Tubuloglomerular feedback: mechanistic insights from gene-manipulated mice
2008; Elsevier BV; Volume: 74; Issue: 4 Linguagem: Inglês
10.1038/ki.2008.145
ISSN1523-1755
AutoresJürgen Schnermann, Josephine P. Briggs,
Tópico(s)Ion channel regulation and function
ResumoTubuloglomerular feedback (TGF) describes a causal and direct relationship between tubular NaCl concentration at the end of the ascending limb of the loop of Henle and afferent arteriolar tone. The use of genetically altered mice has led to an expansion of our understanding of the mechanisms underlying the functional coupling of epithelial, mesangial, and vascular cells in TGF. Studies in mice with deletions of the A or B isoform of NKCC2 (Na,K,2Cl cotransporter) and of ROMK indicate that NaCl uptake is required for response initiation. A role for transcellular salt transport is suggested by the inhibitory effect of ouabain in mutant mice with an ouabain-sensitive α1 Na,K-ATPase. No effect on TGF was observed in NHE2- and H/K-ATPase-deficient mice. TGF responses are abolished in A1 adenosine receptor-deficient mice, and studies in mice with null mutations in NTPDase1 or ecto-5′-nucleotidase indicate that adenosine involved in TGF is mainly derived from dephosphorylation of released ATP. Angiotensin II is a required cofactor for the elicitation of TGF responses, as AT1 receptor or angiotensin-converting enzyme deficiencies reduce TGF responses, mostly by reducing adenosine effectiveness. Overall, the evidence from these studies in genetically altered mice indicates that transcellular NaCl transport induces the generation of adenosine that, in conjunction with angiotensin II, elicits afferent arteriolar constriction. Tubuloglomerular feedback (TGF) describes a causal and direct relationship between tubular NaCl concentration at the end of the ascending limb of the loop of Henle and afferent arteriolar tone. The use of genetically altered mice has led to an expansion of our understanding of the mechanisms underlying the functional coupling of epithelial, mesangial, and vascular cells in TGF. Studies in mice with deletions of the A or B isoform of NKCC2 (Na,K,2Cl cotransporter) and of ROMK indicate that NaCl uptake is required for response initiation. A role for transcellular salt transport is suggested by the inhibitory effect of ouabain in mutant mice with an ouabain-sensitive α1 Na,K-ATPase. No effect on TGF was observed in NHE2- and H/K-ATPase-deficient mice. TGF responses are abolished in A1 adenosine receptor-deficient mice, and studies in mice with null mutations in NTPDase1 or ecto-5′-nucleotidase indicate that adenosine involved in TGF is mainly derived from dephosphorylation of released ATP. Angiotensin II is a required cofactor for the elicitation of TGF responses, as AT1 receptor or angiotensin-converting enzyme deficiencies reduce TGF responses, mostly by reducing adenosine effectiveness. Overall, the evidence from these studies in genetically altered mice indicates that transcellular NaCl transport induces the generation of adenosine that, in conjunction with angiotensin II, elicits afferent arteriolar constriction. Changes in NaCl concentration in the tubular lumen near the tubulovascular contact point at the distal end of the ascending loop of Henle elicit adjustments in glomerular arteriolar resistance, a phenomenon referred to as ‘tubuloglomerular feedback’ (TGF).1.Schnermann J. Briggs J.P. Function of the juxtaglomerular apparatus: control of glomerular hemodynamics and renin secretion.in: Alpern R.J. Hebert S.C. The Kidney Physiology and Pathophysiology. 1. Elsevier Academic Press, Burlington–San Diego–London2008: 589-626Google Scholar As increases in NaCl concentration cause increases of afferent arteriolar resistance and a fall in glomerular filtration rate (GFR), the system is constructed as a negative feedback loop that serves to keep NaCl delivery into the distal parts of the nephron within narrow boundaries. The TGF response is complex, requiring coordinated functional changes in the epithelial, mesangial, and smooth muscle cells, and delineation of the cellular mechanisms responsible for linking the NaCl input with the vascular end points has been relatively slow. Micropuncture has proven to be the most valuable tool in establishing the relationship between luminal NaCl concentration and GFR or glomerular capillary pressure, but this approach has major limitations in resolving the intermediate steps in the transmission pathway. The use of imaging and electrophysiological techniques in isolated perfused tubule/glomerulus preparations has provided an approach for the detailed study of the changes in epithelial function that result from changes in luminal composition, but the relationship between specific changes in epithelial function and the glomerular arteriolar end point has been difficult to study with these preparations.2.Lapointe J.Y. Bell P.D. Cardinal J. Direct evidence for apical Na+:2Cl-:K+ cotransport in macula densa cells.Am J Physiol Renal Physiol. 