PAH Clearance Research Articles

124-OR: Effects of Dapagliflozin Therapy on Renal Hemodynamics in Type 2 Diabetes Patients

Aim: To investigate the renal hemodynamic effects of SGLT-2i therapy in patients with type 2 diabetes [T2D]. Design & Methods: We recruited 24 drug naïve T2D, not receiving ACEi/ARBs. GFR [>100ml/min/1.73m2] was measured with Iohexol clearance and renal plasma (blood) flow [RP(B)F] with PAH clearance divided by (1-Hct). Mean arterial pressure [MAP] and renal vascular resistance [RVR] were determined before and 4 months after dapagliflozin 10mg/day (DAPA, n=12); results were compared to T2D receiving metformin±glipizide to achieve similar glycemic reduction (CONTROL, n=12). Results: HbA1c decreased equally in DAPA (8.2±0.2 to 7.1±0.2%) and CONTROL (8.4±0.3 to 7.2±0.1%); body weight decreased in DAPA (89.9±4.0 to 86.2±4.2 kg) and rose in CONTROL (86.5±6.2 to 88.9±5.5 kg). GFR [ml/min/1.73m2] declined in DAPA (118±5 to 103±5) but not in CONTROL (113±6 vs. 114±7) (p<0.01). RPF [ml/min] did not change in DAPA (668±65 vs. 676±70) and CONTROL (630±40 vs. 629±39), while filtration fraction [FF] decreased in DAPA (18±1 vs. 15±1%) but not in CONTROL (18±2 vs. 18±2%) (p<0.05). MAP [mmHg] declined in DAPA (95.4±2.6 vs. 89.9±2.5) but not in CONTROL (90.3±4.1 vs. 90.7±2.1) (p<0.05). Thus, calculated RVR [mmHg/L/min] in DAPA decreased (87.0±4.2 to 77.6±2.9), whereas it did not change (86.2±3.8 to 86.4±4.2) in CONTROL after 4 months of therapy (p<0.05). Conclusion: Despite equal glycemic control, treatment with dapagliflozin, but not with metformin+glipizide, reduced glomerular filtration fraction and renal vascular resistance. Thus, unlike studies in T1D, our data suggest that in T2D post-glomerular vasodilatation is the predominant reno-protective mechanism of SGLT-2 inhibitors. Concomitant pre-glomerular vasoconstriction, however, cannot be excluded. Disclosure Y.Qin: None. G.Baskoy: None. C.L.Triplitt: Speaker's Bureau; Novo Nordisk. C.Solis-herrera: None. J.M.Adams: None. R.A.Defronzo: Advisory Panel; AstraZeneca, Bayer Inc., Boehringer-Ingelheim, Novo Nordisk, Research Support; AstraZeneca, Boehringer-Ingelheim, Merck & Co., Inc., Speaker's Bureau; AstraZeneca. E.Cersosimo: None. Funding AstraZeneca; Texas Diabetes Institute

Incubation Time Influences Organic Anion Transporter 1 Kinetics and Renal Clearance Predictions.

Accurate predictions of drug uptake transporter involvement in renal excretion of xenobiotics require determination of in vitro transport kinetic parameters under initial-rate conditions. The purpose of the present study was to determine how changing the incubation time from initial rate to steady state influences ligand interactions with the renal organic anion transporter 1 (OAT1), and the impact of the different experimental conditions on pharmacokinetic predictions. Transport studies were performed with Chinese hamster ovary cells expressing OAT1 (CHO-OAT1) and the Simcyp Simulator was used for physiological-based pharmacokinetic predictions. Maximal transport rate and intrinsic uptake clearance (CLint) for PAH decreased with increasing incubation time. The CLint values ranged 11-fold with incubation times spanning from 15 s (CLint,15s, initial rate) to 45 min (CLint,45min, steady state). The Michaelis constant (Km) was also influenced by the incubation time with an apparent increase in the Km value at longer incubation times. Inhibition potency of five drugs against PAH transport was tested using incubation times of either 15 s or 10 min. There was no effect of time on inhibition potency for omeprazole or furosemide, whereas indomethacin was less potent, and probenecid (~2-fold) and telmisartan (~7-fold) more potent with the longer incubation time. Notably, the inhibitory effect of telmisartan was reversible, albeit slowly. A pharmacokinetic model was developed for PAH using the CLint,15s value. The simulated plasma concentration-time profile, renal clearance, and cumulative urinary excretion-time profile of PAH agreed well with reported clinical data, and the PK parameters were sensitive to the time-associated CLint value used in the model.

857-P: Mechanisms of Dapagliflozin to Reduce Intraglomerular Pressure in Patients with Type 2 Diabetes

