Reactive Oxygen Species-Mediated Diabetic Heart Disease: Mechanisms and Therapies.

Significance: Diabetic heart disease (DHD) is the primary cause of mortality in people with diabetes. A significant contributor to the development of DHD is the disruption of redox balance due to reactive oxygen species (ROS) overproduction resulting from sustained high glucose levels. Therapies specifically focusing on the suppression of ROS will hugely benefit patients with DHD. Recent Advances: In addition to the gold standard pharmacological therapies, the recent development of gene therapy provides an exciting avenue for developing new therapeutics to treat ROS-mediated DHD. In particular, microRNAs (miRNAs) are gaining interest due to their crucial role in several physiological and pathological processes, including DHD. Critical Issues: miRNAs have many targets and differential function depending on the environment. Therefore, a proper understanding of the function of miRNAs in specific cell types and cell states is required for the successful application of this technology. In the present review, we first provide an overview of the role of ROS in contributing to DHD and the currently available treatments. We then discuss the newer gene therapies with a specific focus on the role of miRNAs as the causative factors and therapeutic targets to combat ROS-mediated DHD. Future Directions: The future of miRNA therapeutics in tackling ROS-mediated DHD is dependent on a complete understanding of how miRNAs behave in different cells and environments. Future research should also aim to develop conditional miRNA therapeutic platforms capable of switching on and off in response to disruptions in the redox state. Antioxid. Redox Signal. 36, 608-630.

Diabetes induces dysregulation of microRNAs associated with survival, proliferation and self-renewal in cardiac progenitor cells.

AIMS/HYPOTHESIS: Diabetes mellitus causes a progressive loss of functional efficacy in stem cells, including cardiac progenitor cells (CPCs). The underlying molecular mechanism is still not known. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate genes at the post-transcriptional level. We aimed to determine if diabetes mellitus induces dysregulation of miRNAs in CPCs and to test if in vitro therapeutic modulation of miRNAs would improve the functions of diabetic CPCs. METHODS: CPCs were isolated from a mouse model of type 2 diabetes (db/db), non-diabetic mice and human right atrial appendage heart tissue. Total RNA isolated from mouse CPCs was miRNA profiled using Nanostring analysis. Bioinformatic analysis was employed to predict the functional effects of altered miRNAs. MS analysis was applied to determine the targets, which were confirmed by western blot analysis. Finally, to assess the beneficial effects of therapeutic modulation of miRNAs in vitro and in vivo, prosurvival miR-30c-5p was overexpressed in mouse and human diabetic CPCs, and the functional consequences were determined by measuring the level of apoptotic cell death, cardiac function and mitochondrial membrane potential (MMP). RESULTS: Among 599 miRNAs analysed in mouse CPCs via Nanostring analysis, 16 miRNAs showed significant dysregulation in the diabetic CPCs. Using bioinformatics tools and quantitative real-time PCR (qPCR) validation, four altered miRNAs (miR-30c-5p, miR-329-3p, miR-376c-3p and miR-495-3p) were identified to play an important role in cell proliferation and survival. Diabetes mellitus significantly downregulated miR-30c-5p, while it upregulated miR-329-3p, miR-376c-3p and miR-495-3p. MS analysis revealed proapoptotic voltage-dependent anion-selective channel 1 (VDAC1) as a direct target for miR-30c-5p, and cell cycle regulator, cyclin-dependent protein kinase 6 (CDK6), as the direct target for miR-329-3p, miR-376c-3p and miR-495-3p. Western blot analyses showed a marked increase in VDAC1 expression, while CDK6 expression was downregulated in diabetic CPCs. Finally, in vitro and in vivo overexpression of miR-30c-5p markedly reduced the apoptotic cell death and preserved MMP in diabetic CPCs via inhibition of VDAC1. CONCLUSIONS/INTERPRETATION: Our results demonstrate that diabetes mellitus induces a marked dysregulation of miRNAs associated with stem cell survival, proliferation and differentiation, and that therapeutic overexpression of prosurvival miR-30c-5p reduced diabetes-induced cell death and loss of MMP in CPCs via the newly identified target for miR-30c-5p, VDAC1.

