Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD).

Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as arachidonate 5-lipoxygenase (ALOX5) and ALOX5 activating protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6 days training, they exhibit enhanced TNF-α and IL-6 production to lipopolysaccharide (LPS). Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF-α and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF-α after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.

Proteomic analysis of endothelial cells and extracellular vesicles in response to indoxyl sulfate: Mechanisms of endothelial dysfunction in chronic kidney disease.

AIMS: Cardiovascular pathology is the main cause of death in chronic kidney disease (CKD) patients. CKD is associated with the accumulation of uremic toxins in the bloodstream, and indoxyl sulfate (IS) is one of the most abundant uremic toxins found in the blood of CKD patients. We conducted an in vitro study to assess the mechanisms underlying the IS-induced endothelial dysfunction that could lead to cardiovascular diseases. We also studied their extracellular vesicles (EVs) owing to their capacity to act as messengers that transmit signals through their cargo. MAIN METHODS: EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and tetraspanin expression. Cell lysates and isolated EVs were analyzed using liquid chromatography coupled with mass spectrometry, followed by Gene Set Enrichment Analysis to identify the altered pathways. KEY FINDINGS: Proteomic analysis of endothelial cells revealed that IS causes an increase in proteins related to adipogenesis, inflammation, and xenobiotic metabolism and a decrease in proliferation. Extracellular matrix elements, as well as proteins associated with myogenesis, response to UV irradiation, and inflammation, were found to be downregulated in IS-treated EVs. Fatty acid metabolism was also found to be increased along with adipogenesis and inflammation observed in cells. SIGNIFICANCE: The treatment of endothelial cells with IS increased the expression of proteins related to adipogenesis, inflammation, and xenobiotic metabolism and was less associated with proliferation. Furthermore, EVs from cells treated with IS may mediate endothelial dysfunction, since they present fewer extracellular matrix elements, myogenesis, inflammatory factors, and proteins downregulated in response to UV radiation.

Indoxyl Sulfate-Induced Macrophage Toxicity and Therapeutic Strategies in Uremic Atherosclerosis.

Cardiovascular disease (CVD) frequently occurs in patients with chronic kidney disease (CKD), particularly those undergoing dialysis. The mechanisms behind this may be related to traditional risk factors and CKD-specific factors that accelerate atherosclerosis and vascular calcification in CKD patients. The accumulation of uremic toxins is a significant factor in CKD-related systemic disorders. Basic research suggests that indoxyl sulfate (IS), a small protein-bound uremic toxin, is associated with macrophage dysfunctions, including increased oxidative stress, exacerbation of chronic inflammation, and abnormalities in lipid metabolism. Strategies to mitigate the toxicity of IS include optimizing gut microbiota, intervening against the abnormality of intracellular signal transduction, and using blood purification therapy with higher efficiency. Further research is needed to examine whether lowering protein-bound uremic toxins through intervention leads to a reduction in CVD in patients with CKD.

Dietary Phosphorus Levels Influence Protein-Derived Uremic Toxin Production in Nephrectomized Male Rats.

Gut microbiota-derived uremic toxins (UT) accumulate in patients with chronic kidney disease (CKD). Dietary phosphorus and protein restriction are common in CKD treatment, but the relationship between dietary phosphorus, a key nutrient for the gut microbiota, and protein-derived UT is poorly studied. Thus, we explored the relationship between dietary phosphorus and serum UT in CKD rats. For this exploratory study, we used serum samples from a larger study on the effects of dietary phosphorus on intestinal phosphorus absorption in nephrectomized (Nx, n = 22) or sham-operated (sham, n = 18) male Sprague Dawley rats. Rats were randomized to diet treatment groups of low or high phosphorus (0.1% or 1.2% w/w, respectively) for 1 week, with serum trimethylamine oxide (TMAO), indoxyl sulfate (IS), and p-cresol sulfate (pCS) analyzed by LC-MS. Nx rats had significantly higher levels of serum TMAO, IS, and pCS compared to sham rats (all p < 0.0001). IS showed a significant interaction between diet and CKD status, where serum IS was higher with the high-phosphorus diet in both Nx and sham rats, but to a greater extent in the Nx rats. Serum TMAO (p = 0.24) and pCS (p = 0.34) were not affected by dietary phosphorus levels. High dietary phosphorus intake for 1 week results in higher serum IS in both Nx and sham rats. The results of this exploratory study indicate that reducing dietary phosphorus intake in CKD may have beneficial effects on UT accumulation.

