Tubular Transport Research Articles

Identification and characterization of an endogenous biomarker of the renal vectorial transport (OCT2-MATE1).

The renal tubular organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) mediate the vectorial elimination of many drugs and toxins from the kidney, and endogenous biomarkers for vectorial transport (OCT2-MATE1) would allow more accurate drug dosing and help to characterize drug-drug interactions and toxicity. Human serum uptake in OCT2-overexpressing cells and metabolomics analysis were carried out. Potential biomarkers were verified in vitro and in vivo. The specificity of biomarkers was validated in renal transporter overexpressing cells and the sensitivity was investigated by Km . The results showed that the uptake of thiamine, histamine, and 5-hydroxytryptamine was significantly increased in OCT2-overexpressing cells. In vitro assays confirmed that thiamine, histamine, and 5-hydroxytryptamine were substrates of both OCT2 and MATE1. In vivo measurements indicated that the serum thiamine level was increased significantly in the presence of the rOCT2 inhibitor cimetidine, and the level in renal tissue was increased significantly by the rMATE1 inhibitor pyrimethamine. There were no significant changes in the uptake or efflux of thiamine in cell lines overexpressed OAT1, OAT2, OAT3, MRP4, organic anion transporting polypeptide 4C1, P-gp, peptide transporter 2, urate transporter 1, and OAT4. The Km for thiamine with OCT2 and MATE1 were 71.2 and 10.8μM, respectively. In addition, the cumulative excretion of thiamine at 2 and 4h was strongly correlated with metformin excretion (R2 >0.6). Thus, thiamine is preferentially secreted by the OCT2 and MATE1 in renal tubules and can provide a reference value for evaluating the function of the renal tubular OCT2-MATE1.

Tubular phosphate transport: a comparison between different methods of urine sample collection in FGF23-dependent hypophosphatemic syndromes.

Tubular maximum phosphate reabsorption per glomerular filtration rate (TmP/GFR) is used to evaluate renal phosphate reabsorption and it is a useful tool for the differential diagnosis of hypophosphatemic syndromes. TmP/GFR is typically calculated from fasting plasma and second morning void urine samples, obtained 2 h after the first void (TmP/GFR 2 h). The purpose of this study was to evaluate if TmP/GFR calculated from 24 h urine collection (TmP/GFR 24 h) can be used as an alternative for TmP/GFR 2 h in patients with urine phosphate wasting. We enrolled adult patients with X-linked hypophosphatemia (XLH) or tumor-induced osteomalacia (TIO). All patients underwent blood and urine sample collections, to calculate TmP/GFR 24 h and TmP/GFR 2 h. Twenty patients (17 XLH and 3 TIO), aged 24-78 years, were included. All patients had low TmP/GFR 2 h (0.35 mmol/L, IQR 0.24-0.47 mmol/L) and TmP/GFR 24 h (0.31 mmol/L, IQR 0.22-0.43 mmol/L). The concordance correlation coefficient between TmP/GFR 2 h and TmP/GFR 24 h was 0.86 (95 % CI: 0.69-0.93), with a systematic bias of 0.05 mmol/L (95 % limits of agreement:-0.10 to 0.20). Furthermore, in 70 % (i.e.,14 patients out of 20) and 80 % (i.e.,16 patients out of 20) of cases the difference between TmP/GFR 2 h and TmP/GFR 24 h was within ±30 % and ±35 %, respectively. Despite TmP/GFR 2 and 24 h show a relatively suboptimal agreement, the difference between the two parameters appears to be small and not clinically significant in the setting of adult patients with FGF23-dependent urine phosphate wasting and secondary hypophosphatemia.

Primary Role of the Kidney in Pathogenesis of Hypertension.

