Segmental localization of mRNAs encoding Na+-K+-ATPase alpha 1- and beta 1-subunits in diabetic rat kidneys using RT-PCR

P. Scherzer and M. M. Popovtzer

Nephrology and Hypertension Services, Hadassah University Hospital, Jerusalem 91120, Israel


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MATERIALS AND METHODS
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The present study evaluated renal Na+-K+-ATPase activity and mRNA in rats with diabetes mellitus. To localize the segmental alpha 1- and beta 1-mRNAs of Na+-K+-ATPase 1 and 8 days after induction of diabetes, we used the polymerase chain reaction after reverse transcription of the mRNA in microdissected nephron segments. Na+-K+-ATPase activity in the proximal convoluted tubule (PCT) rose on days 1 and 8 by 42 and 23%, respectively. In the medullary thick ascending limb (MTAL), it remained unchanged on day 1 and increased on day 8 by 55%. In the cortical collecting duct (CCD), activity rose by 81 and 45% on days 1 and 8, respectively. In parallel, alpha 1-mRNA in the PCT increased by 52 and 22% on days 1 and 8, respectively. In the MTAL, alpha 1-mRNA remained unchanged on day 1 and rose by 47% on day 8. In the CCD, alpha 1-mRNA increased by 140 and 110% on days 1 and 8, respectively. beta 1-mRNA was unchanged in the PCT throughout the study and was elevated in the MTAL and CCD on days 1 and 8. Thus there was a temporal dissociation between alpha 1- and beta 1-subunit expression. There was a highly significant linear correlation between Na+-K+-ATPase activity and alpha 1-mRNA in all nephron segments throughout the experiment. It appears that microdissection of nephron tubules combined with reverse transcription-polymerase chain reaction defines the molecular identity of the amplified gene product and its segmental distribution in the nephron. We propose that altered gene expression may be the mechanism underlying enhanced Na+ pump activity along the nephron in diabetic rats.

diabetes mellitus; streptozotocin; proximal convoluted tubule; medullary thick ascending limb; cortical collecting duct


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PREVIOUS STUDIES from this laboratory have demonstrated an increase in renal tubular Na+-K+-ATPase activity in microdissected nephron segments from rats with diabetes mellitus (DM) induced by streptozotocin (STZ) (31, 32). In those studies, we proposed that the increase in Na+-K+-ATPase activity in the proximal convoluted tubule (PCT) and proximal straight tubule was related to augmented cotransport of glucose and Na+ in these segments. In the cortical and medullary thick ascending limbs of Henle's loop (MTAL), the increase in enzyme activity closely followed the rise in glomerular filtration rate (GFR) and, consequently, the augmented delivery of Na+ to the loop of Henle. There was a direct linear correlation between GFR and Na+-K+-ATPase activity in the MTAL. In the cortical collecting duct (CCD), the increase in Na+-K+-ATPase activity was aldosterone dependent. Accordingly, a very close correlation between plasma aldosterone concentration and Na+-K+-ATPase activity in the CCD was evident (32).

The increases in Na+-K+-ATPase activity are consistent with enhanced Na+ absorption in the diabetic kidney. These increases could consist of an increased number and/or enhanced insertion of Na+ pumps in the basolateral membrane.

The transporting enzyme Na+-K+-ATPase consists of a heterodimer of an alpha -subunit and a beta -glycoprotein subunit (25). The alpha -subunit is considered to be the catalytic and transporting subunit, inasmuch as it contains binding sites for ATP, Na+, K+, and cardiac glycosides (12, 25). Thus the alpha -subunit is responsible for ATP hydrolysis, which drives the cation transport. The functional role of the glycosylated beta -subunit is much less defined. It has been suggested that the beta -subunit plays a role in the transfer of the alpha -subunit from the alpha - and beta -assembly line in the endoplasmic reticulum, via the Golgi apparatus, to the cell membrane. Therefore, the beta -subunit appears to exert an accessory and modulatory effect on the active enzyme (1, 8, 14, 17, 25).

There are three catalytic isoforms of Na+-K+-ATPase (alpha 1, alpha 2, and alpha 3) and two beta -isoforms (beta 1 and beta 2), expressed and regulated differentially, depending on the type of tissue (12). In the rat kidney, alpha 1- and beta 1-mRNA are coexpressed at a similar level, which varies along the renal tubule according to the cell type (6, 7, 10).

The anatomic intricacy of the nephron has made evaluation of site-specific changes in gene expression of Na+-K+-ATPase at the mRNA level difficult. To overcome this difficulty, micromethods have been used by us (21) and others (5) to assess changes in Na+-K+-ATPase activity in response to various pathophysiological conditions.

