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(Hypertension. 1996;28:1018-1025.)
© 1996 American Heart Association, Inc.

Articles

Renal Na,K-ATPase in Genetic Hypertension

Mara Ferrandi; Grazia Tripodi; Sergio Salardi; Monica Florio; Rossana Modica; Paolo Barassi; Paolo Parenti; Alla Shainskaya; Steven Karlish; Giuseppe Bianchi; Patrizia Ferrari

Prassis-Sigma Tau Research Institute, Settimo M.se, Milan, Italy (M. Ferrandi, G.T., S.S., M. Florio, R.M., P.F.); Division of Nephrology and Hypertension, University of Milan (Italy) and San Raffaele Hospital (G.B.); Department of Physiology and Biochemistry, University of Milan (Italy) (P.B., P.P.); and Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel (A.S., S.K.).

Correspondence to Dr Patrizia Ferrari, Prassis Sigma-Tau Istituto Ricerche, Via Forlanini, 3, 20019 Settimo Milanese, Milano, Italy. E-mail mc3405@mclink.it.

	Abstract

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Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb⁺ occlusion, turnover number, ₁- and ß₁-subunit protein abundance, and ₁ and ß₁ mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb⁺ occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of ₁ and ß₁ mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.

Key Words: Na⁺,K⁺-exchanging ATPase • genetics • rats • kidney • cytoskeleton

	Introduction

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The hypertensive rat strain MHS develops hypertension because of a primary alteration of renal function.¹ The central role of the kidney in the pathogenesis of hypertension in MHS has been clearly demonstrated by kidney cross-transplantation experiments, which showed that MHS kidney produces hypertension in control MNS, even when transplanted from young prehypertensive rats.^{2 3} Much experimental evidence supports the hypothesis that this pressor role of MHS kidney is linked to an enhanced tubular sodium reabsorption: (1) The development of hypertension is preceded by a phase of salt retention⁴ ; (2) despite the fact that glomerular filtration rate is increased in MHS,⁵ their urinary sodium excretion is similar to that of MNS, thus implying a greater tubular reabsorption⁴ ; (3) the interstitial hydrostatic pressure, measured by micropuncture before the development of hypertension and taken as an index of tubular reabsorption, is increased in MHS compared with that in MNS⁶ ; and (4) in both isolated tubules⁷ and basolateral membrane vesicles from kidney cortex,⁸ the activity of the Na-K pump is higher in MHS than in MNS. Moreover, also at the luminal level, the Na-H antiporter in proximal tubules⁹ and the Na-K cotransport in the TAL¹⁰ are faster in MHS. These findings suggest that the renal sodium retention in MHS and the concomitant development of hypertension may be linked to an altered sodium transport across the tubular epithelium. The complex relationship between luminal and basolateral ion transports in renal epithelium makes it difficult to establish whether the primary alteration responsible for the faster transtubular sodium reabsorption resides in the apical exchange, in the cotransport systems, or in the basolateral Na-K pump. However, it is well known that any modification in tubular sodium transport is accompanied by a change in function or synthesis of the Na-K pump, which creates the electrochemical gradient between intracellular and extracellular compartments, allowing sodium to enter the cell.¹¹ In the present study, we approached the problem of the involvement of the renal Na-K pump in MHS hypertension by trying to discriminate between the following two possibilities: (1) The altered Na-K pump rate in MHS is merely due to an enhanced enzymatic activity or turnover rate, or (2) it is due to an enhanced synthesis of Na,K-ATPase ₁- and ß₁-subunits and a higher number of active sites on cell membranes. Moreover, to establish whether the alteration of the Na,K-ATPase precedes or follows the full development of hypertension, we compared the activity, number of active sites, enzymatic turnover number, protein abundance, and mRNA levels of Na,K-ATPase ₁- and ß₁-subunits from renal outer medulla in young 25-day-old and adult 75-day-old MHS and MNS.

