Body mass-specific Na+-K+-ATPase activity in the medullary thick ascending limb: implications for species-dependent urine concentrating mechanisms

Abstract

In general, the mammalian whole body mass-specific metabolic rate correlates positively with maximal urine concentration (U_max) irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass-specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na⁺-K⁺-ATPase activity and Na⁺-K⁺-ATPase α1-gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na⁺-K⁺-ATPase activity (~70%) compared with two rat strains (Sprague-Dawley and Munich-Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na⁺-K⁺-ATPase α1-protein (~4- to 6-fold) and mRNA (~13-fold) and higher TAL mitochondrial volume density (~20%) occur in the kangaroo rat compared with both rat strains. Rat TAL Na⁺-K⁺-ATPase α1-protein expression is relatively unaffected by body hydration status or, shown previously, by dietary Na⁺, arguing against confounding effects from two unavoidably dissimilar diets: grain-based diet without water (kangaroo rat) or grain-based diet with water (rat). We conclude that higher TAL Na⁺-K⁺-ATPase activity contributes to relationships between whole body mass-specific metabolic rate and high U_max. More vigorous TAL Na⁺-K⁺-ATPase activity in kangaroo rat than rat may contribute to its steeper Na⁺ and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.

Keywords: comparative physiology; countercurrent multiplication; loop of Henle; renal outer medulla; sodium transport.

Fig. 1.

Electron micrographs of the thick ascending limb (TAL) from the inner stripe of the outer medulla (ISOM) of the kangaroo rat, Munich-Wistar rat and Sprague-Dawley rat. The mitochondria density in the kangaroo rat is significantly higher compared with the Munich-Wistar and Sprague-Dawley rats. See results for details. Scale bars = 10 μm.

Fig. 2.

Na⁺-K⁺-ATPase activity in homogenates of the ISOM of the kangaroo rat (n = 6), Munich-Wistar rat (n = 8) and Sprague-Dawley rat (n = 9). Individual values (○) and means ± SE (☐) are shown. The statistical significance between sample means was determined with a one-way ANOVA and Tukey’s post hoc test. Mean values that share the same letter are not significantly different from each other.

Fig. 3.

The linearity of the Na⁺-K⁺-ATPase α1-protein immunoblot detection system. The detection of Na⁺-K⁺-ATPase α1-protein by immunoblot was linear for a range of 5–40 µg total protein. A: immunoblot with 5–40 μg of protein from Munich-Wistar rat ISOM applied to each lane. B: quantification of Na⁺-K⁺-ATPase α1 normalized to band intensity measured for 5 µg total protein.

Fig. 4.

Na⁺-K⁺-ATPase α1-protein expression in the ISOM of the kangaroo rat (KR) and the Munich-Wistar rat (MW). A: immunoblots with 20 μg of protein applied to each lane; each lane represents a different animal. B: quantification of Na⁺-K⁺-ATPase α1 immunoblot normalized to β-actin; n = 6 for each species. Kangaroo rat data are expressed relative to the mean of the Munich-Wistar rat, which was set to 1. *Significant difference in protein expression between species by Student’s unpaired t-test.

Fig. 5.

Na⁺-K⁺-ATPase α1-protein expression in the ISOM of the kangaroo rat and the Sprague-Dawley rat, the latter with water ad libitum (control) or water restricted for 72 h (see methods). A: immunoblots with 10 μg of protein applied to each lane; each lane represents a different animal. Black lines denote where blot was cut for rearrangement of lanes. B: quantification of Na⁺-K⁺-ATPase α1-immunoblot normalized to β-actin; n = 5 (kangaroo rat) and n = 3 (Sprague-Dawley rats). Kangaroo rat and water-restricted Sprague-Dawley rat data are expressed relative to the mean of the control Sprague-Dawley rat, which was set to 1. Kangaroo rats were mixed gender. The statistical significance between sample means was determined with a one-way ANOVA and Tukey’s post hoc test. Mean values that share the same letter are not significantly different from each other.

Fig. 6.

Na⁺-K⁺-ATPase α1-mRNA expression in the ISOM of the kangaroo rat compared with the Munich-Wistar rat (A) and kangaroo rat compared with the Sprague-Dawley rat (B), as determined by real-time PCR. β-Actin expression was not significantly different between the two species in each panel. Arbitrary units; means shown in parentheses. *Significant difference in gene expression between species by Student’s unpaired t-test.

Fig. 7.

Na⁺, K⁺ and urea concentrations in the inner and outer medulla of the kangaroo rat and the Munich-Wistar rat. Na⁺, K⁺ and urea concentrations were determined in three medullary zones: the ISOM, outer 50% of the inner medulla (IM1), and inner 50% of the inner medulla (IM2). The statistical significance of differences between sample means was determined with a two-way, repeated-measures, mixed-model ANOVA, and post hoc Bonferroni adjustments were calculated for each tested effect (zone, species or zone*species). *Significant differences between species in each zone; bars sharing the same letter of the same case are not significantly different from one another.