Renal Secretion Research Articles

Pseudo-Worsening of Kidney Function Due to Inhibition of Renal Creatinine Secretion: Quality of Information Provided in Prescribing Information/SmPC.

Determination of serum creatinine concentrations and subsequent calculation of estimated glomerular filtration rates (eGFR) is a cornerstone of clinical medicine. Crucial clinical decisions such as drug treatment discontinuations are based on eGFR calculated from serum creatinine measurements. However, creatinine is not only filtered in the kidneys, but also actively secreted into urine. Creatinine transporters such as OCT2, OCT3, MATE1, MATE2-K, and OAT2 expressed in proximal tubular cells are responsible for active renal secretion of creatinine. Multiple drugs (e.g., oral antitumor drugs) inhibit these transporters thereby causing a pseudo-worsening of kidney function with an increase in serum creatinine concentrations and a decrease in eGFR while other methods for eGFR determination (e.g., by cystatin C) reveal normal kidney function. Since US Prescribing Information (PI) and European Summaries of Product Characteristics (SmPCs) are the most relevant source of information for physicians, we investigated the quality of information in US PI/German SmPCs of drugs with clear evidence for pseudo-worsening of kidney function. 514 drugs putatively interacting with creatinine transporters were identified. For 149 of those drugs, an increase in serum creatinine concentrations has been described. Available data confirmed the existence of pseudo-worsening of kidney function for 30 of those drugs, for the remaining 119 drugs existing data are insufficient. Only 23.5% (12/51) of the 30 drugs' PI/SmPCs contained unambiguous statements on this proven pseudo-worsening of kidney function and gave clear recommendations for clinical management. Taken together, inadequate information provided in PI or SmPCs on the pseudo-worsening of kidney function poses patients at unnecessary risks.

Prediction of Inhibitory Activity against the MATE1 Transporter via Combined Fingerprint- and Physics-Based Machine Learning Models.

Renal secretion plays an important role in excretion of drug from the kidney. Two major transporters known to be highly involved in renal secretion are MATE1/2 K and OCT2, the former of which is highly related to drug-drug interactions. Among published in silico models for MATE inhibitors, a previous model obtained a ROC-AUC value of 0.78 using high throughput percentage inhibition data [J. Med. Chem. 2013, 56(3), 781-795] which we aimed to improve upon here using a combined fingerprint and physics-based approach. To this end, we collected 225 publicly available compounds with pIC50 values against MATE1. Subsequently, on the one hand, we performed a physics-based approach using an Alpha-Fold protein structure, from which we obtained MM-GB/SA scores for those compounds. On the other hand, we built Random Forest (RF) and message passing neural network models using extended-connectivity fingerprints with radius 4 (ECFP4) and chemical structures as graphs, respectively, which also included MM-GB/SA scores as input variables. In a five-fold cross-validation with a separate test set, we found that the best predictivity for the hold-out test was observed in the RF model (including ECFP4 and MM-GB/SA data) with an ROC-AUC of 0.833 ± 0.036; while that of the MM-GB/SA regression model was 0.742. However, the MM-GB/SA model did not show a dependency of the performance on the particular chemical space being predicted. Additionally, via structural interaction fingerprint analysis, we identified interacting residues with inhibitor as identical for those with noninhibitors, including substrates, such as Gln49, Trp274, Tyr277, Tyr299, Ile303, and Tyr306. The similar binding modes are consistent with the observed similar IC50 value inhibitor when using different substrates experimentally, and practically, this can release the experimental scientists from bothering of selecting substrates for MATE1. Hence, we were able to build highly predictive classification models for MATE1 inhibitory activity with both ECFP4 and MM-GB/SA score as input features, which is fit-for-purpose for use in the drug discovery process.

Identification of human-specific amino acid residues governing atenolol transport via organic cation transporter 2

Organic cation transporter 2 (OCT2/SLC22A2) is predominantly localized on the basolateral membranes of renal tubular epithelial cells and plays a crucial role in the renal secretion of various cationic drugs. Although variations in substrate selectivity among renal organic cation transport systems across species have been reported, the characteristics of OCT2 remain unclear. In this study, we demonstrated that atenolol, a β1-selective adrenergic antagonist, is transported almost exclusively by human OCT2, contrasting with OCT2s from other selected species. Using chimeric constructs between human OCT2 (hOCT2) and the highly homologous monkey OCT2 (monOCT2), along with site-directed mutagenesis, we identified non-conserved amino acids Val8, Ala31, Ala34, Tyr222, Tyr245, Ala270, Ile394, and Leu503 as pivotal for hOCT2-mediated atenolol transport. Kinetic analysis revealed that atenolol was transported by hOCT2 with a 12-fold lower affinity than MPP+, a typical OCT2 substrate. The inhibitory effect of atenolol on MPP+ transport was 6200-fold lower than that observed for MPP+ on atenolol transport. Additionally, we observed weaker inhibitory effects on MPP+ transport compared to atenolol transport with ten different OCT2 substrates. Altogether, this study suggests that eight hOCT2-specific amino acids constitute the low-affinity recognition site for atenolol transport, indicating differences in OCT2-mediated drug elimination between humans and highly homologous monkeys. Our findings underscore the importance of understanding species-specific differences in drug transport mechanisms, shedding light on potential variations in drug disposition and aiding in drug development.