	Methods

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Animals
The studies were performed in 25- or 75-day-old inbred MHS/Giuseppe Bianchi (MHS/Gib) and MNS/Gib rats derived from our original stock colony (Prassis Research Institute, Settimo M.se, Italy). Rats were fed a standard diet (Altromin MT, Rieper) containing 2.5 g/kg NaCl and received tap water ad libitum. Systolic pressure was recorded by the tail-cuff method (BP Recorder, U. Basile) in conscious rats. Systolic pressure was similar in young rats (MHS, 129±4 mm Hg; MNS, 125±2) and was significantly increased in adult MHS (167±2) compared with age-matched MNS (136±1.8, P<.001). For each experiment, groups of four to five rats were anesthetized with ether and killed by decapitation. Kidneys were removed and sliced, and the outer medulla was dissected under a stereo microscope at 4°C; tissues were pooled and immediately frozen in liquid nitrogen and stored at -70°C up to the moment of microsome preparation. For mRNA extraction, tissues of kidney outer medulla were pooled, immediately frozen in liquid nitrogen, and stored at -70°C or directly homogenized in guanidine isothiocyanate and stored at -70°C.

Determination of Na,K-ATPase Activity in Renal Total Homogenate and Microsomes
Kidney outer medulla slices were weighed, suspended (1 g/10 mL) in an ice-cold solution (containing [mmol/L] sucrose 250, histidine 30, and disodium EDTA 5) (Sigma Chemical Co) at pH 7.2, and homogenized in a polytron (PCU-Kinematica AG). Each sample was homogenized twice for 15 seconds at setting 5. The homogenate was centrifuged at 6000g for 15 minutes at 4°C (Beckman Instruments); the supernatant fluid was decanted and saved; and the pellet was resuspended in the same solution, homogenized, and centrifuged at 6000g for 15 minutes at 4°C. The second supernatant was decanted, pooled with the first one, and centrifuged at 48 000g for 30 minutes at 4°C. Pellets were resuspended in the sucrose-histidine solution (1:1, wt/vol). The protein content of the microsomes was determined with the method of Lowry et al¹² using bovine serum albumin as standard.

Na,K-ATPase enzymatic activity was determined both in total medulla homogenate and in microsome preparations previously permeabilized with DOC (DOC–Tris base neutralized solution at pH 7.4). To establish the proper DOC concentration able to yield maximal activation of Na,K-ATPase, MHS and MNS total renal homogenate and microsomes (30 µg protein) were treated with increasing DOC concentrations (from 0.1 to 1.2 mg DOC/mg protein) for 30 minutes at room temperature, and the Na,K-ATPase activity was determined. Maximal Na,K-ATPase activity was obtained around 0.65 mg DOC/mg protein for both rat preparations (data not shown), and all subsequent experiments were performed by pretreating homogenate and microsomes with this DOC concentration for 30 minutes at room temperature.

Na,K-ATPase activity was assayed in both total renal medulla homogenate and microsomes after the release of inorganic ³²P from [³²P]ATP. Two micrograms of homogenate or microsomes was preincubated for 10 minutes at 37°C in 110 µL of medium containing (mmol/L) NaCl 140, MgCl₂ 3, HEPES-Tris 50, and ATP 3, pH 7.5, in the presence or absence of 10 mmol/L ouabain. After preincubation, 10 µL of solution containing 10 mmol/L KCl and 20 nCi [³²P]ATP (0.5 to 3 Ci/mmol) was added, and the reaction was continued for 15 minutes at 37°C. The reaction was stopped by addition of 100 µL ice-cold perchloric acid solution (30% vol/vol) and 500 µL activated charcoal. Inorganic ³²P released from [³²P]ATP hydrolysis was separated by centrifugation and radioactivity measured by liquid scintillation counting (Beckman LS 5000 CE ß-counter). Na,K-ATPase activity was calculated as the ouabain-sensitive fraction of total ATPase activity and expressed as micromoles per minute per milligram protein.