ß2 -Adrenoceptors indirectly support impaired ß1 -adrenoceptor responsiveness in the isolated type 2 diabetic rat heart.

NEW FINDINGS: What is the central question of this study? Are there specific contributions of ß1 - and ß2 -adrenoceptor subtypes to the impaired ß-adrenoceptor responsiveness of the type 2 diabetic heart? What is the main finding and its importance? In hearts isolated from the Zucker diabetic fatty rat model of type 2 diabetes, we showed that the ß1 -adrenoceptors are the main subtype to regulate heart rate, contraction and relaxation. Notably, the ß2 -adrenoceptor subtype actions seem to support function in the diabetic heart indirectly. ABSTRACT: Impaired ß-adrenoceptor (ß-AR) responsiveness causes cardiac vulnerability in patients with type 2 diabetes, but the independent contributions of ß1 - and ß2 -AR subtypes to ß-AR-associated cardiac dysfunction in diabetes are unknown. Our aim was to determine the specific ß1 - and ß2 -AR responsiveness of heart rate (HR), contraction and relaxation in the diabetic heart. Isolated Langendorff-perfused hearts of Zucker type 2 diabetic fatty (ZDF) rats were stimulated with the ß-AR agonist isoprenaline (1 × 10-11 to 3 × 10-8  mol l-1 ) with or without the selective ß1 -AR antagonist CGP20712A (3 × 10-8  mol l-1 ) or the ß2 -AR antagonist ICI-118,551 (5 × 10-8  mol l-1 ), and HR, contraction and relaxation were measured. Diabetic hearts showed lower basal HR (non-diabetic 216 ± 17 beats min-1 versus diabetic 151 ± 23 beats min-1 , P < 0.05). However, the ß-AR-induced increase in HR was similar and was completely blocked by the ß1 -AR antagonist, but not by the ß2 -AR antagonist. The ß-AR-induced increase in contraction and acceleration of relaxation was impaired in diabetic hearts, completely blocked by the ß1 -AR antagonist and partly impaired by the ß2 -AR antagonist. Western blots revealed 41% higher phosphorylation levels of AMP kinase (AMPK), a key regulator of cardiac energy metabolism, in diabetic hearts (non-diabetic 1.62 ± 0.19 a.u. versus diabetic 2.30 ± 0.25 a.u., P < 0.05). In conclusion, the ß1 -AR is the main subtype regulating chronotropic, inotropic and lusitropic ß-AR responses in the healthy heart and the type 2 diabetic heart. The ß2 -AR subtype indirectly supports the ß1 -AR functional response in the diabetic heart. This suggests that ß2 -ARs could be an indirect target to improve the function of the heart in type 2 diabetes.

Cardiac ß-adrenergic responsiveness of obese Zucker rats: The role of AMPK.

