Mechanism of Porphyra Yezoensis Polysaccharides in Inhibiting Hyperoxalate-Induced Renal Injury and Crystal Deposition.

Oxidative damage to the kidneys is a primary factor in the occurrence of kidney stones. This study explores the inhibitory effect of Porphyra yezoensis polysaccharides (PYP) on oxalate-induced renal injury by detecting levels of oxidative damage, expression of adhesion molecules, and damage to intracellular organelles and revealed the molecular mechanism by molecular biology methods. Additionally, we validated the role of PYP in vivo using a crystallization model of hyperoxalate-induced rats. PYP effectively scavenged the overproduction of reactive oxygen species (ROS) in HK-2 cells, inhibited the adhesion of calcium oxalate (CaOx) crystals on the cell surface, unblocked the cell cycle, restored the depolarization of the mitochondrial membrane potential, and inhibited cell death. PYP upregulated the expression of antioxidant proteins, including Nrf2, HO-1, SOD, and CAT, while decreasing the expression of Keap-1, thereby activating the Keap1/Nrf2 signaling pathway. PYP inhibited CaOx deposition in renal tubules in the rat crystallization model, significantly reduced high oxalate-induced renal injury, decreased the levels of the cell surface adhesion proteins, improved renal function in rats, and ultimately inhibited the formation of kidney stones. Therefore, PYP, which has crystallization inhibition and antioxidant properties, may be a therapeutic option for the treatment of kidney stones.

Study on Fu-Fang-Jin-Qian-Cao Inhibiting Autophagy in Calcium Oxalate-induced Renal Injury by UHPLC/Q-TOF-MS-based Metabonomics and Network Pharmacology Approaches.

INTRODUCTION: Fu-Fang-Jin-Qian-Cao is a Chinese herbal preparation used to treat urinary calculi. Fu-Fang-Jin-Qian-Cao can protect renal tubular epithelial cells from calcium oxalateinduced renal injury by inhibiting ROS-mediated autopathy. The mechanism still needs further exploration. Metabonomics is a new subject; the combination of metabolomics and network pharmacology can find pathways for drugs to act on targets more efficiently. METHODS: Comprehensive metabolomics and network pharmacology to study the mechanism of Fu-Fang-Jin-Qian-Cao inhibiting autophagy in calcium oxalate-induced renal injury. Based on UHPLC-Q-TOF-MS, combined with biochemical analysis, a mice model of Calcium oxalateinduced renal injury was established to study the therapeutic effect of Fu-Fang-Jin-Qian-Cao. Based on the network pharmacology, the target signaling pathway and the protective effect of Fu- Fang-Jin-Qian-Cao on Calcium oxalate-induced renal injury by inhibiting autophagy were explored. Autophagy-related proteins LC3-II, BECN1, ATG5, and ATG7 were studied by immunohistochemistry. RESULTS: Combining network pharmacology and metabolomics, 50 differential metabolites and 2482 targets related to these metabolites were found. Subsequently, the targets enriched in PI3KAkt, MAPK and Ras signaling pathways. LC3-II, BECN1, ATG5 and ATG7 were up-regulated in Calcium oxalate-induced renal injury. All of them could be reversed after the Fu-Fang-Jin-Qian- Cao treatment. CONCLUSIONS: Fu-Fang-Jin-Qian-Cao can reverse ROS-induced activation of the MAPK signaling pathway and inhibition of the PI3K-Akt signaling pathway, thereby reducing autophagy damage of renal tubular epithelial cells in Calcium oxalate-induced renal injury.

Tight junction and kidney stone disease.