Na,K-ATPase ⁸⁶Rb⁺ Occlusion
The Rb⁺ occlusion assay was performed according to Shani et al.¹³ Sixty microliters of microsomes (3 mg/mL) suspended in the sucrose-histidine/EDTA Tris medium was incubated for 30 minutes at room temperature with 132 µL water or a solution of 10 mmol/L ouabain plus 5 mmol/L P_i Tris, 3 mmol/L MgCl₂ (final concentrations), and 48 µL of 1 mg/mL DOC Tris. Aliquots (30-µL) of the microsomes (45 to 90 µg protein) were incubated for 15 minutes at room temperature with a reaction medium containing RbCl (50 to 1000 µmol/L) plus ⁸⁶Rb⁺ (2x10⁶ cpm per sample, Amersham). Ice-cold sucrose medium (200 mmol/L, 0.5 mL) was added and the suspension then passed through ice-cold Dowex columns prepared by pouring Dowex-50x8, Tris form (Fluka), in Pasteur pipettes preequilibrated with 1 mL of 25 mg/mL bovine serum albumin. ⁸⁶Rb⁺ bound to the microsomes was eluted from the columns with 1.5 mL ice-cold sucrose solution, and the radioactivity was measured by liquid scintillation counting (Beckman LS 5000 CE ß-counter). Occluded Rb⁺ was expressed as nanomoles per milligram of protein. The fraction of Rb⁺ associated with the Na,K-ATPase was calculated as the difference between the Rb⁺ occluded in the absence and presence of ouabain.

Na,K-ATPase ₁- and ß₁-Subunit Abundance
Samples of total medulla homogenate or microsomes (10 µg protein for the -subunit and 50 µg protein for the ß-subunit detection) were denatured in SDS sample buffer (2% mercaptoethanol, 3% SDS, 10% glycerol, 12.5 mmol/L Tris, 100 mmol/L glycine, pH 8.3) applied to SDS-polyacrylamide gradient slab gels (7% to 15% acrylamide) and run overnight at 70 V with the discontinuous buffer system of Laemmli.¹⁴ The amount of protein was chosen after the linearity of detection between 0 and 20 µg for the ₁-subunit and 0 and 80 µg for the ß₁-subunit had been verified. Proteins were transferred from gels to nitrocellulose paper at 90 V for 3 hours according to Towbin et al,¹⁵ and sheets were blocked for 1 hour in 8% bovine serum albumin–PBS and incubated for 2 hours at room temperature with a monoclonal antibody to the ₁-subunit of rat Na,K-ATPase (6H) (kindly obtained from Dr A. McDonough, University of Southern California, School of Medicine, Los Angeles) or with a commercially available polyclonal antibody to the ß₁-subunit of rat Na,K-ATPase (UBI No. 06-170), both diluted 1:1000. After five washes with TTBS (500 mmol/L NaCl in 20 mmol/L Tris-HCl, pH 7.5, and 0.05% Tween 20), sheets were incubated with ¹²⁵I-labeled anti-mouse or anti-rabbit immunoglobulin F(ab)₂ fragment (Amersham Nos. IM 1310 and IM 1340) for 1 hour in the above conditions. After five washes in TTBS, drying of the sheets, and autoradiography, radioactive bands were excised from the paper (90 to 95 kD for the ₁-subunit and 45 to 50 kD for the ß₁-subunit) and counted for precise quantitation of bound antibody (Beckman 5500B gamma counter). Results are expressed as arbitrary units and normalized to young MNS samples of renal microsome preparations, defined as 1.

Glycopeptidase F Treatment of Microsomes
Deglycosylation of the ß₁-subunit to the core protein¹⁶ was performed in both young and adult rats with glycopeptidase F (Sigma No. G-8031). Rat medulla microsomes (100 µg in a volume of 10 µL) were resuspended in 100 µL of 1% beta-mercaptoethanol and 0.5% SDS and boiled for 10 minutes. After cooling, 10 µL of 0.5 mol/L phosphate buffer, pH 7.5, and 10% Nonidet P-40 were added, followed by 3 µL (0.6 IU) glycopeptidase F. The mixture was incubated 24 hours at 37°C and then treated with SDS sample buffer; samples were run on a gel, blotted, and detected with anti-ß₁ Na,K-ATPase (UBI) as above.

