Effect of Chronic Renal Failure on NaK-ATPase

Transcription

1 ffet of Chroni Renal Failure on NaK-ATPase al and a2 mrna Transription in Rat keletal Musle ilvia Bonilla, 1. Annelise Goeke, alvador Bozzo, Miriam Alvo, Luis Mihea, and lisa T. Marusi Department ofphysiology and Biophysis, Faulty ofmediine, University ofchile, Casilla 75, antiago-chile Abstrat Previous studies have suggested that an alteration in the expression of the NaK-ATPase of musle may be an important determinant of enhaned insulin sensitivity in hroni renal failure. Therefore, in the present studies we have examined the effet of uremia on the NaK-ATPase a isoforms in skeletal musle, at the level of mrna expression and enzymati ativity. The ativity of the sodium pump, as measured ouabain-sensitive 'Rb/K uptake in soleus musle, revealed a redution in the ativity in uremia, related to the inrement in plasma reatinine values. The derement in 86Rb uptake by the rat soleus musle of experimental animals was assoiated with hanges on NaK-ATPase gene produt. Northern analysis of mrna revealed isoform-speifi regulation of Na,K-ATPase by uremia in skeletal musle: a derease of - 5% in al subunit Na,K-ATPase mrna, as ompared to ontrols. The derement in al mrna orrelates with the dereased ativity of the Na,K-ATPase in uremia, under basal onditions and with the almost omplete inhibition of the Na,K-ATPase, of uremi tissue by a onentration of 1-' M ouabain. Although the ativity of the a2 isoform pump was not modified by uremia, the 3.4-kb message for this enzyme was inreased 2.2-fold; this disrepany is disussed. Altogether these findings demonstrate that the defetive extrarenal potassium handling in uremia is at least dependent in the expression of a1 subunit of the Na,K-ATPase. (J. Clin. Invest : ) Key words: NaK-ATPase gene * rat soleus * uremia potassium - homeostasis Introdution keletal musle plays an important role in the learane of a potassium load (1) and a dereased musle uptake ofpotassium ontributes to the maintained hyperkalemia observed after meals in hroni renal failure. In fat, we and others (2, 3) have shown a defet in extrarenal potassium disposal in azotemia, due to a less ative sodium pump in skeletal musle (4, 5); whereas an inrement in the number of sodium pumps of the seretory epithelia has been desribed in uremia (6). As it is well established, ell potassium is mainly ontrolled by the Na,K-ATPase. The Na+,K+-ATPase is the intrinsi Address orrespondene to Dr. lisa T. Marusi, Department ofphysiology and Biophysis, Faulty of Mediine, University ofchile, Casilla 75 antiago-chile. Reeived for publiation 6 February 1991 and in revised form 716 August J. Clin. Invest. The Amerian oiety for Clinial Investigation, In /91/12/2137/5 $2. Volume 88, Deember 1991, membrane protein responsible for pumping Na+ and K+ against their onentration gradients. The enzyme is omposed of a atalyti subunit, a, and a smaller, glyosylated subunit, f, whose funtion is unknown. Two isozymes of the a subunit of the enzyme, known as a 1 and a2 (+), were first disovered by weadner (7). Reently, a third, brain-speifi, form designated a3 was disovered using a rat brain DNA library. ven though all three forms are 85% homologous, they possess important - differenes (8). The a2 and a3 forms have a high affinity for the ardia glyoside ouabain (K1o 5 = IO-` M) whereas ali is relatively resistant to the drug (KO.5 = l M). It has also been shown in both rat adipoytes and rat brain synaptosomes that the a 1 and a2 isozymes have different sodium affinities, with K.5 values of mm and mm, respetively. Insulin ativation is mediated by lowering the K;D5 for sodium of the a2 isozyme (9-1). Little is known about the regulation of Na,K-ATPase isoforms in skeletal musle in advaned hroni renal failure. We have reently shown that the addition oforal gluose to a potassium load was more effetive in the transloation of potassium to the intraellular ompartment in uremi animals (4). Further, in vitro studies indiated that insulin aused a relatively greater stimulation ofouabain-sensitive 86Rb uptake in the uremi tissue as ompared to ontrol soleus musle (4). These results suggest hanges in the expression of the Na,K-ATPase. Thus, this