Defects of tight junction (TJ) are involved in many diseases related to epithelial cell functions, including kidney stone disease (KSD), which is a common disease affecting humans for over a thousand years. This review provides brief overviews of KSD and TJ, and summarizes the knowledge on crystal-induced defects of TJ in renal tubular epithelial cells (RTECs) in KSD. Calcium oxalate (CaOx) crystals, particularly COM, disrupt TJ via p38 MAPK and ROS/Akt/p38 MAPK signaling pathways, filamentous actin (F-actin) reorganization and α-tubulin relocalization. Stabilizing p38 MAPK signaling, reactive oxygen species (ROS) production, F-actin and α-tubulin by using SB239063, N-acetyl-L-cysteine (NAC), phalloidin and docetaxel, respectively, successfully prevent the COM-induced TJ disruption and malfunction. Additionally, genetic disorders of renal TJ, including mutations and single nucleotide polymorphisms (SNPs) of CLDN2, CLDN10b, CLDN14, CLDN16 and CLDN19, also affect KSD. Finally, the role of TJ as a potential target for KSD therapeutics and prevention is also discussed.

STAT6 promoting oxalate crystal deposition-induced renal fibrosis by mediating macrophage-to-myofibroblast transition via inhibiting fatty acid oxidation.

OBJECTIVE AND DESIGN: Kidney stones commonly occur with a 50% recurrence rate within 5 years, and can elevate the risk of chronic kidney disease. Macrophage-to-myofibroblast transition (MMT) is a newly discovered mechanism that leads to progressive fibrosis in different forms of kidney disease. In this study, we aimed to investigate the role of MMT in renal fibrosis in glyoxylate-induced kidney stone mice and the mechanism by which signal transducer and activator of transcription 6 (STAT6) regulates MMT. METHODS: We collected non-functioning kidneys from patients with stones, established glyoxylate-induced calcium oxalate stone mice model and treated AS1517499 every other day in the treatment group, and constructed a STAT6-knockout RAW264.7 cell line. We first screened the enrichment pathway of the model by transcriptome sequencing; detected renal injury and fibrosis by hematoxylin eosin staining, Von Kossa staining and Sirius red staining; detected MMT levels by multiplexed immunofluorescence and flow cytometry; and verified the binding site of STAT6 at the PPARα promoter by chromatin immunoprecipitation. Fatty acid oxidation (FAO) and fibrosis-related genes were detected by western blot and real-time quantitative polymerase chain reaction. RESULTS: In this study, we found that FAO was downregulated, macrophages converted to myofibroblasts, and STAT6 expression was elevated in stone patients and glyoxylate-induced kidney stone mice. The promotion of FAO in macrophages attenuated MMT and upregulated fibrosis-related genes induced by calcium oxalate treatment. Further, inhibition of peroxisome proliferator-activated receptor-α (PPARα) eliminated the effect of STAT6 deletion on FAO and fibrosis-associated protein expression. Pharmacological inhibition of STAT6 also prevented the development of renal injury, lipid accumulation, MMT, and renal fibrosis. Mechanistically, STAT6 transcriptionally represses PPARα and FAO through cis-inducible elements located in the promoter region of the gene, thereby promoting MMT and renal fibrosis. CONCLUSIONS: These findings establish a role for STAT6 in kidney stone injury-induced renal fibrosis, and suggest that STAT6 may be a therapeutic target for progressive renal fibrosis in patients with nephrolithiasis.

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.

Carboxymethylated Rhizoma alismatis Polysaccharides Regulate Calcium Oxalate Crystals Growth and Reduce the Regulated Crystals' Cytotoxicity.