It has been proposed that the glomerular hyperfiltration in diabetic kidneys may be due to resetting of tubular glomerular feedback (18, 27, 28). This has been attributed to enhanced Na+ reabsorption in the nephron, and it is reflected by increased Na+-K+- ATPase activity. The present study was undertaken to further investigate the mechanism(s) of Na+ hyperabsorption by exploring the changes in Na+-K+-ATPase at the molecular level and at defined tubular segments by means of microdissection.

We employed reverse transcription (RT) and semiquantitative polymerase chain reaction (PCR) combined with microdissection of individual nephrons in our experiments. To analyze the distribution of Na+-K+-ATPase mRNA subunits along the nephron, we carried out RT-PCR with specific primer pairs for Na+-K+-ATPase alpha 1- and beta 1-subunits on microdissected PCT, MTAL, and CCD.

We correlated the alterations of enzyme activity with the gene expression of alpha 1- and beta 1-subunits of Na+-K+-ATPase in isolated nephron segments in rats 1 and 8 days after induction of DM by administration of STZ.


    MATERIALS AND METHODS
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Animal experiments were approved by the Institutional Animal Welfare Committee. Male rats of the Hebrew University strain, weighing 200-250 g, were used in all experiments. On day 1, the experimental rats were injected with STZ (55 mg/kg; Sigma, St. Louis, MO) in 0.01 M citrate buffer (pH 7.4), and the control rats received the vehicle only. At 24 h or 8 days after induction of DM, blood was drawn for determination of glucose and aldosterone levels. The rats were killed by dislocation of the neck, the kidneys were removed immediately, and the hilus was cut off and microdissected for Na+-K+-ATPase determination and RNA isolation in the nephron tubules.

To determine the direct effect of high glucose on gene activation, we performed an additional set of experiments. Glucose (50%, 2 ml/100 g body wt) was given by an oral gastric tube to four animals, and blood and urinary glucose levels were measured over a 3-h period at 1-h intervals. By the end of the 3-h measurement period, glucosuria appeared, and the animals were killed and the kidneys were immediately removed and microdissected for Na+-K+-ATPase determination and RNA isolation.

Microdissection

Microdissection of the isolated nephron segments was performed as described by Burg et al. (3) and modified by Schmidt and Horster (22); the procedure was described in detail by Scherzer et al. (21). Briefly, 0.5- to 1.0-mm-thick sagittal slices were cut and immediately immersed in dissection fluid (in mM: 136 NaCl, 3 KCl, 1 K2HPO4, 1.2 MgSO4, 2 CaCl2, 4 sodium lactate, 1 sodium citrate, 6 L-alanine, and 5.5 glucose) plus 0.6% collagenase (145 U/mg; Millipore). The slices were incubated for 30 min in a shaking water bath at 37°C and aerated with 100% O2. The slices were then removed and rinsed twice in ice-cold dissection fluid. Cortical hemicircles, cut at the corticomedullary border, and medullary triangles, including the papilla, were used to obtain the appropriate segments. The slices were transferred to a petri dish with dissection fluid and inserted into a cooled Lucite chamber illuminated by a transmission dark-field source, as described by Schmidt and Horster (22). The length of each segment was measured by an eyepiece micrometer. The segment was rinsed in fresh dissection fluid and transferred to a glass ampule for analysis. The PCT, outer MTAL, and CCD were dissected and used for assay of ATPase activity and isolation of RNA.

Determination of ATPase

The method and apparatus for ATPase determination were previously described in detail (21). Briefly, the method is based on the micromodification of the method of Schoner et al. (23), as described by Czaczkes et al. (4). The hydrolysis of ATP was coupled to the transformation of phosphoenolpyruvate to pyruvate by pyruvate kinase. Pyruvate was reduced to lactate in the presence of lactic dehydrogenase. NADH acted as O2 acceptor and was oxidized to NAD+. There was a stoichiometric relationship between the hydrolysis of ATP and the disappearance of NADH (by oxidation to NAD+). This disappearance could be followed fluorometrically.

Na+-K+-ATPase was calculated as the difference between total ATPase and Mg2+-ATPase. The latter was determined by addition of ouabain (G-strophanthin) to a final concentration of 4 mM.

Pairs of segments of the same type from the same rat were used for total ATPase and Mg2+-ATPase determinations. Na+-K+-ATPase activity was the mean of the difference of activities measured in these pairs of segments. From each rat, three to five pairs of segments of the same type were dissected. For total ATPase and Mg2+-ATPase activities, 9-16 samples per segment were isolated from 4 or 5 rats. Na+-K+-ATPase activity was compared among the three groups studied: control and 1 and 8 days after STZ administration.