Previous transplantation studies and the concept of 'nephron underdosing' support the idea that the kidney plays a crucial role in the development of essential hypertension. This suggests that there are genetic factors in the kidney that can either elevate or decrease blood pressure. The kidney normally maintains arterial pressure within a narrow range by employing the mechanism of pressure-natriuresis. Hypertension is induced when the pressure-natriuresis mechanism fails due to both subtle and overt kidney abnormalities. The inheritance of hypertension is believed to be polygenic, and essential hypertension may result from a combination of genetic variants that code for renal tubular sodium transporters or proteins involved in regulatory pathways. The renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system (SNS) are the major regulators of renal sodium reabsorption. Hyperactivity of either the RAAS or SNS leads to a rightward shift in the pressure-natriuresis curve. In other words, hypertension is induced when the activity of RAAS and SNS is not suppressed despite increased salt intake. Sodium overload, caused by increased intake and/or reduced renal excretion, not only leads to an expansion of plasma volume but also to an increase in systemic vascular resistance. Endothelial dysfunction is caused by an increased intracellular Na+ concentration, which inhibits endothelial nitric oxide (NO) synthase and reduces NO production. The stiffness of vascular smooth muscle cells is increased by the accumulation of intracellular Na+ and subsequent elevation of cytoplasmic Ca++ concentration. In contrast to the hemodynamic effects of osmotically active Na+, osmotically inactive Na+ stimulates immune cells and produces proinflammatory cytokines, which contribute to hypertension. When this occurs in the gut, the microbiota may become imbalanced, leading to intestinal inflammation and systemic hypertension. In conclusion, the primary cause of hypertension is sodium overload resulting from kidney dysregulation.

Furosemide attenuates tubulointerstitial injury and allows functional testing of porcine kidneys during normothermic machine perfusion.

Normothermic machine perfusion (NMP) is a promising pretransplant kidney quality assessment platform, but it remains crucial to increase its diagnostic potential while ensuring minimal additional injury to the already damaged kidney. Interventions that alter tubular transport can influence renal function and injury during perfusion. This study aimed to determine whether furosemide and desmopressin affect renal function and injury during NMP. Eighteen porcine kidneys (n = 6 per group) were subjected to 30 min of warm ischemia and 4 h of oxygenated hypothermic perfusion before being subjected to 6 h of NMP. Each organ was randomized to receive no drug, furosemide (750 mg), or desmopressin (16 μg) during NMP. Compared with the other groups, the addition of furosemide resulted in significantly increased urine output, fractional excretion of sodium and potassium, and urea clearance during NMP. Urinary neutrophil gelatinase-associated lipocalin levels decreased significantly with furosemide supplementation compared with the other groups. The addition of desmopressin did not result in any significantly different outcome measurements compared with the control group. This study showed that the addition of furosemide affected renal function while attenuating tubulointerstitial injury during NMP. Therefore, furosemide supplementation may provide renal protection and serve as a functional test for pretransplant kidney viability assessment during NMP.

Tubular dysfunction impairs renal excretion of pseudouridine in diabetic kidney disease.

Plasma nucleosides-pseudouridine (PU) and N2N2-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies.NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N2N2-dimethyl guanosine and diabetic kidney disease.

Coexistence of Neonatal Bartter Syndrome and Congenital Cutis Laxa, in which a new mutation in SLC12A1 was identified

Introduction: Neonatal Bartter Syndrome (NBS) is an extremely rare congenital chronic renal tubular transport disorder characterized by preterm birth, polyhydramnios, polyuria, salt wasting, and severe dehydration. Congenital Cutis Laxa (CCL) is a rare disease characterized by loss of skin flexibility and laxity, and atypical facial appearance.
 Case Presentation: A 1290-gram female baby at 30+6 weeks of gestation was hospitalized due to premature birth and respiratory distress. During physical examination, an atypical facial appearance, wrinkles on the forehead, skin laxity, and philtrum were visible as well as a small chin and arachnodactyly in the fingers and toes. The patient displayed metabolic alkalosis in her blood gases. The patient, with an atypical face and current findings submitted her exome for genetic diagnosis. Genetic analysis revealed the presence of homozygous NM_001184832.2:c.2485+5G>A (rs749269268) in SLC12A1, and NBS Type 1 was diagnosed. A novel heterozygous NM_000501.4:c.352G>T, p.Val118Phe (rs781922544) variant was detected in ELN, and CCL syndrome with autosomal dominant inheritance was diagnosed.
 Conclusions: The rare co-occurrence of NBS and CCL syndrome in our patient was seen as a mere coincidence. Since this is the first reported case in the literature, it was deemed appropriate to present this case.