Aim: To examine the renal hemodynamic effects of SGLT-2i therapy in type 2 diabetes patients [T2D]. Design & Methods: T2D (n=20) with normal GFR measured with 24-hour creatinine clearance [CrCl>100ml/min/1.73m2], who were not receiving ACEi or ARB, had renal blood flow (RBF) measured using PAH clearance divided by (1-Hct) and mean arterial pressure [MAP] to calculate renal vascular resistance [RVR] prior to and 4 months after Dapagliflozin 10mg/day (DAPA, n=10) . Results were compared to T2D treated with metformin ± glipizide to achieve similar glycemic control (Control, n=10) . Results: HbA1c decreased equally in DAPA (8.6±0.5% to 7.4±0.3%) and CONTROL (8.7±0.3 to 7.5±0.2%) . There was a non-significant increase in RBF (ml/min) in DAPA compared to Control. MAP (mmHg) decreased by 8.5% from 98.4±2.9 mmHg to 90.0±2.0 mmHg and RVR by 21.4% from 87.9±5.1 mmHg/l/min to 69.1±5.2 mmHg/l/min in the DAPA group, while MAP & RVR remained unchanged in CONTROL group. Data at baseline [Pre-Rx] and after 4 months of therapy [Post-Rx] are shown in the table. Conclusion: These findings show that, with equivalent glycemic control, dapagliflozin, but not metformin/glipizide, is accompanied by a decrease in renal vascular resistance, without significant change in RBF. Unlike studies in T1D, our results suggest that in T2D patients post-glomerular vasodilatation is the primary reno-protective mechanism of SGLT-2i therapy. Disclosure M.Barkhordarian: None. C.L.Triplitt: Consultant; Bayer AG, Speaker's Bureau; Eli Lilly and Company, Novo Nordisk. C.Solis-herrera: Speaker's Bureau; Novo Nordisk. J.M.Adams: None. R.A.Defronzo: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Intarcia Therapeutics, Inc., Novo Nordisk, Research Support; AstraZeneca, Boehringer Ingelheim International GmbH, Merck & Co., Inc., Speaker's Bureau; AstraZeneca. E.Cersosimo: Research Support; AstraZeneca. Funding AstraZeneca

Diclofenac toxicity in susceptible bird species results from a combination of reduced glomerular filtration and plasma flow with subsequent renal tubular necrosis.

Diclofenac caused the death of millions of vultures on the Asian subcontinent. Other non-steroidal anti-inflammatory drugs (NSAIDs) have since also been shown to be toxic to vultures with the exception of meloxicam. For this study, we evaluated the effect of diclofenac on renal uric acid transport and glomerulus filtration in an acute toxicity model. In a two-phase study with the same birds, healthy chickens (a validated model species) were treated intravenously with para-amino hippuric acid (PAH) and iohexol (IOH) in combination in phase 1. In phase 2, the same PAH and IOH combination was then combined with diclofenac (10 mg/kg). In both phases, blood and faeces were sequentially collected. In phase 1, the birds showed no signs of ill health. Moreover, PAH, IOH and uric acid clearance was rapid. In phase 2, two chickens showed early signs of hyperuricemia 8 hours after exposure and died approximately 24h later. Necropsy showed classic signs of renal damage and gout. Diclofenac had a rapid plasma half-life of elimination of less than 2 hours indicating that toxicity was likely due to an irreversible destruction of a physiological process. All the birds in phase 2 had decreased uric acid, PAH and IOH clearance in comparison to phase 1. The decrease in PAH clearance was variable between the birds (average of 71%) but was near 98% reduced in the two birds that died. It is concluded that diclofenac alters both renal perfusion and renal plasma flow, with death associated with tubular secretion being reduced to negligible functionality for a prolonged period. This would support previous in vitro findings of early cell death from ROS accumulation. However, further evaluation is needed to elucidate this final step.

Angiotensin II-induced natriuresis is attenuated in knockout mice lacking the receptors for tumor necrosis factor-α.

Intravenous infusion of relatively higher doses of angiotensin II (AngII) elicits natriuresis as opposed to its usual anti‐natruretic response. As AngII can induce tumor necrosis factor‐α (TNFα) production which elicits natriuresis via its action on TNFα receptor type 1 (TNFR1), we hypothesize that the concomitant release of TNFα contributes to the natriuretic response to AngII. Responses to AngII infusion (1 ng min−1 g−1 for 75 min, iv) were evaluated in anesthetized knockout (KO) mice lacking TNFR1 (n = 6) and TNFR2 (TNFα receptor type 2; n = 6) and compared these responses with those in wild type (WT; n = 6) mice. Arterial pressure (AP) was recorded from a cannula placed in the carotid artery. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by PAH and inulin clearances, respectively. Urine was collected from a catheter placed in the bladder. AngII caused similar increases (p < 0.05 vs basal values) in AP (WT, 37 ± 5%; TNFR1KO, 35 ± 4%; TNFR2KO, 30 ± 4%) and decreases (p < 0.05) in RBF (WT, −39 ± 5%; TNFR1KO, −28 ± 6%; TNFR2KO, −31 ± 4%) without significant changes in GFR (WT, −17 ± 7%; TNFR1KO, −18 ± 7%; TNFR2KO, −12 ± 7%). However, despite similar changes in AP and renal hemodynamics, AngII induced increases (p < 0.05) in urinary sodium excretion in WT (3916 ± 942%) were less in the KO strains, more or less in TNFR1KO (473 ± 170%) than in TNFR2KO (1176 ± 168%). These data indicate that TNF‐α receptors, particularly TNFR1 are involved in the natriuretic response that occur during acute infusion of AngII and thus, plays a protective role in preventing excessive salt retention at clinical conditions associated with elevated AngII level.

The Development of a Juvenile Porcine Augmented Renal Clearance Model Through Continuous Infusion of Lipopolysaccharides: An Exploratory Study.