RNA Isolation

Total RNA was extracted from pooled isolated nephron segments (PC, MTAL, and CCD) using a commercially available kit (RNeasy, Qiagen, Chatsworth, CA). After microdissection, the pools of tubules (50-200 mm) were transferred with 5-10 µl of microdissection solution into 400 µl of denaturing solution that contains guanidinium isothiocyanate-containing buffer, which immediately inactivates RNases to ensure isolation of intact RNA. The final RNA pellets were resuspended in 30-60 µl of diethylpyrocarbonate-treated water and quantified by absorbance at 260 nm.

cDNA Synthesis and PCR of alpha 1- and beta 1-Subunits of Na+-K+-ATPase

First-strand cDNA was synthesized from 2-4 µl (0.2-0.5 µg) of the total RNA harvested from each of the microdissected tubules. The RNA sample was incubated for 1 h at 37°C in oligo[d(T)21] primer (Eisenberg Bros., Tel-Aviv, Israel), Maloney's murine leukemia virus RT (200 U/µl; Promega, Madison, WI), and buffer supplied by the manufacturer containing 25 mM MgCl2, 200 µM dNTPs, and RNase inhibitor (40 U/µl) in a final reaction volume of 22 µl. The reaction was stopped by incubation at 95°C for 10 min, and the cDNA was stored at -20°C until it was used.

The primers for Na+-K+-ATPase subunits were selected by comparative nucleotide sequence analysis of published cDNA sequences by Shull et al. (24) for alpha 1-subunits and by Mercer et al. (16) for beta 1-subunits. For the alpha 1-subunit, the primer sequence was 5'-CCGGAATTCTGCCTTCCCCTACTCCCTTCTCATC-3' (sense) and 5'-TGCTCTAGACTTCCCCGCTGTCGTCCCCGTCCAC-3' (antisense), and the expected size of the PCR product was 331 bp (nt 3207-3529). For the beta 1-subunit, the primer sequence was 5'-GTCGAATTCCCTTCCGTCCTAATGACCCCAAGA-3' (sense) and 5'-GCGGGATCCGACCAGAGCAGTTCCCCAGCCAGTC-3' (antisense), and the expected size of the PCR product was 237 bp (nt 725-943). The primers for the control gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were purchased from Clontech; the expected length of the PCR product was 930 bp.

PCR

PCR was performed in a thermal cycler (Progene, Techne, Oxford, UK) on 2- to 5-µl aliquots of the first-strand cDNA. The PCR mixture contained 500 ng of each primer, 200 µM of each dNTP, 2.5 U of AmpliTaq polymerase (Promega), and reaction buffer supplied by the manufacturer containing 1.5 mM MgCl2 in a final volume of 25 µl.

Amplification was performed as follows: 95°C for 2 min (initial denaturation), 35 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min and a final extension at 72°C for 7 min.

Negative controls included dissection medium subjected to RT-PCR, water subjected to PCR, and aliquots from each RNA-containing sample without RT. All negative controls failed to generate PCR products.

cDNA samples derived from three rats (one control, one 1 day after STZ treatment, and one 8 days after STZ treatment) were always amplified simultaneously in the same PCR procedure.

Analysis of PCR Products

After amplification, 10 µl of the PCR products were separated by electrophoresis with a 1.5% agarose gel containing ethidium bromide (0.5 µg/ml; Sigma) in 0.04 M Tris acetate-0.001 M EDTA buffer. Ethidium bromide-stained product bands of expected size were visualized under ultraviolet (UV) light, and the size was confirmed by comparison with synthetic DNA size markers (100-bp DNA ladder; MBI Fermentas, Amherst, NY). Band intensities of alpha 1-and beta 1-subunits of Na+-K+-ATPase and the control gene G3PDH were quantified by densitometry (Multi-Analyst/PC-Fluor-S Multi Imager System, Bio-Rad Laboratories, Hercules, CA).

In each experiment, serial dilutions of cDNAs obtained by RT from tubules of control animals and from animals 1 and 8 days after STZ treatment were performed for the control gene G3PDH. The purpose of these dilutions was to ensure a constant reference quantity of the control gene. This served to semiquantify the variations in alpha 1- and beta 1-subunit cDNA vs. control cDNA after simultaneous amplifications with the corresponding primers for the same number of cycles. Thus we were dealing with a fixed amount of the control gene, which served as a standard for comparison, and we assayed the changes in alpha 1- and beta 1-subunits accordingly. The band intensities of alpha 1- and beta 1-subunits of Na+-K+-ATPase in the nephron segments were calculated from samples in which signal intensity of the control gene remained unchanged by UV imaging. The results were calculated as the ratio of intensities of alpha 1- and beta 1-subunits to the intensity of the control gene, which served as the standard. Data are expressed as percent change in cDNA density of the different experimental groups compared with the control group, which served as baseline.