Albumin-Mediated Drug Uptake by Organic Anion Transporter 1/3 Is Real: Implications for the Prediction of Active Renal Secretion Clearance.

Modulation of the transport-mediated active uptake by human serum albumin (HSA) for highly protein-bound substrates has been reported and improved the in vitro-to-in vivo extrapolation (IVIVE) of hepatic clearance. However, evidence for the relevance of such a phenomenon in the case of renal transporters is sparse. In this study, transport of renal organic anion transporter 1 or 3 (OAT1/3) substrates into conditionally immortalized proximal tubular epithelial cells transduced with OAT1/3 was measured in the presence and absence of 1 and 4% HSA while keeping the unbound substrate concentration constant (based on measured fraction unbound, fu,inc). In the presence of 4% HSA, the unbound intrinsic active uptake clearance (CLint,u,active) of six highly protein-bound substrates increased substantially relative to the HSA-free control (3.5- to 122-fold for the OAT1 CLint,u,active, and up to 28-fold for the OAT3 CLint,u,active). The albumin-mediated uptake effect (fold increase in CLint,u,active) was more pronounced with highly bound substrates compared to no effect seen for weakly protein-bound substrates adefovir (OAT1-specific) and oseltamivir carboxylate (OAT3-specific). The relationship between OAT1/3 CLint,u,active and fu,inc agreed with the facilitated-dissociation model; a relationship was established between the albumin-mediated fold change in CLint,u,active and fu,inc for both the OAT1 and OAT3, with implications for IVIVE modeling. The relative activity factor and the relative expression factor based on global proteomic quantification of in vitro OAT1/3 expression were applied for IVIVE of renal clearance. The inclusion of HSA improved the bottom-up prediction of the level of OAT1/3-mediated secretion and renal clearance (CLsec and CLr), in contrast to the underprediction observed with the control (HSA-free) scenario. For the first time, this study confirmed the presence of the albumin-mediated uptake effect with renal OAT1/3 transporters; the extent of the effect was more pronounced for highly protein-bound substrates. We recommend the inclusion of HSA in routine in vitro OAT1/3 assays due to considerable improvements in the IVIVE of CLsec and CLr.

Utilising Endogenous Biomarkers in Drug Development to Streamline the Assessment of Drug-Drug Interactions Mediated by Renal Transporters: A Pharmaceutical Industry Perspective.

The renal secretion of many drugs is facilitated by membrane transporters, including organic cation transporter 2, multidrug and toxin extrusion protein 1/2-K and organic anion transporters 1 and 3. Inhibition of these transporters can reduce renal excretion of drugs and thereby pose a safety risk. Assessing the risk of inhibition of these membrane transporters by investigational drugs remains a key focus in the evaluation of drug-drug interactions (DDIs). Current methods to predict DDI risk are based on generating in vitro data followed by a clinical assessment using a recommended exogenous probe substrate for the individual drug transporter. More recently, monitoring plasma-based and urine-based endogenous biomarkers to predict transporter-mediated DDIs in early phase I studies represents a promising approach to facilitate, improve and potentially avoid conventional clinical DDI studies. This perspective reviews the evidence for use of these endogenous biomarkers in the assessment of renal transporter-mediated DDI, evaluates how endogenous biomarkers may help to expand the DDI assessment toolkit and offers some potential knowledge gaps. A conceptual framework for assessment that may complement the current paradigm of predicting the potential for renal transporter-mediated DDIs is outlined.

Evaluation of 14 PFAS for permeability and organic anion transporter interactions: Implications for renal clearance in humans

Per- and polyfluoroalkyl substances (PFAS) encompass a diverse group of synthetic fluorinated chemicals known to elicit adverse health effects in animals and humans. However, only a few studies investigated the mechanisms underlying clearance of PFAS. Herein, the relevance of human renal transporters and permeability to clearance and bioaccumulation for 14 PFAS containing three to eleven perfluorinated carbon atoms (ηpfc = 3–11) and several functional head-groups was investigated. Apparent permeabilities and interactions with human transporters were measured using in vitro cell-based assays, including the MDCK-LE cell line, and HEK293 stable transfected cell lines expressing organic anion transporter (OAT) 1–4 and organic cation transporter (OCT) 2. The results generated align with the Extended Clearance Classification System (ECCS), affirming that permeability, molecular weight, and ionization serve as robust predictors of clearance and renal transporter engagement. Notably, PFAS with low permeability (ECCS 3A and 3B) exhibited substantial substrate activity for OAT1 and OAT3, indicative of active renal secretion. Furthermore, we highlight the potential contribution of OAT4-mediated reabsorption to the renal clearance of PFAS with short ηpfc, such as perfluorohexane sulfonate (PFHxS). Our data advance our mechanistic understanding of renal clearance of PFAS in humans, provide useful input parameters for toxicokinetic models, and have broad implications for toxicological evaluation and regulatory considerations.

Inhibitory effect of flavonoids on multidrug and toxin extrusion protein 1 function: Implications for food/herb-drug interaction and drug-induced kidney injury.