Na,K-ATPase ₁ and ß₁ mRNA Extraction and Northern Analysis
RNA was prepared from MHS and MNS kidney medulla by the guanidine isothiocyanate/cesium chloride method.¹⁷ Equal amounts of total RNA (10 µg) were size-fractionated by electrophoresis on denaturing 1.3% agarose formaldehyde gels and subsequently transferred in 20x SSC (1x SSC=150 mmol/L NaCl and 15 mmol/L trisodium citrate) to nylon membranes (GeneScreen, DuPont). The total RNA amount was predetermined to be within a linear range of detection between 5 and 30 µg. Hybridizations were performed for 16 hours at 44°C in hybridization buffer (50% deionized formamide, 3x SSC, 10x Denhardt's solution, 0.1% SDS, 100 mg/mL salmon sperm DNA), with an excess of [³²P]dCTP-labeled cDNAs encoding rat Na,K-ATP ₁-subunits¹⁸ and ß₁-subunits¹⁹ and rat 18S²⁰ probes with the use of a multiprimer DNA labeling kit (Amersham). The ₁ cDNA probe consisted of a 2.2-kb Nco I restriction fragment, and the ß₁ cDNA probe consisted of a 0.3-kb HindIII–Pst I restriction fragment. Membranes were washed in 2x SSC (5 minutes at room temperature) followed by increases in wash stringency up to 0.2x SSC/0.1% SDS (50 minutes at 65°C). Before rehybridization with other probes, cDNA probes were removed from membranes by washing with 1% SDS for 1 hour at 70°C. Hybridization of RNA to a radiolabeled rat 18S cDNA probe served to confirm uniform amounts of total RNA in each lane. Na,K-ATPase ₁ and ß₁ mRNAs were visualized and quantified by electronic autoradiography (InstantImager 2024, Packard). Triplicate RNA samples from different kidney medulla pools were analyzed simultaneously on the same nylon filter. Quantification was expressed as arbitrary units normalized for 18S rRNA levels and then related to the samples of MNS renal homogenate, which was given the arbitrary value of 1.

Statistical Analysis
All data are expressed as mean±SE. Statistical comparisons were performed by ANOVA or by Student's t test and the Bonferroni correction for multiple comparisons. A value of P<.05 was regarded as significant. Kinetic parameters (B_max and K_m) for sigmoid saturation curves of the Rb⁺ occlusion experiments were fitted with a nonlinear least-squares program (Enzfitter, Elsevier-Biosoft) with the use of explicit weighted values.

	Results

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Na,K-ATPase Activity
Na,K-ATPase activity was determined under saturating conditions for sodium, potassium, magnesium, and ATP (Fig 1) in permeabilized renal outer medulla microsomes. The Na,K-ATPase activity at V_max was significantly higher in MHS than in MNS at both 25 days (MHS, 1.61±0.1 µmol P_i/min per milligram; MNS, 1.31±0.08; P<.05) and 75 days (MHS, 2.83±0.1; MNS, 2.10±0.06; P<.001) of age. A statistically significant increase of Na,K-ATPase activity was also observed in both strains as a function of age (Fig 1). To measure the total pool size of Na,K-ATPase, we also checked the activity at V_max in outer medulla homogenate from adult MHS and MNS. As in microsomes, the Na,K-ATPase activity at V_max in renal homogenate was significantly higher (P<.01) in MHS (0.918±0.082 µmol P_i/min per milligram, n=8) than in MNS (0.653±0.044, n=7).

View larger version (60K): [in this window] [in a new window]   Figure 1. Na,K-ATPase activity measured in renal outer medulla microsomes from young (25 days) and adult (75 days) MHS (dark hatched bars) and MNS (light hatched bars). Values are mean±SE; n=8 for each group. *P<.05, **P<.001, MHS vs MNS; §§P<.001, young vs adult.

⁸⁶Rb⁺ Occlusion
To determine whether the higher renal Na,K-ATPase activity of MHS could be explained by either an enhanced turnover rate or a higher number of functionally active pump sites, we measured the amount of occluded Rb⁺ ions (congener of K⁺) and the affinity for Rb⁺ ions in permeabilized microsomes. Fig 2 shows the curves of Rb⁺ occlusion, as a function of Rb⁺ concentrations up to 1 mmol/L, obtained with five different preparations of outer renal medulla microsomes from 75-day-old MHS and MNS. The fitted values demonstrate that B_max values of Rb⁺ were significantly higher in MHS than in MNS preparations (0.412±0.02 versus 0.281±0.02 nmol Rb⁺/mg protein, P<.01), whereas K_m values (205±9 versus 158±10 µmol/L) and Hill numbers (1.01 versus 0.98) were similar. Na,K-ATPase activity measured in the same preparations resulted in significantly higher values in MHS, as shown in the Table. Rb⁺ occlusion was also measured in young rats of both strains, but only two separate microsome preparations were analyzed because of the technical difficulties of obtaining sufficient quantities of microsome proteins from renal outer medulla of 25-day-old rats. The B_max values calculated in the two experiments were 0.247 and 0.250 nmol Rb⁺/mg protein in MHS and 0.181 and 0.209 in MNS, which account for an average increase in MHS of 27%.