Linearity of the Primers

Individual reactions were performed for different numbers of cycles with 500 nmol of G3PDH and alpha 1- and beta 1-subunit primers and 0.2-0.5 µg of the first-strand cDNA of each segment studied as a template. The amplified band intensities were determined. The logarithm of intensity was plotted against the number of cycles.

Northern Blot Analysis

Because we used G3PDH as internal control in normal and diabetic rats, we performed Northern blot analysis to determine whether its expression was modified by the diabetic state. Northern blots were performed as previously described (20). Membranes were hybridized to cDNAs of G3PDH or 18S, which served as control.

Southern Blot Analysis

To verify the molecular identities of the G3PDH and alpha 1- and beta 1-subunit PCR products, we performed Southern blots. The gels that were visualized and recorded by densitometry as mentioned above were denatured, neutralized, and transferred according to the protocol of Manniatis et al. (13) to nylon membranes (Gene Screen, NEN Research Products, Boston, MA) using 20× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). The DNA was cross-linked to nylon membranes by UV light (Ultraviolet Transilluminator, UVP). To perform Southern analysis of the PCR products, blots were hybridized for 16-20 h with 32P-labeled cDNA fragments corresponding to alpha 1- and beta 1-subunit Na+-K+-ATPase under stringent conditions. The radioactive probe was prepared with a DNA labeling kit (Rediprime, Amersham). The hybridizations were performed with a PstI/EcoRI fragment of the alpha 1-subunit of Na+-K+-ATPase (nt 3060-3636) and an EcoRI fragment of the beta 1-subunit of Na+-K+-ATPase (nt 343-1600). Membranes were washed and autoradiographed by standard procedures. Bound cDNA probes were removed by 2 min of boiling in 1× SSC, and the same membranes were rehybridized with a control probe synthesized from G3PDH cDNA. The abundance of this cDNA was independent of any of the treatments described in this study. The binding was quantified by phosphorimaging (model BHS 1000, Fujix) and expressed as a ratio of the intensity obtained for G3PDH cDNA. Each result was confirmed by repeating the Southern analysis with PCR products from four different RNA preparations. For all experiments, Southern blots were performed with specific alpha 1- and beta 1-subunits of Na+-K+-ATPase probes that confirmed the identity of the PCR products (data not shown).

Blood Sample Analysis

Blood glucose levels were measured using a Glucometer Elite (Bayer, Elkhart, IN). Aldosterone levels were determined by radioimmunoassay (ALDOCTK-125-M, Sorin Biomedica). All reagents were purchased from Sigma.

Statistics

Values are means ± SE. Analysis of variance was performed for statistical evaluation between the groups. Results of individual groups were compared by a nonpaired Student's t-test with a modified level of significance according to Bonferroni's method (9).


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Linearity of the Primers in PCR

We have established the linear conditions for the PCR experiments with G3PDH and alpha 1- and beta 1-subunit primers. The mixture of primers with the first-strand cDNA of PCT, MTAL, and CCD segments, which served as templates, was amplified with 20-35 cycles, as described above. Figure 1 shows linear conditions for primers in the PCT: G3PDH and alpha 1- and beta 1-subunits. The logarithm of intensity of the amplified primer plotted against the number of cycles shows a very good linearity between 25 and 35 cycles (r = 0.998 for all primers studied), whereas with fewer cycles, the amplification was not apparent. Similar results were obtained for the MTAL and CCD (not shown).


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Fig. 1.   Linearity of primers in PCR in proximal convoluted tubules (PCT). Logarithm of intensity of amplified primer [glyceraldehyde-3-phosphate dehydrogenase (G3PDH), alpha 1-subunit (a), and beta 1-subunit (b)] is plotted against number of cycles. Linear regression of lines was >0.998.

Northern Blot Analysis

The results of Northern blot analysis of G3PDH and 18S, which served as control, are depicted in Fig. 2. DM did not modify G3PDH expression in all nephron segments studied before (control) and 1 and 8 days after induction of DM by STZ administration.


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Fig. 2.   Northern blot analysis for internal control gene G3PDH and 18S in PCT, medullary thick ascending limb (MTAL), and cortical collecting duct (CCD) from control rats (C) and experimental rats 1 and 8 days after induction of diabetes by administration of streptozotocin (1 and 8, respectively). Expression of G3PDH was not modified by diabetes.

