Thomas E. Andreoli

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: I. ADH increases transcellular conductance pathways.

SummaryThis paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (Gcmin) of the transcellular conductance, estimated from Ba++ blockade of apical membrane K+ channels, indicated thatGcmin was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K+ was the major conductive species; and ADH increased the magnitude of a Ba++-sensitive K+ conductance under conditions where net Cl− absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl− conductance; this ADH-dependent increase in basal Cl− conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl− absorption. Cl− removal from luminal solutions had no detectable effect onGe, and net Cl− absorption was reduced at luminal K+ concentrations less than 5mm; thus apical Cl− entry may have been a Na+,K+,2Cl− cotransport process having a negligible conductance. The net rate of K+ secretion was approximately 10% of the net rate of Cl− absorption, while the chemical rate of net Cl− absorption was virtually equal to the equivalent short-circuit current. Thus net Cl− absorption was rheogenic; and approximately half of net Na+ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K+ was the principal conductive species in apical plasma membranes, support the view that the majority of K+ efflux from cell to lumen through the Ba++-sensitive apical K+ conductance pathway was recycled into cells by Na+,K+,2Cl− cotransport.

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl− absorption

SummaryCellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,Ve, which attends net Cl− absorption,JClnet, and to assess how ADH might augment bothJClmet andVe in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba++ for 5mm K+ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,Rc/Rbl, and increasing luminal K+ from 5 to 50mm in the presence of luminal 10−4m furosemide resulted in a 53-mV depolarization of apical membrane voltage,Va. Thus K+ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10−4m luminal furosemide reducedVe andJClnet to near zero values and hyperpolarized bothVa andVbl, the voltage across basolateral membranes; however, the depolarization ofVbl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofVa, Thus ADH-dependent increases inVb were referable to greater depolarizations ofVbl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofVa was referable to a decrease in the K+ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofVbl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl− current. Thus an increase in intracellular Cl− activity may have contributed to the depolarization ofVbl during net Cl− absorption, and the intracellular Cl− activity was likely greater with ADH than without hormone. Since ADH increases apical K+ conductance and since the chemical driving force for electroneutral Na+,K+,2Cl− cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba++-sensitive conductance K+ channels and electroneutral Na+,K+,2Cl− cotransport units in apical plasma membranes.

The effects of antidiuretic hormone (ADH) on solute and water transport in the mammalian nephron

The biochemical and physiological events responsible for osmotic homeostasis range from vasopressin (antidiuretic hormone, ADH) synthesis and release from the central nervous system to the action of ADH on specific renal tubular cells, the latter being ultimately responsible for water conservation by the kidney. Although a general review of this subject would appear timely, the explosion of knowledge in this field over the last decade has been great; thus covering all recent events would permit only a superficial analysis. Consequently, this review has been restricted to an examination of the effects of antidiuretic hormone on solute and volume flows in the mammalian nephron. Although such a choice might appear myopic, we believe it to be justifiable for several reasons. First, an understanding of the physiological effects of ADH on renal tubular epithelia forms a basis for analyzing the pathophysiology of clinical disorders of renal water transport. Second, recent experimental observations on the temperature-dependence of tracer water diffusion and net volume flow in cortical collecting tubules, both in the presence and absence of ADH, provide a way of assigning specific characteristics to the water permeation sites in apical membranes of these tubules, and to the way ADH affects these regions. In addition, this type of analysis demonstrates the importance of explicit knowledge of the major barriers which impede transport across epithelia when interpreting apparent breaks in Arrhenius activation energy relations, either for water transport or for solute transport. Third, the discovery of an ADH-induced NaC1 absorptive mechanism (Hall, 1979; Hall & Varney, 1979; Hebert etal., 1980b) in the mouse medullary thick ascending limb of Henles loop provides a way of investigating the

Cl- channels in basolateral renal medullary membranes. XII. Anti-rbClC-Ka antibody blocks MTAL Cl- channels.

Cl- channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl-concentrations. rbClC-Ka, a ClC Cl- channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl- transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl- uptake and efflux were measured in suspensions of mouse MTAL segments. Cl- uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3,5-cyclic monophosphate (DBcAMP). Cl- efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 ± 20 to 196 ± 36 nmol ⋅ mg protein-1 ⋅ 45 s-1( Pu2009=u20090.003). Cl- efflux was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (154u2009±u200926 vs. 70u2009±u200921 nmol ⋅ mg protein-1 ⋅ 45 s-1, Pu2009=u20090.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in lipid bilayers, reduced Cl- efflux from intact MTAL segments (154u2009±u200928 vs. 53u2009±u200914 nmol ⋅ mg protein-1 ⋅ 45 s-1, Pu2009=u20090.02). These results support the view that rbClC-Ka is the basolateral membrane Cl- channel that mediates vasopressin-stimulated net Cl- transport in the MTAL segment.

PRESIDENT'S COMMENTS

The Federal Motor Carrier Safety Administration’s proposed new hours-of-service rule would cut driving hours, a move that trucking industry leaders said was politically motivated, adding cost and complexity while jeopardizing safety advances made in recent years. The proposed rule, released Dec. 23, was the results of a settlement FMCSA reached with advocacy groups that have twice sued successfully in federal court to block the safety agency’s revision of DepressionEra driving limits. FMCSA’s proposal would restrict drivers’ ability to restart their weekly work cycle with a 34hour rest period, effectively cutting the total work cycle. It also leans toward cutting allowable driving time before a break to 10 hours from the current 11. “When viewed against trucking’s sterling safety record, it’s plan that the Obama administration’s willingness to break something that’s not broken likely has everything to do with politics and little or nothing to do with highway safety or driver health,” said Bill Graves, president of American Trucking Associations. The proposed rule, Graves said, “begs the age-old question: What part of success didn’t you like?” “The current rule has been working quite well by every measure, every assessment,” Graves said, referring to federal data showing that truck-involved traffic fatalities have fallen steadily since 2003 when the first HOS rule was adopted. FMCSA, whose new proposal follows a legal settlement last year between the federal government and a coalition of labor and advocacy groups, will take public comments on the proposal for the next 60 days, with the final rule expected by July 26. While FMCSA stopped short of proposing a 10-hour driving limit to replace the current Trucking Blasts HOS Plan Drive Time May be Cut; Restart Rules Altered