View larger version (14K): [in this window] [in a new window]   Figure 2. Rb+ occlusion at different Rb+ concentrations in renal outer medulla microsomes from adult (75 days) MHS and MNS. DOC-treated microsomes (45 µg) were incubated in standard medium in triplicate with 50 to 1000 µmol/L RbCl plus 86Rb+ in the absence and presence of 10 mmol/L ouabain; occlusion was measured as described in "Methods." Values of the ouabain-sensitive Rb+ occlusion from five different MHS and MNS microsome preparations for each RbCl concentration are reported as mean±SE.

View this table: [in this window] [in a new window]   Table 1. Na,K-ATPase Activity at Vmax, Rb+ Occlusion Bmax, and Turnover Number in Outer Medulla Microsomes From Adult and Young Milan Hypertensive and Normotensive Rats

The validity of this measurement in microsomal preparations has been assessed by calculation of the expected stoichiometry of Rb⁺ occlusion for preparations having an enzyme activity of the microsomes. As experimentally demonstrated by Shani et al¹³ and Karlish et al,²¹ an enzyme with ATPase activity between 15 and 20 µmol P_i/min per milligram binds 3.75 nmol Rb⁺/mg; this corresponds to two K⁺ (or Rb⁺) ions bound for each phosphoenzyme molecule, which is in keeping with the classic stoichiometry of 1ATP:2K:3Na reported for the Na-K pump.¹¹ From these experimental data, the theoretical amount of occluded Rb⁺ expected for a microsomal enzyme with activity between 1.5 and 3 µmol P_i/min per milligram should be between 0.28 and 0.55 nmol Rb⁺/mg. These values are in good agreement with the experimental B_max values calculated in the present study (Fig 2). Moreover, the calculated K_m for Rb⁺ in rat microsome preparations is between 150 and 200 µmol/L, which is in good agreement with that reported previously for pig²¹ and dog²² purified enzyme.

We estimated the relative turnover number for both MHS and MNS microsome preparations using the determination of Rb⁺ occlusion B_max and the appropriate Na,K-ATPase activities. These values are reported in the Table. It can be seen that in both adult and young rats, the Na,K-ATPase V_max and Rb⁺ occlusion B_max values were similarly higher in MHS, whereas the calculated turnover number was not significantly different between the two strains.

In summary, the data on Rb⁺ occlusion confirm that even before the development of hypertension, the increased Na,K-ATPase activity in MHS is due to a higher number of Na-K pump sites able to translocate K⁺ and not to a higher turnover rate. Moreover, as previously shown for the Na,K-ATPase activity, the B_max of Rb⁺ occlusion increased with age in both strains.

Na,K-ATPase ₁- and ß₁-Subunit Abundance
We determined the abundance of Na,K-ATPase subunits by Western blot analysis in some of the microsome preparations used for the Na,K-ATPase activity determination. Representative immunoblots of both young and adult MHS and MNS preparations are shown for the ₁ and ß₁ isoforms in Fig 3. A band corresponding to the ₁-subunit was detected around M_r 95 000 in both strains at both ages (Fig 3, top). The glycosylated form of the ß₁-subunit showed a slight upward shift in the apparent M_r, from 55 000 to 58 000, in young MHS compared with MNS; this shift was not present in adult rats (Fig 3, bottom left). The different mobility shown in Fig 3, bottom, could reflect differences in glycosylation of the ß₁-subunit in young rats; therefore, the enzyme was digested with glycopeptidase F to the ß₁ core protein. As shown in Fig 3, bottom right, the digestion reduced the apparent M_r to 32 000 in MHS and MNS of both ages, suggesting that the observed shift was indeed due to a different glycosylation of the MHS ß₁-subunit. A difference of signal intensity for the glycosylated ß₁-subunit between young and adult rats (Fig 3, bottom) has been observed. This effect may be ascribed to a different affinity of the polyclonal antibody (raised versus a recombinant form of the ß₁-subunit) for the young and adult glycosylated forms of the protein.