Blood Glucose in STZ-Treated Rats

Blood glucose levels in control and STZ-treated rats are shown in Table 1. Blood glucose was high after 24 h (P < 0.001 vs. control) and remained high 8 days after induction of DM (P < 0.001 vs. control).

                              
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Table 1.   Blood glucose and aldosterone levels in control and streptozotocin-treated rats

Aldosterone Levels

Aldosterone levels in control and STZ-treated rats are listed in Table 1. At 1 day after STZ administration, aldosterone levels increased from 29.5 ± 2.5 (control) to 58.2 ± 3.5 ng/100 ml (P < 0.001). After 8 days, aldosterone levels decreased to 45.2 ± 2.5 ng/100 ml but remained above control levels (P < 0.001 vs. control) and were significantly lower than 1 day after STZ administration (P < 0.02 vs. 1 day after STZ).

Effects of STZ on Na+-K+-ATPase Gene Expression

PCT. Typical results of RT-PCR analysis of mRNA expression of alpha 1- and beta 1-subunits of Na+-K+-ATPase in isolated PCT from STZ-treated rats 1 and 8 days after induction of DM are depicted in Fig. 3A. An increase in alpha 1-subunit (331 bp), but not beta 1-subunit (237 bp), product density in the PCT of the diabetic kidney is apparent. Negative controls demonstrated no contamination in the reaction mixture. Expression of the control gene G3PDH (930 bp) was unchanged in control and experimental PCTs.


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Fig. 3.   Expression of alpha 1- and beta 1-subunits of Na+-K+-ATPase and its activity in PCT from control and diabetic rats. A: representative analysis of RT-PCR products on ethidium bromide-stained gel of Na+-K+-ATPase alpha 1- and beta 1-subunits in PCT from control and diabetic rats. Coamplification of cDNA of PCT shows control gene G3PDH (930 bp) and Na+-K+-ATPase alpha 1- and beta 1-subunits (331 and 237 bp, respectively). To show negative controls, PCR was performed without addition of mRNA (CRT) and without addition of reverse transcriptase (CPCR). B: Na+-K+-ATPase alpha 1- and beta 1-mRNA levels and Na+-K+-ATPase activity in PCT from control rats and from experimental rats 1 and 8 days after induction of diabetes. Open and solid bars, abundance of alpha 1- and beta 1-subunit expression in membranes from >= 5 rats for each group that were run on the same blot (means ± SE); grey bars, Na+-K+-ATPase activity in PCT from 10 pairs of segments for each group. *P < 0.05 vs. control (alpha 1-subunit). +P < 0.02 vs. control (Na+-K+-ATPase activity).

Changes in expression of alpha 1- and beta 1-mRNAs of Na+-K+-ATPase was evaluated by comparing the density of the bands of the diabetic animals with that of the control rats.

Southern blots with a alpha 1- and beta 1-mRNA- and G3PDH-specific probes confirmed the identity of the PCR products (data not shown).

Figure 3B summarizes Na+-K+-ATPase activity in isolated PCT of control and experimental rats after 1 and 8 days of STZ treatment and expression of alpha 1- and beta 1-mRNAs of the enzyme in the same animals. Levels of alpha 1- and beta 1-mRNAs are presented as a percentage of that of control rats, which is considered 100%.

At 1 and 8 days after STZ administration, Na+-K+-ATPase activity in the PCT increased by 42 and 23%, respectively. Similarly, expression of alpha 1-mRNA of Na+-K+-ATPase was increased by 52 and 22% at 1 and 8 days after STZ administration, respectively (P < 0.001 and 0.0025, respectively, vs. control). No change in expression of beta 1-mRNA of Na+-K+-ATPase was found in the PCT after 1 and 8 days of DM (Fig. 3).

MTAL. Changes in expression of alpha 1- and beta 1-mRNA of Na+-K+-ATPase are depicted in Fig. 4. In Fig. 4A, a typical representative gel of RT-PCR products from MTAL RNA in control and diabetic rats shows no change in expression of the control gene G3PDH.


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Fig. 4.   Expression of alpha 1- and beta 1-subunits of Na+-K+-ATPase and its activity in MTAL from control and diabetic rats. A: representative analysis of RT-PCR products on ethidium bromide-stained gel of Na+-K+-ATPase alpha 1- and beta 1-subunits in MTAL from control rats and experimental rats 1 and 8 days after induction of diabetes. Coamplification of cDNA of MTAL shows control gene G3PDH (930 bp), Na+-K+-ATPase alpha 1-subunit (331 bp), and Na+-K+-ATPase beta 1-subunit (237 bp). To show negative controls, PCR was performed as described in Fig. 3 legend. B: Na+-K+-ATPase alpha 1- and beta 1-mRNA levels and Na+-K+-ATPase activity in MTAL from control and experimental rats 1 and 8 days after induction of diabetes. See Fig. 3 legend for details. *P < 0.05 vs. control (alpha 1-subunit). +P < 0.02 vs. control (Na+-K+-ATPase activity). ^P < 0.05 vs. control (beta 1-subunit).