Indoxyl Sulfate Aggravates Podocyte Damage through the TGF-ß1/Smad/ROS Signalling Pathway.

INTRODUCTION: Hyperglycaemia induces the production of a large quantity of reactive oxygen species (ROS) and activates the transforming growth factor ß1 (TGF-ß1)/Smad signalling pathway, which is the main initiating factor in the formation of diabetic nephropathy. Indoxyl sulphate (IS) is a protein-binding gut-derived uraemic toxin that localizes to podocytes, induces oxidative stress, and inflames podocytes. The involvement of podocyte damage in diabetic nephropathy through the TGF-ß1 signalling pathway is still unclear. METHODS: In this study, we cultured differentiated rat podocytes in vitro and measured the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA by quantitative real-time PCR (qRT-PCR) and Western blotting after siRNA-mediated TGF-ß1 silencing, TGF-ß1 overexpression, and the presence of the ROS inhibitor acetylcysteine. We detected the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, small mother against decapentaplegic (Smad)2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, NADPH oxidase 4 (NOX4), and ROS levels under high glucose (HG) and IS conditions. RESULTS: The results indicated that nephrin, synaptopodin, CD2AP, and SRGAP2a expressions were significantly upregulated, and α-SMA expression was significantly downregulated in the presence of HG under siRNA-mediated TGF-ß1 silencing or after the addition of acetylcysteine. However, in the presence of HG, the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a were significantly downregulated, and the expression of α-SMA was significantly upregulated with the overexpression of TGF-ß1. IS supplementation under HG conditions further significantly reduced the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a; altered the expressions of Smad2/3, p-Smad2/3, Smad7, and NOX4; and increased ROS production in podocytes. CONCLUSION: This study suggests that IS may modulate the expression of nephrin, synaptopodin, CD2AP, and SRGAP2a by regulating the ROS and TGF-ß1/Smad signalling pathways, providing new theoretical support for the treatment of diabetic nephropathy.

Renoprotective effect of rosmarinic acid by inhibition of indoxyl sulfate-induced renal interstitial fibrosis via the NLRP3 inflammasome signaling.

We previously reported that rosmarinic acid (RA) ameliorated renal fibrosis in a unilateral ureteral obstruction (UUO) murine model of chronic kidney disease. This study aimed to determine whether RA attenuates indoxyl sulfate (IS)-induced renal fibrosis by regulating the activation of the NLRP3 inflammasome/IL-1ß/Smad circuit. We discovered the NLRP3 inflammasome was activated in the IS treatment group and downregulated in the RA-treated group in a dose-dependent manner. Additionally, the downstream effectors of the NLRP3 inflammasome, cleaved-caspase-1 and cleaved-IL-1ß showed similar trends in different groups. Moreover, RA administration significantly decreased the ROS levels of reactive oxygen species in IS-treated cells. Our data showed that RA treatment significantly inhibited Smad-2/3 phosphorylation. Notably, the effects of RA on NLRP3 inflammasome/IL-1ß/Smad and fibrosis signaling were reversed by the siRNA-mediated knockdown of NLRP3 or caspase-1 in NRK-52E cells. In vivo, we demonstrated that expression levels of NLRP3, c-caspase-1, c-IL-1ß, collagen I, fibronectin and α-SMA, and TGF- ß 1 were downregulated after treatment of UUO mice with RA or RA + MCC950. Our findings suggested RA and MCC950 synergistically inhibited UUO-induced NLRP3 signaling activation, revealing their renoprotective properties and the potential for combinatory treatment of renal fibrosis and chronic kidney inflammation.

Indoxyl sulphate-TNFα axis mediates uremic encephalopathy in rodent acute kidney injury.

