Cl- transport in the loop of Henle is responsible for reclamation of 25-40% of the filtered NaCl load and for the formation of dilute urine. Our understanding of the physiologic and molecular mechanisms responsible for Cl- reabsorption in both the thin ascending limb and thick ascending limb of Henle's loop has increased greatly over the last decade. Plasma membrane Cl- channels are known to play an integral role in transcellular Cl- transport in both the thin and thick ascending limbs. This review focuses on the functional characteristics and molecular identities of these Cl- channels, as well as the role of these channels in the pathophysiology of disease.

Renal failure is a common and serious complication of longstanding diabetes mellitus. Diabetes is now the most common cause of end-stage renal failure requiring dialysis in the United States, accounting for almost 40% of all new dialysis patients (1). Moreover, the incidence of renal failure caused by diabetes, particularly type II diabetes, is rising dramatically worldwide (2). Compounding the tragedy of the explosive growth in the incidence renal failure caused by diabetes is the grim reality that the survival of patients with renal failure caused by diabetes is much worse than that of patients with renal failure resulting from other causes. In Germany, for example, Koch et al. (3) reported a 5-year survival of only 5% among patients with type II diabetes undergoing dialysis. Fortunately, progress is being made in our understanding of the pathogenesis of diabetic renal disease and in our ability to delay, or even prevent, this devastating complication. Two studies appearing in this issue of PNAS (4, 5) are illustrative of this progress. Diabetic nephropathy refers to a characteristic set of structural and functional kidney abnormalities that occur in patients with diabetes. Although best described in patients with type I diabetes (6), similar findings are now known to occur in the more common type II diabetic patient (7). Structural abnormalities include hypertrophy of the kidney, an increase in the thickness of glomerular basement membranes, accumulation of extracellular matrix components in the glomerulus (nodular and diffuse glomerulosclerosis), tubular atrophy, and interstitial fibrosis (6, 7). Functional alterations include an early increase in the glomerular filtration rate with intraglomerular hypertension, subsequent proteinuria, systemic hypertension, and eventual loss of renal function (8).

BACKGROUNDAntidiuretic hormone (ADH) or adenosine 3', 5'-cyclic phosphate (cAMP) analogues augment net NaCl absorption in microperfused mouse medullary thick ascending limb (MTAL) segments but not in cortical thick ascending limb (CTAL) segments. This ADH-dependent MTAL effect is due to increased apical Na+/K+/2Cl- admittance and apical K+ recycling accompanied by a rise in calculated intracellular Cl- concentrations and by a threefold rise in basolateral Cl- conductance. rbClC-Ka, a 75.2 member of the ClC family of Cl- channels, mediates net Cl- absorption in the MTAL. The gating characteristics of rbClC-Ka channels from their intracellular surfaces are, to our knowledge, unique among Cl- channels. The channels are activated by small increases in intracellular Cl- (K1/2 = 10 mM Cl-). Adenosine triphosphate plus the catalytic subunit of protein kinase A (ATP + PKA) gate rbClC-Ka when cytosolic Cl- concentrations are 25 mM. Thus, in mouse MTAL segments, ADH-dependent rises in cytosolic Cl- are primarily responsible for basolateral Cl- conductance increases.METHODSThese experiments compared the properties of Cl- channels fused into bilayers from basolaterally enriched vesicles from cultured mouse CTAL cells with rbClC-Ka channels.RESULTSThe key findings were that anti-rbClC-Ka, antibody that recognizes and blocks rbClC-Ka, recognized and blocked basolateral Cl- channels in CTAL cells, that the extracellular faces of the CTAL channels were, like rbClC-Ka, substrate gated with a K1/2 of approximately 170 mM Cl-, and that, unlike rbClC-Ka channels, cytosolic faces of basolateral CTAL Cl- channels were not gated by either increasing cytosolic Cl- concentrations or cytosolic (ATP + PKA). This failure of activation of basolateral CTAL Cl- channels was confirmed using excised patch clamp studies. Finally, on Western blots, anti-rbClC-Ka recognized a 74 kDa band on basolateral CTAL vesicles.CONCLUSIONSBasolateral CTAL Cl- channels probably share a high degree of structural homology and possibly molecular mass with rbClC-Ka channels. However, significant differences between rbClC-Ka channels and CTAL Cl- channels account for the inability of increasing either cytosolic Cl- or (PKA + ATP) to raise Po in CTAL basolateral Cl- channels.

