Psammomys obesus lives in an arid environment and feeds on saltbush. When animals are fed a laboratory diet, urine osmolarity drops. To explore the mechanism(s) of water conservation, we measured renal function, kidney solute content, Na-K-ATPase activity, and mRNA in several groups: group I (saltbush diet, 18 g/day, 4.2 g protein); group II (laboratory diet, 10 g/day, 1.8 g protein); and group III, the same as group I, and group IV, the same as group II, both plus a 1-day fast. Urine osmolarity was 2,223 ± 160, 941 ± 144, 1,122 ± 169 and 648 ± 70.9 mosM in groups I, II, III, and IV, respectively. Tissue osmolarities in cortex, outer medulla, and inner medulla, respectively, were 349 ± 14, 644 ± 63, and 1,152 ± 34 μosM/mg tissue in group I; 317 ± 24, 493 ± 17, and 766 ± 60 μosM/mg tissue in group II; 335 ± 6, 582 ± 15, 707 ± 35 μosM/mg tissue in group III; and 314 ± 18, 490 ± 22, and 597 ± 29 μosM/mg tissue in group IV. There were no differences in Na-K-ATPase activity and mRNA in cortex and in medulla between groups I and II, whereas ingroup III Na-K-ATPase activity and mRNA increased in cortex and outer medulla. These results suggest a key role for urea in corticomedullary osmotic gradient of Psammomys. The absence of differences in Na-K-ATPase activity and mRNA between groups I and II despite differences in tissue sodium concentrations is consistent with Na-K-ATPase-independent Na absorption. Increased Na-K-ATPase activity and mRNA in fasting suggest transition to Na-K-ATPase- dependent Na transport.
- mRNA of α- and β-subunits
the fat sand rat, Psammomys obesus, lives in the most arid areas of Sahara-Arabian deserts, from Algeria to Sudan in North Africa eastward to Arabia. In Israel, it lives in the Negev, the Judean desert, and the “Arava” (7, 26, 27). Psammomys differs from other desert geribillids in that it is diurnally active above ground all year round. The other geribillids are nocturnal animals and remain in burrows during the day (10). Psammomys is able to thrive to reproduce and to grow when feeding entirely on leaves and stems of plants belonging to the Chenopodiaceae family. In the Israeli deserts, it feeds solely on Atriplex halimus, the saltbush (3).
A. halimus has a high moisture content and thus provides much of the needed water. It is also readily available throughout the year. Because the burrows of the Psammomys are at the base of this plant, little energy is expended for foraging. In addition, no other rodents are feeding on A. halimus. The A. halimus saltbush is low in energy content and as such has a relatively low efficiency of energy for maintenance when consumed byPsammomys (15). Thus the animal consumes large amounts of this plant for survival. The saltbush has a high nitrogen and electrolyte content (Table 1).Psammomys scrapes the salt-coated outer layer of the leaves with its teeth before consuming them (14, 15), thus removing many of the electrolytes.
The distinctive and specialized features of Psammomyslifestyle has aroused much interest among physiologists. The structural organization of Psammomys kidney has revealed that, contrary to what has been earlier reported in the literature, that most nephrons have long loops of Henle (18, 24), this species has short (66%) and long (34%) nephrons alike (13). The ultrastructure of the thin limbs of Henle of these short-looped and long-looped nephron segments reveals specialized structure and function (1, 2). From micropuncture studies, it seems that in Psammomys the NaCl and urea addition to the thin descending limb of Henle's loop plays a major part in the concentration mechanism (4, 5).
It is widely accepted that Na-K-ATPase, the enzymatic equivalent of the Na-K pump, which is present in a high concentration in the kidney, is responsible for the active transport of sodium and potassium across the tubular epithelium (16, 23). The importance of this enzyme in creating the hyperosmolarity of the medulla is well established. Thus the highest activity of Na-K-ATPase was found in the thick ascending limb of Henle's loop (11, 16, 22).