NEW FINDINGS: What is the central question of the study? Is the reduced signalling of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis in the heart, responsible for the reduced ß-adrenergic responsiveness of the heart in obesity? What is the main finding and its importance? Inhibition of AMPK in isolated hearts prevented the reduced cardiac ß-adrenergic responsiveness of obese rats, which was accompanied by reduced phosphorylation of AMPK, a proxy of AMPK activity. This suggests a direct functional link between ß-adrenergic responsiveness and AMPK signalling in the heart, and it suggests that AMPK might be an important target to restore the ß-adrenergic responsiveness in the heart in obesity. ABSTRACT: The obesity epidemic impacts heavily on cardiovascular health, in part owing to changes in cardiac metabolism. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis in the heart and is regulated by ß-adrenoceptors (ß-ARs) in normal conditions. In obesity, chronic sympathetic overactivation leads to impaired cardiac ß-AR responsiveness, although it is unclear whether AMPK signalling, downstream of ß-ARs, contributes to this dysfunction. Therefore, we aimed to determine whether reduced AMPK signalling is responsible for the reduced ß-AR responsiveness in obesity. In isolated hearts of lean and obese Zucker rats, we tested ß-AR responsiveness to the ß1 -AR agonist isoprenaline (ISO, 1 × 10-10 to 5 × 10-8  m) in the absence and presence of the AMPK inhibitor, compound C (CC, 10 µm). The ß1 -AR expression and AMPK phosphorylation were assessed by Western blot. ß-Adrenergic responsiveness was reduced in the hearts of obese rats (logEC50 of ISO-developed pressure dose-response curves: lean -8.53 ± 0.13 × 10x  m versus obese -8.35 ± 0.10 × 10x  m ; P < 0.05 lean versus obese, n = 6 per group). This difference was not apparent after AMPK inhibition (logEC50 of ISO-developed pressure curves: lean CC -8.19 ± 0.12 × 10x  m versus obese CC 8.17 ± 0.13 × 10x  m, P < 0.05, n = 6 per group). ß1 -Adrenergic receptor expression and AMPK phosphorylation were reduced in hearts of obese rats (AMPK at Thr172 : lean 1.73 ± 0.17 a.u. versus lean CC 0.81 ± 0.13 a.u., and obese 1.18 ± 0.09 a.u. versus obese CC 0.81 ± 0.16 a.u., P < 0.05, n = 6 per group). Thus, a direct functional link between ß-adrenergic responsiveness and AMPK signalling in the heart exists, and AMPK might be an important target to restore the reduced cardiac ß-adrenergic responsiveness in obesity.

Uric acid: a modulator of prostate cells and activin sensitivity.

Elevated serum uric acid (SUA) or urate is associated with inflammation and gout. Recent evidence has linked urate to cancers, but little is known about urate effects in prostate cancer. Activins are inflammatory cytokines and negative growth regulators in the prostate. A hallmark of prostate cancer progression is activin insensitivity; however, mechanisms underlying this are unclear. We propose that elevated SUA is associated with prostate cancer counteracting the growth inhibitory effects of activins. The expression of activins A and B, urate transporter GLUT9 and tissue urate levels were examined in human prostate disease. Intracellular and secreted urate and GLUT9 expression were assessed in human prostate cancer cell lines. Furthermore, the effects of urate and probenecid, a known urate transport inhibitor, were determined in combination with activin A. Activin A expression was increased in low-grade prostate cancer, whereas activin B expression was reduced in high-grade prostate cancer. Intracellular urate levels decreased in all prostate pathologies, while GLUT9 expression decreased in benign prostatic hyperplasia, prostatitis and high-grade prostate cancer. Activin responsive LNCaP cells had higher intracellular and lower secreted urate levels than activin-insensitive PC3 cells. GLUT9 expression in prostate cancer cells was progressively lower than in prostate epithelial cells. Elevated extracellular urate was growth promoting in vitro, which was abolished by the gout medication probenecid, and it antagonized the growth inhibitory effects of activins. This study shows for the first time that a change in plasma or intracellular urate levels, possibly involving GLUT9 and a urate efflux transporter, has an impact on prostate cancer cell growth, and that lowering SUA levels in prostate cancer is likely to be therapeutically beneficial.

Chronic bilateral renal denervation reduces cardiac hypertrophic remodelling but not ß-adrenergic responsiveness in hypertensive type 1 diabetic rats.