Southern blots with alpha 1- and beta 1-mRNA-specific probes confirmed the identity of Na+-K+-ATPase alpha 1- and beta 1-subunits (data not shown).

In Fig. 4B, alpha 1- and beta 1-mRNA in the MTAL of control and diabetic animals and the activity of the enzyme are quantified. At 1 day after STZ administration, there was no change in Na+-K+-ATPase activity in the MTAL; after 8 days, the enzyme activity increased by 55% (Fig. 4B). There was also no change in alpha 1-mRNA expression in the MTAL after 1 day; however, after 8 days it increased by 47% (P < 0.001 vs. control; Fig. 4B). Expression of beta 1-mRNA of Na+-K+-ATPase in the MTAL of the diabetic rats differed from expression of alpha 1-mRNA. At 1 day after STZ administration, beta 1-mRNA expression was increased by 30% compared with control rats (P < 0.001). This increase in expression of beta 1-mRNA of Na+-K+-ATPase in the MTAL persisted at the same level (30%) 8 days after induction of DM (Fig. 4B).

CCD. Figure 5A shows a representative gel for RT-PCR products of the CCD RNA. An increase in PCR product density of alpha 1- and beta 1-subunits in the CCD of diabetic kidneys is apparent 1 and 8 days after STZ. There was no change in G3PDH levels. The Southern blots with alpha 1- and beta 1-mRNA-specific oligonucleotide probes confirmed the identity of Na+-K+-ATPase alpha 1- and beta 1-subunits (data not shown).


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Fig. 5.   Expression of alpha 1- and beta 1-subunits of Na+-K+-ATPase and its activity in CCD from control and diabetic rats. A: representative analysis of RT-PCR products on ethidium bromide-stained gel of Na+-K+-ATPase alpha 1- and beta 1-subunits in CCD from control rats and experimental rats 1 and 8 days after induction of diabetes. Coamplification of cDNA of CCD tubules shows control gene G3PDH (930 bp), Na+-K+-ATPase alpha 1-subunit (331 bp), and Na+-K+-ATPase beta 1-subunit (237 bp). To show negative controls, PCR was performed as described in Fig. 3 legend. B: Na+-K+-ATPase alpha 1- and beta 1-mRNA levels and Na+-K+-ATPase activity in CCD from control rats and from experimental rats 1 and 8 days after induction of diabetes. See Fig. 3 legend for details. *P < 0.05 vs. control (alpha 1-subunit). +P < 0.02 vs. control (Na+-K+-ATPase activity). ^P < 0.001 vs. control (beta 1-subunit).

Figure 5B summarizes alpha 1- and beta 1-mRNA and Na+-K+-ATPase activity in the CCD. Na+-K+-ATPase activity in the CCD increased by 81 and 45% at 1 and 8 days after STZ, respectively. Similarly, the increments in aldosterone 1 and 8 days after STZ were 95 and 55%, respectively (P < 0.001 vs. control; P < 0.01 vs. 1 day after STZ; Table 1). The abundance of alpha 1-mRNA of Na+-K+-ATPase increased by 140% 1 day after induction of DM compared with control rats. Changes in alpha 1-mRNA paralleled alterations in enzyme activity (Fig. 5B) and aldosterone levels (Table 1). Expression of alpha 1-mRNA 8 days after induction of DM in the CCD increased by 110% and was significantly less than that observed 1 day after of induction of DM (P < 0.001 vs. control and vs. 1 day after STZ). This reduction is similar to the reduction in aldosterone levels after 8 days of DM (Table 1).

Expression of beta 1-mRNA of Na+-K+-ATPase in the CCD increased by 132% at 1 day after STZ administration (P < 0.005 vs. control), and a further increase of 205% above expression of the beta 1-subunit of control rats (P < 0.001) was observed 8 days after induction of DM.

Relationships Between Na+-K+-ATPase Activity and alpha 1-Subunit Expression

The relationships between Na+-K+-ATPase activity and expression of alpha 1-subunits are plotted in Fig. 6 for the PCT, MTAL, and CCD. There was a significant direct linear correlation between Na+-K+-ATPase activity and percent change in alpha 1-mRNA in all nephron segments.