Psammomys feeds on a saltbush diet, excretes urine with high osmolarity, and, when kept in the laboratory and fed a laboratory diet, its urine osmolarity falls dramatically. The above findings raised many questions regarding the effect of environmental factors, and principally dietary composition and consumption of food, on renal concentrating ability in Psammomys. More specifically, we faced the question of what components of the diet may play a key role in the urinary concentrating ability: is it the salt content, nitrogen content, or caloric intake? The present study was undertaken to further explore and define in more detail the renal mechanism(s) of the adaptation of Psammomys to its natural arid environment. Similarly, we find it very important to define the alteration that takes place when the animals are removed from their natural environment and are kept in the laboratory. For this purpose, we employed in the present study dietary manipulations including fasting for 24 h but allowed free access to water. This may allow us to delineate in more detail the dependence of the renal mechanism of urine concentration on dietary regimens. Renal function, aldosterone levels, kidney solute content, Na-K-ATPase activity, and mRNA of α- and β-subunits of Na-K-ATPase were measured in Psammomys fed a saltbush (natural) diet, a laboratory diet, and in fasting animals fed both diets.
MATERIALS AND METHODS
Animal experiments were approved by the Institutional Animal Welfare Committee.
P. obesus pups of the Hebrew University strain were weaned to different diets: A. halimus (SB; group I); laboratory diet (ALD; group II); saltbush diet plus 1-day fast (SB-FA; group III); and laboratoy diet plus 1-day fast (ALD-FA; group IV). The compositions of the diets are given in Table 1.
The animals were kept individually for 6 wk and were fed as above. The saltbush leaves that served as the native diet were picked from their natural habitat, the Dead Sea area. In the fasted groups, the animals were fed their suitable diet before entering a 24-h fast. All groups drank tap water ad libitum.
By the end of the 6-wk period, the day before the experiments the animals were housed individually in metabolic cages, and groups I and II were given access to their respective diets. Fluid and food intake and 24-h urine excretion were measured. Blood was drawn from the bifurcation of the aorta under light ether anesthesia.
All groups were subdivided into three subsections. In one, the kidneys were removed for solute content determination. In the second, the kidneys were removed, immediately decapsulated, weighed, and kept on ice. Slices of cortex, outer medulla, and inner medulla were cut and pooled separately for enzyme preparation. In the third, the kidneys were excised and dissected into cortex, outer medulla, and inner medulla for measurement of cellular mRNA levels of α- and β-subunits of Na-K-ATPase.
Kidney solute content determination.
Psammomys were exsanguinated from the bifurcation of the aorta under light ether anesthesia. The kidneys were removed, and their water and solute composition were determined by the method of Levitin et al. (19). Briefly, slices of cortex, outer medulla, and inner medulla were dissected and processed. Electrolyte and osmolar content were determined from the tissue extracts, and urea was calculated by subtracting measured electrolytes from tissue osmolality: osmoles − (2 Na+ + 2 K+). We also measured urea content directly and found no differences between the measured and calculated urea concentrations.
Preparation of microsomes.
Preparation of the microsomal ATPase was carried out essentially according to Jørgensen and Skou (12).Psammomys tissues pooled from at least four animals were homogenized in 10 vol of a medium containing 0.25 M sucrose and 2 mM EDTA buffered with 5 mM Tris · HCl to a pH of 7.4–7.5. The homogenate was centrifuged at 7,000 g for 15 min; the supernatant was decanted and centrifuged at 48,000 g for 40 min. The pellet was resuspended in an equal volume of the above solution and again homogenized in 10 vol of desoxycholate 0.1% containing 2 mM EDTA and 25 mM Tris · HCl (pH 7.0). After incubation at 37°C for 30 min, the suspension was centrifuged at 25,000 g for 30 min. The pellet was suspended in the above sucrose-EDTA-Tris. This final suspension was frozen at −20°C until assayed.
Assay of ATPase.
