive kidney damage. PH1 is caused by inherent genetic defects of the alanine glyoxylate aminotransferase (AGXT) gene. The invivo repair of disease-causing genes was exceedingly inefficient before the invention of base editors which can efficiently introduce precisely targeted base alterations without double-strand DNA breaks. Adenine base editor (ABE) can precisely convert A·T to G·C with the assistance of specific guide RNA. Here, we demonstrated that systemic delivery of dual adeno-associated virus encoding a split-ABE8e could artificially repair 13% of the pathogenic allele in AgxtQ84X rats, a model of PH1, alleviating the disease phenotype. Specifically, ABE treatment partially restored the expression of alanine-glyoxylate-aminotransferase (AGT), reduced endogenous oxalate synthesis and alleviated calcium oxalate crystal deposition. Western blot and immunohistochemistry confirmed that ABE8e treatment restored AGT protein expression in hepatocytes. Moreover, the precise editing efficiency in the liver remained stable six months after treatment. Thus, our findings provided a prospect of invivo base editing as a personalized and precise medicine for PH1 by directly correcting the mutant Agxt gene.

The Clinical and Pathological Characteristics of Patients with Oxalate Nephropathy.

Key PointsOxalate nephropathy is an underrecognized cause of CKD and ESKDWe present one of the largest native oxalate nephropathy cohorts to date from a tertiary care institution in the United StatesOxalate nephropathy has multiple etiologies and given its clinical course and poor prognosis, attention must be paid to screening for risk factors to guide prompt diagnosis and managementBackgroundOxalate nephropathy (ON) is characterized by deposition of calcium oxalate crystals in the kidney and is commonly under-recognized. Causes of ON include primary hyperoxaluria, enteric hyperoxaluria, and ingestion of excess oxalate or its precursors.MethodsWe report the clinical and pathological characteristics of one of the largest series of native kidney ON to date, from January 2015 to March 2023 at the Cleveland Clinic.ResultsWe identified 60 native biopsies with oxalate deposits and excluded patients with clinically insignificant biopsies (n=12) or lack of data (n=17). Thirty-one patients with native ON were described. The mean age at diagnosis was 66.2 years (±12.1), and 58.1% were female. 87.1% had hypertension, 58.1% had diabetes, 42% had nephrolithiasis, and 77.4% had underlying CKD, with a mean baseline creatinine of 1.8 mg/dl ±1.3.The mean creatinine at biopsy was 5.2 mg/dl ±1.7. Kidney biopsies showed abundant calcium oxalate crystal deposits, and 27 of 31 biopsies had additional diagnoses, the most common of which were acute tubular injuryn=17 (54.8%) and diabetic glomerulosclerosisn=7 (22.6%). Severe and moderate interstitial fibrosis was present in 38.7% (n=12) and 51.6% (n=16) of biopsies, respectively. Ten had a single etiology of ON, ten had a multifactorial etiology (both enteric hyperoxaluria and high precursor intake), and 11 had an unclear etiology. Notably, only seven patients had a history of gastric bypass.The mean duration of follow-up was 26.8 months, and 26 patients had follow-up data &gt;1 year. Of these, 21 required dialysis, and five were dialysis-free at presentation. Five of the 26 were deceased at 1 year, with 12 patients (38.7%) deceased at last follow-up. Seventeen patients received targeted management, while nine patients did not receive targeted treatment, and all nine required hemodialysis. More patients (31.6%) had vitamin C intake after the coronavirus disease 2019 pandemic (2020–2023) versus 16.7% before 2020.ConclusionsON presents as AKI or acute on CKD. The prognosis is poor with most patients requiring dialysis at presentation with high morbidity and mortality. Clinicians need to be aware of the risk factors associated with ON to aid prompt diagnosis and management.PodcastThis article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/K360/2024_01_26_KID0000000000000340.mp3

In vivo Anti-lithiasis activity and antibacterial effect of hydroalcoholic extract and infusion of Punica granatum L. peel.

