Protein bound-uremic toxins (PBUTs) are not efficiently removed by hemodialysis in chronic kidney disease (CKD) patients and their accumulation leads to various co-morbidities via cellular dysfunction, inflammation and oxidative stress. Moreover, it has been shown that increased intrarenal expression of the NLRP3 receptor and IL-1β are associated with reduced kidney function, suggesting a critical role for the NLRP3 inflammasome in CKD progression. Here, we evaluated the effect of PBUTs on inflammasome-mediated IL-1β production in vitro and in vivo. Exposure of human conditionally immortalized proximal tubule epithelial cells to indoxyl sulfate (IS) and a mixture of anionic PBUTs (UT mix) increased expression levels of NLRP3, caspase-1 and IL-1β, accompanied by a significant increase in IL-1β secretion and caspase-1 activity. Furthermore, IS and UT mix induced the production of intracellular reactive oxygen species, and caspase-1 activity and IL-1β secretion were reduced in the presence of antioxidant N-acetylcysteine. IS and UT mix also induced NF-κB activation as evidenced by p65 nuclear translocation and IL-1β production, which was counteracted by an IKK inhibitor. In vivo, using subtotal nephrectomy CKD rats, a significant increase in total plasma levels of IS and the PBUTs, kynurenic acid and hippuric acid, was found, as well as enhanced urinary malondialdehyde levels. CKD kidney tissue showed an increasing trend in expression of NLRP3 inflammasome components, and a decreasing trend in superoxide dismutase-1 levels. In conclusion, we showed that PBUTs induce inflammasome-mediated IL-1β production in proximal tubule cells via oxidative stress and NF-κB signaling, suggesting their involvement in disease-associated inflammatory processes.

The bacterial metabolite 4-methylphenol (para-cresol or p-cresol) and its derivative p-cresyl sulfate (pCS) are elevated in the urine and feces of children with autism spectrum disorder (ASD). It has been shown that p-cresol administration induces social behavior deficits and repetitive behavior in mice. However, the mechanisms of p-cresol, specifically its metabolite pCS that can reach the brain, in ASD remain to be investigated. The pCS has been shown to inhibit LPS-stimulated inflammatory response. A Disintegrin And Metalloprotease 10 (ADAM10) and A Disintegrin And Metalloprotease 17 (ADAM17) are thought to regulate microglial immune response by cleaving membrane-bound proteins. In the present study, a neuroinflammation model of LPS-activated BV2 microglia has been used to unveil the potential molecular mechanism of pCS in ASD pathogenesis. In microglial cells pCS treatment decreases the expression or maturation of ADAM10 and ADAM17. In addition, pCS treatment attenuates TNF-α and IL-6 releases as well as phagocytosis activity of microglia. In in vitro ADAM10/17 inhibition experiments, either ADAM10 or ADAM17 inhibition reduces constitutive and LPS-activated release of TNF-α, TNFR-1 and IL-6R by microglial cells, while it increases constitutive and LPS-activated microglial phagocytotic activity. The in vivo results further confirm the involvement of ADAM10 and ADAM17 in ASD pathogenesis. In in utero VPA-exposed male mice, elevated concentration in serum of p-cresol-associated metabolites pCS and p-cresyl glucuronide (pCG) is associated with a VPA-induced increased ADAM10 maturation, and a decreased ADAM17 maturation that is related with attenuated levels of soluble TNF-α and TGF-β1 in the mice brain. Overall, the present study demonstrates a partial role of ADAM10 and ADAM17 in the derailed innate immune response of microglial cells associated with pCS-induced ASD pathogenesis.

The organic anion transporter-1 (OAT1), located at the basal side of kidney proximal tubule cells, plays a crucial role in metabolic waste excretion. In chronic kidney disease (CKD), the progressive decline in renal function leads to the accumulation of endogenous metabolites in the bloodstream, exacerbating comorbidities. CKD also leads to gut dysbiosis, increasing the production of uremic metabolites and reducing the production of nephroprotective short chain fatty acids (SCFAs), such as acetate, propionate and butyrate, thereby contributing to disease progression. This study investigates the potential of SCFAs to enhance kidney function by modulating OAT1 activity and facilitating the secretion of uremic toxins. Our findings demonstrate that propionate and butyrate significantly enhance OAT1 activity by upregulating SLC22A6/OAT1 gene and protein expression. Butyrate exposure, in particular, enhances the secretion of the uremic toxin indoxyl sulfate (IS) to the luminal compartment in our kidney-on-chip (KoC) system. Notably, SCFAs exert their effect independently of G-protein coupled receptor (GPCR) activation, instead inhibiting gene expression of class II histone deacetylases (HDACs). Transcriptome analysis suggests that this inhibition modulates cyclic adenosine monophosphate (cAMP) signaling pathway, activating CREB1 and PI3K gene expression, both implicated in cell metabolism and resilience against stress, thereby enhancing cellular fitness. These findings highlight the therapeutic potential of SCFAs in enhancing proximal tubule secretory activity, emphasizing their value as nutritional interventions in CKD management.