Contribution of thick ascending limb and distal convoluted tubule to glucose-induced hypercalciuria in healthy controls.

Carbohydrates increase kidney stone risk and increase urine calcium and magnesium. We hypothesize that the effects of glucose as an allosteric modulator of calcium-sensing receptors may mediate this effect. Six healthy subjects were on a low-sodium diet before consuming 100 g of glucose beverage. Timed fasting (3) and postglucose (6) urine and blood samples were collected every 30 min. Urine composition and serum markers were measured and microvesicular abundance of tubular transport proteins (NHE3, NKCC2, NCC, and TRPV5) were quantified. Postglucose, serum glucose, and insulin rose rapidly with a parallel increase in calcium and magnesium excretion and no change in fractional excretion of sodium. Both serum parathyroid hormone (PTH) and urine TRPV5 fell in the postglucose periods. The rise in the calcium and magnesium excretion likely occurred primarily in the thick ascending limb where they are both under control of the calcium-sensing receptor. The fall in PTH and TRPV5 support the role of glucose as an allosteric modulator of calcium-sensing receptor.NEW & NOTEWORTHY Sugar increases urine calcium and magnesium as well as kidney stone and bone disease risk. Our study provided new insights into the underlying mechanism as we gave healthy subjects an oral glucose load and used newer tools such as fractional excretion of lithium, serum parathyroid hormone, and microvesicular abundance of tubular transport proteins to characterize the mechanism and identify the thick ascending limb with possible calcium-sensing receptor mediation as a likely contributor to this mechanism.

Влияние изменений водно-солевого баланса на ионо- и осморегулирующую функции почек у озерной лягушки

Changes in the water-salt balance and excretion of ions by the kidney after subcutaneous injections of hypertonic and isotonic NaCl solutions and dehydration were analyzed in lake frogs (Pelophylax ridibundus). The introduction of 0,75 M NaCl and dehydration for 1 hour increased the serum osmolality and Na+ and Cl– concentrations, and injections of isotonic solution did not affect these parameters. The applied treatments increased the level of Na+ and Cl– in the urine, and renal excretion of these ions. The most pronounced changes, including an increase in diuresis and glomerular filtration rate, were found after 0,75 М NaCl injection. In renal proximal tubules, changes in the pattern of the chloride channel (ClC-5), presumably involved in the process of protein endocytosis, were detected. An increase in the number of ClC-5-immunopositive tubule profiles and the intensity of the fluorescent signal in the apical cytoplasm of epithelial cells were revealed by immunohistochemistry and confocal microscopy. Studied effects of hypernatremia, hypervolemia and dehydration may be useful for further investigations of kidney function and mechanisms of epithelial tubular transport in frog models.

Navigating the kidney organoid: insights into assessment and enhancement of nephron function.