study was designed to determine the moleular basis of the enhaned insulin sensitivity in the uremi skeletal musle. We examined the expression of two isoforms of the Na,K-ATPase, the ali and a2, in soleus musle fibers from ontrol and hroni renal failure rats. The results suggest that tissue-speifi alterations in the expressions of these two isoforms may be the major mehanism for the dereased potassium uptake in uremi musle tissue as ompared to ontrols. Methods Animals. Male prague-dawley rats (- 25 g) fed a standard rat how diet were used. Chroni renal failure was indued by ligating branhes ofthe artery to infart about /4 ofthe kidney; a right nephretomy was performed after 4 d. The experiments were done 3-4 wk after surgery. Plasma reatinine, blood arterial gases, ph, and plasma potassium were ontrolled at the beginning of eah study. 86Rb transport in skeletal musle. The musle was removed from ontrol and uremi animals and then groups of fibers weighing 8-15 mg were inubated in separate vials. The musular fibers were washed for 15 min in 2 ml of KRB ontaining (mm) 4.2 KC1; 1.19 KH2P4; 12 NaCl; 25 NaHCO3; 1.2 MgO4; 1.3 CaCl2; 5 -gluose (ph = 7.4). The buffer was gassed with 95% 2 and 5% CO2. Thereafter, the musles were preinubated for 3 min in KRB in the presene or absene of 1-2 M or l-' M ouabain. Finally, the musles were inubated for 2 min in KRB ontaining 86Rb (.1 Ci/ml) in the presene or absene of 1-2 M or l-' M ouabain. The reation was stopped by transferring the musle into ied KRB; the musles were washed in old buffer and blotted. Radioativ- Na,K-ATPase mrna and Chroni Renal Failure 2137

2 ity of the samples was determined by the Cerenkow radiation in a liquid sintillation ounter. RNA isolation and Northern blot analysis. Upon killing of rats, soleus musles were removed and immediately proessed. Total ellular RNA was extrated from 25 mg of soleus skeletal musle using the guanidinium thyoianate (11). RNA was eletrophoresed on 2.2 M formaldehyde-agarose gels (12), blotted and fixed onto nylon membranes, and then hybridized to DNA probes. The a l and a2 DNA was provided to us by Nelson Ruiz-Opazo, Boston University. Isoform-speifi restrition endonulease Bam HI fragments were prepared from these probes. The a I and a2 DNA probes were labeled with digoxigenin-d UTP, using the nonradioative DNA labeling and detetion kit from Boehringer Mannheim Corp., Indianapolis, IN. Membranes were hybridized to the orresponding probes for 5-6 h at 42C in 5% formamide in 5X C (.3 M NaCl,.3 M sodium itrate) ph 7. with 5% of bloking reagent, aording to instrutions reommended by the kit. Then the membranes were washed at high strigeny twie in 2X C with.1% D for 1 or 2 min eah at room temperature, then twie for 15 min eah in.1 x C with.1% D at 68C to assure the speifiity of the Northern. The hybridized membranes were deteted immediately by the enzyme-linked immunoassay system. The amount of mrna present in eah sample was quantified by omputer sanning densitometry analysis, omparing the intensity of the experimental sample to ontrol rat mrna samples or to the internal standard GAPDH mrna (glyeraldehyde-3-phosphate dehydrogenase) for eah proessed skeletal musle (12). The speifi DNA probe (p RGAPDH- 13) was kindly provided by P. Carvallo, NIH. DNA was determined on 25 mg of musle using the spetrofluorometri method of Labara and Paigen (14). Total ellular RNA was determined by spetrophotometry (12) and protein ontent by the Hartree method (15). Quantifiation dots on Northern blots. Color dots were quantified with a sanning densitometer (Genisan, Fremont, CA) interfaed to an IBM PC 286 omputer with a G-45 sanner and speially written C program, ignifiane was determined with tudent's t test (paired) and aepted at the P <.1 level. Results Charateristis ofthe experimental rats. The harateristis of experimental animals, 3-4 wk after partial nephretomy (3/4), were similar to those previously desribed (4). The experimental rats have body weights and blood ph values similar to ontrol rats. The nephretomized rats with plasma reatinine values above 1. mg/dl were onsidered with established renal insuffiieny. To evaluate the influene of azotemia in the Na,K-ATPase ativity of the