Time-dependent effect of leptin on renal Na +,K +-ATPase activity

Leptin, secreted by adipose tissue, is involved in the pathogenesis of arterial hypertension, however, the mechanisms through which leptin increases blood pressure are incompletely elucidated. We investigated the effect of leptin, administered for different time periods, on renal Na(+),K(+)-ATPase activity in the rat. Leptin was infused under anesthesia into the abdominal aorta proximally to the renal arteries for 0.5-3 h. Leptin administered at doses of 1 and 10 microg/min per kg for 30 min decreased the Na(+),K(+)-ATPase activity in the renal medulla. This effect disappeared when the hormone was infused for > or =1 h. Leptin infused for 3 h increased the Na(+),K(+)-ATPase activity in the renal cortex and medulla. The stimulatory effect was abolished by a specific inhibitor of Janus kinases (JAKs), tyrphostin AG490, as well as by an NAD(P)H oxidase inhibitor, apocynin. Leptin increased urinary excretion of hydrogen peroxide (H(2)O(2)) between 2 and 3 h of infusion. The effect of leptin on renal Na(+),K(+)-ATPase and urinary H(2)O(2) was augmented by a superoxide dismutase mimetic, tempol, and was abolished by catalase. In addition, infusion of H(2)O(2) for 30 min increased the Na(+),K(+)-ATPase activity. Inhibitors of extracellular signal regulated kinases (ERKs), PD98059 or U0126, prevented Na(+),K(+)-ATPase stimulation by leptin and H(2)O(2). These data indicate that leptin, by acting directly within the kidney, has a delayed stimulatory effect on Na(+),K(+)-ATPase, mediated by JAKs, H(2)O(2) and ERKs. This mechanism may contribute to the abnormal renal Na(+) handling in diseases associated with chronic hyperleptinemia such as diabetes and obesity.


Vol. 52 No. 4/2005, 803-809
on-line at: www.actabp.plLeptin, the peptide hormone secreted by white adipose tissue, acts on hypothalamic centres to decrease food intake and stimulate energy expenditure.Leptin has also multiple peripheral effects on carbohydrate and lipid metabolism, hormone secretion, immune and inflammatory response, reproductive and cardiovascular function (Fruhbeck, 2004).Plasma leptin concentration is proportional to the amount of adipose tissue and is markedly increased in obese humans and in animals with dietary-induced obesity.Recent studies indicate that hyperleptinemia is involved in the pathogenesis of obesity-associated cardiovascular complications, including arterial hypertension (Correia & Haynes, 2004).
Renal sodium handling is the main longterm regulator of arterial pressure.The amount of sodium excreted in urine is primarily determined by the rate of its tubular reabsorption, the process driven by Na + ,K + -ATPase contained in the basolateral membrane of tubular cells (Féraille & Doucet, 2001).Abnormal regulation of the renal Na + pump leading to increased sodium reabsorption plays an important role in the pathogenesis of various animal models of hypertension, including obesity-induced hypertension (Hussain et al., 1999).However, the role of leptin in the regulation of renal Na + ,K + -ATPase is poorly understood.Acutely administered leptin increases natriuresis, in part by reducing renal medullary Na + ,K + -ATPase activity (Jackson & Li, 1997;Bełtowski et al., 2004a).The inhibitory effect of leptin on the sodium pump is mediated by phosphatidylinositol-3-kinase (PI3K) (Sweeney et al., 2000;Bełtowski et al., 2004b).In contrast, chronic hyperleptinemia induced by 7-day administration of exogenous hormone increases the Na + ,K + -AT- Pase activity; an effect also observed in dietary-induced obesity (Bełtowski et al., 2004a).Systemic hyperleptinemia could modulate renal Na + ,K + -AT-Pase through multiple indirect mechanisms such as changes in insulin sensitivity, renin-angiotensin system, nitric oxide production etc.On the other hand, in acute studies leptin was administered either as a bolus or was infused for a short time not exceeding 30 min, so it is unclear whether leptin has any direct Na + ,K + -ATPase-stimulatory effect when administered locally in a more prolonged manner.Therefore, in the present study we examined the effect of leptin administered for different time periods on renal Na + , K + -ATPase activity.

