Regulation of solute carriers oct2 and OAT1/3 in the kidney: a phylogenetic, ontogenetic, and cell dynamic perspective.

Over the course of more than 500 million years, the kidneys have undergone a remarkable evolution from primitive nephric tubes to intricate filtration-reabsorption systems that maintain homeostasis and remove metabolic end products from the body. The evolutionarily conserved solute carriers organic cation transporter 2 (OCT2) and organic anion transporters 1 and 3 (OAT1/3) coordinate the active secretion of a broad range of endogenous and exogenous substances, many of which accumulate in the blood of patients with kidney failure despite dialysis. Harnessing OCT2 and OAT1/3 through functional preservation or regeneration could alleviate the progression of kidney disease. Additionally, it would improve current in vitro test models that lose their expression in culture. With this review, we explore OCT2 and OAT1/3 regulation from different perspectives: phylogenetic, ontogenetic, and cell dynamic. Our aim is to identify possible molecular targets both to help prevent or compensate for the loss of transport activity in patients with kidney disease and to enable endogenous OCT2 and OAT1/3 induction in vitro in order to develop better models for drug development.

Tubuloid culture enables long-term expansion of functional human kidney tubule epithelium from iPSC-derived organoids.

Kidney organoids generated from induced pluripotent stem cells (iPSC) have proven valuable for studies of kidney development, disease, and therapeutic screening. However, specific applications have been hampered by limited expansion capacity, immaturity, off-target cells, and inability to access the apical side. Here, we apply recently developed tubuloid protocols to purify and propagate kidney epithelium from d7+18 (post nephrogenesis) iPSC-derived organoids. The resulting 'iPSC organoid-derived (iPSCod)' tubuloids can be exponentially expanded for at least 2.5 mo, while retaining expression of important tubular transporters and segment-specific markers. This approach allows for selective propagation of the mature tubular epithelium, as immature cells, stroma, and undesirable off-target cells rapidly disappeared. iPSCod tubuloids provide easy apical access, which enabled functional evaluation and demonstration of essential secretion and electrolyte reabsorption processes. In conclusion, iPSCod tubuloids provide a different, complementary human kidney model that unlocks opportunities for functional characterization, disease modeling, and regenerative nephrology.

Dietary protein intake and the tubular handling of indoxyl sulfate.

BACKGROUND AND HYPOTHESIS: Chronic kidney disease (CKD) patients are advised to limit their protein intake. A high protein diet is known to induce glomerular hyperfiltration, as well as hypertrophy of the remnant kidney, and glomerulosclerosis. Whether the diet causes changes in kidney tubule transport via gut microbiome metabolites is still unknown. We hypothesized that protein intake affects not only the intestinal generation and absorption, but also the kidney disposal of microbial amino acid metabolites. METHODS: We combined data from animal models and human studies. 5/6th nephrectomy rats were administered a high (HP) or low-protein (LP) diet for 7 weeks. Plasma and urine concentration of the uremic toxins (UTs) indoxyl sulfate (IS), p-cresyl sulfate (PCS), and p-cresyl glucuronide (PCG) were measured. Their fractional excretion (FE) was calculated. The expression of kidney membrane transporters OAT1, OAT3, BCRP, OCT2 and MRP4 was analyzed. Differences in FE of UTs between individuals with higher and lower protein intake in two CKD cohorts were sought. RESULTS: CKD rats on an HP diet showed increased plasma levels of PCS and PCG but not IS compared to rats on a LP diet. Conversely, urinary excretion and FE of IS were higher in the HP CKD group. BCRP, MRP4 and OCT2 were not influenced by the diet. OAT1 and OAT3 were upregulated in the HP CKD group. In two independent cohorts of CKD patients, individuals with a high dietary protein intake showed a significantly higher FE of IS. CONCLUSIONS: A HP diet leads to a higher generation and/or absorption of aminoacid-derived UT precursors in CKD rodent models and humans, most likely via gut microbiome modulation. We demonstrate that dietary protein intake modulates transcription and expression of OAT1 and OAT3, corroborating the existence of the remote sensing and signaling hypothesis. Dietary protein intake influences kidney physiology beyond glomerular filtration.