Acute kidney injury (AKI) is often accompanied by uremic encephalopathy resulting from accumulation of uremic toxins in brain possibly due to impaired blood-brain barrier (BBB) function. Anionic uremic toxins are substrates or inhibitors of organic anionic transporters (OATs). In this study we investigated the CNS behaviors and expression/function of BBB OAT3 in AKI rats and mice, which received intraperitoneal injection of cisplatin 8 and 20 mg/kg, respectively. We showed that cisplatin treatment significantly inhibited the expressions of OAT3, synaptophysin and microtubule-associated protein 2 (MAP2), impaired locomotor and exploration activities, and increased accumulation of uremic toxins in the brain of AKI rats and mice. In vitro studies showed that uremic toxins neither alter OAT3 expression in human cerebral microvascular endothelial cells, nor synaptophysin and MAP2 expressions in human neuroblastoma (SH-SY5Y) cells. In contrast, tumour necrosis factor alpha (TNFα) and the conditioned medium (CM) from RAW264.7 cells treated with indoxyl sulfate (IS) significantly impaired OAT3 expression. TNFα and CM from IS-treated BV-2 cells also inhibited synaptophysin and MAP2 expressions in SH-SY5Y cells. The alterations caused by TNFα and CMs in vitro, and by AKI and TNFα in vivo were abolished by infliximab, a monoclonal antibody designed to intercept and neutralize TNFα, suggesting that AKI impaired the expressions of OAT3, synaptophysin and MAP2 in the brain via IS-induced TNFα release from macrophages or microglia (termed as IS-TNFα axis). Treatment of mice with TNFα (0.5 mg·kg-1·d-1, i.p. for 3 days) significantly increased p-p65 expression and reduced the expressions of Nrf2 and HO-1. Inhibiting NF-κB pathway, silencing p65, or activating Nrf2 and HO-1 obviously attenuated TNFα-induced downregulation of OAT3, synaptophysin and MAP2 expressions. Significantly increased p-p65 and decreased Nrf2 and HO-1 protein levels were also detected in brain of AKI mice and rats. We conclude that AKI inhibits the expressions of OAT3, synaptophysin and MAP2 due to IS-induced TNFα release from macrophages or microglia. TNFα impairs the expressions of OAT3, synaptophysin and MAP2 partly via activating NF-κB pathway and inhibiting Nrf2-HO-1 pathway.

Convenient preparation of indigo from the Ieaves of Baphicacanthus cusia(Nees) Bremek by enzymatic method and its MALDI-TOF-MS and UPLC-Q-TOF/MS analysis.

The manufacturing of indigo naturalis requires prolonged leaf soaking and lime stirring; the resulting indigo purity is less than 3.00% and the yield of indigo (measured in stems and leaves weight) is less than 0.50%, making it unsuitable for use in industrial procedures like printing and dyeing. An enzymatic method of creating indigo without the requirement for lime was investigated in order to generate high purity indigo. Single factor tests were performed to optimize the enzymatic preparation conditions. The findings showed that 60 °C, pH 5.5, 200 mL of leaves extract containing 0.45 mg/mL indican, and a 4:1 ratio of the acidic cellulose (activity: 9000 U/mL, liquid) to indican were the ideal parameters for enzymatic preparation. The yield of indigo was 40.32%, and the contents of indigo and indirubin were 37.37% and 2.30%, respectively. MALDI-TOF-MS in positive ion mode and UPLC-Q-TOF-MS in both positive and negative ion modes were used to analyze indigo extracts from Baphicacanthus cusia(Nees) Bremek by enzymatic preparation. It has been discovered that 13 alkaloids, 5 organic acids, 3 terpenoids, 3 steroids, 2 flavones, and 7 other compounds are present in indigo extracts. The presence of the indigo, indirubin, isorhamnetin, tryptanthrin, indigodole B, and indigodole C determined by UPLC-Q-TOF-MS was verified by MALDI-TOF-MS analysis. The enzymatic preparation of indigo extracts kept the same chemical makeup as conventional indigo naturalis. Thermal analysis and SEM morphology were used to confirm that there was no lime in the indigo extract. During the enzymatic process, Baphicacanthus cusia (Nees) Bremek was employed more effectively, increasing the yield and purity of indigo.

Roles of AhR/CYP1s signaling pathway mediated ROS production in uremic cardiomyopathy.