MATERIALS AND METHODS
Reagents.The specific inhibitor of H + ,K + -AT-Pases, Sch 28080, was provided by the Schering-Plough Research Institute (Kenilworth, NJ, USA).Recombinant human leptin was purchased from Calbiochem-Novabiochem (San Diego, CA, USA).Catalase (from bovine liver, specific activity 2000-5000 units/mg protein) and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Experimental protocol.All studies were performed on adult male Wistar rats weighing 250-300 g.The study protocol was approved by the Bioethics Commi�ee of the Medical University of Lublin.The animals were anaesthetized with pentobarbital (50 mg/kg i.p.) and a thin catheter was inserted through the femoral artery into the abdominal aorta proximally (< 0.5 cm) to the renal arteries for the infusion of the investigated substances.In some animals a second catheter was inserted into the urinary bladder for urine collection.A�er the surgery, 0.9% NaCl was infused at a rate of 66 µl/min (4 ml/h) for 30 min (stabilization period).Then, leptin was infused for different time periods (0.5, 1, 2 or 3 h).Leptin was dissolved in a 15 mM HCl/7.5 mM NaOH mixture (5 : 3, v/v) and infused at a rate of 2 ml/h.Control animals received leptin vehicle solution starting from 31st min of the experiment for the corresponding time periods.When infusion was performed for > 1 h, anesthesia was supplemented at hourly intervals by i.p. injections of 2.5 mg pentobarbital/kg.Rats were placed on a heating pad and the temperature was maintained at 37 o C. Until otherwise stated, compounds expected to block leptin's effect were administered for 60 min, i.e. during the 30-min stabilization period and during leptin infusion in animals treated with leptin for 30 min and between 2 and 3 h of leptin infusion in rats receiving this hormone for 3 h.After the infusion, blood sample (0.5 ml) was withdrawn to an EDTA-containing tube for the measurement of plasma leptin.Then, the kidneys were excised and the animals sacrificed by a lethal dose of pentobarbital.
Na + ,K + -ATPase assay.Microsomal fraction was isolated from the renal cortex and medulla as previously described (Bełtowski et al., 2004a).Na + ,K + -ATPase was assayed by measuring the amount of inorganic phosphate (P i ) liberated from ATP in a buffer containing 100 mM NaCl, 20 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, 40 mM Tris/HCl (pH 7.4) and 3 mM Na 2 ATP, and was calculated as the difference between the activities assayed in the absence and in the presence of 2 mM ouabain.Both samples contained 0.2 mM Sch 28080 to block ouabain-sensitive H + ,K + -ATPase which otherwise interferes with the Na + ,K + -ATPase assay.Enzyme activity was expressed in µmole of P i liberated by 1 mg of microsomal protein during 1 h (µmol/h per mg protein).P i and protein concentrations were assayed by the method of Hurst (1964) and Lowry et al. (1951), respectively.
Other assays.Hydrogen peroxide in urine was measured by the FOX method based on the oxidation of Fe 2+ to Fe 3+ which subsequently forms a blue-violet chromogen with xylenol orange (Yuen & Benzie, 2003).In brief, 200 µl of urine was mixed with 340 µl of FOX reagent (125 µM xylenol orange, 100 mM sorbitol and 250 µM ferrous ammonium sulphate in 25 mM H 2 SO 4 ), incubated at room temperature for 60 min and the absorbance was read at 560 nm.To a separate set of urine samples 0.1 mg catalase was added 5 min before mixing with the FOX reagent.Standards of H 2 O 2 (1-10 µM) were prepared from 30% stock solution and H 2 O 2 concentration in urine was calculated from the difference between the absorbance of the samples assayed without and with catalase.The sensitivity of this assay (mean 0+S.D. of 10 zero standards) was 0.5 µM and the intra-and interassay CVs were < 3% and < 6%, respectively.Plasma leptin was measured using Leptin Enzyme Immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA).