The application of organ-on-chip models for the prediction of human pharmacokinetic profiles during drug development.

Organ-on-chip (OoC) technology has led to in vitro models with many new possibilities compared to conventional in vitro and in vivo models. In this review, the potential of OoC models to improve the prediction of human oral bioavailability and intrinsic clearance is discussed, with a focus on the functionality of the models and the application in current drug development practice. Multi-OoC models demonstrating the application for pharmacokinetic (PK) studies are summarized and existing challenges are identified. Physiological parameters for a minimal viable platform of a multi-OoC model to study PK are provided, together with PK specific read-outs and recommendations for relevant reference compounds to validate the model. Finally, the translation to in vivo PK profiles is discussed, which will be required to routinely apply OoC models during drug development.

Cisplatin-induced nephrotoxicity in childhood cancer: comparison between two countries.

BACKGROUND: Various definitions used to describe cisplatin nephrotoxicity potentially lead to differences in determination of risk factors. This study evaluated incidence of kidney injury according to commonly used and alternative definitions in two cohorts of children who received cisplatin. METHODS: This retrospective cohort study included children from Vancouver, Canada (one center), and Mexico City, Mexico (two centers), treated with cisplatin for a variety of solid tumors. Serum creatinine-based definitions (KDIGO and Pediatric RIFLE (pRIFLE)), electrolyte abnormalities consisted of hypokalemia, hypophosphatemia and hypomagnesemia (based on NCI-CTCAE v5), and an alternative definition (Alt-AKI) were used to describe nephrotoxicity. Incidence with different definitions, definitional overlap, and inter-definition reliability was analyzed. RESULTS: In total, 173 children (100 from Vancouver, 73 from Mexico) were included. In the combined cohort, Alt-AKI criteria detected more patients with cisplatin nephrotoxicity compared to pRIFLE and KDIGO criteria (82.7 vs. 63.6 vs. 44.5%, respectively). Nephrotoxicity and all electrolyte abnormalities were significantly more common in Vancouver cohort than in Mexico City cohort except when using KDIGO definition. The most common electrolyte abnormalities were hypomagnesemia (88.9%, Vancouver) and hypophosphatemia (24.2%, Mexico City). The KDIGO definition provided highest overlap of cases in Vancouver (100%), Mexico (98.6%), and the combined cohort (99.4%). Moderate overall agreement was found among Alt-AKI, KDIGO, and pRIFLE definitions (κ = 0.18, 95% CI 0.1-0.27) in which KDIGO and pRIFLE showed moderate agreement (κ = 0.48, 95% CI 0.36-0.60). CONCLUSIONS: Compared to pRIFLE and KDIGO criteria, Alt-AKI criteria detected more patients with cisplatin nephrotoxicity. pRIFLE is more sensitive to detect not only actual kidney injury but also patients at risk of cisplatin nephrotoxicity, while KDIGO seems more useful to detect clinically significant kidney injury. A higher resolution version of the Graphical abstract is available as Supplementary information.

Bioengineered Kidney Tubules Efficiently Clear Uremic Toxins in Experimental Dialysis Conditions.

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.

Animal Models for Studying Protein-Bound Uremic Toxin Removal-A Systematic Review.

Protein-bound uremic toxins (PBUTs) are associated with the progression of chronic kidney disease (CKD) and its associated morbidity and mortality. The conventional dialysis techniques are unable to efficiently remove PBUTs due to their plasma protein binding. Therefore, novel approaches are being developed, but these require validation in animals before clinical trials can begin. We conducted a systematic review to document PBUT concentrations in various models and species. The search strategy returned 1163 results for which abstracts were screened, resulting in 65 full-text papers for data extraction (rats (n = 41), mice (n = 17), dogs (n = 3), cats (n = 4), goats (n = 1), and pigs (n = 1)). We performed descriptive and comparative analyses on indoxyl sulfate (IS) concentrations in rats and mice. The data on large animals and on other PBUTs were too heterogeneous for pooled analysis. Most rodent studies reported mean uremic concentrations of plasma IS close to or within the range of those during kidney failure in humans, with the highest in tubular injury models in rats. Compared to nephron loss models in rats, a greater rise in plasma IS compared to creatinine was found in tubular injury models, suggesting tubular secretion was more affected than glomerular filtration. In summary, tubular injury rat models may be most relevant for the in vivo validation of novel PBUT-lowering strategies for kidney failure in humans.