ObjectiveUrolithiasis disease is a severe pathology causing painful bleeding in patients and can lead to kidney failure. Current treatments are limited, expensive, invasive with side effects and requires new alternatives, especially those based on natural products. This work aimed to determine the in vivo antilithiasis effect of hydroalcoholic extract (E.PG) and infusion (I.PG) of the Punica granatum L. plant, as well as their antibacterial capacity against the strains responsible of urinary tract infection such as: E. coli, P. Mirabilis, K. pneumoniae and S. Aureus. MethodsA curative protocol was followed, which consisted of inducing calcium oxalate lithiasis in rats by ethylene glycol (75%) and ammonium chloride (1%) solutions. After 15 days of oxalocalcic lithiasis induction, plant extracts were administered to rats at two different doses (200 and 400 mg/kg/day), with potassium citrate (Cit.K) used as a positive control. Water intake, urine volume, pH and body weight were measured with crystalluria and histopathological examinations of the kidneys performed. The antibacterial activity was studied by the disk diffusion method and the minimum inhibitory concentration (MIC) method. The checkerboard technique was used to determine the interactions between the two extracts. ResultsThe results found show a significant effectiveness of the Punica granatum L. extracts, especially the infusion in preventing the formation, development and calcium oxalate crystals deposition. In fact, crystalluria revealed an important diuresis with the presence of a very low crystals number (<50 / mm3) of very small size in the rat's urine treated with EPG and IPG at the concentration of 400 mg/kg/day. Kidney histopathology analysis showed the absence of crystal deposition, as well as an almost normal structure. Moreover, the antimicrobial activity showed the efficiency of the plant extracts in inhibiting the growth of P. mirabilis and S. aureus where the inhibition zones (IZ) revealed by the disk diffusion method are between 14 and 16 mm. I.PG extract displayed a very low MIC of 1.875 against E. coli. Furthermore, the interaction between these two extracts showed partial synergy and an additive effect against S. Aureus and K. pneumoniae. ConclusionsPunica granatum L. plant has promising antilithiasis and antibacterial effects. Therefore, future extensive studies are needed to establish its effectiveness for clinical use.

YAP/ACSL4 Pathway-Mediated Ferroptosis Promotes Renal Fibrosis in the Presence of Kidney Stones.

The potential association between calcium oxalate stones and renal fibrosis has been extensively investigated; however, the underlying mechanisms remain unclear. Ferroptosis is a novel form of cell death characterized by iron-dependent lipid peroxidation and regulated by acyl coenzyme A synthase long-chain family member 4 (ACSL4). Yes-associated protein (YAP), a transcriptional co-activator in the Hippo pathway, promotes ferroptosis by modulating ACSL4 expression. Nevertheless, the involvement of YAP-ACSL4 axis-mediated ferroptosis in calcium oxalate crystal deposition-induced renal fibrosis and its molecular mechanisms have not been elucidated. In this study, we investigated ACSL4 expression and ferroptosis activation in the kidney tissues of patients with calcium oxalate stones and in mice using single-cell sequencing, transcriptome RNA sequencing, immunohistochemical analysis, and Western blot analysis. In vivo and in vitro experiments demonstrated that inhibiting ferroptosis or ACSL4 mitigated calcium oxalate crystal-induced renal fibrosis. Furthermore, YAP expression was elevated in the kidney tissues of patients with calcium oxalate stones and in calcium oxalate crystal-stimulated human renal tubular epithelial cell lines. Mechanistically, in calcium oxalate crystal-stimulated human renal tubular epithelial cell lines, activated YAP translocated to the nucleus and enhanced ACSL4 expression, consequently inducing cellular ferroptosis. Moreover, YAP silencing suppressed ferroptosis by downregulating ACSL4 expression, thereby attenuating calcium oxalate crystal-induced renal fibrosis. Conclusively, our findings suggest that YAP-ACSL4-mediated ferroptosis represents an important mechanism underlying the induction of renal fibrosis by calcium oxalate crystal deposition. Targeting the YAP-ACSL4 axis and ferroptosis may therefore hold promise as a potential therapeutic approach for preventing renal fibrosis in patients with kidney stones.