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Fig. 6.   Correlation between Na+-K+-ATPase activity and alpha 1-mRNA of Na+ pump in PCT (A, n = 10), MTAL (B, n = 10), and CCD (C, n = 10) in control and diabetic rats.

Acute Glucose Load

After acute intragastric glucose load, blood glucose levels were 111 ± 5 (baseline), 498 ± 20 (after 1 h), 400 ± 25 (after 2 h), and 329 ± 30 mg/dl (after 3 h; n = 4 each). Urinary glucose was tested with a dipstick and was negative at baseline, became positive after 2 h, and rose further at 3 h (>200 mg/dl). Table 2 shows Na+-K+-ATPase activity and alpha 1- and beta 1-subunit gene expression for control and glucose-loaded animals. Na+-K+-ATPase activity was significantly increased in the PCT after 3 h in glucose-loaded animals (P < 0.001 vs. control), while it remained unchanged in the MTAL and CCD. The alpha 1- and beta 1-subunits of Na+-K+-ATPase gene expression did not change in all segments that were examined.

                              
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Table 2.   Na+-K+-ATPase activity and alpha 1- and beta 1-subunit gene expression in control and hyperglycemic animals


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our present experiments demonstrated that the rise in Na+-K+-ATPase activity in isolated renal tubules in rats with STZ-induced DM is associated with an increase in the abundance of alpha 1-mRNA of the enzyme along the nephron. The relationship in the nephron segments is illustrated in Fig. 6. There was a highly significant linear correlation between Na+-K+-ATPase activity and the level of alpha 1-mRNA in the PCT, MTAL, and CCD. At variance with the alpha 1-mRNA, there was no change in beta 1-mRNA abundance in the PCT throughout the 8 days of the experimental period. In the MTAL, an early increase in beta 1-mRNA was observed 1 day after induction of DM and was maintained until 8 days after induction of DM. Similarly, in the CCD, an increase in beta 1-mRNA was observed 1 day after STZ induction of DM, and a further rise was apparent 8 days after induction of DM.

In normal rats, after 3 h of glucose load accompanied by glycosuria, an impressive increase in Na+-K+-ATPase activity was observed in the PCT without changes in gene expression of alpha 1- and beta 1-subunits (Table 2). This would suggest that, at this stage, alpha 1- and beta 1-subunits of the enzyme were recruited to the cell membrane and that activation of gene expression by glucose could be ruled out.

Because Na+-K+-ATPase activity generally reflects the rate of Na+ uptake and its vectorial transport across the tubular epithelium, it is plausible to suggest that the rate of Na+ transport may play a role as a major regulator of alpha 1-subunit gene expression. In this regard, it is appropriate to draw an analogy to experiments in isolated renal epithelial cells reported by others. In those experiments, the amount of Na+-K+-ATPase in cell membranes was modulated according to the demand for transport capacity through the influence of Na+ concentration in the cytoplasm, which enhances the transcription rates of Na+-K+-ATPase (10). In suspensions of kidney tubule cells, an increase in the cytoplasmic Na+-to-K+ ratio triggered an early rise in the amount of specific mRNA of Na+-K+-ATPase followed by an increase in the rate of synthesis of alpha 1- and beta 1-subunits (10). Similarly, in our model of STZ-induced DM in rats, an increased filtered and delivered load of Na+ to the tubular epithelial cells resulted in adaptive increases in the abundance of alpha 1-mRNA of Na+-K+-ATPase and a parallel rise in the enzyme activity. Only the alpha 1-subunit contains specific binding sites for Na+, and it is possible that its synthesis could be influenced by Na+ delivery to the PCT and MTAL.

It is noteworthy that the beta 1-mRNA of Na+-K+-ATPase was altered independently of the enzyme activity. Thus, after 24 h, Na+-K+-ATPase activity was totally dissociated from changes in beta 1-subunit gene expression in the PCT and MTAL; similarly, the magnitude of alteration after 8 days was different. Similar to our findings, Ver et al. (29) demonstrated a dissociation between mRNA levels of alpha 1- and of beta 1-subunits in STZ-treated diabetic rats in the kidney and heart 2 and 4 wk after induction of DM. Farman et al. (7) also demonstrated a dissociation between alpha 1- and beta 1-mRNA after adrenalectomy in nondiabetic rats. In this experimental setting, adrenalectomy was followed by a significant decrease in alpha 1-mRNA in the early distal tubule and in the CCD, whereas beta 1-mRNA abundance was unaffected (7).