Augmented renal clearance (ARC) as observed in the critically ill (pediatric) population can have a major impact on the pharmacokinetics and posology of renally excreted drugs. Although sepsis has been described as a major trigger in the development of ARC in human critically ill patients, mechanistic insights on ARC are currently lacking. An appropriate ARC animal model could contribute to reveal these underlying mechanisms. In this exploratory study, a state of ARC was induced in 8-week-old piglets. Conscious piglets were continuously infused over 36 h with lipopolysaccharides (LPS) from Escherichia coli (O111:B4) to induce sepsis and subsequently trigger ARC. To study the dose-dependent effect of LPS on the renal function, three different doses (0.75, 2.0, 5.0 μg/kg/h) were administered (two ♂ piglets/dose, one sham piglet), in combination with fluid administration (0.9% NaCl) at 6 ml/kg/h. Single boluses of renal markers, i.e., creatinine [40 mg/kg body weight (BW)], iohexol (64.7 mg/kg BW), and para-aminohippuric acid (PAH, 10 mg/kg BW) were administered intravenously to evaluate the effect of LPS on the renal function. Clinical parameters were monitored periodically. Blood sampling was performed to determine the effect on hematology, neutrophil gelatinase-associated lipocalin, and prostaglandin E2 plasma levels. All piglets that were continuously infused with LPS displayed an elevated body temperature, heart rhythm, and respiratory rate ~1–3 h after start of the infusion. After infusion, considerably higher total body clearances of iohexol, creatinine, and PAH were observed, independent of the administration of LPS and/or its dose. Since also the sham piglet, receiving no LPS, demonstrated a comparable increase in renal function, the contribution of fluid administration to the development of ARC should be further evaluated.

Erythropoietin alters the pharmacokinetics of organic anions mainly eliminated by the kidney in rats.

Erythropoietin (EPO) is a cytokine originally used for its effects on the hematopoietic system, and is widely prescribed around the world. In the present study, the effects of EPO administration on p-aminohippurate (PAH, a prototype organic anion) pharmacokinetics and on the renal expression of PAH transporters were evaluated. Male Wistar rats were treated with EPO or saline (control group). After 42h, PAH was administered, and plasma samples were obtained at different time points to determine PAH levels. PAH levels in renal tissue and urine were also assessed. The renal expression of PAH transporters was evaluated by Western blotting. EPO-treated rats showed an increase in PAH systemic clearance, in its elimination rate constant, and in urinary PAH levels, while PAH in renal tissue was decreased. Moreover, EPO administration increased the expression of the transporters of the organic anions evaluated. The EPO-induced increase in PAH clearance is accounted for by the increase in its renal secretion mediated by the organic anion transporters. The goal of this study is to add important information to the wide knowledge gap that exists regarding drug-drug interactions. Owing to the global use of EPO, these results are useful in terms of translation into clinical practice.

Glucosuria Interferes With Measurement of Effective Renal Plasma Flow Using para-Aminohippuric Acid, With a Focus on SGLT2 Inhibitors

Abstract Para-aminohippuric acid (PAH)-derived effective renal plasma flow (ERPF) is increasingly being used in parallel with the revived interest in kidney hemodynamic function due to the development of sodium glucose co-transporter 2 (SGLT2) inhibitors. In our trial (Kidney Int 2020;97: 202-12) we measured ERPF by urinary PAH clearance and calculated fractional PAH extraction ratios, which initially were very low during both the hyperglycemic clamps at baseline, as well as the euglycemic conditions following treatment with SGLT2 inhibitor dapagliflozin. Previous literature showed that hyperglycemic i.e. glucosuric conditions reduce urinary PAH concentrations, due to a Schiff base that is formed between the para-amino group of PAH and the aldehyde group of glucose. By using hydrochloric acid (HCl) any PAH that has been glycosylated can be reconverted, independent of storage time. After treatment with HCL we indeed found higher PAH extraction ratios in the expected range. In addition, a negative correlation between storage time and PAH extraction ratio in people treated with SGLT2 inhibitor dapagliflozin during a euglycemic clamp before treatment with HCl. Besides these human data, comparable results were observed in male obese ZSF1 rats with hyperglycemia. Therefore, researchers should be aware that even under euglycemic conditions, SGLT2 inhibitors induce glucosuria that interferes with accurate ERPF measurement due to glycosylation of PAH in stored urine samples. Pre-treatment of urine with NaOH or adding HCl prior to measurement will overcome this problem.

Abstract P147: Attenuation Of Diuretic And Natriuretic Responses To Acute Saline Volume Expansion In Mice Pre-treated With Tumor Necrosis Factor-alpha (TNFα) Inhibitor, Etanercept.

