Impact of hypoxia and reoxygenation on the extra/intracellular metabolome and on transporter expression in a human kidney proximal tubular cell line.

INTRODUCTION: Ischemia-reperfusion injury (IRI) induces several perturbations that alter immediate kidney graft function after transplantation and may affect long-term graft outcomes. Given the IRI-dependent metabolic disturbances previously reported, we hypothesized that proximal transporters handling endo/exogenous substrates may be victims of such lesions. OBJECTIVES: This study aimed to determine the impact of hypoxia/reoxygenation on the human proximal transport system through two semi-targeted omics analyses. METHODS: Human proximal tubular cells were cultured in hypoxia (6 or 24 h), each followed by 2, 24 or 48-h reoxygenation. We investigated the transcriptomic modulation of transporters. Using semi-targeted LC-MS/MS profiling, we characterized the extra/intracellular metabolome. Statistical modelling was used to identify significant metabolic variations. RESULTS: The expression profile of transporters was impacted during hypoxia (y + LAT1 and OCTN2), reoxygenation (MRP2, PEPT1/2, rBAT, and OATP4C1), or in both conditions (P-gp and GLUT1). The P-gp and GLUT1 transcripts increased (FC (fold change) = 2.93 and 4.11, respectively) after 2-h reoxygenation preceded by 24-h hypoxia. We observed a downregulation (FC = 0.42) of y+LAT1 after 24-h hypoxia, and of PEPT2 after 24-h hypoxia followed by 2-h reoxygenation (FC = 0.40). Metabolomics showed that hypoxia altered the energetic pathways. However, intracellular metabolic homeostasis and cellular exchanges were promptly restored after reoxygenation. CONCLUSION: This study provides insight into the transcriptomic response of the tubular transporters to hypoxia/reoxygenation. No correlation was found between the expression of transporters and the metabolic variations observed. Given the complexity of studying the global tubular transport systems, we propose that further studies focus on targeted transporters.

A multi-omics investigation of tacrolimus off-target effects on a proximal tubule cell-line.

INTRODUCTION: Tacrolimus, an immunosuppressive drug prescribed to a majority of organ transplant recipients is nephrotoxic, through still unclear mechanisms. This study on a lineage of proximal tubular cells using a multi-omics approach aims to detect off-target pathways modulated by tacrolimus that can explain its nephrotoxicity. METHODS: LLC-PK1 cells were exposed to 5 µM of tacrolimus for 24 h in order to saturate its therapeutic target FKBP12 and other high-affine FKBPs and favour its binding to less affine targets. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, as well as of the other gluconeogenesis-limiting enzymes FBP1 and FBP2, was measured using RT-qPCR. Cell viability with this concentration of tacrolimus was further checked until 72 h. RESULTS: In our cell model of acute exposure to a high concentration of tacrolimus, different metabolic pathways were impacted including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p < 0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis and acid-base balance control. DISCUSSION: The variations found using a multi-omics pharmacological approach clearly point towards a dysregulation of energy production and decreased gluconeogenesis, a hallmark of chronic kidney disease which may also be an important toxicity pathway of tacrolimus.

Blood-brain barrier perturbations by uremic toxins: Key contributors in chronic kidney disease-induced neurological disorders?