NEW FINDINGS: What is the central question of this study? Can bilateral renal denervation, an effective antihypertensive treatment in clinical and experimental studies, improve cardiac ß-adrenoceptor responsiveness in a diabetic model with underlying hypertension? What is the main finding and its importance? Bilateral renal denervation did not affect ß-adrenergic responsiveness in the diabetic hypertensive rat heart, but denervation reduced the hypertension-induced concentric hypertrophic remodelling. This suggests that the positive haemodynamic changes induced by renal denervation are most likely to reflect an attenuation of sympathetic effects on the systemic vasculature and/or the renal function rather than direct sympathetic modulation of the heart. Bilateral renal denervation (BRD) has been shown to normalise blood pressure in clinical and experimental studies of hypertension by reducing systemic sympathetic output. This study determined the effect of BRD on cardiac ß-adrenoceptor (AR) responsiveness in a diabetic model with underlying hypertension using the transgenic (mRen-2)27 rats. Bilateral renal denervation or sham surgeries were conducted repeatedly at 3, 6 and 9 weeks in Ren-2 rats with or without streptozotocin (STZ)-induced diabetes (4 × n = 7); Sprague-Dawley rats (n = 6) served as control animals. Cardiac function was determined in isolated hearts at 18 weeks of age. Normalised left ventricular developed pressure and relaxation was recorded in response to incremental concentrations of the ß-AR agonist isoprenaline (from 10-10 to 10-7 m) or the ß3 -AR agonist BRL37344 (from 10(-13) to 10(-6 ) m). Expression levels of ß1 -AR were determined by Western blot. Both inotropic and lusitropic ß-AR responsiveness was reduced in the hypertensive diabetic hearts, but these responses were unaltered after BRD. Expression levels of ß1 -AR were increased after BRD (Sham, 0.85 ± 0.11 versus 1.01 ± 0.05 a.u.; BRD, 1.45 ± 0.11 versus 1.46 ± 0.07 a.u.; Ren-2 versus Ren-2 STZ, P < 0.05 versus Sham). No effect of ß3 -AR agonist stimulation with BRL37344 was observed. Interestingly, BRD increased left ventricular diastolic volume in both the Ren-2 and the Ren-2 STZ groups. Bilateral renal denervation did not restore the attenuated cardiac ß-AR responsiveness in the diabetic hypertensive rats, but it reduced the extent of hypertension-induced concentric hypertrophic remodelling. Thus, the haemodynamic protection offered by renal denervation appears to reflect an attenuated sympathetic innervation of the systemic vasculature and/or kidney rather than a direct cardiac effect.

The Role of MicroRNAs in Cardiac Stem Cells.

Stem cells are considered as the next generation drug treatment in patients with cardiovascular disease who are resistant to conventional treatment. Among several stem cells used in the clinical setting, cardiac stem cells (CSCs) which reside in the myocardium and epicardium of the heart have been shown to be an effective option for the source of stem cells. In normal circumstances, CSCs primarily function as a cell store to replace the physiologically depleted cardiovascular cells, while under the diseased condition they have been shown to experimentally regenerate the diseased myocardium. In spite of their major functional role, molecular mechanisms regulating the CSCs proliferation and differentiation are still unknown. MicroRNAs (miRs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the important role of miRs in regulating stem cell proliferation and differentiation, as well as other physiological and pathological processes related to stem cell function. This review summarises the current understanding of the role of miRs in CSCs. A deeper understanding of the mechanisms by which miRs regulate CSCs may lead to advances in the mode of stem cell therapies for the treatment of cardiovascular diseases.

Increased Efferent Cardiac Sympathetic Nerve Activity and Defective Intrinsic Heart Rate Regulation in Type 2 Diabetes.

Elevated sympathetic nerve activity (SNA) coupled with dysregulated ß-adrenoceptor (ß-AR) signaling is postulated as a major driving force for cardiac dysfunction in patients with type 2 diabetes; however, cardiac SNA has never been assessed directly in diabetes. Our aim was to measure the sympathetic input to and the ß-AR responsiveness of the heart in the type 2 diabetic heart. In vivo recording of SNA of the left efferent cardiac sympathetic branch of the stellate ganglion in Zucker diabetic fatty rats revealed an elevated resting cardiac SNA and doubled firing rate compared with nondiabetic rats. Ex vivo, in isolated denervated hearts, the intrinsic heart rate was markedly reduced. Contractile and relaxation responses to ß-AR stimulation with dobutamine were compromised in externally paced diabetic hearts, but not in diabetic hearts allowed to regulate their own heart rate. Protein levels of left ventricular ß1-AR and Gs (guanine nucleotide binding protein stimulatory) were reduced, whereas left ventricular and right atrial ß2-AR and Gi (guanine nucleotide binding protein inhibitory regulatory) levels were increased. The elevated resting cardiac SNA in type 2 diabetes, combined with the reduced cardiac ß-AR responsiveness, suggests that the maintenance of normal cardiovascular function requires elevated cardiac sympathetic input to compensate for changes in the intrinsic properties of the diabetic heart.