ATPase activity was determined by the amount of Pi released during incubation at 37°C in a shaking, thermostatic bath, according to Gutman et al. (9). All assays were run in duplicate. The Pi release was measured with and without K+in the medium. The standard incubation medium consisted of (in mM) 100 NaCl, 10 KCl, 4 MgCl2, and 4 ATP. Enzymatic activity was stopped by the addition of 10% trichloroacetic acid. Piwas determined according to the method of Fiske and Subbarow (6). Enzymatic protein was assayed according to Lowry et al. (20). Na-K-ATPase was estimated as the difference in Pi release in the medium without and with K+.
Measurement of cellular mRNA levels (Northern blots).
RNA was extracted from Psammomys kidney slices, and the levels of mRNA for α- and β-subunits of Na-K-ATPase were measured by Northern blots. After extraction with Tri Reagent kit (Molecular Research Center, Cincinnati, OH), RNA (10 μg) was denatured and ethidum bromide was added to each sample at a concentration of 0.1 mg/ml. The samples were size fractionated by electrophoresis in 1% agarose gels containing formaldehyde and transferred to nylon membrane (Gene Screen; New England Nuclear Research Products, Boston, MA) by diffusion blotting. The integrity of the RNA and the uniformity of RNA transfer to the membrane were determined by ultraviolet (UV) visualization of the ribosomal RNA bands of the gels and the filters. The filters were fixed by UV cross-linking. Membrane strips were hybridized for 16–20 h with 32P-labeled cDNA fragments corresponding to α-Na-K-ATPase and β-Na-K-ATPase under stringent conditions. The radioactive probe was prepared with a Rediprime DNA labeling kit (Amersham). The hybridizations were performed withPst I/EcoR I fragment of the α-subunit of Na-K-ATPase (nucleotide 3060–3636) and EcoR I fragment of β-subunit of Na-K-ATPase (nucleotide 343–1600). Membranes were washed and autoradiographed by standard procedures. Bound cDNA probes were removed by 1–2 min of boiling in 1× standard sodium citrate+0.1% sodium dodecyle sulfate, and the same membranes were hybridized with a control probe synthesized from a cloned fragment of β-actin cDNA. The abundance of this cDNA/RNA species was independent of any of the treatments described in this study. Bindings were quantified by phosphorimaging (Fujix, BHS 1000) and expressed as the ratio of intensities obtained by hybridizing the same stripe with the cDNA studied and β-actin cDNA, respectively, as control gene. Each result was confirmed by repeating the Northern hybridization with four different RNA preparations.
Blood, urine, and tissue extracts were analyzed for sodium, potassium, and osmolality. Blood and urine samples were also analyzed for creatinine, whereas blood was taken also for aldosterone level determinations. Sodium and potassium were analyzed by flame photometry (Instrumentation Laboratory). Osmolality was measured by a Fiske osmometer (Fiske Associates). Urea concentration was determined by automated enzymatic ultraviolet test, urease/glutamate dehydrogenase (GLDH) method, and creatinine concentration was determined by an automated picric acid method, with autoanalyzer of Cobas Mira (Hoffmann-La Roche and Limited Diagnostica, Basel, Switzerland). Aldosterone levels in the plasma were determined by radioimmunoassay (Coat-A-Count, Diagnostic Products, Los Angeles, CA).
Creatinine clearance and urinary excretion of sodium and potassium were calculated. Data are presented as statistical evaluation among the four groups. Results were presented as means ± SE for the enzymatic assay, four determinations from different membrane preparations, each pooled from four animals. Results between individual groups were compared by a nonpaired Student's t-test with a modified level of significance according to the Bonferroni method (8). All reagents were purchased from Sigma, St. Louis, MO.
