The distribution of vacuolar H'ATPase in rat kidney was examined by immunocytochemistry using affinity-purified antibodies against the 31-, 56-, and 70-kD subunits of the bovine kidney proton pump. Proximal convoluted tubules were labeled over apical plasma membrane invaginations, and in the initial part of the thin descending limb, apical and basolateral plasma membranes were moderately stained. Thick ascending limbs and distal convoluted tubules were apically stained although the intensity was greater in the distal convoluted tubule. Collecting duct principal cells were virtually unlabeled, but intercalated cells had intense staining with an apical, basolateral or diffuse pattern in the cortex, and exclusively apical staining in the medulla. These results (a) show the presence of an H'ATPase in the apical plasma membrane of the proximal tubule that may contribute to H+ transport in this segment; (b) provide direct evidence that the intercalated cell contains most of the H'ATPase detectable in the collecting duct, supporting its proposed role in H+ transport; (c) demonstrate that subpopulations of cortical intercalated cells have opposite polarities of an H'ATPase, consistent with the presence of both proton-and bicarbonate-secreting cells; and (d) suggest a role for the H+ATPase in acid/base regulation or H+ transport in segments other than the collecting duct and the proximal tubule.

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.

Proteinuria is a common laboratory finding in outpatients and should not be discounted. When it is due to a glomerular disease, early diagnosis is important to prevent further renal damage. Proteinuria may also be a marker for progressive atherosclerosis.

We have isolated a cDNA encoding the 31-kDa subunit of the bovine kidney vacuolar H+-ATPase. The composite sequence contains 1219 base pairs, which includes the entire 678-base-pair coding region. A lysine-rich sequence previously found in the Na',K+-ATPase a subunit and the H+,K+-ATPase was identified in the 31-kDa subunit. An Proton-translocating ATPases acidify a variety of intracellular compartments in the vacuolar system (1). In the specialized acid-secreting kidney intercalated cell, the vacuolar H+-ATPase (proton pump) is also found in high concentrations on the plasma membrane, where its polarized distribution effects transepithelial proton secretion (2). Recently, this laboratory reported the isolation and functional reconstitution of the vacuolar H+-ATPase from bovine kidney microsomes (3, 4). The intact purified enzyme has a molecular mass of =580 kDa and has over 10 component subunits [i.e., one each of 70, 45, 42, 38, 33, 31, 15, 14, and 12 kDa and several of -56 kDa (3, 4)].Vacuolar proton pumps partially purified from Neurospora vacuoles (5), plant tonoplast (6, 7), chromaffin granules (8), and coated vesicles (9) also are large molecular mass enzymes with subunits of t70, =56, -17 and, generally, -30 kDa (5,6,8,9). Several features suggest that the complex structure of these enzymes and of the kidney H +-ATPase is similar to that of FoF1 H + -ATPases, such as the mitochondrial H + -ATPase. Rapid-freeze and thinsection electron micrographs of the vacuolar H+-ATPase show a 9.5-nm cytoplasmic domain ("stud") bound to the membrane (10), which is morphologically similar to FoF, ATPase structures. Antibodies specific for the 70-, 56-, or 31-kDa kidney H+-ATPase subunits all localize to this cytoplasmic domain (37). The %70-(5) and -56-kDa (7) subunits have been implicated in the enzyme's catalytic activity, whereas the dicyclohexylcarbodiimide-binding '17-kDa subunit (5, 7) may form part of an integral membrane channel.The cytosolic domains of several membrane proteins are necessary for sorting and control of removal by endocytosis (11,12). The H+-ATPase's cytoplasmic domain might, in addition to having a role in catalytic activity, function in enzyme regulation or sorting to different membrane com-

Background: Blood pressure (BP) reduction in patients with chronic kidney disease (CKD), particularly with a renin-angiotensin system inhibitor (RASI), commonly leads to an initial decrease in glomerular filtration rate. The current clinical guideline, based on studies with single RASIs, is to tolerate an increase in the serum creatinine only up to 30%. This guideline has aptly guided CKD care for over a decade, but should be updated in the contemporary context of more aggressive RASIand diuretic use. Methods: This study is a retrospective review of 48 mostly African-American patients with CKD treated with multiple and/or high-dose renin-angiotensin system (RAS) inhibition and diuretics, targeting both low BP and reduction of urine protein. RASI was not reduced in response to initial increases in serum creatinine greater than 30%. Results: A clinically well-tolerated increase in serum creatinine over 30% during the first year occurred in 41% of the patients. Treatment was unaltered, and target goals for BP and urine protein were typically achieved. After the point of maximal serum creatinine in the first year, these patients had minimal progression of disease over the next 6 years, with a long-term estimated glomerular filtration rate slope of only –0.52 ml/min/year/1.73 m². Only 25% progressed to end-stage renal disease or death. Conclusion: The 30% limitation to initial increases in the serum creatinine still pertains for single RASI at usual doses. However, favorable long-term outcomes suggest that initial increases over 30% should be tolerated in the context of dual goal-directed, more aggressive RASI and diuretic use.

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Lowering both blood pressure and urinary albumin excretion to specific goals may slow the progression of proteinuric chronic kidney disease. However, this dualgoal approach needs to be validated prospectively.

Optimal treatment of chronic kidney disease requires flexible approaches towards achieving the goals for systolic blood pressure and reduction of urinary protein loss.