Kidney organoids are three-dimensional structures generated from pluripotent stem cells (PSCs) that are capable of recapitulating the major structures of mammalian kidneys. As this technology is expected to be a promising tool for studying renal biology, drug discovery, and regenerative medicine, the functional capacity of kidney organoids has emerged as a critical question in the field. Kidney organoids produced using several protocols harbor key structures of native kidneys. Here, we review the current state, recent advances, and future challenges in the functional characterization of kidney organoids, strategies to accelerate and enhance kidney organoid functions, and access to PSC resources to advance organoid research. The strategies to construct physiologically relevant kidney organoids include the use of organ-on-a-chip technologies that integrate fluid circulation and improve organoid maturation. These approaches result in increased expression of the major tubular transporters and elements of mechanosensory signaling pathways suggestive of improved functionality. Nevertheless, continuous efforts remain crucial to create kidney tissue that more faithfully replicates physiological conditions for future applications in kidney regeneration medicine and their ethical use in patient care.NEW & NOTEWORTHY Kidney organoids are three-dimensional structures derived from stem cells, mimicking the major components of mammalian kidneys. Although they show great promise, their functional capacity has become a critical question. This review explores the advancements and challenges in evaluating and enhancing kidney organoid function, including the use of organ-on-chip technologies, multiomics data, and in vivo transplantation. Integrating these approaches to further enhance their physiological relevance will continue to advance disease modeling and regenerative medicine applications.

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

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. This study aimed to determine the impact of hypoxia/reoxygenation on the human proximal transport system through two semi-targeted omics analyses. Human proximal tubular cells were cultured in hypoxia (6 or 24h), 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. 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. 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.

Analysis of syndrome of inappropriate anti-diuresis and cerebral salt wasting in patients with acute stroke

Hyponatremia in hospitalized patients can significantly alter the morbidity, mortality, especially in chronic neurological patients. Most commonly, it is attributed to SIADH, and in stroke it occurs due to AVP secretion inappropriate to the osmotic threshold. The suppressed proximal renal tubular transport in this condition can lead on to bicarbonaturia and hypouricemia. CSW, on the other hand, is essentially a volume depleted state, which occurs due to the combined effects of decreased sympathetic outflow and increased natriuretic peptides. This resultant natriuresis leads to volume depletion and an appropriate AVP response. The treatment for SIADH AND CSW varies, and the outcomes vary considerably too. Hence, we performed this study to study the clinical profile of stroke patients with hyponatremia, and to identify the factors associated with the same.

Targeted deletion of von-Hippel-Lindau in the proximal tubule conditions the kidney against early diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development.

Gliflozins, Erythropoietin, and Erythrocytosis: Is It Renal Normoxia- or Hypoxia-Driven?

The introduction of gliflozins in the management of type 2 diabetes mellitus leads to a better control of hyperglycemia, obesity, hypertension, dyslipidemia, and fluid retention. Most importantly, it also improves renal survival and reduces major cardiovascular events and mortality. Gliflozins were also found to induce erythropoietin (EPO) synthesis, leading to reticulocytosis and erythropoiesis. The mechanism(s) by which gliflozins induce erythropoiesis is a matter of debate. Although the canonical pathway of triggering EPO synthesis is through renal tissue hypoxia, it has been suggested that improved renal oxygenation may facilitate EPO synthesis via non-canonical routes. The latter proposes that the recovery of peritubular interstitial fibroblasts producing erythropoietin (EPO) is responsible for enhanced erythropoiesis. According to this hypothesis, enhanced glucose/sodium re-uptake by proximal tubules in uncontrolled diabetes generates cortical hypoxia, with injury to these cells. Once transport workload declines with the use of SGLT2i, they recover and regain their capacity to produce EPO. In this short communication, we argue that this hypothesis may be wrong and propose that gliflozins likely induce EPO through the documented intensification of renal hypoxia at the corticomedullary junction, related to the translocation of tubular transport from cortical segments to medullary thick ascending limbs. We propose that gliflozins, through intensified hypoxia in this region, trigger local EPO synthesis in peritubular interstitial cells via the canonical pathway of blocking HIF-prolyl hydroxylases (that initiate HIF alpha degradation), with the consequent stabilization of HIF-2 signal and an apocrinic induction of EPO in these same cells.