Food intake of Psammomys fed the saltbush and laboratory diet is given in Table 2. The data on food intake are normalized to animal food intake per animal. There is a marked difference in fresh material intake of SB animals compared with ALD animals (18.10 ± 0.64 vs. 10.92 ± 1.14 g/day, P < 0.001). Total protein intake of SB animals was 43% higher than in ALD animals (2.39 ± 0.085 compared with 1.68 ± 0.17 g/day, P < 0.005). Water intake, however, did not differ between the two groups, whether fed or fasting. In group I it was 10.2 ± 3.5 ml/24 h, in group II 10.3 ± 1.52 ml/24 h, in group III 12.2 ± 3.14 ml/24 h, and in group IV14.5 ± 3.2 ml/24 h.
The NaCl content of the natural saltbush was obviously strikingly higher than that of laboratory diet.
Blood aldosterone, sodium, potassium, and creatinine levels in the experimental groups are given in Table 3. This is the first report on aldosterone levels in Psammomysfed on their native diet. Aldosterone levels were significantly lower (2.20 ± 0.57 ng%) in Psammomys fed on their native saltbush diet compared with animals fed the laboratory diet (37 ± 7.3, P < 0.001). In ALD-FA, aldosterone levels remained elevated (30.4 ± 7.9 ng%), whereas in the SB-FA animals fed their native food, the saltbush leaves, it rose markedly compared with fed animals (14.2 ± 5.38 ng%, P < 0.05).
It is of interest that hypokalemia was observed in fasting animals,groups III and IV. This could reflect the kaliuretic effect of aldosterone in the absence of dietary intake of potassium. The serum sodium of the fasted animals of both groups was significantly lower compared with fed animals (P < 0.001). Blood creatinine and creatinine clearance (see also Tables4 and 5) remained unchanged, indicating the lack of effect of fasting on glomerular function.
Metabolic data of SB and SB-FA Psammomys are listed in Table4. Creatinine clearance did not differ between fed and fasted animals. Reduction by 50% was observed in the urine osmolarity of the SB-FA animals (1,122 ±169 compared with 2,223 ± 160 mosM,P < 0.001). This reduction was not associated with a significant decrease in the diuresis of these animals. Marked natriuresis and kaliuresis were found in Psammomys eating saltbush compared with those fasting.
Metabolic data for ALD and ALD-FA Psammomys are presented in Table 5. Again, as in the SB and SB-FA animals, there were no differences in the creatinine clearance between fed and fasted animals maintained on a laboratory diet. A decrease in urinary osmolarity in ALD-FA animals was observed but did not reach a statistically significant difference.
Renal solute composition.
There are differences in the osmotic gradients from the cortex to inner medulla in the four groups. There is a marked increase in the osmotic gradient from cortex to inner medulla in the kidneys of animals fed on a saltbush diet and a moderate increase in Psammomys fed on a laboratory diet; in ALD-FA animals (group IV) and SB-FA animals (group III), the osmotic gradient was reduced.
In the cortex, osmolarity and electrolyte and urea contents are very similar among SB and ALD animals or in ALD-FA. By contrast, in the SB-FA animals (group III), the sodium content increased and urea content decreased (Table 6, Fig. 1).
In the outer medulla of ALD and ALD-FA animals (groups IIand IV, respectively), the osmotic gradient is decreased compared with in SB and SB-FA animals (groups I andIII, respectively). The sodium and urea contents in ALD and ALD-FA animals were lower compared with SB animals. The osmolarity of the outer medulla of SB-FA animals (group III) is still high, with sodium content even higher than that found in SB animals (group I) (249 ± 13 compared with 209 ± 25 μosM/mg tissue, P not significant). At the same proportion, the urea content in this group of animals is decreased compared with fed animals (180 ± 14 and 309 ± 30 μosM/mg, respectively, P < 0.005). Thus, in the face of reduced tissue osmolarity, there is a change in the solute composition featuring decreased urea and increased NaCl content in SB-FA animals (group III).
It appears, therefore, that in SB-FA animals the decrement in urea is partly replaced with an increment in salt content, which helps preserve the tissue osmolarity.
There is marked reduction in the osmotic gradient of the inner medulla of ALD-FA Psammomys compared with all other groups of animals. In this group, the osmolarity and sodium and urea contents were decreased compared with the other three groups (Fig. 1, Table 6).