High salt intake induces an immune response that activates the mononuclear phagocyte system (MPS) cells to produce TNFα. We have previously demonstrated that intravenous infusion of TNFα in mice induces diuresis and natriuresis by inhibiting renal tubular sodium reabsorption. We hypothesize that the intravenous saline infusion can also induce the production of TNFα from MPS cells and such increase in TNFα influences saline induced natriuresis in the kidney. To examine this hypothesis, we measured the changes in TNFα levels in plasma and in urinary excretion rate (U TNFα V) during intravenous infusion of isotonic saline (0.9% NaCl), first at euvolemic condition (3 μL/min for 60 min; Baseline period) and then at an enhanced infusion rate (12 μL/min for 90 min; Volume expansion period) in anesthetized mice (n=5). Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by PAH and inulin clearances respectively. TNFα concentration in plasma and urine samples were determined using ELISA kit (Ebioscience, Woburn, MA). TNFα level in plasma collected during baseline period was undetectable, however, TNFα was increased to 3.7±1.3 pg/mL during volume expansion (VE) period. Baseline U TNFα V level was 0.01±0.002 pg/min/g (kidney wt), which was increased to 0.11±0.03 pg/min/g (P&lt;0.05) during VE period. VE increased urine flow (V, 5.4±0.5 to 22.0±5.1 μL/min/g; P&lt;0.05) and sodium excretion (U Na V; 0.54±0.19 to 4.84 ±1.16 μmol/min/g; P&lt;0.05) without significant changes in RBF (7.1±1.0 to 6.0±1.8 mL/min/g) and GFR (1.18±0.11 to 0.94±0.28 mL/min/g). Interestingly, the diuretic and natriuretic responses to VE were markedly attenuated in mice (n=5) pretreated with a TNFα inhibitor, etanercept (ETN; 0.5 mg/kg intraperitoneally once daily for 3 days prior to the experiment day). VE induced changes in ETN treated mice are as follows: V, 5.9±0.65 to 7.9 ±1.6 μL/min/g; U Na V, 0.43±0.6 to 1.1±0.28 μmol/min/g; RBF, 5.4±0.4 to 6.6±0.9 mL/min/g and GFR, 0.87±0.09 to 0.98±0.13 mL/min/g. These findings demonstrate for the first time that an intravenous saline volume infusion resulted an increase in TNFα level in plasma and in urine. These results strongly suggest a physiological role for TNFα in regulating renal excretory function during saline volume expansion.

Conventional Pig as Animal Model for Human Renal Drug Excretion Processes: Unravelling the Porcine Renal Function by Use of a Cocktail of Exogenous Markers.

Over recent years, pigs have been promoted as potential animal model due to their anatomical and physiological similarities with humans. However, information about the contribution of distinct renal elimination processes [glomerular filtration rate (GFR), effective renal plasma flow (ERPF), tubular secretion, and reabsorption] in pigs is currently limited. Therefore, a cocktail of renal markers, consisting of iohexol (GFR), para-aminohippuric acid (ERPF and net tubular anion secretion), pindolol (net tubular cation secretion), and fluconazole (net tubular reabsorption) was administered intravenously to 7-week-old male conventional pigs. Plasma and urinary concentrations were determined using validated analytical methods. The clearance of iohexol (GFR) was 97.87 ± 16.05 ml/min/m² (mean ± SD). The ERPF, calculated as the renal clearance of PAH, was 226.77 ± 62.45 ml/min/m², whereas the net tubular secretion of PAH was 130.28 ± 52.62 ml/min/m². The net tubular secretion of R-pindolol and S-pindolol was 13.53 ± 12.97 and 18.01 ± 39.23 ml/min/m², respectively. The net tubular reabsorption of fluconazole was 78.32 ± 13.52 ml/min/m². Overall, this cocktail of renal markers was considered to be safe for use in pigs since no adverse effects were observed. Iohexol, PAH and fluconazole were considered suitable renal marker to assess the porcine renal function. Pindolol seems less appropriate due to the high degree of nonrenal clearance in pigs. The values of GFR, ERPF, and anion secretion are within the same range for both human and pig. Regarding the tubular reabsorption of fluconazole, slightly higher values were obtained for pigs. Nevertheless, these results indicate the conventional pig could be an appropriate animal model to study renal drug elimination processes in humans.

Renal sinus fat and renal hemodynamics: a cross-sectional analysis

ObjectivesIncreased renal sinus fat (RSF) is associated with hypertension and chronic kidney disease, but underlying mechanisms are incompletely understood. We evaluated relations between RSF and gold-standard measures of renal hemodynamics in type 2 diabetes (T2D) patients.MethodsFifty-one T2D patients [age 63 ± 7 years; BMI 31 (28–34) kg/m2; GFR 83 ± 16 mL/min/1.73 m2] underwent MRI-scanning to quantify RSF volume, and subcutaneous and visceral adipose tissue compartments (SAT and VAT, respectively). GFR and effective renal plasma flow (ERPF) were determined by inulin and PAH clearances, respectively. Effective renal vascular resistance (ERVR) was calculated.ResultsRSF correlated negatively with GFR (r = − 0.38; p = 0.006) and ERPF (r = − 0.38; p = 0.006) and positively with mean arterial pressure (MAP) (r = 0.29; p = 0.039) and ERVR (r = 0.45, p = 0.001), which persisted after adjustment for VAT, MAP, sex, and BMI. After correction for age, ERVR remained significantly related to RSF.ConclusionsIn T2D patients, higher RSF volume was negatively associated to GFR. In addition, RSF volume was positively associated with increased renal vascular resistance, which may mediate hypertension and CKD development. Further research is needed to investigate how RSF may alter the (afferent) vascular resistance of the renal vasculature.