PURPOSE: Uremic cardiomyopathy (UCM) is the leading cause of chronic kidney disease (CKD) related mortality. Uremic toxins including indoxyl sulfate (IS) play important role during the progression of UCM. This study was to explore the underlying mechanism of IS related myocardial injury. METHODS: UCM rat model was established through five-sixths nephrectomy to evaluate its effects on blood pressure, cardiac impairment, and histological changes using echocardiography and histological analysis. Additionally, IS was administered to neonatal rat cardiomyocytes (NRCMs) and the human cardiomyocyte cell line AC16. DHE staining and peroxide-sensitive dye 2',7'-dichlorofluorescein diacetate (H2DCFDA) was conducted to assess the reactive oxygen species (ROS) production. Cardiomyocyte hypertrophy was estimated using wheat germ agglutinin (WGA) staining and immunofluorescence. Aryl hydrocarbon receptor (AhR) translocation was observed by immunofluorescence. The activation of AhR was evaluated by immunoblotting of cytochrome P450 1 s (CYP1s) and quantitative real-time PCR (RT-PCR) analysis of AHRR and PTGS2. Additionally, the pro-oxidative and pro-hypertrophic effects were evaluated using the AhR inhibitor CH-223191, the CYP1s inhibitor Alizarin and the ROS scavenger N-Acetylcysteine (NAC). RESULTS: UCM rat model was successfully established, and cardiac hypertrophy, accompanied by increased blood pressure, and myocardial fibrosis. Further research confirmed the activation of the AhR pathway in UCM rats including AhR translocation and downstream protein CYP1s expression, accompanied with increasing ROS production detected by DHE staining. In vitro experiment demonstrated a translocation of AhR triggered by IS, leading to significant increase of downstream gene expression. Subsequently study indicated a close relationship between the production of ROS and the activation of AhR/CYP1s, which was effectively blocked by applying AhR inhibitor, CYP1s inhibitor and siRNA against AhR. Moreover, the inhibition of AhR/CYP1s/ROS pathway collectively blocked the pro-hypertrophic effect of IS-mediated cardiomyopathy. CONCLUSION: This study provides evidence that the AhR/CYP1s pathway is activated in UCM rats, and this activation is correlated with the uremic toxin IS. In vitro studies indicate that IS can stimulate the AhR translocation in cardiomyocyte, triggering to the production of intracellular ROS via CYP1s. This process leads to prolonged oxidative stress stimulation and thus contributes to the progression of uremic toxin-mediated cardiomyopathy.

How renal toxins respond to renal function deterioration and oral toxic adsorbent in pH-controlled releasing capsule.

The number of patients with chronic kidney disease (CKD) is increasing. Oral toxin adsorbents may provide some value. Several uremic toxins, including indoxyl sulfate (IS), p-cresol (PCS), acrolein, per- and poly-fluoroalkyl substances (PFAS), and inflammation markers (interleukin 6 [IL-6] and tumor necrosis factor [TNF]-alpha) have been shown to be related to CKD progression. A total of 81 patients taking oral activated charcoal toxin adsorbents (AC-134), which were embedded in capsules that dissolved in the terminal ileum, three times a day for 1 month, were recruited. The renal function, hemoglobulin (Hb), inflammation markers, three PFAS (PFOA, PFOS, and PFNA), and acrolein were quantified. Compared with the baseline, an improved glomerular filtration rate (GFR) and significantly lower acrolein were noted. Furthermore, the CKD stage 4 and 5 group had significantly higher concentrations of IS, PCS, IL-6, and TNF but lower levels of Hb and PFAS compared with the CKD Stage 3 group at baseline and after the intervention. Hb was increased only in the CKD Stage 3 group after the trial (p = .032). Acrolein did not differ between the different CKD stage groups. Patients with improved GFR (responders) (about 77%) and nonresponders had similar baseline GFR. Responders had higher acrolein and PFOA levels throughout the study and a more significant reduction in acrolein, indicating a better digestion function. Both the higher PFOA and lower acrolein may be related to improved eGFR (and possibly to improvements in proteinuria, which we did not measure. Proteinuria is associated with PFAS loss in the urine), AC-134 showed the potential to improve the GFR and decrease acrolein, which might better indicate renal function change. Future studies are needed with longer follow-ups.

Role of Organic Anion Transporter NPT4 in Renal Handling of Uremic Toxin 3-indoxyl Sulfate.

Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3-/- mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3-/- mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3-/- mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated Km and Vmax values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3-/- mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion.

Indigofera cryptantha-induced pigmenturia in cattle in South Africa.