Statistics.Data are presented as mean ± S.E.M. from 8 animals in each group.Statistical significance was evaluated by Student's t-test or ANOVA followed by Duncan's multiple range test for comparison of 2 or > 2 groups, respectively.P < 0.05 was considered significant.

Time-dependent effect of leptin on renal Na + ,K + -AT-Pase: role of PI3K and JAKs
Consistently with our previous study (Bełtowski et al., 2004b), leptin infused for 30 min at 0.1-10 µg/min per kg had no effect on the Na + ,K + -Leptin and renal Na + ,K + -ATPase ATPase activity in the renal cortex but reduced its activity in the renal medulla in a dose-dependent manner (Fig. 1).However, this inhibitory effect was transient and disappeared if leptin infusion was continued for a longer time period.A�er 1 h of infusion, a significant decrease in medullary Na + ,K + -AT-Pase activity was observed only in animals receiving the highest dose of leptin.Infusion of leptin for 3 h resulted in stimulation of Na + ,K + -ATPase in both regions of the kidney in animals receiving either 1 or 10 µg leptin/min per kg (Fig. 1).
Plasma leptin in control animals was 3.96 ± 0.32 ng/ml.Infusion of leptin at 10 µg/min per kg for 30 min, 1, 2 and 3 h increased plasma leptin to 6.76 ± 0.74, 6.21 ± 0.53, 5.95 ± 0.57 and 6.42 ± 0.58 ng/ml, respectively.Lower doses of leptin had no effect on plasma leptin concentration.To exclude extrarenal effects, further experiments were performed with leptin at the dose 1 µg/min per kg, which did not elevate the systemic hormone level but exerted significant effects on renal Na + ,K + -ATPase.
Upon binding to plasma membrane receptor, leptin activates cytosolic protein tyrosine kinases belonging to the JAK family (Hegyi et al., 2004).In the present study the specific JAK inhibitor, tyrphostin AG490, blocked both the stimulatory and the inhibitory effect of leptin (Fig. 2).

Effect of apocynin and tempol on Na + ,K + -ATPase stimulation by leptin
Nitric oxide decreases renal Na + ,K + -ATPase activity, however, its effect may be curtailed by intrarenally produced superoxide anion radical (O 2 •-).NAD(P)H oxidase is a main source of O 2 •-in the cardiovascular system and kidney (Kalinowski & Maliński, 2004).Previously we have demonstrated that an NAD(P)H oxidase inhibitor, apocynin, and a superoxide dismutase mimetic, tempol, decrease renal medullary Na + ,K + -ATPase activity in an NO-dependent manner, suggesting that endogenous O 2 •tonically stimulates the Na + pump by scavenging NO (Bełtowski et al., 2004c).Leptin stimulates reactive oxygen species (ROS) formation in various cell types (Boulomie et al., 1999;Yamagishi et al., 2001).In addition, up-regulation of renal Na + ,K + -ATPase in rats treated with leptin for 7 days was prevent-  Leptin was infused at a dose of 1 µg/min per kg for either 30 min or 3 h.In separate groups of animals, AG490 (10 -7 mol/min per kg) was administered for 60 min, i.e. for 30 min before and throughout leptin infusion in animals treated with leptin for 30 min, or for the last 60 min of leptin infusion in rats receiving this hormone for 3 h.*P < 0.05 vs. control group not treated with either leptin or AG490.