Viscoelastic Chondroitin Sulfate and Hyaluronic Acid Double-Network Hydrogels with Reversible Cross-Links.

Viscoelastic hydrogels are gaining interest as they possess necessary requirements for bioprinting and injectability. By means of reversible, dynamic covalent bonds, it is possible to achieve features that recapitulate the dynamic character of the extracellular matrix. Dually cross-linked and double-network (DN) hydrogels seem to be ideal for the design of novel biomaterials and bioinks, as a wide range of properties required for mimicking advanced and complex tissues can be achieved. In this study, we investigated the fabrication of chondroitin sulfate/hyaluronic acid (CS/HA)-based DN hydrogels, in which two networks are interpenetrated and cross-linked with the dynamic covalent bonds of very different lifetimes. Namely, Diels-Alder adducts (between methylfuran and maleimide) and hydrazone bonds (between aldehyde and hydrazide) were chosen as cross-links, leading to viscoelastic hydrogels. Furthermore, we show that viscoelasticity and the dynamic character of the resulting hydrogels could be tuned by changing the composition, that is, the ratio between the two types of cross-links. Also, due to a very dynamic nature and short lifetime of hydrazone cross-links (∼800 s), the DN hydrogel is easily processable (e.g., injectable) in the first stages of gelation, allowing the material to be used in extrusion-based 3D printing. The more long-lasting and robust Diels-Alder cross-links are responsible for giving the network enhanced mechanical strength and structural stability. Being highly charged and hydrophilic, the cross-linked CS and HA enable a high swelling capacity (maximum swelling ratio ranging from 6 to 12), which upon confinement results in osmotically stiffened constructs, able to mimic the mechanical properties of cartilage tissue, with the equilibrium moduli ranging from 0.3 to 0.5 MPa. Moreover, the mesenchymal stromal cells were viable in the presence of the hydrogels, and the effect of the degradation products on the macrophages suggests their safe use for further translational applications. The DN hydrogels with dynamic covalent cross-links hold great potential for the development of novel smart and tunable viscoelastic materials to be used as biomaterial inks or bioinks in bioprinting and regenerative medicine.

Organs-on-chip technology: a tool to tackle genetic kidney diseases.

Chronic kidney disease (CKD) is a major healthcare burden that takes a toll on the quality of life of many patients. Emerging evidence indicates that a substantial proportion of these patients carry a genetic defect that contributes to their disease. Any effort to reduce the percentage of patients with a diagnosis of nephropathy heading towards kidney replacement therapies should therefore be encouraged. Besides early genetic screenings and registries, in vitro systems that mimic the complexity and pathophysiological aspects of the disease could advance the screening for targeted and personalized therapies. In this regard, the use of patient-derived cell lines, as well as the generation of disease-specific cell lines via gene editing and stem cell technologies, have significantly improved our understanding of the molecular mechanisms underlying inherited kidney diseases. Furthermore, organs-on-chip technology holds great potential as it can emulate tissue and organ functions that are not found in other, more simple, in vitro models. The personalized nature of the chips, together with physiologically relevant read-outs, provide new opportunities for patient-specific assessment, as well as personalized strategies for treatment. In this review, we summarize the major kidney-on-chip (KOC) configurations and present the most recent studies on the in vitro representation of genetic kidney diseases using KOC-driven strategies.

Differentiated kidney tubular cell-derived extracellular vesicles enhance maturation of tubuloids.