skeletal musle we measured ouabain sensible 86Rb uptake by the soleus musle of rats with different degrees of renal insuffiieny, as determined by plasma reatinine values. Fig. 1 inludes the results on 86Rb uptake of the isolated soleus fibers. As shown in the figure, there is an inverse relationship between both parameters: as plasma reatinine inreases, the ativity of the sodium pump reahes very low values. These results onfirm a defet in the Na,K-ATPase ativity in hroni renal failure and are indiative that this defet is related to the degree of renal insuffiieny. Regulation of Na,K-ATPase mrnas in skeletal musle. Next, we examined the expression of the two a isoforms of the Na,K-ATPase known to be present in musle, from ontrol and uremi rats. In this study, and subsequent experiments, mean plasma reatinine values were.4±.1 mg/dl and 1.6±.4 mg/dl, respetively. Preliminary results were indiative of dramati hange in both isoforms mrna with uremia. a, 34 3: C I.-_ NC 2( I O le i Plasma reatinine (mg/dl) Figure 1. Correlation of plasma reatinine with ouabain-sensitive '*Rb/K uptake by isolated soleus musle fibers. Values represent individual rats, either sham operated animals (n = 3) or rats with different degrees of hroni renal insuffiieny as measured by plasma reatinine onentration (n = 11). 'Rb uptake was measured with or without 1-2 M ouabain, as desribed in Methods, in tripliate samples. The orrelation between these two parameters is highly signifiant (r =.83; P <.1). Therefore, we assayed musle weight, ontent of DNA, RNA, and protein. Table I shows the results: musle weight did not vary signifiantly between both groups, nor did nulei aids or protein ontent. Next, we examined the effet of hroni renal failure on the relative amount ofthe a 1 and a2 isoforms mrna by omparing the results with GADPH mrna, used as an mrna marker (13). As shown in the Northern blots of Fig. 2 there are no signifiant differenes in GAPDH mrna ontent between ontrol and the experimental groups. Therefore, we used GAPDH mrna as a referene to measure the relative abundane of a 1 and a2 isoforms mrna, as ompared to the marker. The 3.4-kb a2 transript is the major one in musle, whereas in brain the 5.3-kb speies is the predominant form; nevertheless, both transripts enode the same protein. Quantifiation of the levels of Na,K-ATPase a isoforms mrna was made in separate samples for a I and for 3.4-kb a2. The results obtained in several experiments are shown in Fig. 3; mean values± for eah isoform are expressed as the relative olor intensity of eah a isoform dot blot ompared to GADPH. As shown in Fig. 3 A, a 1 mrna levels for the uremi tissue dereased signifiantly to one-half of the ontrol values. When total RNA isolated from the skeletal musle of uremi rats was probed with the a2-speifi DNA probe, the amount of the 3.4-kb transript was found to be inreased over those Table L Musle Analysis Group Control Uremi oleus weight, mg 132±6 136±5 Total DNA Aig/g tissue 14±17 146±21 Total RNA ug/g tissue 213±2 25±18 Total protein ug/g tissue 23.3± ±1.2 Values are mean±; n = 6 for all groups Bonilla, I. A. Goeke,. Bozzo, M. Alvo, L. Mihea, and. T. Marusi

3 A GADPH -- C Ul U2-28s - 18s B Figure 2. oleus musle Na,K-ATPase a isoforms mrna levels and GAPDH mrna in re- -28 sponse to hroni renal j 2 failure. Northern blot - 8 analysis of skeletal musle RNA (25,gg/lane) GADPH - from individual rats was performed as indiated under experimental proedures. These blots are representative of five C U U2 separate experiments. present in normal rats (Fig. 3 B). ine the size of the probes was different no diret omparison between the amount of a 1 vs. a2 was made (aal = 1,7 bp; a2 = 7 bp). Data derived from this analysis suggest that the two known a isoforms expressed in skeletal musle are modified with uremia. At least part of the altered Na,K-ATPase ativity in skeletal musle is at the level ofthe mrna. It annot be determined from these experiments whether the pretranslational regulation of the sodium pump expression is at the level of initiation of transription or stability of the mrna. These possibilities are urrently under investigation. Comparison ofouabain sensitivity in skeletal musle ofuremi versus ontrol rats. From the 86Rb/K pumping data it has been established that in the range of I M ouabain, a2 is 9% saturated with ouabain, whereas a 1 has - 1% Table II. ffet ofouabain in the Musle Na,K-A TPase Ativity Ouabain-sensitive '6Rb uptake Controls nmol/g wet wt per min CRF Basal 161.7± ±7.5* Plus 1-5 M ouabain 91.3± ±4.4 Perent inhibition 43.2± ±4.1* Basal ativity of the pump was measured as the differene of total ativity minus 1o-2 M ouabain. 