View larger version (170K): [in this window] [in a new window]   Figure 3. Na,K-ATPase 1- and ß1-subunit protein abundance in renal outer medulla microsomes from young (25 days) and adult (75 days) MHS and MNS. Top, Immunoblot of microsomes (10 µg per lane) probed with monoclonal antibody to 1 rat Na,K-ATPase and labeled with 125I–anti-mouse immunoglobulin F(ab)2 fragment. Bottom, Immunoblot of microsomes (50 µg per lane) probed with polyclonal antibody to ß1 rat Na,K-ATPase and labeled with 125I–anti-rabbit immunoglobulin F(ab)2 fragment. Right panels show effect of glycanase (glycopeptidase F) digestion on ß1-subunit apparent Mr. Representative autoradiograms are shown. Ny indicates young MNS; Hy, young MHS; Na, adult MNS; and Ha, adult MHS.

The results obtained in three and six different microsome preparations from young and adult rats, respectively, of both strains are summarized in Fig 4. Na,K-ATPase ₁- and ß₁-subunits were both 50% higher (₁ and ß₁ in young) and 23% (₁ in adult) and 31% (ß₁ in adult) higher in MHS than in age-matched MNS.

View larger version (41K): [in this window] [in a new window]   Figure 4. Na,K-ATPase 1-subunit (A) and ß1-subunit (B) protein levels in renal outer medulla microsomes of young (25 days) and adult (75 days) MHS (dark hatched bars) and MNS (light hatched bars). Values are expressed as arbitrary units normalized to samples of renal microsome preparations from young MNS, defined as 1, and are reported as mean±SE (n=3 for young and n=6 for adult rats of both strains). *P<.05, **P<.01, MHS vs MNS.

We also measured the total pool size of the ₁-subunit in renal medulla homogenate from adult MHS and MNS and found it to be significantly higher (P<.05) in MHS (3.23±0.3 arbitrary units, n=8) than in MNS (2.46±0.19, n=6). The ß₁-subunit was not measurable because the ß₁ polyclonal antibody gave a very faint signal compared with the background when used on total medulla homogenate by Western blot.

Na,K-ATPase ₁ and ß₁ mRNA Levels
To assess whether the increase in Na,K-ATPase activity, Rb⁺ occlusion, and ₁- and ß₁-subunit abundance detected in MHS is associated with pretranslational regulation of subunit synthesis, we extracted total RNA from renal outer medulla of young and adult MHS and MNS and determined the relative abundance of Na,K-ATPase ₁ and ß₁ mRNA by Northern blot analysis (Fig 5). Three and four pools of total outer medulla extracts were processed for young and adult rats, respectively, of both strains. Results are expressed as arbitrary units normalized for a standard renal homogenate extract. The amount of ₁ mRNA was significantly increased in MHS compared with MNS at both 25 and 75 days of age (28±9% and 39±9%, respectively; P<.05 for both ages) (Fig 5A). ß₁ mRNA levels (Fig 5B) were also 23±7% higher in young and 57±10% higher in adult MHS than in MNS, the increase being statistically significant only in adult rats (P<.02).

View larger version (36K): [in this window] [in a new window]   Figure 5. Na,K-ATPase 1 (A) and ß1 (B) mRNA levels in renal outer medulla from young (25 days) and adult (75 days) MHS (dark hatched bars) and MNS (light hatched bars). Values are expressed as arbitrary units normalized to renal homogenate preparation from MNS, defined as 1, and are reported as mean±SE (n=3 for young and n=4 for adult rats of both strains). *P<.05, **P<.01, MHS vs MNS.

	Discussion

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Kidney outer medulla is mainly formed by the TAL of Henle, where selective Na⁺ reabsorption drives the mechanism of countercurrent and the formation of hypertonic urine.²³ This active transport of Na⁺ out of the tubule is strictly linked to the Na,K-ATPase activity, which is present in relatively high amounts in this portion of the nephron.²⁴ Several studies have demonstrated that an excessive tubular Na⁺ reabsorption, an impaired natriuretic capacity, or both play a crucial role in the development of genetic and experimental forms of hypertension.^{25 26} In this study, we show that MHS are characterized by an increased enzymatic activity of the outer medulla Na,K-ATPase. This increase is already present before the development of hypertension and is due to a higher number of functionally active pump sites on the cell membrane surface supported by an enhanced synthesis of the ₁- and ß₁-subunits, as demonstrated in renal outer medulla by the increased ₁ and ß₁ mRNA levels, Na,K-ATPase V_max activity, and ₁ and ß₁ protein abundance.