The prevalence of end-stage kidney disease (ESKD) is rapidly increasing with the need for regenerative therapies. Adult stem cell derived kidney tubuloids have the potential to functionally mimic the adult kidney tubule, but still lack the expression of important transport proteins needed for waste removal. Here, we investigated the potential of extracellular vesicles (EVs) obtained from matured kidney tubular epithelial cells to modulate in vitro tubuloids functional maturation. We focused on organic anion transporter 1 (OAT1), one of the most important proteins involved in endogenous waste excretion. First, we show that EVs from engineered proximal tubule cells increased the expression of several transcription factors and epithelial transporters, resulting in improved OAT1 transport capacity. Next, a more in-depth proteomic data analysis showed that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation. Moreover, we demonstrated that the combination of EVs and tubuloid-derived cells can be used as part of a bioartificial kidney to generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from kidney tubular epithelial cells can phenotypically improve in vitro tubuloid maturation, thereby enhancing their potential as functional units in regenerative or renal replacement therapies.

Remote sensing and signaling in kidney proximal tubules stimulates gut microbiome-derived organic anion secretion.

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

A robust, accurate, sensitive LC-MS/MS method to measure indoxyl sulfate, validated for plasma and kidney cells.

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.

Extrahepatic metabolism of ibrutinib.

Ibrutinib is a first-in-class Bruton's kinase inhibitor used in the treatment of multiple lymphomas. In addition to CYP3A4-mediated metabolism, glutathione conjugation can be observed. Subsequently, metabolism of the conjugates and finally their excretion in feces and urine occurs. These metabolites, however, can reach substantial concentrations in human subjects, especially when CYP3A4 is inhibited. Ibrutinib has unexplained nephrotoxicity and high metabolite concentrations are also found in kidneys of Cyp3a knockout mice. Here, a mechanism is proposed where the intermediate cysteine metabolite is bioactivated. The metabolism of ibrutinib through this glutathione cycle was confirmed in cultured human renal proximal tubule cells. Ibrutinib-mediated toxicity was enhanced in-vitro by inhibitors of breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) and multidrug resistance protein (MRP). This was a result of accumulating cysteine metabolite levels due to efflux inhibition. Finally, through inhibition of downstream metabolism, it was shown now that direct conjugation was responsible for cysteine metabolite toxicity.

A Randomized Trial of Distal Diuretics versus Dietary Sodium Restriction for Hypertension in Chronic Kidney Disease.

BACKGROUND: Distal diuretics are considered less effective than loop diuretics in CKD. However, data to support this perception are limited. METHODS: To investigate whether distal diuretics are noninferior to dietary sodium restriction in reducing BP in patients with CKD stage G3 or G4 and hypertension, we conducted a 6-week, randomized, open-label crossover trial comparing amiloride/hydrochlorothiazide (5 mg/50 mg daily) with dietary sodium restriction (60 mmol per day). Antihypertension medication was discontinued for a 2-week period before randomization. We analyzed effects on BP, kidney function, and fluid balance and related this to renal clearance of diuretics. RESULTS: A total of 26 patients (with a mean eGFR of 39 ml/min per 1.73 m2) completed both treatments. Dietary sodium restriction reduced sodium excretion from 160 to 64 mmol per day. Diuretics produced a greater reduction in 24-hour systolic BP (SBP; from 138 to 124 mm Hg) compared with sodium restriction (from 134 to 129 mm Hg), as well as a significantly greater effect on extracellular water, eGFR, plasma renin, and aldosterone. Both interventions resulted in a similar decrease in body weight and NT-proBNP. Neither approaches decreased albuminuria significantly, whereas diuretics did significantly reduce urinary angiotensinogen and ß2-microglobulin excretion. Although lower eGFR and higher plasma indoxyl sulfate correlated with lower diuretic clearance, the diuretic effects on body weight and BP at lower eGFR were maintained. During diuretic treatment, higher PGE2 excretion correlated with lower free water clearance, and four patients developed mild hyponatremia. CONCLUSIONS: Distal diuretics are noninferior to dietary sodium restriction in reducing BP and extracellular volume in CKD. Diuretic sensitivity in CKD is maintained despite lower diuretic clearance. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: DD-study: Diet or Diuretics for Salt-sensitivity in Chronic Kidney Disease (DD), NCT02875886.

Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis.