SUN-281 Electrically stimulated acupuncture improve muscle function and increases renal blood flow through exosomes-carried miR-181

our previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) can prevent muscle atrophy by increasing muscle protein anabolism in mouse models of chronic kidney disease and diabetes. Previous other studies have found that Acu/LFES decreases urinary albumin secretion and improves renal blood flow and GFR in nephropathy patients and animals. We hypothesized that Acu/LFES has benefits on both skeletal muscle and kidney. Normal C56BL6 mice were awake without any anesthesia and appeared to be comfortable throughout the Acu/LFES. Acupuncture points selected were according to the WHO Standard Acupuncture guidelines. The mice were treated with Acu/LFES (anode: Yang Ling Quan, GB34 and cathode: Zu San Li, ST36) daily for 15 days. Renal plasma flow was measured by p-Aminohippuric acid infusion (PAH Colorimetric Assay Kit, BioVision Inc.). Glomerular filtration rate was determined using inulin infusion and measured with a FIT-GFR kit (BioPhysics Assay Lab, Inc.). Muscle function was measured using a mouse grip strength meter. Exosomes were isolated by serial centrifugations. A miR deep sequencing assay and qPCR were used to identify microRNA expression in exosomes. Skeletal muscle function and protein anabolic markers were increased after Acu/LFES treatment. Interestingly, PAH clearance was increased by 45% in the mice after 15 minutes of Acu/LFES versus mice with sham Acu/LFES (sham 6.1 ± 1.3 vs. Acu/LFES 8.9 ± 2.7 ml/min/g BW, P = 0.009, n=8/group). In additional, inulin clearance was increased 39.8% (sham 65.9 ± 16.3 vs. Acu/LFES108.9 ± 19.7 ul/min/BW, P = 0.014). We found that Acu/LFES increases serum exosome abundance (Figure 1). When exosome secretion was blocked using GW4869, the Acu/LFES-induced increase in renal blood flow and GFR were limited. To identify how exosomes regulate renal blood flow, we performed microRNA deep sequencing of exosomes isolated from mouse serum and found that 34 microRNAs were altered by Acu/LFES. Notably, miR-181d-5p, a microRNA that is involved in regulating translation of renin, was significantly increased in the serum exosomes of Acu/LFES treated mice. Using a luciferase reporter assay, we demonstrated that miR-181 directly inhibits angiotensinogen, which provides potential evidence of Acu/LFES regulation of renal blood flow. Acu/LFES not only improves muscle function, but also increases miR-181 in serum exosomes and increases renal blood flow, suggesting that miR181 may regulate renal blood flow possibly by influencing the renin-angiotensin system. This study provides new insights about the mechanism(s) of muscle-organ cross talk through exosome-derived microRNA.

1128-P: SGLT2 Inhibition Increases Serum Copeptin in Adults with Type 1 Diabetes (T1D)

Elevated copeptin, a stable surrogate of vasopressin, is recognized to be higher in T1D and predicts diabetic kidney disease and cardiovascular (CV) mortality. Vasopressin increases renal oxygen consumption through enhanced sodium reabsorption and may result in renal hypoxia. Given the natriuretic and cardiorenal protective effects of SGLT2 inhibition (SGLT2i) in clinical trials, our aim was to: 1) examine serum copeptin in adults with T1D after SGLT2i with empagliflozin (EMPA) for 8-weeks; and 2) determine the relationship between copeptin and metabolic, renal and system hemodynamic parameters. In this post hoc, exploratory analysis, serum copeptin concentration, glomerular filtration rate (GFR) by inulin clearance, effective renal plasma flow (ERPF) by PAH clearance, vascular stiffness parameters, plasma RAAS markers, HbA1c, 24-hour urine volume and sodium and glucose excretion were measured in 40 participants with T1D (24.1±5.0 years, HbA1c 8.0±0.9%) during eu- and hyperglycemia at baseline and after 25 mg of daily EMPA. Copeptin concentrations increased under both eu- and hyperglycemic conditions (4.0±2.1 to 5.1±6.0 pmol/L, p=0.005 and p&amp;lt;0.0001, respectively) in response to EMPA. Higher copeptin concentrations were associated with higher HbA1c (β±SE: 0.97±0.30, p=0.003), lower 24h urine volume (β±SE: -3.08±1.05, p=0.006) and fractional sodium excretion (β±SE: -8.30±2.73, p=0.004), after correcting for age, sex, systolic blood pressure (SBP), and HbA1c. There were no significant relationships between copeptin and RAAS markers, vascular stiffness, renal or systemic hemodynamic parameters in fully adjusted models. SGLT2i increased serum copeptin concentrations, which positively correlated with HbA1c and inversely with urine output and natriuresis, but not with renal or system hemodynamic parameters. Further work is required to evaluate the clinical implications of elevated copeptin with SGLT2i; whether it is simply a marker of diuresis or may contribute to CV disease long-term. Disclosure Y. Lytvyn: None. P. Bjornstad: Advisory Panel; Self; Horizon, XORTX. Consultant; Self; Bayer US, Boehringer Ingelheim International GmbH, Bristol-Myers Squibb Company. A. Katz: None. S.K. Singh: None. L.C. Godoy: None. L.T. Chung: None. C.L. Vinovskis: None. B.A. Perkins: Advisory Panel; Self; Abbott, Boehringer Ingelheim International GmbH, Boehringer Ingelheim International GmbH, Insulet Corporation. Research Support; Self; Boehringer Ingelheim International GmbH. Other Relationship; Self; Abbott, Boehringer Ingelheim International GmbH, Lilly Diabetes, Medtronic, Novo Nordisk Inc., Sanofi. D. Cherney: Other Relationship; Self; AbbVie Inc., AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Janssen Pharmaceuticals, Inc., Merck &amp; Co., Inc., Mitsubishi Tanabe Pharma Corporation, Prometic Life Sciences Inc., Sanofi.