OBJECTIVE: This study explored the effects of polysaccharides (RAPD) extracted from the traditional anti-stone Chinese medicine Rhizoma alismatis and their carboxymethylated derivatives (RAPs) on the crystal phase, morphology, and size of calcium oxalate (CaOx). It also determined the damaging ability of the regulated crystals on human renal tubular epithelial cells (HK-2). METHODS: RAPD carboxymethylation with a carboxyl group (-COOH) content of 3.57% was carried out by the chloroacetic acid solvent method. The effects of -COOH content in RAPs and RAP concentration on the regulation of CaOx crystal growth were studied by controlling the variables. Cell experiments were conducted to explore the differences in the cytotoxicity of RAP-regulated crystals. RESULTS: The -COOH contents of RAPD, RAP1, RAP2, and RAP3 were 3.57%, 7.79%, 10.84%, and 15.33%, respectively. RAPs can inhibit the growth of calcium oxalate monohydrate (COM) and induce the formation of calcium oxalate dihydrate (COD). When the -COOH content in RAPs was high, their ability to induce COD formation was enhanced. In the crystals induced by RAPs, a high COD content can lower the damage to cells. In particular, the cytotoxicity of the crystals induced by RAP3 was the lowest. When the concentration of RAP3 increased, the cytotoxicity gradually increased due to the reduced size of the formed COD crystals. An interaction was observed between RAPs and crystals, and the number of RAPs adsorbed in the crystals was positively correlated with the -COOH content in RAPs. CONCLUSIONS: RAPs can reduce the damage of CaOx to HK-2 cells by regulating the crystallization of CaOx crystals and effectively reducing the risk of kidney stone formation. RAPs, especially RAP3 with a high carboxyl group content, has the potential to be developed as a novel green anti-stone drug.

Synergistic inhibition of calcium oxalate crystal formation and synergistic protection of HK-2 cells from crystal damage by sulfated Laminarin polysaccharide and potassium citrate.

Objective: The first objective is to study the synergistic inhibition of calcium oxalate (CaOx) formation by Laminarin polysaccharides (DLP and SDLP, before and after sulfation) and potassium citrate (K3cit) and determine the synergistic protection of renal epithelial cells (HK-2 cells) caused by CaOx crystal damage. The second objective is to explore new ways to prevent and treat kidney stones. Methods: The CaOx crystals regulated by five additives (K3cit group, DLP group, SDLP group, DLP-K3cit synergistic group and SDLP-K3cit synergistic group) were characterized by FT-IR, XRD, SEM, zeta potential, ICP, and TGA. The protective effect of each additive group on HK-2 cells damaged by nano-calcium oxalate monohydrate (nano-COM) was compared by detecting cell viability, the cell reactive oxygen species level, the cell survival rate, and mitochondrial membrane potential. Results: When DLP or SDLP acted synergically with K3cit, the synergistic group induced the same amount of COD at a lower concentration or more COD formation at the same concentration, highlighting the synergistic enhancement effect of 1 + 1 > 2. At 0.3 g L-1, the COD contents induced by DLP, SDLP, K3cit, DLP-K3cit, and SDLP-K3cit synergistic groups were 20.3%, 75.8%, 75.4%, 87.3%, and 100%, respectively. The synergistic group increased the concentration of soluble Ca2+ ions in the supernatant, increased the absolute value of the zeta potential on the surface of CaOx crystals, and inhibited the aggregation among the crystals. TGA and DTG analyses established the adsorption of polysaccharides in the crystals. Cell experiments showed the ability of the synergistic group to significantly inhibit the damage of nano-COM crystals on HK-2 cells, reduce the level of reactive oxygen species and mortality, and improve cell viability and the mitochondrial membrane potential. Conclusions: The synergistic group can more effectively induce COD formation and cell protection than the standalone polysaccharide group or K3cit group. The synergistic groups, especially SDLP-K3cit, may be a potential drug for inhibiting the formation of CaOx kidney stones.

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.

Hyperoside Ameliorates Renal Tubular Oxidative Damage and Calcium Oxalate Deposition in Rats through AMPK/Nrf2 Signaling Axis.