BACKGROUND: Mutations in the gene that encodes the lysosomal cystine transporter cystinosin cause the lysosomal storage disease cystinosis. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The most severe phenotype, nephropathic cystinosis, manifests during the first months of life, as renal Fanconi syndrome. The cystine-depleting agent cysteamine significantly delays symptoms, but it cannot prevent progression to ESKD and does not treat Fanconi syndrome. This suggests the involvement of pathways in nephropathic cystinosis that are unrelated to lysosomal cystine accumulation. Recent data indicate that one such potential pathway, lysosome-mediated degradation of autophagy cargoes, is compromised in cystinosis. METHODS: To identify drugs that reduce levels of the autophagy-related protein p62/SQSTM1 in cystinotic proximal tubular epithelial cells, we performed a high-throughput screening on the basis of an in-cell ELISA assay. We then tested a promising candidate in cells derived from patients with, and mouse models of, cystinosis, and in preclinical studies in cystinotic zebrafish. RESULTS: Of 46 compounds identified as reducing p62/SQSTM1 levels in cystinotic cells, we selected luteolin on the basis of its efficacy, safety profile, and similarity to genistein, which we previously showed to ameliorate other lysosomal abnormalities of cystinotic cells. Our data show that luteolin improves the autophagy-lysosome degradative pathway, is a powerful antioxidant, and has antiapoptotic properties. Moreover, luteolin stimulates endocytosis and improves the expression of the endocytic receptor megalin. CONCLUSIONS: Our data show that luteolin improves defective pathways of cystinosis and has a good safety profile, and thus has potential as a treatment for nephropathic cystinosis and other renal lysosomal storage diseases.

Extracellular Vesicles as a Therapeutic Tool for Kidney Disease: Current Advances and Perspectives.

Extracellular vesicles (EVs) have been described as important mediators of cell communication, regulating several physiological processes, including tissue recovery and regeneration. In the kidneys, EVs derived from stem cells have been shown to support tissue recovery in diverse disease models and have been considered an interesting alternative to cell therapy. For this purpose, however, several challenges remain to be overcome, such as the requirement of a high number of EVs for human therapy and the need for optimization of techniques for their isolation and characterization. Moreover, the kidney's complexity and the pathological process to be treated require that EVs present a heterogeneous group of molecules to be delivered. In this review, we discuss the recent advances in the use of EVs as a therapeutic tool for kidney diseases. Moreover, we give an overview of the new technologies applied to improve EVs' efficacy, such as novel methods of EV production and isolation by means of bioreactors and microfluidics, bioengineering the EV content and the use of alternative cell sources, including kidney organoids, to support their transfer to clinical applications.

The Impact of Genetic Polymorphisms in Organic Cation Transporters on Renal Drug Disposition.

A considerable number of drugs and/or their metabolites are excreted by the kidneys through glomerular filtration and active renal tubule secretion via transporter proteins. Uptake transporters in the proximal tubule are part of the solute carrier (SLC) superfamily, and include the organic cation transporters (OCTs). Several studies have shown that specific genetic polymorphisms in OCTs alter drug disposition and may lead to nephrotoxicity. Multiple single nucleotide polymorphisms (SNPs) have been reported for the OCT genes (SLC22A1, SLC22A2 and SLC22A3), which can influence the proteins' structure and expression levels and affect their transport function. A gain-in-function mutation may lead to accumulation of drugs in renal proximal tubule cells, eventually leading to nephrotoxicity. This review illustrates the impact of genetic polymorphisms in OCTs on renal drug disposition and kidney injury, the clinical significances and how to personalize therapies to minimize the risk of drug toxicity.

Phase I/II Trial of a Combination of Anti-CD3/CD7 Immunotoxins for Steroid-Refractory Acute Graft-versus-Host Disease.