Two field cases of reddish-black pigmenturia occurred where cattle grazed on an established Cenchrus ciliaris (blue buffalo grass) pasture in South Africa. The pasture was noticeably invaded by Indigofera cryptantha, which was heavily grazed. Apart from the discolored urine, no other clinical abnormalities were detected. Urinalysis revealed hemoglobinuria, proteinuria and an alkaline pH. When the animals were immediately removed from the infested pasture, they made an uneventful recovery. However, a bull died when one of the herds could not be removed from the I. cryptantha-infested pasture. Macroscopically, the kidneys were dark red in color and the urinary bladder contained the dark pigmented urine. Microscopically, the renal tubules contained eosinophilic, granular pigment casts in the lumen. In addition, many renal tubular epithelial cells were attenuated with granular cytoplasm and were detached from the basement membranes. Chemical analysis was performed on dried, milled plant material and two urine samples collected during the field investigations. Qualitative UPLC-UV-qTOF/MS analysis revealed the presence of indican (indoxyl-ß-glucoside) in the stems, leaves and pods of I. cryptantha and indoxyl sulfate was identified, and confirmed with an analytical standard, in the urine samples. It is proposed that following ingestion of I. cryptantha, indican will be hydrolysed in the liver to indoxyl and conjugated with sulfate. Indoxyl sulfate will then be excreted in relatively high concentrations in the urine. In the alkaline urine, two indoxyl molecules might dimerize to form leucoindigo with subsequent oxidation to indigo, thus, contributing to the dark pigmentation of the urine. It is also possible that indoxyl sulfate contributed to the renal failure and death of the bull. Although I. suffruticosa-induced hemoglobinuria has been described in Brazil, this is the first report of I. cryptantha-induced pigmenturia in cattle in South Africa.

Indoxyl sulphate-initiated activation of cardiac fibroblasts is modulated by aryl hydrocarbon receptor and nuclear factor-erythroid-2-related factor 2.

In the last decade, extensive attention has been paid to the uremic toxin indoxyl sulphate (IS) as an inducer of cardiac fibroblast (cFib) activation and cardiac fibrosis in chronic kidney disease. At cellular level, IS engages aryl hydrocarbon receptor (AhR) and regulates many biological functions. We analysed how AhR inhibition by CH-223191 (CH) and overexpression of non-functional (dominant negative, DN) nuclear factor-erythroid-2-related factor 2 (NRF2), a transcription factor recruited by AhR, modulate the response of neonatal mouse (nm) cFib to IS. We also evaluated nm-cardiomyocytes after incubation with the conditioned medium (CM) of IS±CH-treated nm-cFib. IS induced activation, collagen synthesis, TLR4 and-downstream-MCP-1, and the genes encoding angiotensinogen, angiotensin-converting enzyme, angiotensin type 1 receptor (AT1r) and neprilysin (Nepr) in nm-cFib. CH antagonized IS-initiated nm-cFib activation, but did not affect or even magnified the other features. IS promoted NRF2 nuclear translocation and expression the NRF2 target Nqo1. Both pre-incubation with CH and transfection of DN-NRF2 resulted in loss of NRF2 nuclear localization. Moreover, DN-NRF2 overexpression led to greater TLR4 and MCP-1 levels following exposure to IS. The CM of IS-primed nm-cFib and to a larger extent the CM of IS+CH-treated nm-cFib upregulated AT1r, Nepr and TNFα and myostatin genes in nm-cardiomyocytes. Hence, IS triggers pro-inflammatory activation of nm-cFib partly via AhR, and AhR-NRF2 counteract it. Strategies other than AhR inhibition are needed to target IS detrimental actions on cardiac cells.

Indoxyl sulfate contributes to colorectal cancer cell proliferation and increased EGFR expression by activating AhR and Akt.

Although patients with chronic kidney disease (CKD) have a higher risk of colorectal cancer (CRC) aggravation, the connection between these two diseases is not well understood. Recent studies have shown that both CKD and CRC aggravation are closely related to an increased abundance of indole-producing Fusobacterium nucleatum in the gut. The indole absorbed from the gut is eventually metabolized to indoxyl sulfate in the liver. Since indoxyl sulfate is involved not only in accelerating CKD progression but also in the initiation and development of its associated complications, the present study aimed to clarify whether indoxyl sulfate induces the proliferation of CRC cells. This study found that indoxyl sulfate induced the proliferation of CRC-derived HCT-116 cells by activating the aryl hydrocarbon receptor (AhR) and the proto-oncogene Akt. The AhR antagonist CH223191 and Akt inhibitor MK2206 suppressed indoxyl sulfate-induced proliferation of HCT-116 cells. We also found that indoxyl sulfate upregulated epidermal growth factor receptor (EGFR) expression, which is associated with poor prognosis of CRC, whereas CH223191 and MK2206 repressed EGFR expression. Furthermore, indoxyl sulfate increased the sensitivity of CRC cells to EGF by upregulating EGFR expression. These findings suggest that indoxyl sulfate may be an important link between CKD and CRC aggravation.