Background: Nephrolithiasis is a common disease that seriously affects the health and life quality of patients. Despite the reported effect of hyperoside (Hyp) against nephrolithiasis, the specific mechanism has not been clarified. Therefore, this study is aimed at investigating the effect and potential mechanism of Hyp on renal injury and calcium oxalate (CaOx) crystal deposition. Methods: Rat and cell models of renal calculi were constructed by ethylene glycol (EG) and CaOx induction, respectively. The renal histopathological damage, CaOx crystal deposition, and renal function damage of rats were assessed by HE staining, Pizzolato staining, and biochemical detection of blood and urine parameters. MTT and crystal-cell adhesion assays were utilized to determine the activity of HK-2 cells and crystal adhesion ability, biochemical detection and enzyme-linked immunosorbent assay (ELISA) to measure the levels of oxidative stress-related substances and inflammatory factors, and western blot to test the expression levels of proteins related to the AMPK/Nrf2 signaling pathway. Results: Briefly speaking, Hyp could improve the renal histopathological injury and impaired renal function, reduce the deposition of CaOx crystals in the renal tissue of rats with renal calculi, and decrease the adhesion of crystals to CaOx-treated HK-2 cells. Besides, Hyp also significantly inhibited oxidative stress response. Furthermore, Hyp was associated with the downregulation of malondialdehyde, lactate dehydrogenase, and reactive oxygen species and upregulation of superoxide dismutase activity. Additionally, Hyp treatment also suppressed inflammatory response and had a correlation with declined levels of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor. Further exploration of mechanism manifested that Hyp might play a protective role through promoting AMPK phosphorylation and nuclear translation of Nrf2 to activate the AMPK/Nrf2 signaling pathway. Conclusion: Hyp can improve renal pathological and functional damage, decrease CaOx crystal deposition, and inhibit oxidative stress and inflammatory response. Such effects may be achieved by activating the AMPK/Nrf2 signaling pathway.

NEAT1 Regulates Calcium Oxalate Crystal-Induced Renal Tubular Oxidative Injury via miR-130/IRF1.

Aims: Calcium oxalate (CaOx) crystal deposition induces damage to the renal tubular epithelium, increases epithelial adhesion, and contributes to CaOx nephrocalcinosis. The long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) is thought to be involved in this process. In this study, we aimed to investigate the mechanism by which NEAT1 regulates renal tubular epithelium in response to inflammatory and oxidative injury triggered by CaOx crystals. Results: As CaOx crystals were deposited in mouse kidney tissue, the expression of NEAT1 was significantly elevated and positively correlated with interferon regulatory factor 1 (IRF1), Toll-like receptor 4 (TLR4), and NF-κB. NEAT1 targets and inhibits miR-130a-3p as a competitor to endogenous RNA. miR-130 binds to and exerts inhibitory effects on the 3'-untranslated region of IRF1. After transfected with silence-NEAT1, IRF1, TLR4, and NF-κB were also variously inhibited, and oxidative damage in renal calcinosis was subsequently attenuated. When we simultaneously inhibited NEAT1 and miR-130, renal tubular injury was exacerbated. Innovation and Conclusion: We found that the lncRNA NEAT1 can enhance IRF1 signaling through targeted repression of miR-130a-3p and activate TLR4/NF-κB pathways to promote oxidative damage during CaOx crystal deposition. This provides an explanation for the tubular epithelial damage caused by CaOx crystals and offers new ideas and drug targets for the prevention and treatment of CaOx nephrocalcinosis. Antioxid. Redox Signal. 38, 731-746.

Ferrostatin­1 alleviates oxalate­induced renal tubular epithelial cell injury, fibrosis and calcium oxalate stone formation by inhibiting ferroptosis.