The marked differences of the inner medulla osmolarity among the different groups are in sodium and urea content. The osmolarity in the inner medulla of the SB-FA Psammomys (group III) accrued from the high sodium content compared with in ALD-FA animals (whereas the urea content is similar).
Na-K-ATPase activity in the cortex, outer medulla, and inner medulla of the four groups is depicted in Fig. 2. There was no difference in enzyme activity between both fed animalgroups I and II.
The most striking change is observed in the outer medulla of SB-FA animals (group III), in which a fivefold increase in enzyme activity compared with SB-fed animals was seen. The enzyme activity in this group of animals was also increased in the cortex and in the inner medulla (Fig. 2).
No changes in enzyme activity were observed in animals on ALD-FA (group IV).
Na-K-ATPase gene expression.
In both fasted groups (groups III and IV), there was an increase in α-subunit mRNA expression in cortex and in outer medulla, although it did not reach statistical significance ingroup IV (Fig. 3). In the inner medulla, there were no changes in gene expression in ALD-FA animals (Fig. 3). β-Subunit mRNA expression (Fig. 4) increased in the cortex and in the outer medulla (P < 0.005) of SB-FA animals (group III) but not in ALD-FA animals (group IV).
Figure 5 shows Northern blot analysis for α- and β-subunits of mRNA with regard to Na-K-ATPase and β-actin mRNA expression in the outer medulla of the saltbush-fed and fastedPsammomys in groups I and III.
The present study examined the renal function, aldosterone levels, kidney solute content, Na-K-ATPase activity, and gene expression of its α- and β-subunits in Psammomys. The studies were conducted both in animals nourished with native and with laboratory diets, both in fed and fasting animals. As expected, urinary osmolarity correlated directly with the magnitude of kidney tissue osmotic gradient. It is noteworthy that the osmotic concentration gradient was determined by the type of diet. Thus animals that were fed native saltbush leaves had a higher osmotic tissue concentration gradient than those that were fed with a laboratory diet.
A saltbush diet (A. halimus) is high in water and nitrogen (Table 1) (15, 26) but low in energy content, which makesPsammomys consume large quantitites for maintenance of energy balance. Thus this plant creates a paradox forPsammomys. On one hand, Psammomys receives much preformed water from the saltbush, but, on the other hand, the high nitrogen content of the plant requires much water for excretion.
Animals fed on a saltbush diet consumed significantly more protein than animals fed a laboratory diet. The animals fed a saltbush diet consumed 2.39 ± 0.85 g/day of protein compared with laboratory-diet-fed animals that consumed 1.18 ± 0.17 g/day of protein. Thus the nitrogen content in the saltbush diet is 40% higher than in the laboratory diet (Table 2).
The osmolarity of the outer medulla of saltbush-fed animals is high compared with animals fed a laboratory diet. This was accounted for by a 40% increase in NaCl and 20% rise in urea content (Fig. 1, Table6).
Although animals fed on native saltbush exhibited a medullary osmotic concentration that was 1.5 times higher than that in laboratory-diet-fed animals, surprisingly no significant difference was found between the two groups with regard to Na-K-ATPase activity.
The differences in medullary osmolarity without parallel changes in Na-K-ATPase activities lend support to the thesis that the mechanism which produces these disparities in osmotic gradients ofPsammomys kidney medulla is primarily a Na-K-ATPase-independent sodium absorption mechanism.
Thus it may be proposed that in the animals fed saltbush the high dietary protein content with higher urea concentration in the medulla may account for a Na-K-ATPase-independent sodium absorption mechanism that can reflect a passive mechanism of urinary concentration inPsammomys (17, 25).
A model of a passive urinary concentrating mechanism, which assigns a major role for urea, was published by Stephenson (25) and Kokko and Rector (17). Urea in the medullary interstitium extracts salt-free water from the descending limb, causing the concentration of NaCl in the descending limb to rise above that in the surrounding interstitium, setting the stage for the passive outward movement of NaCl from the thin ascending limb and removing the necessity for an energy-requiring transepithelial pump in the thin ascending limb.