In the present study, the number of functional pump sites was quantified by application of the Rb⁺ occlusion method to rat microsome preparations.^{13 22 27} The catalytic -subunit of the Na,K-ATPase contains the loci for ATP and cation binding.²⁸ Therefore, the quantification of the Rb⁺ occlusion B_max in crude microsomal preparations can furnish a precise measure of the number of functionally active Na,K-ATPase -ß dimers inserted on the cell membrane. The increased Rb⁺ occlusion B_max demonstrated in both young and adult MHS (Fig 2) suggests that the higher Na,K-ATPase activity, measured at V_max (Fig 1), is not due to an enhanced turnover rate of the enzyme but to a higher number of Na-K pump sites in the TAL renal tubules of MHS than in those of MNS. The age-dependent variations of the Na,K-ATPase activity, Rb⁺ occlusion B_max, and ₁ and ß₁ protein abundance demonstrate that all these parameters increase in parallel during growth in both strains and are in line with other data demonstrating that the Na,K-ATPase activity increases in rat TAL between 12 and 40 days of age and represents the main determinant of the maturation of urinary concentrating capacity during development.²⁹

The higher level of functionally active pump sites in MHS is further supported by the greater abundance of ₁ and ß₁ protein subunits detected by immunoblotting in microsomal preparations from this strain (Fig 4). As already observed for Na,K-ATPase activity, the increase in protein expression precedes the development of hypertension and is characterized by a qualitative difference in the glycosylation pattern of the ß₁-subunit in young MHS compared with age-matched MNS and adult rats (Fig 3, bottom).

The different apparent M_r of Na,K-ATPase ß₁-subunit in young MHS (Fig 3, bottom) is clearly linked to a different glycosylation pattern,³⁰ since reduction to the core protein with glycopeptidase F nullifies the difference between the two strains. The ß₁ shift in M_r may be due to either a qualitative or quantitative difference in the type or number of N-glycans, and it may be related to a genetic difference between the two strains.

The increased activity and expression of Na,K-ATPase in MHS renal medulla microsomes is paralleled by enhanced ₁ and ß₁ mRNA levels (Fig 5), increased Na,K-ATPase activity at V_max, and enhanced ₁ protein abundance in renal outer medulla homogenate. This suggests not only that a different distribution of the Na-K pumps between MHS and MNS may be present on the cell surface but that a primary involvement of the pretranslational process determines the increase of the total pool size of Na,K-ATPase in MHS. Many reasons could account for an upregulation of Na,K-ATPase expression. An increase of intracellular sodium, due to either a primarily greater entrance from the apical side or a secondary inhibition of the basolateral extrusion (for instance, inhibition of the Na-K pump due to low K⁺, ouabain, ouabainlike factor, etc), can stimulate the synthesis of new Na-K pumps.^{31 32 33 34} It is also known that the hormonal steroid activation of the Na,K-ATPase, which is accompanied by a transient increase in intracellular sodium, besides an early effect of recruitment of a latent pool of Na-K pumps,³⁵ is sustained in the long run by a stimulus to the de novo Na-K pump synthesis.^{36 37} The apical sodium entry measured in membrane vesicles from both proximal⁹ and TAL¹⁰ tubules is higher in MHS than in MNS, whereas the proximal tubular cell intracellular sodium, measured by electron probe analysis, is lower in MHS than in MNS.³⁸ These two apparently opposite aspects of renal cell sodium handling in MHS can be explained assuming that a primary increase in Na-K pump activity (or expression) drives an increased transepithelial sodium transport and resets, in the long run, the intracellular sodium concentration to a lower level in tubular cells of MHS than in those of MNS.