The present study aimed to evaluate the role and mechanism of ferrostatin­1 (Fer­1) in oxalate (Ox)­induced renal tubular epithelial cell injury, fibrosis, and calcium oxalate (CaOx) stone formation. A CaOx model in mice kidneys was established via intraperitoneal injection of 80 mg/kg glyoxylic acid for 14 days. The mice were randomly divided into three groups (n=6), namely, the control (Con), the CaOx group, and the CaOx + Fer­1 group. Cultured human renal tubular epithelial cells (HK­2 cells) were randomly divided into three groups (n=3), namely, the control (Con), the Ox group, and the Ox + Fer­1 group. The levels of heme oxygenase 1 (HO­1), superoxide dismutase 2 (SOD2), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) were assessed by immunofluorescence and western blot analysis. Renal tubular injury and apoptosis were evaluated by H&E and TUNEL staining. Kidney interstitial fibrosis was evaluated by Masson and Sirius red staining, and the levels of E­cadherin, vimentin and α­SMA were detected by immunofluorescence or western blot analysis. Mitochondrial structure was observed using a transmission electron microscope. The levels of reactive oxygen species (ROS) were determined by flow cytometry and CaOx stone formation was evaluated by von Kossa staining. The results revealed that in comparison with the Con group, mitochondrial injury under glyoxylic acid treatment was observed by TEM. The expression of GPX4 and SLC7A11 in the CaOx and Ox groups was downregulated (P<0.05), whereas the expression of HO­1 and SOD2 was upregulated (P<0.05). Renal tissue damage, apoptosis of renal tubular epithelial cells, and interstitial fibrosis were increased in the CaOx and Ox groups (P<0.05). In comparison with the CaOx or Ox group, the expression of GPX4 and SLC7A11 in the CaOx + Fer­1 or Ox + Fer­1 group was upregulated (P<0.05), whereas that of HO­1 and SOD2 was downregulated (P<0.05). Renal tissue damage, apoptosis of renal tubular epithelial cells and interstitial fibrosis were decreased following Fer­1 treatment (P<0.05). The ROS level was also decreased following Fer­1 treatment. Moreover, CaOx stone formation was decreased in the CaOx + Fer­1 group (P<0.05). In conclusion, Fer­1 alleviated Ox­induced renal tubular epithelial cell injury, fibrosis, and CaOx stone formation by inhibiting ferroptosis.

Protective effects of interleukin-22 on oxalate-induced crystalline renal injury via alleviating mitochondrial damage and inflammatory response.

Oxalate-induced crystalline kidney injury is one of the most common types of crystalline nephropathy. Unfortunately, there is no effective treatment to reduce the deposition of calcium oxalate crystals and alleviate kidney damage. Thus, proactive therapeutic is urgently needed to alleviate the suffering it causes to patient. Here, we investigated whether IL-22 exerted nephroprotective effects to sodium oxalate-mediated kidney damage and its potential mechanism. Crystalline kidney injury models were developed in vitro and in vivo that was often observed in clinic. We provided evidence that IL-22 could effectively decrease the accumulation of ROS and mitochondrial damage in cell and animal models and reduce the death of TECs. Moreover, IL-22 decreased the expression of the NLRP3 inflammasome and mature IL-1ß in renal tissue induced by sodium oxalate. Further studies confirmed that IL-22 could play an anti-inflammatory role by reducing the levels of cytokines such as IL-1ß, IL-18, and TNF-α in serum. In conclusion, our study confirmed that IL-22 has protective effects on sodium oxalate-induced crystalline kidney injury by reducing the production of ROS, protecting mitochondrial membrane potential, and inhibiting the inflammatory response. Therefore, IL-22 may play a potential preventive role in sodium oxalate-induced acute renal injury. KEY POINTS: • IL-22 could reduce sodium oxalate-mediated cytotoxicity and ameliorate renal injury. • IL-22 could alleviate oxidative stress and mitochondrial dysfunction induced by sodium oxalate. • IL-22 could inhibit inflammatory response of renal injury caused by sodium oxalate.

Exosomes derived from calcium oxalate-treated macrophages promote apoptosis of HK-2 cells by promoting autophagy.