Proximal tubular damage is an important prognostic determinant in various chronic kidney diseases (CKDs). Currently available diagnostic methods do not allow for early disease detection and are neither efficient. Indoxyl sulfate (IS) is an endogenous metabolite and protein‐bound uremic toxin that is eliminated via renal secretion, but accumulates in plasma during tubular dysfunction. Therefore, it may be suitable as a tubular function marker. To evaluate this, a fast bioanalytical method was developed and validated for IS in various species and a kidney cell line using LC–MS/MS. An isotope‐labeled IS potassium salt as an internal standard and acetonitrile (ACN) as a protein precipitant were used for sample pretreatment. The analyte was separated on a Polaris 3 C18‐A column by gradient elution using 0.1% formic acid in water and ACN, and detected by negative electrospray ionization in selected reaction monitoring mode. The within‐day (≤ 4.0%) and between‐day (≤ 4.3%) precisions and accuracies (97.7 to 107.3%) were within the acceptable range. The analyte showed sufficient stability at all conditions investigated. Finally, applying this assay, significantly higher plasma and lower urine concentrations of IS were observed in mice with diabetic nephropathy with tubular damage, which encourages validation toward its use as a biomarker.

Patients with end-stage kidney disease (ESKD) suffer from high levels of protein-bound uremic toxins (PBUTs) that contribute to various comorbidities. Conventional dialysis methods are ineffective in removing these PBUTs. A potential solution could be offered by a bioartificial kidney (BAK) composed of porous membranes covered by proximal tubule epithelial cells (PTECs) that actively secrete PBUTs. However, BAK development is currently being hampered by a lack of knowledge regarding the cytocompatibility of the dialysis fluid (DF) that comes in contact with the PTECs. Here, we conducted a comprehensive functional assessment of the DF on human conditionally immortalized PTECs (ciPTECs) cultured as monolayers in well plates, on Transwell^® inserts, or on hollow fiber membranes (HFMs) that form functional units of a BAK. We evaluated cell viability markers, monolayer integrity, and PBUT clearance. Our results show that exposure to DF did not affect ciPTECs’ viability, membrane integrity, or function. Seven anionic PBUTs were efficiently cleared from the perfusion fluid containing a PBUTs cocktail or uremic plasma, an effect which was enhanced in the presence of albumin. Overall, our findings support that the DF is cytocompatible and does not compromise ciPTECs function, paving the way for further advancements in BAK development and its potential clinical application.

Kidney tubular damage is an important prognostic determinant in diabetic kidney disease (DKD). A vital homeostatic function of the proximal tubule is active tubular secretion of waste products via organic anion transporters (OATs), including protein-bound uremic toxins (PBUTs) that accumulate in plasma in tubular dysfunction. We here hypothesize that PBUT clearance may be a sensitive tubular function marker, and tested this in a DKD mouse model and in type 2 diabetic patients. Among the PBUTs with the highest OAT affinity (i.e., indoxyl sulfate (IS), hippuric acid (HA) and kynurenic acid (KA)), plasma concentrations were higher and urinary excretions were lower 6 and 8 months after DKD induction in mice. These parameters correlated better with tubular atrophy, f4/80 scores and tubular injury markers than conventional filtration markers. In patients, the clearance of IS, HA, KA and p-cresyl sulfate (PCS) was associated with urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), independent of eGFR. In multiple regression analysis, additionally adjusted for relevant risk factors for tubular injury, the clearance of IS, HA and PCS remained significantly associated with urinary NGAL. In conclusion, IS, HA, KA and PCS clearance may represent a biomarker of kidney tubular function in DKD.