The purpose of this brief commentary is to provide a description of how renal physiology, and more particularly, renal tubular physiology, has evolved over the past thirty years, since the occasion, obviously, is a celebration of the Thirtieth Course on Advances in Nephrology and Dialysis. My arguments will begin by quoting from Homer Smith's book, The Kidney, and merging Smith's observations, which were integrative in the sense that they did not specify detail, with detailed incursions into tubular physiology, first at a cellular level and then at a molecular level. For convenience, the nephron is divided into four functional segments: the proximal nephron; the ascending limb; the distal nephron; and the collecting duct. Each of these carries out a specific function. The proximal nephron absorbs about two-thirds of filtered sodium, without dissociating salt and water absorption. The thick ascending limb absorbs 25% of filtered Na+, but no water. The distal nephron absorbs 10% of filtered Na+ in close relation with K+ and, to some extent, H+ secretion. Finally, the collecting duct includes three kinds of cells: the cortical collecting duct, which is responsible not only for Na+ absorption and K+ secretion, but also for the bulk of the absorption of free water; the outer medullary collecting duct (OMCD), which is largely responsible for the final steep drop in urine pH which occurs between cortex and papilla; and the inner medullary collecting duct (IMCD), whose major function is the final absorption of approximately 5% of filtered Na+.

Background. This article reports studies on the kinetics of chloride (Cl-) conductance in Cl- channels fused into bilayers from basolaterally enriched vesicles from rabbit outer medulla. A considerable body of evidence indicates that these channels represent rbClC-Ka, a 77 kDa kidney-specific protein of the CIC family of Cl- channels. rbClC-Ka, a candidate channel for mediating net Cl- absorption in the medullary thick ascending limb (MTAL), has been cloned from rabbit outer medulla and localized by immunofluorescence to basolateral membranes of the MTAL. Thus, this is the first account, to our knowledge, of the kinetics of ion permeation through a renal Cl- channel mediating net basolateral Cl- absorption in the thick ascending limb of Henle (TALH), and this channel may represent rbClC-Ka.Methods. The electrophysiological properties of these channels were studied by fusing basolaterally enriched MTAL vesicles into planar bilayer membranes.Results. Cl- conductance through these channels was concentration dependent and saturable. The relationship between ga (pS) and symmetrical aqueous Cl- concentrations could be expressed in terms of the Michaelis equation with a limiting conductance (G(Cl)(max), pS) of 114 pS at infinitely high aqueous Cl- concentrations and a Kin of 163 mM Cl-. A log-log plot of the conductance-Cl- concentration relations, in the nonsaturating Cl- concentration range, had a slope of 0.91, that is, virtually unity. The relatively impermeant anion I- produced a voltage-dependent conductance blockade that could be overcome at high electric field strengths.Conclusions. The experimental data described earlier here fulfill the traditional criteria for a first-order process with a single Cl- ion occupying these channels at a given time. Although the channels may contain multiple ion binding sites, the latter function, in integral kinetic terms, as a single rate-limiting locus.

Chloride transport by the medullary thick ascending limb is a major determinant of urinary concentrating and diluting power and also of tubuloglomerular feedback. In this review, we discuss the electrophysiologic, regulatory, and molecular features of a Cl- channel present in basolateral membranes from the medullary thick ascending limb. Functional and immunohistochemical data support the view that rbCIC-Ka, a CIC Cl- channel cloned from rabbit kidney medulla, may be the Cl- channel which mediates basolateral Cl- efflux.