Calcium oxalate (CaOx) crystals are the main component of kidney stones. Macrophages have the function of eliminating these crystals, and the underlying mechanism remains unclear. Here, we attempted to determine the role of macrophage-derived exosomes exposed to CaOx crystals in regulating apoptosis of human proximal tubular cells (HK-2). Exosomes (CaOx-Exo) were isolated from CaOx-treated macrophages and then incubated with HK-2 cells. CaOx-Exo treatment reduced cell viability and promoted apoptosis of HK-2 cells. The expression of Caspase-3 and Bax was increased, and Bcl-2 expression was decreased in HK-2 cells following CaOx-Exo treatment. Moreover, CaOx-Exo treatment caused an increase of LC3-II/LC3-I ratio and Beclin-1 expression and a downregulation of p62 in HK-2 cells. GFP-LC3 puncta were increased in HK-2 cells following CaOx-Exo treatment. Additionally, CaOx-Exo-treated HK-2 cells were treated with 3-methyladenine (3-MA) to inhibit autophagy activity. 3-MA treatment weakened the impact of CaOx-Exo on cell viability and apoptosis of HK-2 cells. 3-MA treatment also reduced the LC3-II/LC3-I ratio and Beclin-1 expression and enhanced p62 expression in CaOx-Exo-treated HK-2 cells. In conclusion, these data demonstrated that exosomes derived from CaOx-treated macrophages promote apoptosis of HK-2 cells by promoting autophagy. Thus, this work suggests that macrophage-derived exosomes may play a vital role in CaOx-induced human proximal tubular cell damage.

Premature Senescence and Telomere Shortening Induced by Oxidative Stress From Oxalate, Calcium Oxalate Monohydrate, and Urine From Patients With Calcium Oxalate Nephrolithiasis.

Oxidative stress, a well-known cause of stress-induced premature senescence (SIPS), is increased in patients with calcium oxalate (CaOx) kidney stones (KS). Oxalate and calcium oxalate monohydrate (COM) induce oxidative stress in renal tubular cells, but to our knowledge, their effect on SIPS has not yet been examined. Here, we examined whether oxalate, COM, or urine from patients with CaOx KS could induce SIPS and telomere shortening in human kidney (HK)-2 cells, a proximal tubular renal cell line. Urine from age- and sex-matched individuals without stones was used as a control. In sublethal amounts, H2O2, oxalate, COM, and urine from those with KS evoked oxidative stress in HK-2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity, but urine from those without stones did not. The proportion of senescent HK-2 cells, as indicated by SA-ßgal staining, increased after treatment with H2O2, oxalate, COM, and urine from those with KS. Expression of p16 was higher in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than it was in cells treated with urine from those without stones and untreated controls. p16 was upregulated in the SA-ßgal positive cells. Relative telomere length was shorter in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than that in cells treated with urine from those without stones and untreated controls. Transcript expression of shelterin components (TRF1, TRF2 and POT1) was decreased in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS, in which case the expression was highest. Urine from those without KS did not significantly alter TRF1, TRF2, and POT1 mRNA expression in HK-2 cells relative to untreated controls. In conclusion, oxalate, COM, and urine from patients with CaOx KS induced SIPS and telomere shortening in renal tubular cells. SIPS induced by a lithogenic milieu may result from upregulation of p16 and downregulation of shelterin components, specifically POT1, and might contribute, at least in part, to the development of CaOx KS.

Sirt1 inhibits kidney stones formation by attenuating calcium oxalate-induced cell injury.

Cell injury is a necessary and critical event during CaOx kidney stone formation. Sirt1 exerts a number of pleiotropic effects, protecting against renal cell injury. This study aims to explore the relationship between Sirt1 and CaOx kidney stone formation and the underlying mechanism. Sirt1 expression in renal tissues or HK-2 cells was detected by Western blot, immunohistochemistry and immunofluorescence. Apoptosis in renal tissues was examined by TUNEL staining. Renal pathological changes and the crystals deposition were detected by hematoxylin-eosin and Von Kossa staining. Crystal-cell adhesion and cell injury in HK-2 cells were assessed by atomic absorption spectrometry and flow cytometry, respectively. Sirt1 expression in nephrolithiasis patients was downregulated and the level of apoptosis was increased. Further study found that Sirt1 expression was decreased in both in vivo and in vitro models. Interestingly, the levels of cell injury were elevated in vivo and in vitro models. Suppressing Sirt1 expression promoted COM-induced crystal-cell adhesion and exacerbated cell injury. In contrast, increasing the expression of Sirt1 by lentivirus transfection in vitro and resveratrol administration in vivo, alleviated crystal deposition and cell damage. Our findings suggest that Sirt1 could inhibit kidney stone formation, at least in part, through attenuating CaOx -induced cell injury.