Multidrug and toxin extrusion protein 1 (MATE1), an efflux transporter mainly expressed in renal proximal tubules, mediates the renal secretion of organic cationic drugs. The inhibition of MATE1 will impair the excretion of drugs into the tubular lumen, leading to the accumulation of nephrotoxic drugs in the kidney and consequently potentiating nephrotoxicity. Screening and identifying potent MATE1 inhibitors can predict or minimize the risk of drug-induced kidney injury. Flavonoids, a group of polyphenols commonly found in foodstuffs and herbal products, have been reported to cause transporter-mediated food/herb-drug interactions. Our objective was to investigate the inhibitory effects of flavonoids on MATE1 in vitro and in vivo and to assess the effects of flavonoids on cisplatin-induced kidney injury. Thirteen flavonoids exhibited significant transport activity inhibition (>50%) on MATE1 in MATE1-MDCK cells. Among them, the six strongest flavonoid inhibitors, including irisflorentin, silymarin, isosilybin, sinensetin, tangeretin, and nobiletin, markedly increased cisplatin cytotoxicity in these cells. In cisplatin-induced in vivo renal injury models, irisflorentin, isosilybin, and sinensetin also increased serum creatinine and blood urea nitrogen levels to different degrees, especially irisflorentin, which exhibited the most potent nephrotoxicity with cisplatin. The pharmacophore model indicated that the hydrogen bond acceptors at the 3, 5, and 7 positions may play a critical role in the inhibitory effect of flavonoids on MATE1. Our findings provide helpful information for predicting the potential risks of flavonoid-containing food/herb-drug interactions and avoiding the exacerbation of drug-induced kidney injury via MATE1 mediation.

Distal Renal Tubular Acidosis Mutants of the Kidney Anion Exchanger Disrupt Cytosolic pH and Cause Abnormal Autophagy in Renal Epithelial Cells

Background: Distal renal tubular acidosis (dRTA) is a disease caused by impaired renal acid secretion leading to metabolic acidosis and subsequent biochemical derangements which facilitate formation of kidney stones. This can result from a mutation in the kidney anion exchanger 1 (kAE1) protein, a chloride bicarbonate exchanger in the alpha intercalated cells (A-ICs) of the kidney collecting duct. Studies in human dRTA patients and mouse dRTA models have shown an unexplained depletion in A-ICs. dRTA mouse kidney sections also showed an accumulation of autophagy markers in the remaining A-ICs. Objective and Hypothesis: To better understand the link between dRTA kAE1 mutant expression and A-IC depletion, we hypothesised that dRTA mutant expression activates IC loss through abnormal autophagy and apoptosis. Methods: We used mouse inner medullary collecting duct (mIMCD3) cells expressing two dRTA kAE1 mutants (R589H or S525F) for in vitro studies and two dRTA kAE1 knockin mice L919X and R607H, the murine equivalents of human R901X and R589H mutations, respectively for in vivo studies. We assessed intracellular pH using the ratiometric pH-sensitive fluorescent probe BCECF-AM. Using live cell confocal microscopy, we measured autophagy flux with the eGFP-RFP-LC3 construct. Real time measurements of extracellular acidification and oxygen consumption rates were used to determine cellular glycolytic and oxidative ATP production rates. Results: In vitro, kAE1 R589H and S525F dRTA mutants had significantly more alkaline intracellular pH compared to wild type (WT). Both mutants had higher autophagy induction and accumulation of autolysosomes at steady state. Chemically acidifying cytosolic pH of mutants reversed abnormal autophagy in mutant cells. The kAE1 R589H mutant had significantly lower oxidative ATP production rate whereas S525F mutant had significantly lower glycolytic ATP production rate compared to WT. In vivo, dRTA R607H homozygous mutant mice showed reduced abundance of the vacuolar proton ATPase and of mature Cathepsin D compared to WT. Conclusion: These findings confirm that kAE1 mutant expression alters cytosolic pH and induces abnormal autophagy as a result of reduced overall ATP production and defective lysosomal function. Future work will analyse the processing/traffcking of lysosomal cathepsins and mitochondrial structure and function in mutant cells, and assessing collecting duct lysosome numbers in kidney sections by immunohistochemistry. Our work provides a lead explaining the loss of A-ICs observed in dRTA patients. Canadian Institutes of Health Research (CIHR), Graduate Student Engagement Scholarship — University of Alberta. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Investigation of Basolateral Targeting Micelles for Drug Delivery Applications in Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a complex disorder characterized by uncontrolled renal cyst growth, leading to kidney function decline. The multifaceted nature of ADPKD suggests that single-pathway interventions using individual small molecule drugs may not be optimally effective. As such, a strategy encompassing combination therapy that addresses multiple ADPKD-associated signaling pathways could offer synergistic therapeutic results. However, severe off-targeting side effects of small molecule drugs pose a major hurdle to their clinical transition. To address this, we identified four drug candidates from ADPKD clinical trials, bardoxolone methyl (Bar), octreotide (Oct), salsalate (Sal), and pravastatin (Pra), and incorporated them into peptide amphiphile micelles containing the RGD peptide (GRGDSP), which binds to the basolateral surface of renal tubules via integrin receptors on the extracellular matrix. We hypothesized that encapsulating drug combinations into RGD micelles would enable targeting to the basolateral side of renal tubules, which is the site of disease, via renal secretion, leading to superior therapeutic benefits compared to free drugs. To test this, we first evaluated the synergistic effect of drug combinations using the 20% inhibitory concentration for each drug (IC20) on renal proximal tubule cells derived from Pkd1flox/-:TSLargeT mice. Next, we synthesized and characterized the RGD micelles encapsulated with drug combinations and measured their in vitro therapeutic effects via a 3D PKD growth model. Upon both IV and IP injections in vivo, RGD micelles showed a significantly higher accumulation in the kidneys compared to NT micelles, and the renal access of RGD micelles was significantly reduced after the inhibition of renal secretion. Specifically, both Bar+Oct and Bar+Sal in the RGD micelle treatment showed enhanced therapeutic efficacy in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre) with a significantly lower KW/BW ratio and cyst index as compared to PBS and free drug-treated controls, while other combinations did not show a significant difference. Hence, we demonstrate that renal targeting through basolateral targeting micelles enhances the therapeutic potential of combination therapy in genetic kidney disease.