86Rb uptake was measured at 2 min in parallel samples. (n = 7). *P <.1. bound (9). Thus, we used a onentration of 1- M ouabain to inhibit a2 isoform almost exlusively, while at 1-2 M ouabain omplete inhibition of both a I and a2 isoforms should be obtained. Therefore, we have studied the ativity ofthe enzyme as an Rb+ pump, under normal onditions by soleus musle in the presene of the above indiated onentrations of ouabain (Table II). As shown in Results, the soleus musle of ontrol rats, inubated in normal buffer, have 43% of a2 mediated uptake; this value is similar to that reently desribed by Hsu and Guidotti for intat soleus fibers (16). However, in the uremi tissue the pumping ativity of a2 isoform is inreased as ompared to aal mediated transport. Nevertheless, the absolute values of the a2 isoform were 75 mmol/min per g in both normal and uremi musles, and the enhaned sensitivity to ouabain is mainly due to the derement a 1 isoform from 9 to 2 mmol/min per g. This last value is onsistent with the derement in ali mrna observed in the musle of hroni renal failure rats. Disussion One of the most prominent transport proteins in nearly all animal ells is the Na,K-ATPase. Under physiologial onditions, this enzyme mediates the transport of three Na ions out ofthe ell and two K ions into the ell per ATP moleule that is split. By this ative transport, transmembranes gradients for Na and K are maintained that are essential for a large variety of ellular funtions. In epithelial ells, the pump is involved in potassium seretion and whole body potassium homeostasis. It is known that in hroni renal failure, as renal mass is diminished, there is a remarkable adaptive inrease in potassium exretion by the remaining funtioning nephrons (6). It has also been reported that in uremia, there is inreased gastrointestinal potassium seretion resulting in an elevated stool potassium ontent. Also, during hroni potassium load there is an inrement in the number of Na,K-ATPase units in the kidney and oloni tissue with a onomitant inreased seretion of potassium (17, 18). As indiated above (6), in both uremi tissues, there is an adaptative inrement in the number of Na pumps in epithelial tissue, whereas intraellular Na is elevated a) * -. Ct :3 z O a) Alpha Alpha 2 Figure 3. Relative a subunits mrna abundane in rat soleus musle. The dots obtained, as shown in Fig. 2, were densitometrially sanned and quantified. An arbitrary value of 1 was given to GAPDH mrna abundane of eah animal. The data are presented as the mean± of a 1 and a2 subunits in ontrol () and uremi rats m. (n = 6 for eah mean value). a1) u 'O C. z._ a Na,K-ATPase mrna and Chroni Renal Failure 2139

4 in erythroytes, leukoytes, and musle ells of patients with hroni renal failure (19). In fat, as shown here, and in previous work (4), a 5% redution of the sodium pump ativity was found in the uremi musle. everal forms ofrat Na,K-ATPase have been desribed that are distint with respet to sodium affinity (1) regulation by thyroid hormones (2), sensitivity to ardia glyosides (9) and insulin (1). These funtional differenes have been orrelated with the presene oftwo or more isoforms ofthe a subunit. The tissue distribution studies of the multiple forms of rat Na,K-ATPase indiate that a I isoform is predominantly present in kidney epithelial tissue, whereas a greater proportion of a2 is present in the skeletal musle (21). peifi DNA probes have been developed for the a isoforms of rat Na,K-ATPase (22). Therefore, the tissue-speifi expression of mrnas enoding the a isoforms in the rat skeletal musle were assessed in the present work. These studies demonstrated that in uremia there is a hange in the proportional expression of the a mrna isoforms in skeletal musle. In fat, in