Calcium oxalate monohydrate crystal disrupts tight junction via F-actin reorganization.

Tight junction is an intercellular protein complex that regulates paracellular permeability and epithelial cell polarization. This intercellular barrier is associated with actin filament. Calcium oxalate monohydrate (COM), the major crystalline composition in kidney stones, has been shown to disrupt tight junction but with an unclear mechanism. This study aimed to address whether COM crystal disrupts tight junction via actin deregulation. MDCK distal renal tubular epithelial cells were treated with 100 µg/ml COM crystals for 48 h. Western blot analysis revealed that level of a tight junction protein, zonula occludens-1 (ZO-1), significantly decreased, whereas that of ß-actin remained unchanged after exposure to COM crystals. Immunofluorescence study showed discontinuation and dissociation of ZO-1 and filamentous actin (F-actin) expression at the cell border. In addition, clumping of F-actin was found in some cytoplasmic areas of the COM-treated cells. Moreover, transepithelial resistance (TER) was reduced by COM crystals, indicating the defective barrier function of the polarized cells. All of these COM-induced defects could be completely abolished by pretreatment with 20 µM phalloidin, an F-actin stabilizer, 2-h prior to the 48-h crystal exposure. These findings indicate that COM crystal does not reduce the total level of actin but causes tight junction disruption via F-actin reorganization.

Regulation of endoplasmic reticulum stress on the damage and apoptosis of renal tubular epithelial cells induced by calcium oxalate crystals.

This study aimed to observe whether calcium oxalate (CaOx) crystals can induce the activation of endoplasmic reticulum (ER) stress in human renal cortex proximal tubule epithelial (HK-2) cells and to explore the regulatory of ER stress on the damage and apoptosis of HK-2 cells induced by CaOx crystals. We detected the optimal CaOx crystal concentration and intervention time by Western blot. ER stress modifiers tunicamycin (TM) and 4-phenylbutyric acid (4-PBA) were used to regulate the ER stress of HK-2 cells. The activities of ER stress marker proteins GRP78 and CHOP were evaluated by Western blot and immunohistochemistry. Western blot and TUNEL staining were used to detect cell apoptosis. We observed cell-crystal adhesion with an optical microscope. Lactate dehydrogenase (LDH) test kit and IL-1ß enzyme-linked immunosorbent assay kit were used to detect and evaluate HK-2 cell damage. We found that the expression of ER stress marker proteins GRP78 and CHOP gradually increased with the increase in CaOx crystal concentration and intervention time and reached the maximum at 2.0 mmol/L and 24 h. The use of ER stress modifiers TM and 4-PBA can effectively regulate the ER stress level induced by CaOx crystals, and the level of apoptosis is positively correlated with the level of ER stress. 4-PBA pretreatment remarkably reduced cell-crystal adhesion and the secretions of IL-1ß and LDH, whereas the results of TM pretreatment were the opposite. In summary, the damage and apoptosis of HK-2 cells induced by CaOx crystals are closely related to the level of ER stress. Inhibiting the ER stress of HK-2 cells can substantially reduce the cell damage and apoptosis induced by CaOx crystals.

The protective role of corilagin on renal calcium oxalate crystal-induced oxidative stress, inflammatory response, and apoptosis via PPAR-γ and PI3K/Akt pathway in rats.