2005
A. Marciniak and others ed by tempol and apocynin (Bełtowski et al., 2005).Therefore, we hypothesized that the stimulation of Na + ,K + -ATPase observed in the present study could also result from O 2 •-dependent scavenging of NO.Apocynin or tempol administered alone decreased the Na + ,K + -ATPase activity in the renal medulla but had no effect on the cortical enzyme (Fig. 3).However, whereas apocynin abolished the effect of leptin in both cortex and medulla, tempol even augmented the leptin-induced stimulation of cortical and medullary Na + ,K + -ATPase (Fig. 3).Polyethylene glycol-coupled superoxide dismutase infused at a dose of 500 U/min per kg had a similar effect, whereas 3-carbamoyl-proxyl, a compound structurally related to tempol but devoid of O 2 •scavenging properties, failed to augment the effect of leptin (not shown).These results indicate that the effect of tempol results from the scavenging of O 2 •-. Leptin stimulated Na + ,K + -ATPase also in animals in which the activity of the NO-cGMP pathway was artificially stimulated by the administration of 8-bromo-cGMP.As previously reported (Bełtowski et al., 2004c), 8-bromo-cGMP (100 nmol/ min per kg for 60 min) decreased the Na + ,K + -ATPase activity in the renal medulla to 13.1 ± 0.8 µmol/min per kg (P < 0.01 vs. control) but not in the renal cortex (17.9 ± 1.6 µmol/min per kg).However, the Na + ,K + -ATPase activity in animals receiving leptin (1 µg/min per kg for 3 h) and 8-bromo-cGMP (for the last hour of leptin infusion) was higher than in rats treated with 8-bromo-cGMP alone (cortex: 24.7 ± 1.8 µmol/min per mg, medulla: 18.8 ± 1.6 µmol/ min per mg, both P < 0.05 vs. 8-bromo-cGMP alone).Taken together, these results suggest that NAD(P)H oxidase-derived ROS other than superoxide mediate the stimulatory effect of leptin and that scavenging of NO is not involved.
Role of mitogen-activated protein kinases in Na + ,K + -ATPase regulation by leptin H 2 O 2 stimulates mitogen-activated protein kinases (MAPKs) in many cell types and leptin has been demonstrated to activate MAPKs in an H 2 O 2dependent manner (Cao et al., 2004).Therefore, we investigated if MAPKs are involved in the regulation of renal Na + ,K + -ATPase by leptin.The specific inhibitor of extracellular signal-regulated kinases (ERK1/p44MAPK and ERK2/p42MAPK), PD98059, abolished the leptin-induced as well as the H 2 O 2induced increase in Na + ,K + -ATPase activity (Fig. 5).A similar effect was observed if a structurally unrelated ERK inhibitor, U0126, was infused with leptin.In contrast, a specific inhibitor of p38 MAPK, SB203580, failed to a�enuate the leptin-or H 2 O 2 -induced increase in Na + ,K + -ATPase activity (Fig. 5).Neither of the MAPK inhibitors had any effect on Na + ,K + -ATPase in animals not treated with leptin.PD98059 and U0126 did not affect H 2 O 2 excretion in rats infused with leptin (not shown), suggesting that ERKs are downstream to H 2 O 2 in the Na + ,K + -ATPase-stimulating signaling cascade.PD98059 did not a�enuate the increase in cortical Na + ,K + -ATPase activity induced by either dibutyryl-cAMP or phorbol 12,13-dibutyrate, activators of protein kinase A and C, respectively, indicating that its effect is not due to nonspecific toxicity or inhibition of other protein kinases (not shown).

DISCUSSION
The results of this study indicate that the decrease in the Na + ,K + -ATPase activity in the renal medulla induced by short-term leptin infusion is only transient and is replaced by stimulation of the cortical and medullary enzyme when leptin is administered for > 2 h.The stimulatory effect is mediated by JAKs, H 2 O 2 and MAPK belonging to the ERK subfamily.This is not the first study demonstrating opposite short-and long-term effects of leptin.For example, in cultured rat cardiomyocytes leptin stimulates or inhibits adenylate cyclase a�er 30-min or 18-h exposure, respectively (Iliano et al., 2002).