Sirtuin1 inhibits calcium oxalate crystal-induced kidney injury by regulating TLR4 signaling and macrophage-mediated inflammatory activation

Sirtuin1 (Sirt1) activation significantly attenuated calcium oxalate (CaOx) crystal deposition and renal inflammatory injury by regulating renal immune microenvironment. Here, to elucidate the molecular mechanism underlying the therapeutic effects of Sirt1 on macrophage related inflammation and tubular epithelial cells (TECs) necrosis, we constructed a macrophage and CaOx monohydrate (COM)-stimulated tubular cell co-culture system to mimic immune microenvironment in kidney and established a mouse model of CaOx nephrocalcinosis in wild-type and myeloid-specific Sirt1 knockout mice. Target prediction analyses of Gene Expression Omnibus Datasets showed that only miR-34b-5p is regulated by lipopolysaccharides and upregulated by SRT1720 and targets the TLR4 3′-untranslated region. In vitro, SRT1720 suppressed TLR4 expression and M1 macrophage polarization and decreased reactive oxygen species (ROS) production and mitochondrial damage in COM-stimulated TECs by targeting miR-34b-5p. Mechanically, Sirt1 promoted miR-34b-5p expression by suppressing the tri-methylation of H3K27, which directly bound to the miR-34b-5p promoter and abolished the miR-34b-5p transcription. Furthermore, loss of Sirt1 aggravated CaOx nephrocalcinosis-induced inflammatory and oxidative kidney injury, while AgomiR-34b reversed these effects. Therefore, our data suggested that Sirt1 inhibited TLR4 signaling and M1 macrophage polarization and decreased inflammatory and oxidative injury of TECs in vitro and in vivo.

Sanjin Paishi Decoction improves the imbalance of gut microbiota and regulates MAPK signaling pathway to inhibit calcium oxalate stones in rats.

Sanjin Paishi Decoction (SJPSD) has positive effects on stone prevention; however, there is a lack of convincing evidence in the prevention of calcium oxalate stones. This study aimed investigates the effect of SJPSD on calcium oxalate stones and to explore its mechanism. The rat model of calcium oxalate stones was established and rats were treated with different doses of SJPSD. The pathological damage of kidney tissues was observed by HE staining, the deposition of calcium oxalate crystals in kidney tissues was examined by Von Kossa staining, and the levels of creatinine (CREA), urea (UREA), calcium (Ca), phosphorus (P), and magnesium (Mg) in serum were analyzed biochemically, the levels of IL-1β, IL-6, and TNF-α in serum were measured by ELISA, and the protein expression of Raf1, MEK1, p-MEK1, ERK1/2, p-ERK1/2, and Cleaved caspase-3 in kidney tissues was analyzed by Western blot. Moreover, the changes in gut microbiota were analyzed by 16S rRNA sequencing. SJPSD attenuated the pathological damage of renal tissues, reduced the levels of CREA, UREA, Ca, P, and Mg, and inhibited the expression of Raf1, p-MEK1, p-ERK1/2, and Cleaved caspase-3 in renal tissues (P < 0.05). SJPSD treatment affected the composition of intestinal microbiota in rats with calcium oxalate stones. The mechanism of SJPSD inhibition of calcium oxalate stone injury in rats may be related to the inhibition of the MAPK signaling pathway and regulation of gut microbiota imbalance.

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

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

Metformin ameliorates calcium oxalate crystallization and stone formation by activating the Nrf2/HO-1 signaling pathway: Two birds with one stone.

Deposition of calcium oxalate (CaOx) crystals and oxidative stress-induced injury of renal tubular epithelial cell are the primary pathogenic factors of nephrolithiasis. In this study we investigated the beneficial effects of metformin hydrochloride (MH) against nephrolithiasis and explored the underlying molecular mechanism. Our results demonstrated that MH inhibited the formation of CaOx crystals and promoted the transformation of thermodynamically stable CaOx monohydrate (COM) to more unstable CaOx dihydrate (COD). MH treatment effectively ameliorated oxalate-induced oxidative injury and mitochondrial damage in renal tubular cells and reduced CaOx crystal deposition in rat kidneys. MH also attenuated oxidative stress by lowering MDA level and enhancing SOD activity in HK-2 and NRK-52E cells and in a rat model of nephrolithiasis. In both HK-2 and NRK-52E cells, COM exposure significantlylowered the expressions of HO-1 and Nrf2, which was rescued by MH treatment even in the presence of Nrf2 and HO-1 inhibitors. In rats with nephrolithiasis, MH treatment significantly rescued the down-regulation of the mRNA and protein expression of Nrf2 and HO-1 in the kidneys. These results demonstrate that MH can alleviate CaOx crystal deposition and kidney tissue injury in rats with nephrolithiasis by suppressing oxidative stress and activating the Nrf2/HO-1 signaling pathway, suggesting the potential value of MH in the treatment of nephrolithiasis.