Use of a Double-Transfected System to Predict hOCT2/hMATE1-Mediated Renal Drug-Drug Interactions.

Accurate predictions of renal drug-drug interactions (DDIs) mediated by the human organic cation transporter 2 (hOCT2) and multidrug and toxin extrusion proteins (hMATEs) remain challenging. Current DDI evaluation using plasma maximal unbound inhibitor concentrations (Imax,u) and IC50 values determined in single transporter-transfected cells frequently leads to false or overprediction especially for hMATE1. Emerging evidence suggests intracellular unbound inhibitor concentration may be more relevant for hMATE1 inhibition in vivo. However, determination of intrarenal inhibitor concentrations is impractical. Here, we explored the use of hOCT2/hMATE1 double-transfected Madin-Darby canine kidney (MDCK) cells as a new in vitro tool for DDI risk assessment. Our results showed that potent in vitro hMATE1 inhibitors (hydroxychloroquine, brigatinib, and famotidine) failed to inhibit metformin B-to-A flux in the double-transfected system. On the other side, the classic hOCT2/hMATE1 inhibitors, pyrimethamine and cimetidine, dose-dependently inhibited metformin apparent B-to-A permeability (Papp). The different behaviors of these hMATE1 inhibitors in the double-transfected system can be explained by their different ability to gain intracellular access either via passive diffusion or transporter-mediated uptake. A new parameter (IC50,flux) was proposed reflecting the inhibitor's potency on overall hOCT2/hMATE1-mediated tubular secretion. The IC50,flux values significantly differ from the IC50 values determined in single transporter-transfected cells. Importantly, the IC50,flux accurately predicted in vivo DDIs (within 2-fold) when used in a static model. Our data demonstrated that the IC50,flux approach circumvents the need to measure intracellular inhibitor concentrations and more accurately predicted hOCT2/hMATE1-mediated renal DDIs. This system represents a new approach that could be used for improved DDI assessment during drug development. SIGNIFICANCE STATEMENT: This study demonstrated that flux studies in double-transfected MDCK cells and the IC50,flux represents a better approach to assess in vivo DDI potential for the renal organic cation secretion system. This study highlights the importance of inhibitor intracellular accessibility for accurate prediction of hMATE1-mediated renal DDIs. This approach has the potential to identify in vitro hMATE1 inhibitors that are unlikely to result in in vivo DDIs, thus reducing the burden of unnecessary and costly clinical DDI investigations.

Significance of Organic Anion Transporter 2 and Organic Cation Transporter 2 in Creatinine Clearance: Mechanistic Evaluation Using Freshly Prepared Human Primary Renal Proximal Tubule Cells.

Creatinine, a clinical marker for kidney function, is predominantly cleared by glomerular filtration, with active tubular secretion contributing to about 30% of its renal clearance. Recent studies suggested the potential involvement of organic anion transporter (OAT)2, in addition to the previously known organic cation transporter (OCT)2-mediated basolateral uptake, in creatinine active secretion. Here we characterized the transport mechanisms of creatinine using transfected human embryonic kidney (HEK)293 cells and freshly prepared human primary renal proximal tubule epithelial cells (hPTCs). Creatinine showed transport by OAT2 in transfected HEK293 cells. In addition, both creatinine and metformin showed transport by OCT2 and multidrug and toxin extrusion pump (MATE)1 and MATE2K, while penciclovir was selective for OAT2. Time-dependent cell accumulation was observed for creatinine and metformin in hPTCs. Their accumulation was increased by pyrimethamine but inhibited by decynium-22, likely due to differential inhibition of OCT2 versus MATEs. Additionally, indomethacin (an OAT2 inhibitor) reduced penciclovir uptake (∼75%) in hPTCs illustrating functional OAT2 activity. However, no modulation of creatinine and metformin cell accumulation was apparent with indomethacin. Creatinine transport characteristics in the presence of inhibitors approached those of metformin, an OCT2/MATE substrate, but were distinct from those of penciclovir, an OAT2-selective substrate. Moreover, indomethacin showed no significant effect on the basolateral-to-apical transport and net secretion of creatinine across hPTC monolayers. Collectively, the functional studies suggest OCT2 as the primary basolateral uptake mechanism and that OAT2 has a minimal role, in creatinine renal secretion. Our results highlight the utility of hPTCs to enable the functional assessment of renal transport mechanisms. SIGNIFICANCE STATEMENT: Our results obtained with primary hPTCs indicate that OCT2/MATE (vs. OAT2) play a major role in the active renal secretion of creatinine. Quantitative pharmacokinetic models should therefore focus on OCT2/MATE when describing serum creatinine and creatinine clearance modulation by inhibitor drugs and genotype- or disease-related activity changes. The present study highlights the utility of freshly isolated hPTCs to support solute carrier phenotyping to enable the functional assessment of renal transport mechanisms.