Molecular mechanism of an adverse drug-drug interaction of allopurinol and furosemide in gout treatment.

Gout patients receiving a combination of allopurinol and furosemide require higher allopurinol doses to achieve the target serum urate (SU) of <6 mg/dl (Stamp et al., 2012) [1]. Our study aimed to identify the molecular basis for this observation. We used a fluorimetric assay to determine the impact of furosemide and oxypurinol (the active metabolite of allopurinol) on xanthine oxidase (XO) activity. Immunoblot analysis quantified expression of XO and AMP-kinase (AMPK) in drug-treated human liver (HepG2) and primary kidney (HRCE) cells. In silico analysis identified miR-448 as a potential XO-regulator, whose expression level in HepG2 cells was examined by qPCR. Fluorimetric experiments revealed no direct interactions between XO and furosemide, nor did the combination of oxypurinol/furosemide alter the XO inhibition profile of oxypurinol. In HepG2 cells, we found a significant decrease in XO protein expression following oxypurinol treatment, which was abolished after co-incubation with furosemide. Probenecid alone or in combination with furosemide reduced XO protein expression significantly. qPCR analysis of miR-448 in HepG2 cells mirrored the drug-dependent changes in XO protein expression. In addition, oxypurinol and the combination of oxypurinol/furosemide significantly down-regulated AMPK protein expression in HRCE cells. In conclusion, we show for the first time that besides the established effects of allopurinol on the purine synthetic pathway the efficiency of allopurinol treatment of gout patients is based on two further complementary mechanisms, the direct inhibition of XO activity by the allopurinol metabolite oxypurinol and a down-regulation of XO protein expression. The latter is compromised by addition of furosemide and might explain why patients receiving furosemide therapy require higher allopurinol doses. miR-448 was identified as a potential drug-dependent XO regulator. Finally, down-regulation of AMPK protein expression in HRCE cells by administration of oxypurinol/furosemide reveals a possible new mechanism of renal drug-induced hyperuricemia.

Impaired relaxation despite upregulated calcium-handling protein atrial myocardium from type 2 diabetic patients with preserved ejection fraction.

BACKGROUND: Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function. METHODS: Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and non-diabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and ß-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of ß1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining. RESULTS: In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to ß-adrenergic stimulation, despite no change in ß1-adrenoreceptor expression levels. CONCLUSIONS: Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during ß-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory calcium-handling mechanism in diabetes in an attempt to maintain diastolic function at rest despite impaired relaxation in the diabetic fibrotic atrial myocardium. Our study addresses important aspects of the underlying mechanisms of diabetes-associated diastolic dysfunction, which is crucial to developing new therapeutic treatments.

URAT1 mutations cause renal hypouricemia type 1 in Iraqi Jews.

BACKGROUND: Hereditary renal hypouricemia may be complicated by nephrolithiasis or exercise-induced acute renal failure. Most patients described so far are of Japanese origin and carry the truncating mutation W258X in the uric acid transporter URAT1 encoded by SLC22A12. Recently, we described severe renal hypouricemia in Israeli patients with uric acid transporter GLUT9 (SLC2A9) loss-of-function mutations. Renal hypouricemia in Iraqi Jews has been previously reported, but its molecular basis has not been ascertained. METHODS: Three Jewish Israeli families of Iraqi origin with hereditary hypouricemia and hyperuricosuria were clinically characterized. DNA was extracted and the URAT1 gene was sequenced. Transport studies into Xenopus laevis oocytes were utilized to evaluate the function of URAT1 mutants found. RESULTS: A missense URAT1 mutation, R406C, was detected in all three families. Two affected siblings were found to carry in addition a homozygous missense URAT1 mutation, G444R. Both mutations dramatically impaired urate uptake into X. laevis oocytes. Moreover, we demonstrate for the first time that URAT1 facilitates urate efflux, which was abolished in the mutants, indicating also a secretion defect. Homozygous patients had serum uric acid concentrations of 0.5-0.8 mg% and a fractional excretion of uric acid of 50-85%. Most individuals studied were asymptomatic, two had nephrolithiasis and none developed exercise-induced acute renal failure. CONCLUSIONS: The URAT1 R406C mutation detected in all three families is likely to be the founder mutation in Iraqi Jews. Our findings contribute to a better definition of the different types of hereditary renal hypouricemia and suggest that the phenotype of this disorder depends mainly on the degree of inhibition of uric acid transport.

