Renal ammonia excretion in response to hypokalemia: effect of collecting duct-specific Rh C glycoprotein deletion

Abstract

Lee HW, Verlander JW, Bishop JM, Handlogten ME, Han KH, Weiner ID. Renal ammonia excretion in response to hypokalemia: effect of collecting duct-specific Rh C glycoprotein deletion. Am J Physiol Renal Physiol 304: F410 -F421, 2013. First published December 5, 2012; doi:10.1152/ajprenal.00300.2012.-The Rhesus factor protein, Rh C glycoprotein (Rhcg), is an ammonia transporter whose expression in the collecting duct is necessary for normal ammonia excretion both in basal conditions and in response to metabolic acidosis. Hypokalemia is a common clinical condition associated with increased renal ammonia excretion. In contrast to basal conditions and metabolic acidosis, increased ammonia excretion during hypokalemia can lead to an acid-base disorder, metabolic alkalosis, rather than maintenance of acid-base homeostasis. The purpose of the current studies was to determine Rhcg's role in hypokalemia-stimulated renal ammonia excretion through the use of mice with collecting duct-specific Rhcg deletion (CD-Rhcg-KO). In mice with intact Rhcg expression, a K ϩ -free diet increased urinary ammonia excretion and urine alkalinization and concurrently increased Rhcg expression in the collecting duct in the outer medulla. Immunohistochemistry and immunogold electron microscopy showed hypokalemia increased both apical and basolateral Rhcg expression. In CD-Rhcg-KO, a K ϩ -free diet increased urinary ammonia excretion and caused urine alkalinization, and the magnitude of these changes did not differ from mice with intact Rhcg expression. In mice on a K ϩ -free diet, CD-Rhcg-KO increased phosphate-dependent glutaminase (PDG) expression in the outer medulla. We conclude that hypokalemia increases collecting duct Rhcg expression, that this likely contributes to the hypokalemia-stimulated increase in urinary ammonia excretion, and that adaptive increases in PDG expression can compensate for the absence of collecting duct Rhcg.acid-base; ammonia; collecting duct; hypokalemia; potassium RENAL AMMONIA METABOLISM is a primary method through which the kidneys maintain acid-base homeostasis, and involves integrated contributions of both intrarenal ammoniagenesis and renal epithelial cell ammonia transport.1 Increasingly, the "traditional" theory of passive NH 3 diffusion and NH 4 ϩ trapping is being replaced by one in which specific membrane proteins transport NH 3 or NH 4 ϩ , enabling facilitated and regulated membrane NH 3 and NH 4 ϩ transport (32, 33). Prominent among these ammonia-transporting proteins are the Rh glycoproteins. Three mammalian Rh glycoproteins have been identified, Rh A glycoprotein, which is expressed specifically in erythroid cells, and the nonerythroid Rh glycoproteins, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), which are expressed widely in ammonia-transporting tissues, including the kidney (29,30,32,33).In the kidney, Rhcg is expressed in the renal distal nephron and collecting duct, from the distal convoluted tubule (DCT) through the inner medullary collecting duct (8,10,20,28), where 60 -80% of final...

Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

“…Gels were then transferred electrophoretically to nitrocellulose membranes. We confirmed that protein loading and transfer efficiency did not differ significantly between samples using Ponceau S staining, as we have previously described (1,2,16,33,35,36). Membranes were then blocked with 5 g/dl nonfat dry milk in Blotto buffer (50 mM Tris, 150 mM NaCl, 5 mM Na 2EDTA, and 0.05% Tween 20; pH 7.6) and incubated at 4°C overnight with primary antibody diluted in nonfat dry milk.…”

Expression of the ammonia transporter family member, Rh B Glycoprotein, in the human kidney

“…We generated these mice using Cre-loxP techniques, using mice with floxed Rhbg genes we characterized previously (2, 3) and using mice expressing Cre-recombinase under control of the Ksp-cadherin promoter (14,29). In previous studies examining Rhcg, we showed that the Ksp-cadherin promoter yields collecting duct-specific gene deletion (19,21). Examination of mice homozygous for floxed Rhbg alleles and expressing Ksp-cadherin-Cre using mouse Rhbg-specific antibodies confirmed collecting duct-specific Rhbg deletion.…”

“…Mice with floxed Rhbg gene generated in our laboratory and described previously (2, 3) and expressing Cre-recombinase under control of the Ksp-cadherin promoter (19,21,29) were used to generate mice with collecting duct-specific Rhbg knockout (CD-Rhbg-KO). Littermates that did not express Cre-recombinase were used as control mice.…”

Ammonia transport in the kidney by Rhesus glycoproteins

“…Hypokalemia increases Rhcg expression, consistent with Rhcg having an important role in the increased ammonia excretion. However, the pattern of changes differs from those seen with metabolic acidosis (8,34,58). Rhcg expression increases in OMCD intercalated cells, where there is more discrete apical Rhcg expression in intercalated cells and a marked increase in apical plasma membrane immunolabel compared with control animals (34).…”