Iron status and the Pi/FGF-23-axis in HFrEF in relation to renal function

Abstract Background Recent studies linked dysregulated phosphate metabolism, fibroblast growth factor-23 (FGF-23) and iron deficiency (ID) with worse clinical outcome and mortality in heart failure with reduced ejection fraction (HFrEF). These factors are intertwined as FGF-23 regulates inorganic phosphate (Pi) through increased renal phosphate secretion and is stimulated by elevated Pi levels and ID. This interplay has recently gained attention as intravenous iron supplementation has hypophosphataemia as a side effect. The relationship between ID and the Pi/FGF-23-axis, particularly concerning renal function, has not been investigated in detail. Purpose We aimed to evaluate the relationship between iron status, Pi and FGF-23 in relation to renal function in HFrEF. Methods Stable HFrEF patients were enrolled at our outpatient clinic between 2018 and 2023 in a prospective registry. Baseline characteristics, medications and laboratory values were documented. For this study patients with complete phosphate, intact FGF-23 and iron status were analyzed (n = 429). Patients were divided into three groups based on their renal function determined by estimated glomerular filtration rate (GFR): GFR > 60, GFR 60-30, GFR < 30. Results The median age was 65 (IQR 52-74), 310 (72%) patients were male, median NT-proBNP was 1657 pg/ml (IQR 512-4116) and median GFR was 57 ml/min/BSA (IQR 37-74). Of the 429 patients, 180 (42%) suffered from ID. Pi levels did not vary based on NYHA stage and showed only a weak correlation with NT-proBNP (r = 0.13, p = 0.008). In contrast, FGF-23 increased with NYHA stage (NYHA I vs NYHA II vs NYHA III/IV: 69.9 (IQR 50-90.2) vs 82.4 (IQR 58.9-115.8) vs 104.2 (IQR 77-199.5), p<0,0001) and correlated positively with NT-proBNP (r = 0.43, p<0.0001) (Figure 1 a, b). Patients with a GFR < 30ml/min/BSA had significantly higher concentrations of Pi and FGF-23 compared to GFR 30-60 ml/min/BSA or > 60ml/min/BSA [Pi: 1.36 mmol/l (IQR 1.15-1.61) vs 1.145 mmol/l (IQR 0.96-1.27) vs 1.1 mmol/l (IQR 0.98-1.25), p<0.0001; FGF-23: 253.1 pg/ml (141.1-936.2) vs 94.3 pg/ml (IQR 75.43-121.5) vs 71.7 pg/ml (IQR 53.18-89.33), p<0.0001)] (Figure 1 c, d). While there was no difference regarding mortality according to Pi levels, patients in the highest FGF-23 tertile had excess mortality (p=ns and p = 0.0002, log rank test) (Figure 1 e). Interestingly, Pi and FGF-23 levels did not differ between patients without and with ID (p=ns for both, Figure 2 a). No correlation could be shown between transferrin saturation and Pi or FGF-23 (p=ns, Figure 2 b). Conclusion The regulation of Pi and FGF-23 is dependent on renal function. Patients with a GFR < 30ml/min/BSA exhibited the highest levels of both FGF-23 and Pi. The phosphaturic effect of FGF-23 seems to rely on renal function, as elevated FGF-23 cannot sufficiently decrease Pi in patients with a GFR < 30ml/min/BSA. No correlation between iron status and FGF23/Pi could be identified for HFrEF.Figure 1Figure 2

Development of 4-Pyridoxic Acid PBPK Model to Support Biomarker-Informed Evaluation of OAT1/3 Inhibition and Effect of Chronic Kidney Disease.

Monitoring endogenous biomarkers is increasingly used to evaluate transporter-mediated drug-drug interactions (DDIs) in early drug development and may be applied to elucidate changes in transporter activity in disease. 4-pyridoxic acid (PDA) has been identified as the most sensitive plasma endogenous biomarker of renal organic anion transporters (OAT1/3). Increase in PDA baseline concentrations was observed after administration of probenecid, a strong clinical inhibitor of OAT1/3 and also in patients with chronic kidney disease (CKD). The aim of this study was to develop and verify a physiologically-based pharmacokinetic (PBPK) model of PDA, to predict the magnitude of probenecid DDI and predict the CKD-related changes in PDA baseline. The PBPK model for PDA was first developed in healthy population, building on from previous population pharmacokinetic modeling, and incorporating a mechanistic kidney model to consider OAT1/3-mediated renal secretion. Probenecid PBPK model was adapted from the Simcyp database and re-verified to capture its dose-dependent pharmacokinetics (n = 9 studies). The PBPK model successfully predicted the PDA plasma concentrations, area under the curve, and renal clearance in healthy subjects at baseline and after single/multiple probenecid doses. Prospective simulations in severe CKD predicted successfully the increase in PDA plasma concentration relative to healthy (within 2-fold of observed data) after accounting for 60% increase in fraction unbound in plasma and additional 50% decline in OAT1/3 activity beyond the decrease in glomerular filtration rate. The verified PDA PBPK model supports future robust evaluation of OAT1/3 DDI in drug development and increases our confidence in predicting exposure and renal secretion in patients with CKD.