Organic anion transporter 3 (OAT3) and renal transport of the metal chelator 2,3-dimercapto-1-propanesulfonic acid (DMPS).

Recent investigations involving intact rabbit renal proximal tubules indicated that organic anion transporter 3 (OAT3) may be involved in the transport of 2,3-dimercapto-1-propanesulfonic acid (DMPS). Therefore, we evaluated the interaction of OAT3 with DMPS to determine the effect of OAT3 on basolateral DMPS uptake. We used stably transfected HEK293 cells expressing human and rabbit orthologs of the exchanger OAT1 and OAT3. Using 6-carboxyfluorescein (6-CF) as a substrate, the IC50 determinations for reduced DMPS (DMPSH) revealed a stronger interaction with OAT1 than with OAT3 (rbOAT1, 123.3 +/- 13.7; hOAT1, 85.1 +/- 8.8; rbOAT3, 171.7 +/- 22.3; and hOAT3, 172.2 +/- 36.4 micromol/L). However, inhibition of 6-CF uptake by the oxidized form of DMPS (DMPSS), the main form of DMPS in the blood, showed a greater affinity for OAT3 (rbOAT1, 237.4 +/- 23; hOAT1, 104.6 +/- 13.1; rbOAT3, 52.4 +/- 7.6; and hOAT3, 31.6 +/- 6.6 micromol/L). To determine whether DMPSH and DMPSS are substrates for OAT3, we performed efflux studies with [14C]glutarate and inwardly directed gradients of glutarate. The inhibitors trans-stimulated the efflux of [14C]glutarate, suggesting that OAT3 may be able to transport both forms of DMPS. On the basis of the substantial interaction of OAT3 with DMPSS, we conclude that OAT3 represents the dominant basolateral player in renal detoxification processes resulting from use of DMPS.

Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy.

Aristolochic acid (AA), present in Aristolochia species, is the major causative agent in the development of severe renal failure and urothelial cancers in patients with AA nephropathy. It may also be a cause of Balkan endemic nephropathy. Epithelial cells of the proximal tubule are the primary cellular target of AA. To study whether organic anion transporters (OATs) expressed in proximal tubule cells are involved in uptake of AA, we used human epithelial kidney (HEK293) cells stably expressing human (h) OAT1, OAT3 or OAT4. AA potently inhibited the uptake of characteristic substrates, p-aminohippurate for hOAT1 and estrone sulfate for hOAT3 and hOAT4. Aristolochic acid I (AAI), the more cytotoxic and genotoxic AA congener, exhibited high affinity for hOAT1 (K(i)=0.6 microM) as well as hOAT3 (K(i)=0.5 microM), and lower affinity for hOAT4 (K(i)=20.6 microM). Subsequently, AAI-DNA adduct formation (investigated by (32)P-postlabelling) was used as a measure of AAI uptake. Significantly higher levels of adducts occurred in hOAT-expressing cells than in control cells: this effect was abolished in the presence of the OAT inhibitor probenecid. In Xenopus laevis oocytes hOAT-mediated efflux of p-aminohippurate was trans-stimulated by extracellular AA, providing further molecular evidence for AA translocation by hOATs. Our study indicates that OATs can mediate the uptake of AA into proximal tubule cells and thereby participate in kidney cell damage by this toxin.

Functional differences in steroid sulfate uptake of organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1) in human placenta.