7-LB: Sodium–Glucose Cotransporter-2 Inhibitors (SGLT2i) Decrease Kidney Oxygen Consumption in Adults with Type 2 Diabetes (T2D)—A Randomized Clinical Trial Using 11C-Acetate Positron Emission Tomography (PET) Imaging

Kidney hypoxia has been proposed as key pathophysiological mechanism in the development of chronic kidney disease, potentially stemming from a mismatch between oxygen delivery and oxygen consumption. SGLT2i are kidney protective drugs that initially lower glomerular filtration rate (GFR). Accordingly, we hypothesized that SGLT2i lower kidney oxygen consumption by reducing GFR and associated tubular workload. Twenty adults with T2D (sex 80% male, age 68±6 years, BMI 30±4 kg/m2, HbA1c 7.5±0.9%, eGFR 76 ± 11 mL/min/1.73m2) received a 4-week treatment with SGLT2i ertugliflozin (ERTU) and matched placebo (PLB) in a randomized, double-blind cross-over study. Participants were treated with metformin and received maximal tolerable dose of an angiotensin receptor blocker. Whole-kidney oxygen consumption (Kmono) was measured by PET using 11C-acetate. GFR was measured by gold-standard iohexol clearance. Tubular sodium transport (TNa) was calculated by kidney sodium load (([arterial Na]*mGFR) - urinary sodium excretion). Kidney efficiency calculated as TNa/ Kmono. GFR was lower during ERTU (94 ± 14 mL/min) vs. PLB (99 ± 15 mL/min) treatment (p=0.02). Kmono was 0.086 ± 0.006 min-1 during ERTU and 0.091 ± 0.009 min-1 during PLB (p<0.01). Kidney sodium load (12.9 ± 1.90 vs 13.7 ± 2.22 mmol/min) and TNa (12.7 ± 1.87 vs 13.6 ± 2.18 mmol/min) were lower during ERTU (p=0.02 for both), while urinary sodium excretion (p=0.1) and TNa/ Kmono remained unchanged (p=0.7). TNa strongly associated with Kmono (r=0.50; p<0.0001). In conclusion, SGLT2i lowers mGFR, TNa, and kidney oxygen consumption, potentially decreasing hypoxia risk. SGLT2i treatment did not lower overall kidney efficiency, despite the ATP efficiency of cortical sodium transport. Additional data on cortex and medulla-specific oxygen consumption and multiparametric kidney MRI data will be presented at ADA. Disclosure A. Hesp: None. L. I. P. Snel: None. P. Schober: None. L. A. Schwarte: None. R. Boellaard: None. P. Bjornstad: Advisory Panel; AstraZeneca. Consultant; Bayer Inc., Bristol-Myers Squibb Company. Advisory Panel; Novo Nordisk, Lilly, Horizon Therapeutics plc, Boehringer Ingelheim (Canada) Ltd., LG Chem. D. van Raalte: Consultant; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck & Co., Inc. Research Support; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck & Co., Inc. Funding MSD

#3386 PROTON-PUMP INHIBITORS AND SERUM CONCENTRATIONS OF UREMIC TOXINS IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Use of proton-pump inhibitors (PPIs) is common in patients with chronic kidney disease (CKD). PPIs and many uremic toxins (UTs) are eliminated by a kidney tubular organic anion transporter system. In a cross-sectional study, we sought to evaluate the association between PPI prescription and serum concentrations of various UTs. Method We studied a randomly selected subgroup of participants in the CKD-REIN prospective cohort (adult patients with a confirmed diagnosis of CKD and estimated glomerular filtration rate (eGFR) <60ml/min/1.73m²) with available frozen samples collected at baseline. PPI prescription was recorded at baseline. Serum concentrations of 10 UTs were measured using a validated liquid chromatography tandem mass spectrometry technique. Multiple linear regression was performed, with the log UT concentration as the dependent variable. Results Of the 680 included patients [median age: 68 years; median eGFR: 32 ml/min/1.73 m2], 31% had PPI prescriptions at baseline. Patients using PPIs had higher levels of certain UTs in comparison to other patients, including total and free indoxyl sulfate (IS), total and free p-cresylsulfate, total and free p-cresylglucuronide (PCG), phenylacetylglutamine (PAG), free kynurenine, and free hippuric acid. After adjustment for baseline comorbidities, number of co-prescribed drugs and laboratory data including eGFR, associations between PPI prescription and elevated serum concentrations of free and total IS, free and total PCG, and PAG remained significant. Conclusion Our results indicate that PPI prescription is independently associated with serum UT retention. These findings indicate a potential mechanism for side effect of PPIs in CKD patients that will need to be confirmed by longitudinal studies.