Preliminary study on the material basis and mechanism underlying uric acid reduction by Thlaspi arvense L.

Ethnopharmacological relevanceIn the Tibetan region of China, Thlaspi arvense L. is utilized for the prevention and treatment of hyperuricemia (HUA). Thlaspi arvense has been shown to lower uric acid levels in HUA rats in preliminary studies. However, the active components and mechanisms that account for its therapeutic effects remain elusive. Aim of studyNetwork pharmacology, ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS), mRNA-sequencing, and quantitative reverse transcription PCR (RT-PCR) were used to investigate the active ingredients of Thlaspi arvense against HUA in rats and elucidate the underlying mechanisms in this study. Materials and methodsA HUA rat model was established by a combination of intraperitoneal injection of potassium oxonate and intragastric administration of yeast extract. In the control and model groups, gastric gavage was performed to administer a normal saline solution, 4.5 mg kg−1 benzbromarone in the positive drug group, and 3.5 g kg−1Thlaspi arvense in the Thlaspi arvense group. After which network pharmacology and UPLC-Q-TOF-MS were employed to explore the active ingredients underlying the lowering of uric acid in Thlaspi arvense. In addition, mRNA-sequencing, network pharmacology and RT-PCR were applied to uncover Thlaspi arvense’s mechanism of uric acid reduction. ResultsThe results showed that a two-week administration of the effective constituents of Thlaspi arvense led to a significant improvement in HUA rats, including lower serum levels of uric acid (UA), xanthine oxidase (XOD), creatinine (CREA), carbamide (UREA), aspartate aminotransferase (AST), alanine transaminase (ALT), and liver tissue activities of XOD, ADA, super (MDA). A network pharmacological analysis revealed 40 active compounds, including organic acids and flavonoids, that act on HUA therapeutic targets. These targets primarily focus on pathways related to uric acid metabolism modulation, such as XOD, SLC22A12, ABCG2, SLC22A8, and others, reducing HUA. The analysis of mRNA-sequencing as well as RT-PCR data from renal tissue demonstrated that the targets modulating uric acid metabolism were SLC22A8, SLC12A1, and SLC16A7. ConclusionIn summary, organic acids and flavonoids may be the active components in Thlaspi arvense that alleviate HUA. The principal mechanisms are as follows: inhibition of XOD activity in the serum to reduce uric acid production, regulation of renal reabsorption and secretion of uric acid to increase uric acid excretion, and alleviation of oxidative stress reaction to decrease systemic damage and, eventually, treatment of HUA.

#5849 RENAL RENIN IS UPREGULATED BY THE COMBINATION TREATMENT OF EMPAGLIFLOZIN AND RAS BLOCKADE IN EXPERIMENTAL DIABETIC NEPHROPATHY

Abstract Background and Aims Sodium-glucose cotransporter 2 (SGLT2) inhibitors have proven to delay diabetic nephropathy (DN) progression on top of renin-angiotensin system (RAS) blockade. This protection is mainly attributed to improvement in renal hemodynamics, although direct effects on the kidney cannot be ruled out. The present study aimed to identify renal proteins differentially expressed between vehicle-treated diabetic mice and mice treated with empagliflozin, ramipril, or their combination, that could help explain the protective mechanisms of the drugs. Method Twelve weeks old diabetic db/db mice were given empagliflozin (10 mg/Kg/day), ramipril (8 mg/Kg/day), or the combination of both drugs during 8 weeks. Vehicle-treated db/db and db/m mice were used as controls. Serum glucose, blood pressure, GFR, and albuminuria were measured at baseline and at the end of the study. After 8 weeks, mice were euthanized, and the kidneys and serum were saved. A differential high-throughput proteomic analysis by mass spectrometry using isobaric tandem mass tags (TMT labelling) was performed in kidney cortex. Results Vehicle-treated db/db mice showed increased glycemia during the whole experiment, and empagliflozin reduced blood glucose. Ramipril treatment decreased blood pressure. Vehicle-treated diabetic mice also showed incipient DN with increased mesangial matrix and albuminuria compared to their non-diabetic littermates. All the treatments reduced mesangial matrix expansion and albuminuria. Only 13 proteins were differentially expressed (false discovery rate <5% and Log2FC ≥1 or ≤ −1) when comparing treated mice vs vehicle-treated db/db mice. The differentially expressed proteins were only identified between the mice treated with the combination of empagliflozin and ramipril and the vehicle-treated diabetic mice. Ramipril or empagliflozin alone did not produce significant changes in renal proteins. Kidney renin was evidently increased by the combination therapy with empagliflozin and ramipril, along with the tubular transporter scaffolding protein MAP17. The results were further validated through renin staining and renal renin concentration measurement. Renal renin concentration was increased by ramipril and further increased by the combination therapy with empagliflozin and ramipril when compared to vehicle-treated db/db mice (55 pg/μg (IQR:49–64), 94 pg/μg (IQR:71–102), and 112 pg/μg (IQR 104–116) in the groups treated with vehicle, ramipril and the combination of empagliflozin and ramipril respectively). However, renin serum concentration was similar between mice treated with ramipril and mice treated with the combination. Conclusion The combined therapy of empagliflozin with ramipril upregulated renin in the kidney of a diabetic mouse model. The increase in kidney renin suggests that other mechanisms different from RAS act in the regulation of glomerular hemodynamics and arteriolar tone. Moreover, the increased sodium delivery to the macula densa does not inhibit renal renin secretion.