Effective therapies for treating patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD), particularly strategies that reduce the duration of immunosuppression following remission, are urgently needed. The investigated immunotoxin combination consists of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT), which induces in vivo depletion of T cells and natural killer (NK) cells and suppresses T cell receptor activation. We conducted a phase I/II trial to examine the safety and efficacy of CD3/CD7-IT in 20 patients with SR-aGVHD; 17 of these patients (85%) had severe SR-aGVHD, and all 20 patients had visceral organ involvement, including 18 (90%) with gastrointestinal (GI) involvement and 5 (25%) with liver involvement. A validated 2-biomarker algorithm classified the majority of patients (11 of 20) as high risk. On day 28 after the start of CD3/CD7-IT therapy, the overall response rate was 60% (12 of 20), with 10 patients (50%) achieving a complete response. The 6-month overall survival rate was 60% (12 of 20), including 64% (7 of 11) classified as high risk by biomarkers. The 1-week course of treatment with CD3/CD7-IT caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse TCR Vß repertoire, and preservation of Epstein-Barr virus- and cytomegalovirus-specific T cell clones. Furthermore, our results indicate that CD3/CD7-IT appeared to be safe and well tolerated, with a relatively low prevalence of manageable and reversible adverse events, primarily worsening of hypoalbuminemia, microangiopathy, and thrombocytopenia. These encouraging results suggest that CD3/CD7-IT may improve patient outcomes in patients with SR-aGVHD.

Organic anion transporters 1 and 3 influence cellular energy metabolism in renal proximal tubule cells.

Organic anion transporters (OATs) 1 and 3 are, besides being uptake transporters, key in several cellular metabolic pathways. The underlying mechanisms are largely unknown. Hence, we used human conditionally immortalized proximal tubule epithelial cells (ciPTEC) overexpressing OAT1 or OAT3 to gain insight into these mechanisms. In ciPTEC-OAT1 and -OAT3, extracellular lactate levels were decreased (by 77% and 71%, respectively), while intracellular ATP levels remained unchanged, suggesting a shift towards an oxidative phenotype upon OAT1 or OAT3 overexpression. This was confirmed by increased respiration of ciPTEC-OAT1 and -OAT3 (1.4-fold), a decreased sensitivity to respiratory inhibition, and characterized by a higher demand on mitochondrial oxidative capacity. In-depth profiling of tricarboxylic acid (TCA) cycle metabolites revealed reduced levels of intermediates converging into α-ketoglutarate in ciPTEC-OAT1 and -OAT3, which via 2-hydroxyglutarate metabolism explains the increased respiration. These interactions with TCA cycle metabolites were in agreement with metabolomic network modeling studies published earlier. Further studies using OAT or oxidative phosphorylation (OXPHOS) inhibitors confirmed our idea that OATs are responsible for increased use and synthesis of α-ketoglutarate. In conclusion, our results indicate an increased α-ketoglutarate efflux by OAT1 and OAT3, resulting in a metabolic shift towards an oxidative phenotype.

Quantitative Translation of Microfluidic Transporter in Vitro Data to in Vivo Reveals Impaired Albumin-Facilitated Indoxyl Sulfate Secretion in Chronic Kidney Disease.

Indoxyl sulfate (IxS), a highly albumin-bound uremic solute, accumulates in chronic kidney disease (CKD) due to reduced renal clearance. This study was designed to specifically investigate the role of human serum albumin (HSA) in IxS renal secretion via organic anion transporter 1 (OAT1) in a microfluidic system and subsequently apply quantitative translation of in vitro data to predict extent of change in IxS renal clearance in CKD stage IV relative to healthy. Conditionally immortalized human proximal tubule epithelial cells overexpressing OAT1 were incubated with IxS (5-200 µM) in the HSA-free medium or in the presence of either HSA or CKD-modified HSA. IxS uptake in the presence of HSA resulted in more than 20-fold decrease in OAT1 affinity (Km,u) and 37-fold greater in vitro unbound intrinsic clearance (CLint,u) versus albumin-free condition. In the presence of CKD-modified albumin, Km,u increased four-fold and IxS CLint,u decreased almost seven-fold relative to HSA. Fold-change in parameters exceeded differences in IxS binding between albumin conditions, indicating additional mechanism and facilitating role of albumin in IxS OAT1-mediated uptake. Quantitative translation of IxS in vitro OAT1-mediated CLint,u predicted a 60% decrease in IxS renal elimination as a result of CKD, in agreement with the observed data (80%). The findings of the current study emphasize the role of albumin in IxS transport via OAT1 and explored the impact of modifications in albumin on renal excretion via active secretion in CKD. For the first time, this study performed quantitative translation of transporter kinetic data generated in a novel microfluidic in vitro system to a clinically relevant setting. Knowledge gaps and future directions in quantitative translation of renal drug disposition from microphysiological systems are discussed.