the uremi tissue we found a diminished al transript as ompared to those present in normal skeletal musle (approximately one-half). By ontrast, as illustrated in Fig. 2 B, the 3.4-kb mrna enoding the a2 subunit in the uremi tissue is more abundant than ontrol: the diminished a 1 mrna is onsistent with the redued pumping ativity of the uremi skeletal musle. In fat, as shown by Lytton et al. (9), while al is pumping at about 1/2 its maximum apaity under basal onditions, a2 is pumping at only 1/2 ofits apabilities. Therefore a derement in a 1 isoform ould aount for the dereased uptake of 56Rb/K in the uremi musle. Nevertheless, there is no suh relationship in the ase of a2 isoform. ven though we have not measured intraellular eletrolytes, it is known that in uremia ell sodium is inreased (23,24); the inrement in ellular sodium would favour the a2 isoform ativity (1). Reently, Hsu and Guidotti (24) also desribed a divergeny between the amount of the a2 isoform and the transriptional regulation of the a2 gene in the skeletal musles of hypokalemi rats. One possibility for the lak of orrelation between the amounts of mrnas and ative enzyme is that the transripts are not translated (25). Another possibility is that the a2 transripts are translated and the a2 polypeptides do not mature into funtional enzyme due to an insuffiient amount ofthe,b hain (26, 27). A third possibility is that the a2 transripts are translated, and the polypeptide is broken down more rapidly than usual. Nevertheless, further studies need to be direted to measure the number of units of eah isoform of the Na,K-ATPase by speifi antibodies (28), before final onlusions ould be drawn. It should be taken into aount that we have measured the pumping ativity ofthe rat soleus musle under normal onditions; as it has been shown by others (16, 29) the ativity of the (Na+, K+) pump in isolated fibers inubated in a normal buffer is a fration of the total possible ativity. Due to the low and variable enzyme reovery. (.2-8.9%) we have not studied the pump ativity in isolated sarolemma; suh subellular preparations may give misleading data on the pump ativity ofskeletal musle ells, as pointed out by Hansen and Clausen (29). These results provide evidene that uremia indues genespeifi modulation in skeletal sodium pump. Furthermore, the modulation of NaK-ATPase is isoform-speifi and the downregulation ofthe a 1 mrna may represent a deindution of gene expression eliited in response to stimuli from some intraellular hanges that ourred in hroni renal failure. A simultaneous upregulation of a2 subunit mrna expression ould be a related phenomena, but no evidene exists at the present time to support this hypothesis. Herrera et al. (22) have shown a 2- to 3-fold inrease in a I mrna and a 3- to 15-fold derease in a2 mrna in the aorta and left ventriule of hypertensive animals. The hanges in amounts of mrna ould represent transriptional regulation, although hanges in mrna stability annot be ruled out. Determination of the mehanisms by whih speifi Na,K-ATPase a subunits genes are modulated in uremia may provide insight into the moleular mehanisms involved in the defetive extrarenal handling of potassium. Aknowledgments We would like to thank Dr. Juan Olate for ritial reading ofthe manusript, Ms. Lupe Ferrer who oordinated the laboratory work, and Ms. Rosario Flores for her expert tehnial assistane. This work was supported by a grant from Fondeyt. Referenes 1. Bia, M. J., and R. A. De Fronzo xtrarenal potassium homeostasis. Am. J. Physiol. 24:F257-F Alvo, M., P. Krsulovi, V. Fernandez, A. M. spinoza, M. sobar, and. T. Marusi ffet of a simultaneous potassium and arbohydrate load on extrarenal K homeostasis in end-stage renal failure. Nephron. 53: Fernindez, J., J. R. Oster, and G.. PKrez Impaired extrarenal disposal ofan aute oral potassium load in patients with end stage renal disease on hroni hemodialysis. Miner. letrolyte Metab. 12: Goeke, I. A.,. Bonilla,. T. Marusi, and M. Alvo nhaned insulin sensitivity in extrarenal potassium handling in uremi rats. Kidney Int. 39: Druml, W., R. A. Kelly, R. C. May, and W.. Mith Abnormal ation transport in uremia. J. Clin. 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