1990; 258: F1466-F1469PubMed Google Scholar,3.Schlatter E. Salomonsson M. Persson A.E. et al.Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl-K+ cotransport.Pflugers Arch. 1989; 414: 286-290Crossref PubMed Scopus (127) Google Scholar The use of gene-manipulated mice has generated a new venue to further explore the mechanisms responsible for TGF. As micropuncture can be relatively easily adapted to this species, the effect of targeted deletions of gene products potentially involved in TGF can be studied without some of the uncertainties associated with pharmacological interventions. In this review, we are focusing on two areas where gene-manipulated mice have facilitated major progress in understanding the mechanisms. Substantial experimental evidence supports the notions that luminal NaCl concentration initiates TGF responses by changes in tubular NaCl transport, and that the signal arising from changes in NaCl transport is transmitted across the juxtaglomerular interstitium by the generation of paracrine messengers. The availability of animals with defined transport deficits and with targeted deficiencies in the generation or action of potential mediators has permitted new insights in both of these areas of TGF function. There is general agreement that the primary mechanism mediating the transduction of luminal NaCl concentration into a propagated signal across the juxtaglomerular interstitium is activation of the Na,K,2Cl cotransporter, NKCC2, in the apical membrane of macula densa (MD) cells. This basic tenet rests on the observation that a number of loop diuretics, including furosemide, bumetanide, piretanide, ethacrynic acid, triflocin, or L-ozolinone,1.Schnermann J. Briggs J.P. Function of the juxtaglomerular apparatus: control of glomerular hemodynamics and renin secretion.in: Alpern R.J. Hebert S.C. The Kidney Physiology and Pathophysiology. 1. Elsevier Academic Press, Burlington–San Diego–London2008: 589-626Google Scholar,4.Wright F.S. Schnermann J. Interference with feedback control of glomerular filtration rate by furosemide, triflocin, and cyanide.J Clin Invest. 1974; 53: 1695-1708Crossref PubMed Scopus (176) Google Scholar produce complete TGF inhibition, and on the good quantitative agreement between the inhibitor concentrations causing half-maximal inhibition of transport and TGF.5.Mason J. Takabatake T. Olbricht C. et al.The early phase of experimental acute renal failure. III. Tubuloglomerular feedback.Pflugers Arch. 1978; 373: 69-76Crossref PubMed Scopus (43) Google Scholar Furosemide also blocks TGF responses during retrograde perfusion, indicating that TGF inhibition does not depend upon a metabolic product of the thick ascending limb (TAL) transmitted to the MD cells.6.Schnermann J. Briggs J.P. Concentration-dependent sodium chloride transport as the signal in feedback control of glomerular filtration rate.Kidney Int. 1982; 22: S82-S89Google Scholar The direct evidence of an NaCl transport-dependency of renin secretion, the other end point of luminal NaCl concentration changes, is not as strong but is nevertheless highly suggestive.1.Schnermann J. Briggs J.P. Function of the juxtaglomerular apparatus: control of glomerular hemodynamics and renin secretion.in: Alpern R.J. Hebert S.C. The Kidney Physiology and Pathophysiology. 1. Elsevier Academic Press, Burlington–San Diego–London2008: 589-626Google Scholar NKCC2 has been found to be expressed in MD cells, and its inhibition causes cellular hyperpolarization and reductions in cytosolic Na and Cl concentrations.2.Lapointe J.Y. Bell P.D. Cardinal J. Direct evidence for apical Na+:2Cl-:K+ cotransport in macula densa cells.Am J Physiol Renal Physiol. 1990; 258: F1466-F1469PubMed Google Scholar,3.Schlatter E. Salomonsson M. Persson A.E. et al.Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl-K+ cotransport.Pflugers Arch. 1989; 414: 286-290Crossref PubMed Scopus (127) Google Scholar The salt-losing phenotype of mice with deficiencies in the proximal fluid transporters NHE3 or AQP1 is relatively mild despite the fact that the reabsorption of a substantial amount of filtered NaCl and water depends upon these transport pathways.7.Lorenz J.N. Schultheis P.J. Traynor T. et al.Micropuncture analysis of single-nephron function in NHE3-deficient mice.Am J Physiol Renal Physiol. 1999; 277: F447-F453PubMed Google Scholar,8.Schnermann J. Chou C-L. Ma T. et al.Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice.Proc Natl Acad Sci USA. 1998; 95: 9660-9664Crossref PubMed Scopus (365) Google Scholar TGF-mediated reductions of GFR and filtered NaCl have been observed in both sodium/hydrogen exchanger (NHE3−/−) and Aquaporin 1 (AQP1−/−) mice, and it has been surmised that reductions in filtered NaCl load by TGF are a major reason for the ability of mice with proximal transport defects to achieve Na balance.9.Schnermann J. NaCl transport deficiencies—hemodynamics to the rescue.Pflugers Arch. 2000; 439: 682-690Crossref PubMed Scopus (20) Google Scholar In contrast, mice with complete inactivation of the NKCC2 gene display the severe salt-losing phenotype of antenatal Bartter syndrome.10.Takahashi N. Chernavvsky D.R. Gomez R.A. et al.Uncompensated polyuria in a mouse model of Bartter's syndrome.Proc Natl Acad Sci USA. 2000; 97: 5434-5439Crossref PubMed Scopus (197) Google Scholar Although TGF responses have not been directly assessed in these mice, inactivation of TGF is suggested by the apparent inability to respond to the elevated distal NaCl load with a reduction in GFR. In mice heterozygous for the NKCC2-null mutation, NKCC2 protein expression was found to be normal, and mice were indistinguishable from wild type.11.Takahashi N. Brooks H.L. Wade J.B. et al.Posttranscriptional compensation for heterozygous disruption of the kidney-specific NaK2Cl cotransporter gene.J Am Soc Nephrol. 2002; 13: 604-610PubMed Google Scholar Further functional exploration of the consequences of NKCC2 deficiency has become possible with the generation of mice with targeted disruption of single NKCC2 isoforms.12.Oppermann M. Mizel D. Huang G. et al.Macula densa control of renin secretion and preglomerular resistance in mice with selective deletion of the B isoform of the Na,K,2Cl co-transporter.J Am Soc Nephrol. 2006; 17: 2143-2152Crossref PubMed Scopus (57) Google Scholar,13.Oppermann M. Mizel D. Kim S.M. et al.Renal function in mice with targeted disruption of the A isoform of the Na-K-2Cl co-transporter.J Am Soc Nephrol. 2007; 18: 440-448Crossref PubMed Scopus (67) Google Scholar The existence of three different full-length variants of NKCC2, first reported by Payne and Forbush14.Payne J.A. Forbush B. Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differently distributed within the rabbit kidney.Proc Nat Acad Sci USA. 1994; 91: 4544-4548Crossref PubMed Scopus (238) Google Scholar in the rabbit kidney, has been confirmed in all species studied.15.Igarashi P. Vanden Heuvel G.B. Payne J.A. et al.Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.Am J Physiol. 1995; 269: F405-F418PubMed Google Scholar,16.Vargas-Poussou R. Feldmann D. Vollmer M. et al.Novel molecular variants of the Na-K-2Cl cotransporter gene are responsible for antenatal Bartter syndrome.Am J Hum Genet. 1998; 62: 1332-1340Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar,17.Yang T. Huang Y.G. Singh I. et al.Localization of bumetanide- and thiazide-sensitive Na-K-Cl cotransporters along the rat nephron.Am J Physiol. 1996; 271: F931-F939PubMed Google Scholar These isoforms, called NKCC2B, NKCC2A, and NKCC2F, are derived from differential splicing of the variable exon 4 of the Slc12a1 gene, a short 96-bp exon that encodes for the second transmembrane domain and parts of the adjacent intracellular loop of the transporter.14.Payne J.A. Forbush B. Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differently distributed within the rabbit kidney.Proc Nat Acad Sci USA. 1994; 91: 4544-4548Crossref PubMed Scopus (238) Google Scholar NKCC2 isoform expression shows a distinct distribution pattern with F found exclusively in the medullary thick ascending limb, A in both the outer medulla and cortex, and B in the cortex.15.Igarashi P. Vanden Heuvel G.B. Payne J.A. et al.Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.Am J Physiol. 1995; 269: F405-F418PubMed Google Scholar,17.Yang T. Huang Y.G. Singh I. et al.Localization of bumetanide- and thiazide-sensitive Na-K-Cl cotransporters along the rat nephron.Am J Physiol. 1996; 271: F931-F939PubMed Google Scholar,18.Gimenez I. Isenring P. Forbush B. Spatially distributed alternative splice variants of the renal Na-K-Cl cotransporter exhibit dramatically different affinities for the transported ions.J Biol Chem. 2002; 277: 8767-8770Crossref PubMed Scopus (87) Google Scholar MD cells of the mouse have been found to express both the B- and A-isoforms of the cotransporter.12.Oppermann M. Mizel D. Huang G. et al.Macula densa control of renin secretion and preglomerular resistance in mice with selective deletion of the B isoform of the Na,K,2Cl co-transporter.J Am Soc Nephrol. 