#4394TACROLIMUS BUT NOT VOCLOSPORIN INHIBITS KIDNEY TUBULAR CALCIUM AND MAGNESIUM REABSORPTION IN RATS AT CLINICALLY THERAPEUTIC DOSES

Abstract Background and Aims Clinically, the calcineurin inhibitor tacrolimus frequently causes hypercalciuria and hypomagnesemia by inhibiting kidney tubular calcium and magnesium reabsorption. Voclosporin, a novel calcineurin inhibitor approved in the USA and Europe for the treatment of adults with active lupus nephritis, has fewer side-effects including less hypomagnesemia. However, the differences between the kidney tubular effects of tacrolimus and voclosporin are unknown. To address this, we compared the effects of tacrolimus, voclosporin, and vehicle in rats. Method Tacrolimus (0.5 mg/kg) and voclosporin (0.5 mg/kg) were administrated by daily intraperitoneal injections in male Wistar rats and were compared against vehicle (Cremophor EL: 95% ethanol:saline (5:5:90, v:v:v)) for 28 days (n = 8–9/group). Dosages were determined based on pharmacokinetic studies in rats and aimed to achieve the area under the concentration-time curve observed in clinical studies. At day 18, blood and 24 h urine were collected to measure trough levels of the drugs and to analyze the fractional excretions of calcium and magnesium. At sacrifice, kidneys were harvested for immunoblotting of tubular proteins. Results Both tacrolimus and voclosporin reached clinically therapeutic doses with trough levels of 2.4 μg/L ± 0.6 μg/L and 25.8 μg/L ± 9.6 μg/L, respectively. Compared to vehicle, tacrolimus caused significantly higher fractional excretions of calcium (+348% ± 127%, P < 0.001) and magnesium (+60% ± 38%, P < 0.01) and also caused hypomagnesemia (plasma magnesium 0.65 mmol/L ± 0.04 mmol/L vs. 0.81 mmol/L ± 0.02 mmol/L, P < 0.001). Compared to vehicle, voclosporin only caused a slight, but non-statistically significant, increase in fractional calcium excretion (+ 44% ± 38%, P = 0.08) and did not cause higher fractional magnesium excretion (+5% ± 30%, P = 0.96) or hypomagnesemia (plasma magnesium 0.8 mmol/L ± 0.04 mmol/L, P = 0.9). Compared to vehicle, tacrolimus caused an 11-fold decrease in the protein abundance of the cytosolic calcium-binding protein calbindin-D28K and a 2-fold decrease in the abundance of the sodium-chloride cotransporter (NCC). In contrast, voclosporin did not decrease the protein abundances of calbindin-D28K and NCC. No differences were observed between vehicle, tacrolimus, and voclosporin in the calcium channel TRPV5 and magnesium channel TRPM6 (Figure 1). Conclusion In contrast to the calcineurin inhibitor tacrolimus, voclosporin does not inhibit the kidney tubular reabsorption of calcium and magnesium and therefore does not cause hypercalciuria or hypomagnesemia. A possible explanation for this difference is that tacrolimus but not voclosporin affects tubular transport in the distal convoluted tubule, which was further supported by the selective inhibition of calbindin-D28K and NCC by tacrolimus. Our data show that tubulotoxicity of tacrolimus is not apparent with voclosporin treatment, at clinically relevant doses.