In the CCD, changes in alpha 1-mRNA abundance correlated with the enzyme activity. However, in this segment of the nephron, enzyme activity and gene expression appear to be regulated by aldosterone levels and are less affected by variations in Na+ delivery and uptake. Expression of alpha 1-mRNA of Na+-K+-ATPase and the enzyme activity increased significantly in the CCD 1 day after induction of DM. At this stage, GFR was not increased (31, 32) and the enzyme activity was unchanged in the MTAL. However, changes in alpha 1-mRNA in the CCD were seen 1 day after induction of DM, and they closely paralleled changes in aldosterone levels. Thus it appears that in the CCD, as opposed to the MTAL, alterations in alpha 1-mRNA and enzyme activity are induced primarily by aldosterone levels, while in the MTAL the changes mainly depend on variations in GFR and the accompanying rise in Na+ delivery (32). Verrey et al. (30) demonstrated that aldosterone alters the gene expression of the alpha 1-subunit in a manner that is independent of Na+ entry into A6 kidney cells, which bear some similarity to epithelial cells of the CCD. Similarly, it appears that the effect of aldosterone on CCD alpha 1-mRNA in diabetic rats in the present experiments was dissociated from changes in Na+ uptake in this nephron segment. Using a different experimental model, Tsuchiya et al. (26) presented similar results. In their experiments, adrenalectomy selectively decreased alpha 1-mRNA in rat CCD. They concluded that regulation of Na+-K+-ATPase in the CCD in vivo can be attributed, at least in part, to mineralocorticoid-dependent control of Na+-K+- ATPase alpha 1-mRNA abundance.

Furthermore, it has been speculated that aldosterone could have a direct effect on the splicing and stability of mRNA. It is also of interest that, in the CCD, aldosterone in the presence of triiodothyronine increases the amount of Na+-K+-ATPase by de novo synthesis of new pump units independent of changes in cytoplasmic Na+ (2).

Increasing evidence has accumulated suggesting that the beta -subunit plays a role in regulating the assembly, oligomerization, and functional maturation of the Na+ pump (1, 8, 14, 17, 25). Because there is an excess of alpha 1-mRNA, beta 1-subunit mRNA becomes the limiting factor, which is presumably regulated by mineralocorticoids (8). In our present experiments in rats with STZ-induced DM, the level of aldosterone was increased 1 day after induction of DM, and at that time a substantial increase in beta 1-mRNA was detected in the CCD. Thus it is possible that aldosterone played a key role in enhancing synthesis of beta 1-subunit mRNA in this nephron segment. Changes in beta 1-subunit gene expression did not exactly follow the variations in aldosterone levels, Na+-K+-ATPase activity, and alpha 1-subunit gene expression. This discrepancy between alpha 1- and beta 1-mRNA is not surprising, since the alpha 1- and beta 1-subunit genes are located on different chromosomes (11, 33) and display distinct tissue-specific expression (19). The regulatory sequences on each gene might be different, which might explain the differential effect of aldosterone on the transcription of alpha 1- and beta 1-subunit genes.

An increase in beta 1-mRNA in the MTAL preceded changes in enzyme activity and alpha 1-mRNA, but 8 days after induction of DM, enzyme activity and alpha 1- and beta 1-subunit gene expression displayed a similar increment. The early increase in beta 1-subunit gene expression was not associated with change in Na+-K+-ATPase activity and, thus, probably was not associated with altered active Na+ transport. Therefore, it appears that the diabetic state and, possibly, hyperglycemia could have a direct impact on beta 1-subunit gene expression in this segment. It is also suggested that synthesis of the beta 1-subunit precedes that of the alpha 1-subunit, which is in agreement with the findings of McDonough et al. (15).

In summary, our present experiments, based on RT-PCR methodology in isolated nephron segments, helped delineate alterations in Na+-K+-ATPase alpha 1- and beta 1-mRNA in correlation with enzyme activity in experimental DM. The most striking finding of the present study was a highly significant linear correlation between Na+-K+-ATPase activity and the abundance of alpha 1-mRNA in diabetic rats. We and others proposed that the glomerular hyperfiltration may be due to resetting of tubuloglomerular feedback in the nephrons of the diabetic kidney (18, 27, 28). This has been linked with Na+ hyperabsorption in the nephron as reflected by enhanced Na+-K+-ATPase activity. The present study further explored this phenomenon using methods of molecular biology. These results suggest that altered gene expression may be the underlying mechanism for enhanced activity of the Na+ pump and Na+ absorption along the nephron in STZ-treated diabetic rats.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Scherzer, Nephrology and Hypertension Services, Hadassah University Hospital, POB 12000, Jerusalem 91120, Israel (E-mail: henrietta{at}hadassah.org.il).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00053.2001

Received 20 February 2001; accepted in final form 28 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 282(3):F492-F500
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