Effect of High Sodium Intake on Gut Tight Junctions' Structure and Permeability to Bacterial Toxins in a Rat Model of Chronic Kidney Disease.

INTRODUCTION: Uremic toxicity changes the gut structure and permeability, allowing bacterial toxins to translocate from the lumen to the blood during chronic kidney failure (CKD). Clinical fluid overload and tissue edema without uremia have similar effects but have not been adequately demonstrated and analyzed in CKD. AIMS: To investigate the effect of sodium intake on the plasma concentration of gut-derived uremic toxins, indoxyl sulfate (IS), and p-cresyl sulfate (pCS) and the expression of genes and proteins of epithelial gut tight junctions in a rat model of CKD. METHODS: Sham-operated (control group, CG) and five-sixths nephrectomized (5/6Nx) Sprague-Dawley rats were randomly assigned to low (LNa), normal (NNa), or high sodium (HNa) diets., Animals were then sacrificed at 8 and 12 weeks and analyzed for IS and pCS plasma concentrations, as well as for gene and protein expression of thigh junction proteins, and transmission electron microscopy (TEM) in colon fragments. RESULTS: The HNa 5/6Nx groups had higher concentrations of IS and pCS than CG, NNa, and LNa at eight and twelve weeks. Furthermore, HNa 5/6Nx groups had reduced expression of the claudin-4 gene and protein than CG, NNa, and LNa. HNa had reduced occludin gene expression compared to CG. Occludin protein expression was more reduced in HNa than in CG, NNa, and LNa. The gut epithelial tight junctions appear dilated in HNa compared to NNa and LNa in TEM. CONCLUSION: Dietary sodium intake and fluid overload have a significant role in gut epithelial permeability in the CKD model.

Uremic toxins mediate kidney diseases: the role of aryl hydrocarbon receptor.

Aryl hydrocarbon receptor (AhR) was originally identified as an environmental sensor that responds to pollutants. Subsequent research has revealed that AhR recognizes multiple exogenous and endogenous molecules, including uremic toxins retained in the body due to the decline in renal function. Therefore, AhR is also considered to be a uremic toxin receptor. As a ligand-activated transcriptional factor, the activation of AhR is involved in cell differentiation and senescence, lipid metabolism and fibrogenesis. The accumulation of uremic toxins in the body is hazardous to all tissues and organs. The identification of the endogenous uremic toxin receptor opens the door to investigating the precise role and molecular mechanism of tissue and organ damage induced by uremic toxins. This review focuses on summarizing recent findings on the role of AhR activation induced by uremic toxins in chronic kidney disease, diabetic nephropathy and acute kidney injury. Furthermore, potential clinical approaches to mitigate the effects of uremic toxins are explored herein, such as enhancing uremic toxin clearance through dialysis, reducing uremic toxin production through dietary interventions or microbial manipulation, and manipulating metabolic pathways induced by uremic toxins through controlling AhR signaling. This information may also shed light on the mechanism of uremic toxin-induced injury to other organs, and provide insights into clinical approaches to manipulate the accumulated uremic toxins.

Indoxyl Sulfate-Induced Valve Endothelial Cell Endothelial-to-Mesenchymal Transition and Calcification in an Integrin-Linked Kinase-Dependent Manner.