What I have tried to describe in these brief introductory statements are the key elements stimulating renal sodium retention mediated by arterial underfilling, as illustrated by the volume repletion reaction (Fig. 3). They may be summarized by saying that edematous states characterized by underfilling-most notably systolic pump failure-represent, in fact, suicidal arterial filling. This point of view is illustrated in Figure 8. Essentially, as shown in Figure 8, the original reduction in filling of the arterial tree, by provoking both hemodynamic changes and diminished sodium avidity, has the net effect of increasing end diastolic volume and, for a time, cardiac output, but at two great expenses: an increased afterload and a an increased preload. In the end, these latter two effects produce a reduced ejection fraction which obviously propagates the syndrome of systolic failure. Thus, suicidal arterial filling represents a vicious cycle in which homeostatic mechanisms set into play an attempt to compensate for inadequate arterial filling, which inevitably leads to increasing degrees of cardiac dysfunction and, hence, to deterioration of the patient. I have enjoyed presenting these introductory remarks. I am certain that subsequent talks in this Conference will consider more explicitly the provocative issue of the "overfilling" hypothesis as a mechanism for sodium retention in edematous states such as the nephrotic syndrome and cirrhosis.

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 (P = 0.003). Cl- efflux was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (154 +/- 26 vs. 70 +/- 21 nmol.mg protein-1.45 s-1, P = 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in lipid bilayers, reduced Cl- efflux from intact MTAL segments (154 +/- 28 vs. 53 +/- 14 nmol.mg protein-1.45 s-1, P = 0.02). 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.

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.

The experiments reported herein compared Cl- channels fused into bilayers from rabbit outer medullary vesicles with Cl- channels in excised patches of basolateral membranes from cultured mouse medullary thick ascending limb (MTAL) cells and evaluated whether the latter were plausible candidates for the Cl- channels mediating net NaCl absorption in microperfused mouse MTAL segments. The unique signature characteristics of Cl- channels incorporated into lipid bilayers from outer medullary vesicles include activation of open probability (Po) by increases in the Cl- concentrations bathing intracellular faces; activation of Po by protein kinase A (PKA) + ATP, when the Cl- concentrations bathing intracellular faces are low; and no effect of PKA + ATP on Po with high cytoplasmic-face Cl- concentrations. These same properties were observed in Cl- channels studied using excised patches of basolateral membranes from mouse MTAL cells. Moreover, in both bilayers and in excised patches, the sharpest fractional increase in Cl- channel Po occurred with cytosolic-face Cl- concentration increases to values similar to the antidiuretic hormone (ADH)-dependent values of intracellular Cl- activity in microperfused mouse MTAL segments, and these fractional Po increases were adequate to account quantitatively for the ADH-dependent increase in basolateral membrane Cl- conductance in microperfused mouse MTAL segments. Thus the excised-patch basolateral Cl- channels reported here are reasonable candidates for those mediating net Cl- absorption in the MTAL.

These experiments were intended to identify candidate cDNAs which might encode basolateral membrane Cl- channels of the mTAL using a homology-based cloning strategy. We prepared a cDNA library using a 1.8 to 3.2 kb mRNA fraction from rabbit outer medulla that induces a Cl- conductance in cellular membranes of Xenopus laevis oocytes. The cDNA library was screened with two 32P-oligonucleotide probes corresponding to highly conserved sequences in other Cl- channels. We isolated two cDNAs: rbClC-Ka and rbClC-Kb. The protein sequences deduced from these two cDNAs had 99% homology. Using RT-PCR technology, cultured mouse mTAL cells were found to contain mRNA corresponding to those two cDNAs. Expression of the mRNAs corresponding to these two cDNAs was kidney-specific and was greater in rabbit renal medulla than rabbit renal cortex. Finally, by using RT-PCR technology in combination with microdissected glomeruli or tubule segments, we found mRNA for rbClC-Ka in glomeruli, proximal convoluted tubules, mTAL and cortical collecting tubules.