Human trophoblasts depend on the supply of external precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16alpha-OH-DHEA-S for synthesis of estrogens. Recently, we have characterized the uptake of DHEA-S by isolated mononucleated trophoblasts and identified different transporter polypeptides involved in this process. Immunohistochemistry of 1st and 3rd trimester placenta detected organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1, former name OATP-B) in cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast, indicating that both transporter polypeptides are involved in placental uptake of foetal derived steroid sulfates. In the present study we have characterized and compared the kinetics of DHEA-S and estrone sulfate (E(1)S) uptake by these transporters stably expressed in FlpIn -HEK293 cells using the Flp recombinase-mediated site-specific recombination. Uptake of E(1)S by OAT4- and OATP2B1-transfected cells was highly increased compared to the non-transfected cells. In contrast, DHEA-S uptake was only highly increased in OAT4 (40 times), but only weakly enhanced in OATP2B1 cells. The uptake of DHEA-S and E(1)S by OAT4 was partly Na(+)-dependent (about 50%), whereas uptake of DHEA-S by OATP2B1 was Na(+)-independent. Kinetic analysis of the initial uptake rates of E(1)S by OAT4 and OATP2B1 gave very similar values for K(m) (about 20microM) and V(max) (about 600pmol/(minxmg protein)). In contrast, the affinity of DHEA-S towards OATP2B1 was about 10 times lower (K(m)>200microM) then for OAT4 (K(m)=29microM). Our results suggest different physiological roles of the two transporter polypeptides in placental uptake of foetal derived steroid sulfates. OATP2B1 seems not to be involved in de novo synthesis of placental estrogens but may contribute to the clearance of estrogen sulfates from foetal circulation.

Identification of a new urate and high affinity nicotinate transporter, hOAT10 (SLC22A13).

The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.

Gender differences in kidney function.

Sex hormones influence the development of female (F) and male (M) specific traits and primarily affect the structure and function of gender-specific organs. Recent studies also indicated their important roles in regulating structure and/or function of nearly every tissue and organ in the mammalian body, including the kidneys, causing gender differences in a variety of characteristics. Clinical observations in humans and studies in experimental animals in vivo and in models in vitro have shown that renal structure and functions under various physiological, pharmacological, and toxicological conditions are different in M and F, and that these differences may be related to the sex-hormone-regulated expression and action of transporters in the apical and basolateral membrane of nephron epithelial cells. In this review we have collected published data on gender differences in renal functions, transporters and other related parameters, and present our own microarray data on messenger RNA expression for various transporters in the kidney cortex of M and F rats. With these data we would like to emphasize the importance of sex hormones in regulation of a variety of renal transport functions and to initiate further studies of gender-related differences in kidney structure and functions, which would enable us to better understand occurrence and development of various renal diseases, pharmacotherapy, and drug-induced nephrotoxicity in humans and animals.

Human renal organic anion transporter 4 operates as an asymmetric urate transporter.

Human organic anion transporter 4 (hOAT4) is located at the apical membrane of proximal tubule cells and involved in renal secretion and reabsorption of endogenous substances as well as many drugs and xenobiotics. This study reevaluated the physiologic role, transport mode, and driving forces of hOAT4. 6-Carboxyfluorescein (6-CF) uptake into HEK293 cells that stably expressed hOAT4 was saturable, resulting in a K(m) of 108 muM. 6-CF as well as [(3)H]estrone sulfate ([(3)H]ES) accumulation by HEK293-hOAT4 cells were abolished by ES, dehydroepiandrosterone sulfate, sulfinpyrazone, benzbromarone, and probenecid, whereas several OA, including p-aminohippurate (PAH), lactate, pyrazinoate, nicotinate, glutarate, and the diuretic hydrochlorothiazide (HCTZ) exhibited a slight or a NS inhibitory effect. PAH and glutarate are not taken up by HEK293-hOAT4 cells, but they trans-stimulated 6-CF and [(3)H]ES uptake, indicating an asymmetric interaction of hOAT4 with these substrates. In chloride-free medium, HEK293-hOAT4-mediated [(3)H]PAH efflux was almost abolished, whereas addition of ES restored it comparable to Ringer solution, consistent with a physiologic ES/PAH or PAH/Cl(-) exchange mode of hOAT4. Moreover, an acidification of the uptake medium increased 6-CF as well as [(3)H]ES uptake, which was reduced by nigericin, suggesting that hOAT4 also can operate as an OA/OH(-) exchanger. hOAT4 facilitates substantial uptake of [(14)C]urate, which was elevated 2.6-fold by intracellular HCTZ. Thus, hOAT4 is the long-postulated, low-affinity apical urate anion exchanger that facilitates HCTZ-associated hyperuricemia.