Electrically‐stimulated acupuncture improves muscle function and increases renal blood flow through exosomes‐carried miR‐181

Introductionour previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) can prevent muscle atrophy by increasing muscle protein anabolism in mouse models of chronic kidney disease, diabetes and denervation. Previous other studies have found that Acu/LFES improves renal blood flow and GFR in nephropathy patients and animals. We hypothesized that Acu/LFES benefits both skeletal muscle and kidney possibly through exosome carried microRNA.MethodsNormal C57BL6 mice were awake without any anesthesia and appeared to be comfortable throughout the Acu/LFES. Acupuncture points selected were according to the WHO Standard Acupuncture guidelines. The mice were treated with Acu/LFES (anode: Yang Ling Quan, GB34 and cathode: Zu San Li, ST36) daily for 15 days. Renal plasma flow was measured by p‐Aminohippuric acid infusion. Glomerular filtration rate was determined using inulin infusion and measured with a FIT‐GFR kit. Exosomes were isolated by serial centrifugations. A miR deep sequencing assay and qPCR were used to identify microRNA expression in exosomes.ResultsSkeletal muscle grip function and muscle regeneration markers were increased after Acu/LFES treatment. Acu/LFES increased muscle cross‐sectional area in normal mice vs. sham Acu‐LFES mice. The abundance of Pax7, MyoD, myogenin and embryo myosin heavy chain was significantly increased by Acu‐LFES in soleus, extensor digitorum longus (EDL) and gastrocnemius muscle. The number of central nuclei was increased in Acu‐LFES treated muscle fibers. Interestingly, PAH clearance was increased by 45% in the mice after 15 minutes of muscle Acu/LFES versus mice with sham Acu/LFES (sham 6.1 ± 1.3 vs. Acu/LFES 8.9 ± 2.7 ml/min/g BW, P = 0.009, n=8/group). Inulin clearance was increased 39.8% (sham 65.9 ± 16.3 vs. Acu/LFES108.9 ± 19.7 ul/min/BW, P = 0.014). We found that Acu/LFES increases serum exosome abundance. When exosome secretion was blocked using GW4869, the Acu/LFES‐induced increase in renal blood flow and GFR were limited. To identify how exosomes regulate renal blood flow, we performed microRNA deep sequencing of exosomes isolated from mouse serum and found that 34 microRNAs were altered by Acu/LFES. Notably, miR‐181d‐5p, a microRNA that is involved in regulating translation of renin, was significantly increased in the serum exosomes of Acu/LFES treated mice. Using a luciferase reporter assay, we demonstrated that miR‐181 directly inhibits angiotensinogen, which provides potential evidence of Acu/LFES regulation of renal blood flow.ConclusionsAcu/LFES not only improves muscle function, but also increases miR‐181 in serum exosomes and increases renal blood flow, suggesting that miR181 may regulate renal blood flow possibly by influencing the renin‐angiotensin system. This study provides new insights about the mechanism(s) of muscle‐organ cross talk through exosome‐delivered microRNA.Support or Funding InformationNIH R01 AR060268 and AHA 17IBDG33780000This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Fructose Overconsumption since Early Age on Renal Function and Sympathetic Nerve Activity

Increased consumption of ready‐to‐eat foods and beverages that contain high fructose content may contribute to the onset of cardiovascular and renal diseases. Moreover, fructose overload in diet since prenatal period may increase the risk of the offspring to diseases later in life. Thus, the objective of this study was to evaluate the effects of increased consumption of fructose since prenatal period until adulthood on: blood pressure, renal function and renal sympathetic nerve activity of the offspring at adulthood.MethodsFemales Wistar rats were randomly assigned to groups: Control (C) and Fructose (F). The C group received food and drinking water ad libitum, and F group received food and D‐fructose solution (20%) for drinking ad libitum. In F, the offer of D‐fructose solution begun 1 week before mating and continued during pregnancy and lactation. After birth, the progeny was limited to 8 offspring and designated as Control Offspring (CO) and Fructose Offspring (FO). At 21st day of life, male offspring were placed in collective cages and received the same treatment of the dams. The experiments were performed at 4 months old rats. Direct measurement of systolic arterial blood pressure and mean arterial blood pressure (SAP and MAP), and the registration of the renal sympathetic nerve activity (RSNA) were performed in 6 animals of each group (CO and FO). The renal function parameters were evaluated in other 6 rats of each group. Renal parameters evaluated: glomerular filtration rate (GFR ‐ inulin clearance), renal plasma flow (RPF ‐ PAH clearance), protein excretion (EProt), urine osmolarity (Uosm), urinary excretion/24 h (V), sodium (ENa+) and potassium (EK+) excretion. The study was approved by the Ethical Research Committee of the Universidade Federal de São Paulo (protocol: 2757270117).ResultsCompared with CO group, FO presented higher values of SAP (FO: 165 ± 5 vs CO: 141 ± 6 mmHg) and MAP (FO: 130 ± 5; CO: 110 ± 4 mmHg). FO had decreased GFR (FO: 5.1 ± 0.7; CO: 8.4 ± 0.5 ml/min/kg), Uosm (FO: 305 ± 60; CO: 1522 ± 81 mosm/l) and potassium excretion (FO: 182 ± 18; CO: 302 ± 28 mM/24 h). The urine excretion and protein excretion were increased in FO compared with CO group (V: FO: 52 ± 1; CO: 14 ± 0.7 ml/24 h; EProt: FO: 10.3 ± 1; CO: 6.4 ± 1 mg/24 h). There were no significant differences between the groups regarding the baseline RSNA (FO: 59 ± 4; CO: 71 ± 14 spikes/s), RPF (FO: 15.0 ± 2; CO: 16.2 ± 1 ml/min/kg) and sodium excretion (FO: 91.2 ± 15; CO: 85.7 ± 15 mM/24 h). Values are presented as mean ± sem, p ≤ 0.05.ConclusionsOur data show that increased consumption of fructose since early age leads to increase in blood pressure and alterations in renal function in the offspring (reduced: GFR, urine osmolarity and potassium excretion; and increased protein excretion).Support or Funding InformationCAPESThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial mineralocorticoid receptor ablation does not alter blood pressure, kidney function or renal vessel contractility.