Calcific Aortic Valve Disease (CAVD) is a significant concern for cardiovascular health and is closely associated with chronic kidney disease (CKD). Aortic valve endothelial cells (VECs) play a significant role in the onset and progression of CAVD. Previous research has suggested that uremic toxins, particularly indoxyl sulfate (IS), induce vascular calcification and endothelial dysfunction, but the effect of IS on valve endothelial cells (VECs) and its contribution to CAVD is unclear. Our results show that IS reduced human VEC viability and increased pro-calcific markers RUNX2 and alkaline phosphatase (ALP) expression. Additionally, IS-exposed VECs cultured in pro-osteogenic media showed increased calcification. Mechanistically, IS induced endothelial-to-mesenchymal transition (EndMT), evidenced by the loss of endothelial markers and increased expression of mesenchymal markers. IS triggered VEC inflammation, as revealed by NF-kB activation, and decreased integrin-linked kinase (ILK) expression. ILK overexpression reversed the loss of endothelial phenotype and RUNX2, emphasizing its relevance in the pathogenesis of CAVD in CKD. Conversely, a lower dose of IS intensified some of the effects in EndMT caused by silencing ILK. These findings imply that IS affects valve endothelium directly, contributing to CAVD by inducing EndMT and calcification, with ILK acting as a crucial modulator.

AST-120 improved uremic pruritus by lowering indoxyl sulfate and inflammatory cytokines in hemodialysis patients.

BACKGROUND AND HYPOTHESIS: Pruritus is a common and distressing symptom that affects patients with chronic kidney disease. The concentration of protein bounded uremic toxin was associated with the uremic pruritus. The aim is to assess the efficacy of AST-120 for uremic pruritus in hemodialysis patients. MATERIALS AND METHODS: The participants were enrolled and then divided into the AST-120 treatment group and control group with a ratio of 2:1. All participants underwent pre-observation screenings two weeks before the study with three visits. In the treatment phase (week 1 to week 4), the treatment group added 6g/day of AST-120 along with routine anti-pruritic treatment. Visual analog scale (VAS) and biochemical parameters were measured. RESULTS: The VAS score began to be lower in the AST-120 treatment group after the 5th visiting (p < 0.05). The reduction in indoxyl sulfate (IS) at 5th week along with TNF-alpha. The reduction ratio of indoxyl sulfate correlated with reduction of parathyroid hormone. CONCLUSION: This study has demonstrated that the four-week treatment of AST-120 decreased the severity of uremic pruritus in patients with ESRD. The concentration of IS and TNF-alpha decreased in the AST-120 treatment group. The reduction of iPTH correlated with the reduction of IS in the AST-120 treatment.

Chemoenzymatic indican for light-driven denim dyeing.

Blue denim, a billion-dollar industry, is currently dyed with indigo in an unsustainable process requiring harsh reducing and alkaline chemicals. Forming indigo directly in the yarn through indican (indoxyl-ß-glucoside) is a promising alternative route with mild conditions. Indican eliminates the requirement for reducing agent while still ending as indigo, the only known molecule yielding the unique hue of blue denim. However, a bulk source of indican is missing. Here, we employ enzyme and process engineering guided by techno-economic analyses to develop an economically viable drop-in indican synthesis technology. Rational engineering of PtUGT1, a glycosyltransferase from the indigo plant, alleviated the severe substrate inactivation observed with the wildtype enzyme at the titers needed for bulk production. We further describe a mild, light-driven dyeing process. Finally, we conduct techno-economic, social sustainability, and comparative life-cycle assessments. These indicate that the presented technologies have the potential to significantly reduce environmental impacts from blue denim dyeing with only a modest cost increase.

Donor-Derived Engineered Microvessels for Cardiovascular Risk Stratification of Patients with Kidney Failure.

Cardiovascular disease is the cause of death in ≈50% of hemodialysis patients. Accumulation of uremic solutes in systemic circulation is thought to be a key driver of the endothelial dysfunction that underlies elevated cardiovascular events. A challenge in understanding the mechanisms relating chronic kidney disease to cardiovascular disease is the lack of in vitro models that allow screening of the effects of the uremic environment on the endothelium. Here, a method is described for microfabrication of human blood vessels from donor cells and perfused with donor serum. The resulting donor-derived microvessels are used to quantify vascular permeability, a hallmark of endothelial dysfunction, in response to serum spiked with pathophysiological levels of indoxyl sulfate, and in response to serum from patients with chronic kidney disease and from uremic pigs. The uremic environment has pronounced effects on microvascular integrity as demonstrated by irregular cell-cell junctions and increased permeability in comparison to cell culture media and healthy serum. Moreover, the engineered microvessels demonstrate an increase in sensitivity compared to traditional 2D assays. Thus, the devices and the methods presented here have the potential to be utilized to risk stratify and to direct personalized treatments for patients with chronic kidney disease.