Renal expression of organic anion transporter OAT2 in rats and mice is regulated by sex hormones.

The renal reabsorption and/or excretion of various organic anions is mediated by specific organic anion transporters (OATs). OAT2 (Slc22a7) has been identified in rat kidney, where its mRNA expression exhibits gender differences [females (F) > males (M)]. The exact localization of OAT2 protein in the mammalian kidney has not been reported. Here we studied the expression of OAT2 mRNA by RT-PCR and its protein by Western blotting (WB) and immunocytochemistry (IC) in kidneys of adult intact and gonadectomized M and F, sex hormone-treated castrated M, and prepubertal M and F rats, and the protein in adult M and F mice. In adult rats, the expression of OAT2 mRNA was predominant in the outer stripe (OS) tissue, exhibiting 1) gender dependency (F > M), 2) upregulation by castration and downregulation by ovariectomy, and 3) strong downregulation by testosterone and weak upregulation by estradiol and progesterone treatment. A polyclonal antibody against rat OAT2 on WB of isolated renal membranes labeled a approximately 66-kDa protein band that was stronger in F. By IC, the antibody exclusively stained brush border (BB) of the proximal tubule S3 segment (S3) in the OS and medullary rays (F > M). In variously treated rats, the pattern of 66-kDa band density in the OS membranes and the staining intensity of BB in S3 matched the mRNA expression. The expression of OAT2 protein in prepubertal rats was low and gender independent. In mice, the expression pattern largely resembled that in rats. Therefore, OAT2 in rat (and mouse) kidney is localized to the BB of S3, exhibiting gender differences (F > M) that appear in puberty and are caused by strong androgen inhibition and weak estrogen and progesterone stimulation.

Uptake of chemically reactive, DNA-damaging sulfuric acid esters into renal cells by human organic anion transporters.

The procarcinogen 1-methylpyrene is activated by hepatic enzymes via 1-hydroxymethylpyrene to 1-sulfooxymethylpyrene (1-SMP), a highly reactive and mutagenic metabolite. Previously, high levels of 1-SMP DNA adducts were observed in rat kidneys after intraperitoneal administration of 1-hydroxymethylpyrene or 1-SMP. This study examined whether organic anion transporters (OAT) that are expressed at the basolateral membrane of proximal tubule cells are involved in uptake of SMP. Human epithelial kidney (HEK293) cells that stably express human OAT1 (hOAT1) and hOAT3 were used. Stable isomers of 1-SMP, (2-SMP and 4-SMP) competitively inhibited the uptake of characteristic substrates p-aminohippurate for hOAT1 and estrone sulfate for hOAT3. Both inhibitors exhibited high affinity for hOAT1 (K(i) = 4.4 microM for 2-SMP; K(i) = 5.1 microM for 4-SMP) as well as hOAT3 (K(i) = 1.9 microM for 2-SMP; K(i) = 2.1 microM for 4-SMP). The uptake rate of 4-SMP (at a concentration of 10 microM) by hOAT1- and hOAT3-expressing cells was 3.0 and 1.6 times higher, respectively, than in control cells. Uptake of the reactive isomer 1-SMP was investigated using as the end point the level of DNA adducts that were formed in the cells. After exposure to 1-SMP (10 microM), the DNA adduct level was 4.6 and 3.0 times higher in hOAT1- and hOAT3-expressing cells, respectively, than in control cells. The enhanced DNA adduct formation in hOAT-expressing cells was abolished in the presence of the OAT inhibitor probenecid. This study indicates that OAT can mediate the basolateral uptake of reactive sulfuric acid esters into proximal tubule cells and thereby participate in kidney cell damage by these compounds.