Aldosterone blockade confers substantial cardiovascular and renal protection. The effects of aldosterone on mineralocorticoid receptors (MR) expressed in endothelial cells (EC) within the renal vasculature have not been delineated. We hypothesized that lack of MR in EC may be protective in renal vasculature and examined this by ablating the Nr3c2 gene in endothelial cells (EC-MR) in mice. Blood pressure, heart rate and PAH clearance were measured using indwelling catheters in conscious mice. The role of the MR in EC on contraction and relaxation was investigated in the renal artery and in perfused afferent arterioles. Urinary sodium excretion was determined by use of metabolic cages. EC-MR transgenics had markedly decreased MR expression in isolated aortic endothelial cells as compared to littermates (WT). Blood pressure and effective renal plasma flow at baseline and following AngII infusion was similar between groups. No differences in contraction and relaxation were observed between WT and EC-MR KO in isolated renal arteries during baseline or following 2 or 4 weeks of AngII infusion. The constriction or dilatations of afferent arterioles between genotypes were not different. No changes were found between the groups with respect to urinary excretion of sodium after 4 weeks of AngII infusion, or in urinary albumin excretion and kidney morphology. In conclusion, deletion of the EC-MR does not confer protection towards the development of hypertension, endothelial dysfunction of renal arteries or renal function following prolonged AngII-infusion.

Drug Clearance from Cerebrospinal Fluid Mediated by Organic Anion Transporters 1 (Slc22a6) and 3 (Slc22a8) at Arachnoid Membrane of Rats

Although arachnoid mater epithelial cells form the blood-arachnoid barrier (BAB), acting as a blood-CSF interface, it has been generally considered that the BAB is impermeable to water-soluble substances and plays a largely passive role. Here, we aimed to clarify the function of transporters at the BAB in regulating CSF clearance of water-soluble organic anion drugs based on quantitative targeted absolute proteomics (QTAP) and in vivo analyses. Protein expression levels of 61 molecules, including 19 ATP-binding-cassette (ABC) transporters and 32 solute-carrier (SLC) transporters, were measured in plasma membrane fraction of rat leptomeninges using QTAP. Thirty-three proteins were detected; others were under the quantification limits. Expression levels of multidrug resistance protein 1 (Mdr1a/P-gp/Abcb1a) and breast cancer resistance protein (Bcrp/Abcg2) were 16.6 and 3.27 fmol/μg protein (51.9- and 9.82-fold greater than in choroid plexus, respectively). Among those organic anion transporters detected only at leptomeninges, not choroid plexus, organic anion transporter 1 (oat1/Slc22a6) showed the greatest expression (2.73 fmol/μg protein). On the other hand, the protein expression level of oat3 at leptomeninges was 6.65 fmol/μg protein, and the difference from choroid plexus was within two-fold. To investigate oat1's role, we injected para-aminohippuric acid (PAH) with or without oat1 inhibitors into cisterna magna (to minimize the contribution of choroid plexus function) of rats. A bulk flow marker, FITC-inulin, was not taken up from CSF up to 15 min, whereas uptake clearance of PAH was 26.5 μL/min. PAH uptake was completely blocked by 3 mM cephalothin (inhibits both oat1 and oat3), while 17% of PAH uptake was inhibited by 0.2 mM cephalothin (selectively inhibits oat3). These results indicate that oat1 and oat3 at the BAB provide a distinct clearance pathway of organic anion drugs from CSF independently of choroid plexus.

Inhaled nitric oxide prevents NSAID-induced renal impairment in pseudo-normovolaemic piglets.

ObjectiveInhaled nitric oxide (iNO) is commonly used as a treatment of pulmonary hypertension. Its action is purported to be specific to the lung, but extrapulmonary effects have been reported. The objective of this study was to evaluate if iNO could compensate the renal impairment induced by ketoprofen, a conventional non-steroidal anti-inflammatory drug (NSAID), during general anaesthesia.MethodsUnder pseudo-normovolaemic condition, thirty piglets were randomly assigned into 5 equal groups and equipped for renal and systemic parameters measurements. A first experiment was carried out to validate methods and reproduce the renal effects of iNO (40 ppm) in comparison with a placebo (100% oxygen). In a second experiment, iNO was inhaled for 120 minutes right after NSAID treatment (ketoprofen 2 mg×kg-1 IV, and 40 ppm iNO; group KiNO) and its effects were compared to ketoprofen alone (2 mg×kg-1 IV; group K) and placebo (saline; group C).ResultsIn this model, iNO increased significantly renal blood flow measured by ultrasonic (RBFUL: +53.2±17.2%; p = 0.008) and by PAH clearance (RBFPAH:+78.6±37.6%; p = 0.004) methods, glomerular filtration rate (GFR: +72.6±32.5%; p = 0.006) and urinary output (UO: +47.4±24.2%; p = 0.01). In the second experiment, no significant temporal variation was noted for renal parameters in groups KiNO and C, whereas a significant and constant decrease was observed in the group K for RBFUL (max -19.0±7.1%), GFR (max -26.6±10.4%) and UO (max -30.3±10.5%).Clinical significanceOur experiments show that iNO, released from its transport forms after its inhalation, can improve renal safety of NSAIDs. This result is promising regarding the use of NSAIDs in critical conditions, but needs to receive clinical confirmation.