Among many other possibilities, the following can also be considered for explaining the sustained upregulation of Na-K pump expression in MHS: (1) a molecular alteration of the ₁- and/or ß₁-subunit, linked to a genetic polymorphism, which affects the behavior and/or expression of the enzyme; (2) a hormonal modulation, linked to altered steroidogenesis; and (3) a higher rate of pump synthesis due to a molecular difference of some other membrane component that affects cell structure and protein turnover. The first point cannot be completely excluded on the basis of the available data. ₁ cDNA has been fully sequenced, and no difference has been found between MHS and MNS (G.T., unpublished results, 1995); however, the ß₁-subunit has not yet been investigated, and therefore, if some polymorphism were present, particularly in the promoter region, this could modulate the expression of the protein. Concerning the second point, it is known that the developmental pattern of Na,K-ATPase in TAL is under the control of glucocorticoid^{29 39} and mineralocorticoid²⁹ hormones. Adrenal steroidogenesis is increased in both young and adult MHS,⁴⁰ and this may participate in the higher Na,K-ATPase expression, especially in 25-day-old MHS. However, it seems unlikely that the pump upregulation observed in adult MHS can be entirely attributed to enhanced levels of aldosterone⁴⁰ because TAL segments are relatively insensitive to this hormone.^{29 41}

The third possibility, that a genetically determined molecular difference between MHS and MNS in some cell membrane component could affect Na-K pump turnover and expression, seems to be the most probable. Recent experimental evidence has documented that a polymorphism of the cytoskeletal protein adducin between MHS and MNS⁴² plays a specific role in modulating cell membrane structure and ion transport. Adducin is a heterodimer composed of - and ß-subunits⁴³ whose cDNA full-length sequences in MHS and MNS revealed the presence of a missense mutation in both the subunits.⁴² We have previously demonstrated that both the mutations are associated with hypertension in MHS.⁴² Moreover, the polymorphism of -adducin differently affects the actin polymerization and bundling in a cell-free system as well as the actin-cytoskeleton organization and the Na-K pump rate in rat epithelial cells transfected with normal (MNS) or mutated (MHS) adducin.⁴⁴ In particular, the V_max activity of the Na-K pump is significantly increased in cells transfected with MHS adducin.⁴⁴

Many data have been collected demonstrating that the actin-cytoskeleton directly affects the processes of epithelial cell polarization,⁴⁵ regulates the activity of ion channels in the apical side of these cells,^{46 47} modulates the Na-K pump activity at the basolateral side,⁴⁸ and influences the retention time of the Na,K-ATPase protein on the membrane surface.⁴⁹ Furthermore, it has been recently demonstrated that actin directly stimulates the Na,K-ATPase activity in a cell-free system⁵⁰ and adducin behaves like an actin barbed-end capping protein by inhibiting the elongation and depolymerization of actin filaments.⁵¹ Currently, we do not know the precise molecular mechanism that links the polymorphism of adducin to the upregulation of Na-K pump expression observed in MHS kidney. However, the available experimental data favor the following hypothesis: The cytoskeletal rearrangement caused by the MHS mutated adducin could produce an altered interaction between some cytoskeletal proteins (for instance, actin) and the Na-K pump. This may influence not only the enzymatic activity (increase) and distribution of the MHS Na-K pumps but also their retention time on the cell membrane surface, which might be shorter than in MNS, for instance because of an accelerated process of internalization. This mechanism may represent the signal that, at pretranslational levels, leads to an enhanced synthesis of Na,K-ATPase in MHS to avoid cell depletion of the enzyme.

In conclusion, these data are in keeping with the previously demonstrated increase of tubular sodium reabsorption observed in MHS and the consequent transient sodium retention and increase of blood pressure. They also favor the hypothesis that the in vivo upregulation of the Na-K pump in MHS is linked to a genetic alteration of adducin that by affecting the actin-cytoskeletal structure, influences the transmembrane ion transports.

	Selected Abbreviations and Acronyms

 

DOC = deoxycholic acid MHS = Milan hypertensive rat(s) MNS = Milan normotensive rat(s) Pi = inorganic phosphate SDS = sodium dodecyl sulfate TAL = thick ascending limb

	Acknowledgments

 
The authors acknowledge Dr Cristina Reina and Elena Minotti for expert technical assistance.

Received May 29, 1996; first decision June 14, 1996; accepted July 30, 1996.

	References

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