Previously, we have reported that chronic hyperleptinemia leads to an increase in renal Na + ,K + -ATPase activity and that this effect is abolished by apocynin and tempol (Bełtowski et al., 2005).
Although apocynin blocked the stimulatory effect of leptin also in the present study, the mechanism downstream to NAD(P)H oxidase seems to be different in both cases.Since leptin reduced and tempol restored urinary excretion of nitric oxide metabolites and cGMP in rats treated with leptin for 7 days (Bełtowski et al., 2005), one may suggest that hyperleptinemia stimulated renal Na + ,K + -ATPase by O 2 •--dependent scavenging of NO.In contrast, in the present study tempol augmented the stimulatory effect of leptin, indicating that H 2 O 2 rather than O 2 •-is involved.In addition, the effect of leptin was preserved in animals treated with 8-bromo-cGMP, suggesting that the increase in the Na + ,K + -ATPase activity was not associated with inhibition of the NO-cGMP pathway.
At present we can not explain why the mechanisms involved in the up-regulation of Na + ,K + -AT-Pase are differed in both studies.One may speculate that in the present 3-h experiment superoxide was efficiently scavenged to H 2 O 2 by SOD, which shi�ed the Na + ,K + -ATPase-stimulatory mechanism from an O 2 •--NO-dependent toward an H 2 O 2 -ERK dependent one, whereas in 7-day hyperleptinemia the O 2 •--scavenging system became ineffective.Reduced expression and/or activity of various SOD isoforms was reported in models of hypertension associated with chronic intrarenal oxidative stress such as spontaneously hypertensive rat (Zhan et al., 2004), 5/6 nephrectomy (Vaziri et al., 2003) or angiotensin II-induced hypertension (Chabrashvili et al., 2003).Moreover, H 2 O 2 metabolizing enzymes, glutathione peroxidase and catalase, are upregulated in the kidney of spontaneously hypertensive rat.Thus, chronic intrarenal oxidative stress may favour a higher O 2 •-/ H 2 O 2 ratio.In addition, chronic hyperleptinemia is associated with hypertension and positive Na + bal- ance (Bełtowski et al., 2005), conditions which per se reduce renal SOD expression (Kitiyakara et al., 2003).It remains to be established if chronic hyperleptinemia induces changes of the expression of antioxidant enzymes similarly to other oxidative stress-dependent forms of hypertension, however, such changes are unlikely to occur in animals receiving leptin for only 3 h.
The second possibility is that NO availability contributes to the different mechanisms involved in the stimulation of renal Na + ,K + -ATPase.In particular, 7-day hyperleptinemia is associated with NO deficiency (Bełtowski et al., 2005).Although this may result from scavenging of NO by superoxide, other mechanisms such as altered expression of nitric oxide synthase can not be excluded.NO deficiency will render hyperleptinemic animals more sensitive to further NO shortage by excess superoxide.In contrast, acutely administered leptin stimulates NO production (Fruhbeck, 2004), therefore, scavenging of NO might not be rate-limiting for the regulation of renal Na + ,K + -ATPase in the present study.
Another interesting finding of the present study is that H 2 O 2 increases renal Na + ,K + -ATPase activity.The role of H 2 O 2 in blood pressure regulation is controversial.Most studies indicate that H 2 O 2 dilates blood vessels and may serve as an endothelium-derived hyperpolarizing factor in some vascular beds (Shimokawa & Matoba, 2004).On the other hand, increased plasma H 2 O 2 level has been reported in animal and human hypertension (Swei et al., 1997;Lacy et al., 2000), and mice overexpressing catalase are characterized by reduced pressor response to various vasoconstrictors (Yang et al., 2003).These findings suggest that in vivo H 2 O 2 might be prohypertensive.Infusion of H 2 O 2 into the renal medullary interstitium reduces natriuresis and diuresis and elevates blood pressure in the long run (Makino et al., 2003;Chen et al., 2003).The results presented here suggest that increased Na + ,K + -ATPase-dependent tubular Na + reabsorption may contribute to the hypertensive effect of H 2 O 2 .