#5535 CALCIUM EXCRETION FRACTION AS AN EMERGING PARAMETER FOR AN ACCURATE DIFFERENTIAL DIAGNOSIS OF ACUTE KIDNEY INJURY

Abstract Background and Aims Urinary parameter determination is an essential tool for differentiating acute kidney injury (AKI) etiology. The most used ion in clinical practice is sodium (and its excretion fraction, NaEF), followed by urea (UreaEF) (that is useful in certain circumstances such as diuretic treatment). However, tubular transport includes other anions and cations that have not been widely studied. In the present study, we aim to evaluate the relation between AKI etiology (i.e. functional vs acute tubular necrosis) and calcium excretion fraction (CaEF). Method This is a transversal study including consecutive patients with diagnosis of AKI. Patients with suspicion of non-functional or ATN etiology (such as those with proteinuria, hematuria) were excluded. At admission, we collected epidemiological data, comorbidities and active treatments. AKI etiology was determined based on urinary parameters (sodium, urea) and recovery pattern of kidney function after treatment instauration. We compared CaEF in patients with ATN and functional AKI in the whole sample and among patients with and without previous CKD and diuretic prescription. Results We included 94 AKI episodes (55% female, 76±15 years). Forty-five (48%) patients had chronic kidney disease (CKD) at baseline and 56 (60%) were receiving treatment with diuretics. Of the 94 AKI, 78 (83%) presented a functional etiology and 16 (17%) were catalogued as ATN. Median CaEF was 1.09 (0.63-2.58) %. CaEF was associated to the etiology of AKI. In patients with functional AKI, median of CaEF was 0.95 (0.60-1.84) % in contrast to ATN (2.33 [1.22-7.27] %)(p<0.001). CaEF presented a positive and significant correlation to NaEF (ρ 0.574, p<0.001) and UreaEF (ρ 0.409, p<0.001). An adjusted regression model including the presence of CKD at baseline and diuretic treatment showed that CaEF independently predicted ATN (OR per 1% increase 1.42, 95%CI [1.16-1.75), p<0.001]. In patients without CKD, CaEF predicted ATN (OR 1.61, 95%CI [1.13-2.28], p = 0.008), in contrast to the cohort with CKD where only a trend was demonstrated (OR 1.29, 95%CI [0.99-1.69], p = 0.057) (Figure 1). CaEF also predicted ATN irrespective of the diuretic prescription at baseline (OR 1.35, 95%CI [1.05-1.74], p = 0.018 for diuretic users and OR 1.57, 95%CI [1.06-2.32], p = 0.025 for non-diuretic users) (Figure 1). Receiving operator curves (ROC) demonstrated a 0,712 area under the curve (AUC) of CaEF >1.6% for ATN, that was the best value for diagnosis in terms of sensitivity and specificity. CaEF>1,6% predicted ATN (OR 8.09, 95%CI [2.32-28.2], p = 0.001) after adjusting for the presence of CKD at baseline and diuretic treatment. Conclusion CaEF could help in the identification of the AKI etiology as higher values (i.e. >1.6%) are independently associated with ATN.