#2989 SELECTIVE RENAL DISPOSITION OF THE CALCINEURIN INHIBITORS VOCLSOPORIN, CYCLOSPORINE, AND TACROLIMUS

Abstract Background and Aims The calcineurin inhibitors (CNI) cyclosporine (CSA) and tacrolimus (TAC) were revolutionary immunosuppressants when first introduced for solid organ transplantation in the 1980s. Voclosporin (VCS), a novel CNI, recently became the first oral therapy approved in the United States, Great Britain, and Europe for the treatment of active lupus nephritis based on positive results from Phase 2 and 3 clinical trials. Unlike CSA and TAC, VCS has demonstrated a consistent pharmacokinetic and pharmacodynamic profile, eliminating the need for therapeutic drug monitoring. Further, VCS is associated with a more favorable metabolic profile and has not been associated with electrolyte disturbances. Emerging evidence indicates small molecule therapeutics may display differential disposition within organ tissues. This suggests that CNIs may be differentially distributed and retained in the kidney, potentially explaining the difference in their efficacy and safety profiles. To evaluate renal disposition of CSA, TAC, and VCS, we assessed in mice and humans the disposition of each CNI in the kidney relative to its systemic drug exposure. Method Single 30 mg/kg doses of CSA, TAC and VCS were administered intravenously to mice. Following intravenous administration, kidneys were collected at 15 minutes, 30 minutes, 1 hour and 2 hours, flash frozen in liquid nitrogen, and stored at −20 °C until sectioning. Sections of 10 μm kidney tissue were mounted on indium tin oxide coated glass slides. Matrix of 10 mg/mL α-Cyano-4-hydroxycinnamic acid in 85% acetonitrile/13% ethanol + 2% water + 0.1% trifluoroacetic acid was sprayed on the tissue using an HTX tissue sprayer, dried for 10 minutes in the vacuum, and subjected to Matrix-assisted Laser Desorption and Ionization Mass Spectrometry Imaging (MALDI-MSI). The systemic and renal clearance in humans of CSA and TAC were obtained from the literature; pharmacokinetic data on VCS was obtained from data on file. Renal secretion of each drug was compared to its expected passive filtration based on glomerular filtration rate (GFR), fraction unbound in plasma (fu), and respective systemic drug exposure. Results MALDI-MSI demonstrated significantly higher concentrations of drug and more diffuse tissue disposition of CSA in mouse kidney compared to VCS (Figure 1). CSA was retained in all kidney tissues up to 2 hours post-administration. Higher concentrations and more diffuse disposition of TAC was also noted compared to VCS at 15 and 30 minutes; TAC was distinctively retained in the cortex and medulla. VCS had moderate distribution in the cortex and was rapidly excreted with low levels of drug present in the kidney after 1 hour. According to published data, CSA has a measured renal clearance of 1.48 mL/min in healthy human subjects, representing approximately 10% of expected passive filtration of 12.5 mL/min (Table 1). TAC has a renal clearance of 0.014 mL/min representing <2% of expected passive filtration of 1.25 mL/min. VCS has a renal clearance of 7.82 mL/min representing approximately 200% of its expected passive filtration rate of 3.75 mL/min. Conclusion MALDI-MSI revealed differential retention and distribution of CSA, TAC and VCS in mice, consistent with their respective renal clearances in humans. Higher drug exposure and >90% renal reabsorption was observed for both CSA and TAC in this study, whereas the renal handling of VCS suggested a significant component of tubular secretion. The higher rate of secretion and lower overall exposure of kidney tissue to VCS may be associated with an improved safety profile when compared to the more diffuse distribution and greater renal retention of CSA and TAC.

Inhibitory interaction of flavonoids with organic cation transporter 2 and their structure-activity relationships for predicting nephroprotective effects.

Organic cation transporter 2 (OCT2) is mainly responsible for the renal secretion of various cationic drugs, closely associated with drug-induced acute kidney injury (AKI). Screening and identifying potent OCT2 inhibitors with little toxicity in natural products in reducing OCT2-mediated AKI is of great value. Flavonoids are enriched in various vegetables, fruits, and herbal products, and some were reported to produce transporter-mediated drug-drug interactions. This study aimed to screen potential inhibitors of OCT2 from 96 flavonoids, assess the nephroprotective effects on cisplatin-induced kidney injury, and clarify the structure-activity relationships of flavonoids with OCT2. Ten flavonoids exhibited significant inhibition (>50%) on OCT2 in OCT2-HEK293 cells. Among them, the six most potent flavonoid inhibitors, including pectolinarigenin, biochanin A, luteolin, chrysin, 6-hydroxyflavone, and 6-methylflavone markedly decreased cisplatin-induced cytotoxicity. Moreover, in cisplatin-induced renal injury models, they also reduced serum blood urea nitrogen (BUN) and creatinine levels to different degrees, the best of which was 6-methylflavone. The pharmacophore model clarified that the aromatic ring, hydrogen bond acceptors, and hydrogen bond donors might play a vital role in the inhibitory effect of flavonoids on OCT2. Thus, our findings would pave the way to predicting the potential risks of flavonoid-containing food/herb-drug interactions in humans and optimizing flavonoid structure to alleviate OCT2-related AKI.