Kidney stones, also known as calcium oxalate (CaOx) nephrolithiasis, are often asymptomatic, leading to kidney injury and renal failure complications. Corilagin is a gallotannin found in various plants and is known to elicit various biological activities. The present study aimed to elucidate the renoprotective effect of corilagin against the rats' renal stones deposition. The rats were induced for nephrolithiasis (CaOx deposition) using 0.75% ethylene glycol in their drinking water. Then, they were treated with corilagin at 50 and 100 mg/kg/day for 4 weeks. At the end of the experimental period, the rats were killed; blood and renal tissues were collected for various histological, biochemical, and gene expression analyses. The results demonstrated that the rats had renal calculi displaying a significant increase in serum creatinine (59.39 µmol/L) and blood urea nitrogen (19.03 mmol/L) levels compared with controls. Moreover, the malondialdehyde (13.29 nmol/mg) level was found to increase with a profound reduction in antioxidants' activities with upregulated inflammatory cytokines. In contrast, the RT-PCR and immunohistochemistry analysis demonstrated a substantial reduction in cell survival markers PPAR-γ and PI3K/Akt with an apparent increase in apoptosis markers genes expressions in rats suffering from renal stones. Thus, the present study results suggest that corilagin could suppress renal CaOx crystal-induced oxidative stress, inflammatory response, and apoptosis via PPAR-γ and PI3K/Akt-mediated pathway.

Comprehensive study of altered proteomic landscape in proximal renal tubular epithelial cells in response to calcium oxalate monohydrate crystals.

BACKGROUND: Calcium oxalate monohydrate (COM), the major crystalline composition of most kidney stones, induces inflammatory infiltration and injures in renal tubular cells. However, the mechanism of COM-induced toxic effects in renal tubular cells remain ambiguous. The present study aimed to investigate the potential changes in proteomic landscape of proximal renal tubular cells in response to the stimulation of COM crystals. METHODS: Clinical kidney stone samples were collected and characterized by a stone component analyzer. Three COM-enriched samples were applied to treat human proximal tubular epithelial cells HK-2. The proteomic landscape of COM-crystal treated HK-2 cells was screened by TMT-labeled quantitative proteomics analysis. The differentially expressed proteins (DEPs) were identified by pair-wise analysis. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEPs were performed. Protein interaction networks were identified by STRING database. RESULTS: The data of TMT-labeled quantitative proteomic analysis showed that a total of 1141 proteins were differentially expressed in HK-2 cells, of which 699 were up-regulated and 442 were down-regulated. Functional characterization by KEGG, along with GO enrichments, suggests that the DEPs are mainly involved in cellular components and cellular processes, including regulation of actin cytoskeleton, tight junction and focal adhesion. 3 high-degree hub nodes, CFL1, ACTN and MYH9 were identified by STRING analysis. CONCLUSION: These results suggested that calcium oxalate crystal has a significant effect on protein expression profile in human proximal renal tubular epithelial cells.

H3 relaxin protects against calcium oxalate crystal-induced renal inflammatory pyroptosis.

OBJECTIVES: Calcium oxalate (CaOx) crystals can activate inflammatory cytokines by triggering inflammasomes, which cause damage to the adhered epithelium, a dysfunctional microenvironment and even renal failure. However, a comprehensive and in-depth understanding of the mechanisms underlying the effects of these crystals on damage and cytokine function in renal tubular epithelial cells (TECs) remains limited and to be explored. MATERIALS AND METHODS: We detected the pyroptosis of TECs induced after exposure to CaOx crystals and demonstrated the significance of cytokine activation in the subsequent inflammatory processes through a proteomic study. We then conducted animal and cell experiments to verify relevant mechanisms through morphological, protein, histological and biochemical approaches. Human serum samples were further tested to help explain the pathophysiological mechanism of H3 relaxin. RESULTS: We verified that crystal-induced extracellular adenosine triphosphate (ATP) upregulation via the membrane purinergic 2X7 receptor (P2X7 R) promotes ROS generation and thereby activates NLRP3 inflammasome-mediated interleukin-1ß/18 maturation and gasdermin D cleavage. Human recombinant relaxin-3 (H3 relaxin) can act on the transmembrane receptor RXFP1 to produce cAMP and subsequently improves crystal-derived damage via ATP consumption. Additionally, endogenous relaxin-3 was found to be elevated in patients with renal calculus and can thus serve as a biomarker. CONCLUSIONS: Our results provide previously unidentified mechanistic insights into CaOx crystal-induced inflammatory pyroptotic damage and H3 relaxin-mediated anti-inflammatory protection and thus suggest a series of potential therapeutic targets and methods for but not limited to nephrocalcinosis.