#4032 RAPAMYCIN AGGRAVATES KIDNEY LESIONS IN RATS FED HIGH PHOSPHATE

Abstract Background and Aims Rapamycin is currently used to prevent rejection after kidney transplantation. mTOR blockade by rapamycin may slow the progression of diabetic kidney disease. Rapamycin promotes phosphaturia by inhibiting renal tubular transport of phosphate. Excessive tubular load of phosphate produces kidney damage by inducing tubular injury and interstitial fibrosis. The aim of this work is to evaluate the effect of rapamycin on kidney pathology in rats fed different phosphate levels. Method Thirty four female Wistar rats were used in this experiment. Rats, aged 2 months at the beginning of the studies, were allotted to 6 groups (n = 4-6). Rats with intact renal function were fed a standard diet containing either nomal (0.6%, NP), low (0.2%, LP) or high (1.2%, HP) phosphate. Half of the groups received placebo treatment (NP, LP, HP) and the other half were treated with rapamycin (NP-Rapa, LP-Rapa, HP-Rapa) at a dose of 1.3 mg/kg/day, for 22 days. Plasma concentrations of urea, creatinine, and phosphate as well as urine creatinine and phosphate were measured by spectrophotometry. Microscopic evaluation of the kidneys was performed in tissue sections fixed with formaldehyde solution and embedded in paraffin. Kidney sections were stained with hematoxylin-eosin, Masson Trichrome, periodic acid Schiff, and Von Kossa staining. Thirty non overlapping fields were evaluated. Chronic interstitial fibrosis and renal calcification were measured with a point counting grid and the result was expressed in percentage. Atrophic tubules were counted and the result was expressed as number of atrophic tubuli/field. Values are expressed as the mean ± standard error (SE). The difference between means was assessed by ANOVA. Fisher LSD test was used as a post-hoc procedure. A correlation study was carried out using the Pearson test. p<0.05 was considered significant. Results Kidney lesions were more severe in rats treated with rapamycin and fed high phosphate diet. Areas of chronic interstitial fibrosis, with interstitial fibrosis and lymphoplasmacytic infiltrate, as well as atrophic tubules with thickened and wrinkled tubular basement membrane, loss of the brush border and flattening of the tubular cell cytoplasm, were observed. Calcification foci, mainly located in the corticomedullary junction, were identified with Von Kossa staining. Treatment with rapamycin did not influence renal function parameters in NP and LP rats but increased plasma creatinine and urea in HP rats. Plasma P concentration tended to decrease after rapamycin treatment. Fractional excretion of P was higher in all the groups treated with rapamycin, and the differences reached significance in HP-Rapa rats. Fractional excretion of P showed an excellent correlation with kidney lesions: chronic interstitial fibrosis (r = 0.822), calcification (r = 0.827) and tubular atrophy (r = 0.855). Conclusion The phosphaturic action of rapamycin aggravates renal lesions in rats fed high phosphate diets. Thus, rapamycin could be deleterious for the kidneys in the context of high phosphorus intake.

#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.

Nonselective and A2a-Selective Inhibition of Adenosine Receptors Modulates Renal Perfusion and Excretion Depending on the Duration of Streptozotocin-Induced Diabetes in Rats.

Long-lasting hyperglycaemia may alter the role of adenosine-dependent receptors (P1R) in the control of kidney function. We investigated how P1R activity affects renal circulation and excretion in diabetic (DM) and normoglycaemic (NG) rats; the receptors' interactions with bioavailable NO and H2O2 were also explored. The effects of adenosine deaminase (ADA, nonselective P1R inhibitor) and P1A2a-R-selective antagonist (CSC) were examined in anaesthetised rats, both after short-lasting (2-weeks, DM-14) and established (8-weeks, DM-60) streptozotocin-induced hyperglycaemia, and in normoglycaemic age-matched animals (NG-14, NG-60, respectively). The arterial blood pressure, perfusion of the whole kidney and its regions (cortex, outer-, and inner medulla), and renal excretion were determined, along with the in situ renal tissue NO and H2O2 signals (selective electrodes). The ADA treatment helped to assess the P1R-dependent difference in intrarenal baseline vascular tone (vasodilation in DM and vasoconstriction in NG rats), with the difference being more pronounced between DM-60 and NG-60 animals. The CSC treatment showed that in DM-60 rats, A2aR-dependent vasodilator tone was modified differently in individual kidney zones. Renal excretion studies after the ADA and CSC treatments showed that the balance of the opposing effects of A2aRs and other P1Rs on tubular transport, seen in the initial phase, was lost in established hyperglycaemia. Regardless of the duration of the diabetes, we observed a tonic effect of A2aR activity on NO bioavailability. Dissimilarly, the involvement of P1R in tissue production of H2O2, observed in normoglycaemia, decreased. Our functional study provides new information on the changing interaction of adenosine in the kidney, as well as its receptors and NO and H2O2, in the course of streptozotocin diabetes.