Monoanionic 99mTc-tricarbonyl-aminopolycarboxylate complexes with uncharged pendant groups: Radiosynthesis and evaluation as potential renal tubular tracers

Introduction99mTc(CO)3-nitrilotriacetic acid, 99mTc(CO)3(NTA), is a new renal tubular agent with pharmacokinetic properties comparable to those of 131I-OIH but the clearance of 99mTc(CO)3(NTA) and 131I-OIH is still less than the clearance of PAH, the gold standard for the measurement of effective renal plasma flow. At physiological pH, dianionic 99mTc(CO)3(NTA) has a mononegative inner metal-coordination sphere and a mononegative uncoordinated carboxyl group. To evaluate alternate synthetic approaches, we assessed the importance of an uncoordinated carboxyl group, long considered essential for tubular transport, by evaluating the pharmacokinetics of three analogs with the 99mTc(CO)3(NTA) metal-coordination sphere but with uncharged pendant groups. Methods99mTc(CO)3 complexes with N-(2-acetamido)iminodiacetic acid (ADA), N-(2-hydroxyethyl)iminodiacetic acid (HDA) and N-(fluoroethyl)iminodiacetic acid (FEDA) were prepared using a tricarbonyl kit and isolated by HPLC. The pharmacokinetics were evaluated in Sprague–Dawley rats, with 131I-OIH as an internal control; urine was analyzed for metabolites. Plasma protein binding and erythrocyte uptake were determined from the 10min blood samples. Re(CO)3(FEDA), the analog of 99mTc(CO)3(FEDA), was prepared and characterized. Results99mTc(CO)3(ADA), 99mTc(CO)3(HDA) and 99mTc(CO)3(FEDA) were efficiently prepared as a single species with high radiochemical purities (>99%). These new monoanionic 99mTc(CO)3 tracers with uncharged dangling groups all showed rapid blood clearance and high specificity for renal excretion. Activity in the urine, as a percent of 131I-OIH at 10 and 60min, was 96% and 99% for ADA, 96% and 100% for HDA, and 100% and 99% for FEDA, respectively. Each new tracer was excreted unchanged in the urine. The Re(CO)3(FEDA) structure adds compelling evidence that such 99mTc(CO)3(NTA) analogs have metal-coordination spheres identical to that of 99mTc(CO)3(NTA). ConclusionsNew tracers lacking the negatively charged pendant carboxyl group previously thought to be essential for rapid renal extraction, 99mTc(CO)3(ADA), 99mTc(CO)3(HDA) and 99mTc(CO)3(FEDA), exhibit pharmacokinetics in rats comparable to those of 99mTc(CO)3(NTA) and 131I-OIH. Furthermore, these encouraging results in rats warrant evaluation of this new tracer type in humans.

OP.7D.04] CHANGES IN RENAL HEMODYNAMIC AND TUBULAR SODIUM HANDLING ONE YEAR AFTER TRANSCATHETER RENAL DENERVATION IN RESISTANT HYPERTENSION

Objective: The sympathetic nervous system is implicated in most form of hypertension. In resistant hypertension, transcatheter renal denervation (RDN) has been proposed as an adjunctive therapy to antihypertensive drugs at maximum tolerated doses. The results on blood pressure (BP) reduction have been the source of debate. Few studies have explored the effect on renal hemodynamics and tubular sodium handling. The objective was to assess these two parameters before RND and after 1 and 12 months. Design and method: Patients with confirmed resistant hypertension and no contraindications for RDN could be included in the study. Systemic hemodynamic, renal hemodynamic using inulin and PAH clearances, sodium and endogenous lithium clearances (a marker of proximal sodium reabsorption), hormones were measured in standardized condition before RDN and after 1 and 12 months. Variables were compared with an analysis of variance. Results: 13 patients were included in the study. Mean age was 56.1 ± 9.9 years, body mass index was 30.9 ± 9.9 Kg/m2. Mean number of ablation points was 4.7 ± 1.2 points on the left artery and 5.0 ± 1.6 points on the right artery. The following table displays the systemic and renal hemodynamics, the sodium tubular handling and the hormonal profile before RDN and after 1 and 12 months.BP and heart rate remained constant while glomerular filtration rate and filtration fraction decreased. Proximal sodium reabsorption, assed by lithium clearances, increased with time. Plasma renin activity tended to increase which could not be explained by the use of diurectics which was similar before and 12 months after RDN. Conclusions: One year after renal denervation, renal hemodynamics and tubular sodium handling seem to be altered with increased filtration fraction and proximal sodium reabsorption. The long term effects of these changes on renal function and blood pressure need to be assed in long term follow-up studies.