Haemoglobin response modelling under erythropoietin treatment: Physiological model‐informed machine learning method

AbstractPatients with renal anaemia are usually treated with recombinant human erythropoietin (EPO) because of insufficient renal EPO secretion. The establishment of a good haemoglobin (Hgb) response model is a necessary condition for dose optimization design. The purpose of this paper is to apply physics‐informed neural networks (PINN) to build the Hgb response model under EPO treatment. Neural network training is guided by a physiological model to avoid overfitting problems. During the training process, the parameters of the physiological model can be estimated simultaneously. To handle differential equations with impulse inputs and time delays, we propose approximate model equations for the pharmacokinetic (PK) model and the pharmacodynamic (PD) model, respectively. The modified PK/PD model was incorporated into PINN for training. Tests on simulated data and clinical data show that the proposed method has better performance than data‐driven modelling methods and the traditional physiological modelling based on the least squares method.

Regional Handling and Prognostic Performance of Circulating Insulin-Like Growth Factor Binding Protein-7 in Heart Failure

BackgroundRegional handling and the prognostic performance of insulin-like growth factor binding protein (IGFBP)-7, in contrast or in combination with other candidate biomarkers, in chronic heart failure (CHF) remain uncertain. ObjectivesThe authors investigated the regional handling of plasma IGFBP-7 and its association with long-term outcomes in CHF in comparison with selected circulating biomarkers. MethodsPlasma concentrations of IGFBP-7, N-terminal pro–B-type natriuretic peptide (NT-proBNP), high-sensitivity troponin-T, growth differentiation factor-15, and high-sensitivity C-reactive protein were measured prospectively in a cohort with CHF (n = 863). The primary outcome was the composite of heart failure (HF) hospitalization or all-cause mortality. In a separate non-HF cohort (n = 66) undergoing cardiac catheterization, transorgan gradients of plasma IGFBP-7 concentrations were evaluated. ResultsAmong 863 patients (age 69 ± 14 years, 30% female, 36% HF with preserved ejection fraction), IGFBP-7 (median: 121 [IQR: 99-156] ng/mL) related inversely to left ventricular volumes but directly to diastolic function. Above the optimal cutoff, IGFBP-7 ≥110 ng/mL was independently associated with 32% increased hazard of the primary outcome: 1.32 (95% CI: 1.06-1.64). Among the 5 markers, IGFBP-7 had the highest hazard for a proportional increment in plasma concentrations independent of HF phenotype in single- and double-biomarker models, and provided incremental prognostic value beyond clinical predictors plus NT-proBNP, high-sensitivity troponin-T, and high-sensitivity C-reactive protein (P < 0.05). Assessment of regional concentrations indicated renal secretion of IGFBP-7 in contrast to renal extraction of NT-proBNP, possible cardiac extraction of IGFBP-7 in contrast to secretion of NT-proBNP, and common hepatic extraction of both peptides. ConclusionsTransorgan regulation of IGFBP-7 is distinct from NT-proBNP. Circulating IGFBP-7 independently predicts adverse outcomes in CHF with a strong prognostic performance when compared with other well-recognized cardiac-specific or noncardiac prognostic markers.

Disorder of purine metabolism in the etiopathogenesis of urate nephrolithiasis

Urinary stone disease (USD) is a polyetiological urological disease caused by both exogenous and endogenous factors, including hereditary ones. It is characterized by the appearance of stones in the kidneys and urinary tract, and a tendency to relapse, often with a severe course. Almost 25 % of stones consist of uric acid (UA). The leading role in the pathogenesis of urate nephrolithiasis (UN) is played by disorders of purine metabolism, which are characterized by the development of hyperuricemia (HU) and hyperuricuria. The aim of the work is to review modern literary sources on the role of purine metabolism disorders in the etiopathogenesis of UN. Results. The development of UN depends on the constancy of the acidic urine pH, as well as on a decrease in diuresis, HU and hyperuricuria. UA is the final metabolite of purine metabolism and the main stone-forming substance in patients with UN. HU develops both due to uncompensated disorders of purine metabolism with a decrease in renal secretion and intestinal uricolysis (excretion pathway) and excessive intake of purine bases in the body and their increased synthesis in vivo (metabolic pathway). Citric acid, as one of the main metabolites of the tricarboxylic acid (TCA) cycle, is connected through the corresponding substrates to the formation of purines and the metabolite of amino acid metabolism, glutamine. TCA is connected to the cycles of urea, glyoxylate and purine bases through α-ketaglutaric acid. It is a substrate of citric acid, and it affects the synthesis of glutamate, which combines with ammonia to form glutamine, used in the cycle of purine synthesis. Conclusions. The role and diagnostic value of purine metabolism upsets, disorders of the TCA (citric acid), amino acid metabolism (glutamine), the activity of xanthine oxidase is a key enzyme in purine synthesis which passes through TCA with the participation of its metabolite α-ketaglutarate, have been established. TCA is bound to glutamine, rich in nitrogen, which is necessary for the synthesis of purine bases.