H 2 O 2 stimulates various MAPKs, including ERKs (Lee & Esselman, 2002).Several recent studies suggest a role of ERKs in the regulation of renal Na + ,K + -ATPase.ERK1/2 are involved in the stimulation of Na + ,K + -ATPase by dopamine D 2 receptor agonists in the rat proximal tubules (Narkar et al., 2002), by vasopressin and aldosterone in the principal cells of mouse cortical collecting duct (Michlig et al., 2004) and by proinsulin C-peptide in cultured human renal tubular cells (Zhong et al., 2004).Outside the kidney, ERKs mediate the stimulatory effect of angiotensin II on the Na + pump in vascular smooth muscle cells (Isenovic et al., 2004).In addition, ERK1/2 are involved in the stimulatory effect of calcitonin and β-adrenergic agonists on a related enzyme, ouabain-resistant H + ,K + -ATPase, in α-and β-intercalated cells of the cortical collecting duct, respectively (Laroche-Joubert et al., 2002;2003).This study indicates that leptin may be another mediator which utilizes ERKs for the stimulation of Na + ,K + -ATPase.ERKs have been demonstrated to phosphorylate the α 1 subunit of Na + ,K + -ATPase (Al-Khalili et al., 2004), however, the precise mechanism of ERKdependent regulation of its activity remains to be established.
Because the stimulatory effect of leptin on Na + ,K + -ATPase was observed only a�er 3 h of hormone infusion, one can not exclude that it was caused by an increase in enzyme expression.Further studies are needed to clarify whether leptin stimulates gene expression of Na + ,K + -ATPase subunits.However, because the stimulatory effect of leptin was mimicked by short-term H 2 O 2 infusion, and the effect of the la�er was abolished by PD98059, one may conclude that the increase in the Na + ,K + -ATPase activity mediated by the H 2 O 2 -ERK pathway can not be a�ributed solely to a genomic mechanism.
In conclusion, we have demonstrated that leptin has a short-term PI3K-mediated inhibitory effect on renal medullary Na + ,K + -ATPase followed by PI3K-independent, H 2 O 2 and ERK-dependent stimulation of the enzyme when the hormone is infused for > 2 h.Both the inhibitory and the stimulatory effect are mediated by JAKs.These results suggest that prolonged hyperleptinemia associated with obesity or diabetes mellitus may contribute to the development of arterial hypertension by increasing renal Na + ,K + -ATPase activity and promoting Na + retention.

Figure 1 .
Figure 1.Time-dependent effect of leptin on Na + ,K + -AT-Pase activity in the renal cortex (top panel) and medulla (bo�om panel).Control animals (closed squares) received 0.9% NaCl infusion for 30 min, followed by leptin vehicle solution for additional 0.5, 1, 2 or 3 h.Other groups received leptin at doses of 0.1 (closed triangles), 1 (open squares) or 10 (open triangles) µg/min per kg.Na + ,K + -ATPase was assayed in isolated microsomal fraction and is expressed in µmole of inorganic phosphate liberated by the enzyme contained in 1 mg of microsomal protein in 1 h (µmol P i / h per mg protein).N = 8 rats in each group.*P < 0.05, **P < 0.01 vs. time-matched control group.

Figure 2 .
Figure 2. The effect of JAK inhibitor, tyrphostin AG490, on Na + ,K + -ATPase regulation by leptin.Leptin was infused at a dose of 1 µg/min per kg for either 30 min or 3 h.In separate groups of animals, AG490 (10 -7 mol/min per kg) was administered for 60 min, i.e. for 30 min before and throughout leptin infusion in animals treated with leptin for 30 min, or for the last 60 min of leptin infusion in rats receiving this hormone for 3 h.*P < 0.05 vs. control group not treated with either leptin or AG490.