Chronic kidney disease is multifactorial and estimated to affect more than 840 million people worldwide constituting a major global health crisis. The number of patients will continue to rise mostly because of the aging population and the increased prevalence of comorbidities such as diabetes and hypertension. Patients with advanced stages display a loss of kidney function leading to an accumulation of, a.o. protein-bound uremic toxins that are poorly eliminated by renal replacement therapies. This systemic retention of toxic metabolites, known as the uremic syndrome, affects other organs. Indeed, neurological complications such as cognitive impairment, uremic encephalopathy, and anxiety have been reported in chronic kidney disease patients. Several factors are involved, including hemodynamic disorders and blood-brain barrier (BBB) impairment. The BBB guarantees the exchange of solutes between the blood and the brain through a complex cellular organization and a diverse range of transport proteins. We hypothesize that the increased exposure of the brain to protein-bound uremic toxins is involved in BBB disruption and induces a perturbation in the activity of endothelial membrane transporters. This phenomenon could play a part in the evolution of neurological disorders driven by this kidney-brain crosstalk impairment. In this review, we present chronic kidney disease-induced neurological complications by focusing on the pathological relationship between the BBB and protein-bound uremic toxins. The importance of mechanistically delineating the impact of protein-bound uremic toxins on BBB integrity and membrane drug transporter expression and function in brain endothelial capillary cells is highlighted. Additionally, we put forward current knowledge gaps in the literature.

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale.

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. µs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.

Pharmacological hypothesis: A recombinant probiotic for taming bacterial ß-glucuronidase in drug-induced enteropathy.

Advances in pharmacomicrobiomics have shed light on the pathophysiology of drug-induced enteropathy associated with the therapeutic use of certain non-steroidal anti-inflammatory drugs, anticancer chemotherapies and immunosuppressants. The toxicity pathway results from the post-glucuronidation release and digestive accumulation of an aglycone generated in the context of intestinal dysbiosis characterized by the expansion of ß-glucuronidase-expressing bacteria. The active aglycone could trigger direct or indirect inflammatory signaling on the gut epithelium. Therefore, taming bacterial ß-glucuronidase (GUS) activity is a druggable target for preventing drug-induced enteropathy. In face of the limitations of antibiotic strategies that can worsen intestinal dysbiosis and impair immune functions, we hereby propose the use of a recombinant probiotic capable of mimicking repressive conditions of GUS through an inducible plasmid vector.

Is Intestinal Dysbiosis-Associated With Immunosuppressive Therapy a Key Factor in the Pathophysiology of Post-Transplant Diabetes Mellitus?

Post-transplant diabetes mellitus (PTDM) is one of the most common and deleterious comorbidities after solid organ transplantation (SOT). Its incidence varies depending on the organs transplanted and can affect up to 40% of patients. Current research indicates that PTDM shares several common features with type 2 diabetes mellitus (T2DM) in non-transplant populations. However, the pathophysiology of PTDM is still poorly characterized. Therefore, ways should be sought to improve its diagnosis and therapeutic management. A clear correlation has been made between PTDM and the use of immunosuppressants. Moreover, immunosuppressants are known to induce gut microbiota alterations, also called intestinal dysbiosis. Whereas the role of intestinal dysbiosis in the development of T2DM has been well documented, little is known about its impacts on PTDM. Functional alterations associated with intestinal dysbiosis, especially defects in pathways generating physiologically active bacterial metabolites (e.g., short-chain fatty acids, trimethylamine N-oxide, indole and kynurenine) are known to favour several metabolic disorders. This publication aims at discussing the potential role of intestinal dysbiosis and dysregulation of bacterial metabolites associated with immunosuppressive therapy in the occurrence of PTDM.

Insights into the structure and function of the human organic anion transporter 1 in lipid bilayer membranes.

The human SLC22A6/OAT1 plays an important role in the elimination of a broad range of endogenous substances and xenobiotics thus attracting attention from the pharmacological community. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about hOAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by µs-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called "charge-relay system" that works as molecular switches modulating the conformation. The principal element of the event points at interactions of charged residues that appear crucial for the transporter dynamics and function. Moreover, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein interactions. MD simulations supported the pivotal role of phosphatidylethanolamine components to the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.

Perfusate Metabolomics Content and Expression of Tubular Transporters During Human Kidney Graft Preservation by Hypothermic Machine Perfusion.