In agreement with above observations respective to Na-K-ATPase activity no differences were observed between the two subgroups (Iand II) with regard to gene expression of α- and β-subunits of sodium pump (Figs. 3 and 4).
It is of interest that Ohtaka et al. (21) found that urea does not increase Na-K-ATPase α1- and β1-subunit mRNA accumulation in primary cultures of inner medullary collecting duct cells of the rat. On the other hand, they observed upregulation of Na-K-ATPase α1- and β1- subunit mRNA by hyperosmolarity induced by poorly permeating solutes other than urea. This indicated that an increase in osmolarity per se does not induce Na-K-ATPase. These findings may be pertinent to our results that failed to show changes in enzyme activity despite changes in tissue osmolarity.
To further assess the role of dietary nitrogen in urinary concentrating mechanism, we used a different experimental manipulation. The animals were not fed for 24 h, thus depriving them of their source of urea. No significant changes in tissue osmolarity of kidney cortex were noticed between fed and fasted animals consuming the same diet. The composition of solutes in SB-FA animals (group III), however, was changed; urea was replaced with equivalent osmoles of sodium (Fig. 1, Table 6). These alterations in solute composition were associated with a commensurate increase in Na-K-ATPase activity (Fig.2). The osmolarity in the outer medulla of SB-FA animals (group III) was higher than that in ALD-FA animals (group IV). This difference between the two fasted groups was primarily due to increased salt content in the former group. Furthermore, the salt content in the SB-FA animals was even higher than that in SB animals. The Na-K-ATPase activity changed accordingly (Fig. 2). Moreover, the gene expression of α- and β-subunits in the cortex and in outer medulla increased significantly in SB-FA animals (Figs. 3-5).
There was no change in Na-K-ATPase activity in ALD-FA animals (group IV), and the increase in α-subunit failed to reach statistical significance (Fig. 3). We cannot rule out, however, the possibility that longer fasting of these animals could eventually increase the enzyme activity and its gene expression.
As opposed to outer medulla, the inner medulla tissue osmolarity decreased in the fasting state both in saltbush- and laboratory diet-consuming animals. This fall in osmolarity reflected decreases in both sodium and urea content. This phenomenon can be explained by the anatomic differences between the outer and inner medullary portions of Henle's loop (2, 4, 13). In the outer medullary segments, the abundance of sodium pumps plays an important role in driving sodium transport, whereas in the inner medullary portions that mainly consist of thin limbs, sodium absorption is mediated mainly by Na-K-ATPase-independent mechanisms. This is reflected in very low enzyme activity measured in the inner medulla and the presumed dependence of sodium transport on a passive mechanism related to urea (17, 25). Therefore, presumably the shortage of dietary nitrogen in the fasting state reduced the urea content (Table 6, Fig.1) necessary for Na-K-ATPase independent sodium reabsorption, resulting in a decline in tissue osmolarity.
Taken together, these results suggest that intergroup differences in dietary protein may account for the increased urea content of the medulla and the higher tissue and higher urine osmolarity in SB animals (group I) compared with ALD animals (group II). Thus urea could play a key role in the corticomedullary gradient inPsammomys. The absence of differences in Na-K-ATPase activity and its gene expression between groups I andII despite marked differences in renal tissue sodium and urea concentrations is consistent with predominance of Na-K-ATPase-independent sodium absorption mechanism inPsammomys kidney. By contrast, in SB-FA animals (group III), the observed increases in Na-K-ATPase activity and in its gene expression suggest transition to a Na-K-ATPase-dependent sodium transport mechanism. This change may help renal concentrating ability in the face of reduced urea supply due to food and protein deprivation.
Address for reprint requests and other correspondence: P. Scherzer, Nephrology and Hypertension Services, PO Box 12000, Jerusalem, Israel 91120 (E-mail:.).
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- Copyright © 2000 the American Physiological Society