BACKGROUND: Ischemia-related injury during the preimplantation period impacts kidney graft outcome. Evaluating these lesions by a noninvasive approach before transplantation could help us to understand graft injury mechanisms and identify potential biomarkers predictive of graft outcomes. This study aims to determine the metabolomic content of graft perfusion fluids and its dependence on preservation time and to explore whether tubular transporters are possibly involved in metabolomics variations. METHODS: Kidneys were stored on hypothermic perfusion machines. We evaluated the metabolomic profiles of perfusion fluids (n = 35) using liquid chromatography coupled with tandem mass spectrometry and studied the transcriptional expression of tubular transporters on preimplantation biopsies (n = 26), both collected at the end of graft perfusion. We used univariate and multivariate analyses to assess the impact of perfusion time on these parameters and their relationship with graft outcome. RESULTS: Seventy-two metabolites were found in preservation fluids at the end of perfusion, of which 40% were already present in the native conservation solution. We observed an increase of 23 metabolites with a longer perfusion time and a decrease of 8. The predictive model for time-dependent variation of metabolomics content showed good performance (R 2 = 76%, Q 2 = 54%, accuracy = 41%, and permutation test significant). Perfusion time did not affect the mRNA expression of transporters. We found no correlation between metabolomics and transporters expression. Neither the metabolomics content nor transporter expression was predictive of graft outcome. CONCLUSIONS: Our results call for further studies, focusing on both intra- and extratissue metabolome, to investigate whether transporter alterations can explain the variations observed in the preimplantation period.

Effects of Ischemia-Reperfusion on Tubular Cell Membrane Transporters and Consequences in Kidney Transplantation.

Ischemia-reperfusion (IR)-induced acute kidney injury (IRI) is an inevitable event in kidney transplantation. It is a complex pathophysiological process associated with numerous structural and metabolic changes that have a profound influence on the early and the late function of the transplanted kidney. Proximal tubular cells are particularly sensitive to IRI. These cells are involved in renal and whole-body homeostasis, detoxification processes and drugs elimination by a transporter-dependent, transcellular transport system involving Solute Carriers (SLCs) and ATP Binding Cassettes (ABCs) transporters. Numerous studies conducted mainly in animal models suggested that IRI causes decreased expression and activity of some major tubular transporters. This could favor uremic toxins accumulation and renal metabolic alterations or impact the pharmacokinetic/toxicity of drugs used in transplantation. It is of particular importance to understand the underlying mechanisms and effects of IR on tubular transporters in order to improve the mechanistic understanding of IRI pathophysiology, identify biomarkers of graft function or promote the design and development of novel and effective therapies. Modulation of transporters' activity could thus be a new therapeutic opportunity to attenuate kidney injury during IR.

Cyclosporine A inhibits MRTF-SRF signaling through Na+/K+ ATPase inhibition and actin remodeling.

Calcineurin inhibitors (CNI) are the pillars of immunosuppression in transplantation. However, they display a potent nephrotoxicity whose mechanisms remained widely unsolved. We used an untargeted quantitative proteomic approach (iTRAQ technology) to highlight new targets of CNI in renal proximal tubular cells (RPTCs). CNI-treated RPTCs proteome displayed an over-representation of actin-binding proteins with a CNI-specific expression profile. Cyclosporine A (CsA) induced F-actin remodeling and depolymerization, decreased F-actin-stabilizing, polymerization-promoting cofilin (CFL) oligomers, and inhibited the G-actin-regulated serum response factor (SRF) pathway. Inhibition of CFL canonical phosphorylation pathway reproduced CsA effects; however, S3-R, an analogue of the phosphorylation site of CFL prevented the effects of CsA which suggests that CsA acted independently from the canonical CFL regulation. CFL is known to be regulated by the Na+/K+-ATPase. Molecular docking calculations identified two inhibiting sites of CsA on Na+/K+-ATPase and a 23% decrease in Na+/K+-ATPase activity of RPTCs was observed with CsA. Ouabain, a specific inhibitor of Na+/K+-ATPase also reproduced CsA effects on actin organization and SRF activity. Altogether, these results described a new original pathway explaining CsA nephrotoxicity.