2006; 17: 2143-2152Crossref PubMed Scopus (57) Google Scholar Marked differences in ion affinities have been identified in in vitro heterologous expression studies in Xenopus laevis oocytes: F was found to have much lower Na+ and Cl− affinities than the A or the B isoform.18.Gimenez I. Isenring P. Forbush B. Spatially distributed alternative splice variants of the renal Na-K-Cl cotransporter exhibit dramatically different affinities for the transported ions.J Biol Chem. 2002; 277: 8767-8770Crossref PubMed Scopus (87) Google Scholar To create NKCC2B- and NKCC2A-deficient mice, the alternate exons 4B or 4A were modified by the introduction of in-frame stop codons resulting in the premature termination of translation. Thus, these strains of isoform-specific knockout mice lack both the full-length and the corresponding truncated NKCC2 isoforms. In vivo microperfusion of loops of Henle showed that in NKCC2B-deficient mice, Cl− reabsorption was significantly reduced at low flow rates,12.Oppermann M. Mizel D. Huang G. et al.Macula densa control of renin secretion and preglomerular resistance in mice with selective deletion of the B isoform of the Na,K,2Cl co-transporter.J Am Soc Nephrol. 2006; 17: 2143-2152Crossref PubMed Scopus (57) Google Scholar whereas the lack of NKCC2A resulted in reduced Cl− absorption at high perfusion rates.13.Oppermann M. Mizel D. Kim S.M. et al.Renal function in mice with targeted disruption of the A isoform of the Na-K-2Cl co-transporter.J Am Soc Nephrol. 2007; 18: 440-448Crossref PubMed Scopus (67) Google Scholar These in vivo data are in line with the notion that TAL reabsorption at low NaCl concentrations relies on the activity of the high Cl−-affinity NKCC2B isoform, whereas NKCC2A comes into play when higher salt concentrations are achieved by high loop perfusion flow. Assessment of TGF responses has confirmed that MD signaling function depends on the successive engagement of NKCC2B and NKCC2A (Figure 1). In the low flow range, NKCC2B-deficient mice were less responsive than wild-type animals, whereas TGF responses of NKCC2A−/− mice were reduced at high flow rates. Thus, V1/2, the flow rate causing half-maximum TGF responses, increased from 6.5 nl min−1 in mice expressing only NKCC2B to 15.5 nl min−1 in mice possessing only NKCC2A. These data suggest that the successive activation of the high Cl−-affinity NKCC2B and the lower Cl−-affinity NKCC2A is responsible for the surprisingly wide range of NaCl concentrations over which TGF operates. Whereas the inhibitory effect of luminal barium on TGF responses was diminished by a pronounced direct vascular constrictor action,19.Schnermann J. Effects of barium ions on tubuloglomerular feedback.Am J Physiol Renal Physiol. 1995; 268: F960-F966Google Scholar the retrograde application of the K+ channel blocker U37883A caused an almost complete inhibition of TGF responsiveness.20.Vallon V. Osswald H. Blantz R.C. et al.Potential role of luminal potassium in tubuloglomerular feedback.J Am Soc Nephrol. 1997; 8: 1831-1837PubMed Google Scholar This effect is mediated by renal outer medullary potassium (ROMK) type K+ channels, as TGF responses were largely absent in mice with targeted ROMK deletion.21.Lorenz J.N. Baird N.R. Judd L.M. et al.Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter's syndrome.J Biol Chem. 2002; 277: 37871-37880Crossref PubMed Scopus (146) Google Scholar The finding of a significantly reduced but not abolished TGF response has subsequently been confirmed in mice in which selective breeding of surviving animals has generated ROMK-deficient mice with less compromised kidney function and reasonably well-maintained blood pressure.22.Lu M. Wang T. Yan Q. et al.Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice.J Biol Chem. 2002; 277: 37881-37887Crossref PubMed Scopus (145) Google Scholar As shown in Figure 2, the mean TGF response of 11.3±1.2 mm Hg in wild-type mice was reduced to 2.2±0.6 mm Hg in ROMK-deficient mice (P<0.001). The observation that inhibition of NKCC2 and ROMK has similar effects on TGF responses argues against a specific ‘sensor’ function of the actual transport proteins, suggesting instead a critical role of some consequence of MD NaCl transport. As ambient distal K+ concentrations near the MD are close to the K+ affinity of the cotransporter, variations in luminal K+ may regulate TGF response magnitude.20.Vallon V. Osswald H. Blantz R.C. et al.Potential role of luminal potassium in tubuloglomerular feedback.J Am Soc Nephrol. 1997; 8: 1831-1837PubMed Google Scholar Detailed studies in the isolated perfused juxtaglomerular apparatus (JGA) preparation of the rabbit have clearly documented the existence of Na/H exchange activity in both the apical and basolateral membranes of MD cells. Increases in luminal NaCl concentration cause increased hydrogen efflux and subsequent alkalinization of the MD cell cytosol.24.Fowler B.C. Chang Y.S. Laamarti A. et al.Evidence for apical sodium proton exchange in macula densa cells.Kidney Int. 1995; 47: 746-751Abstract Full Text PDF PubMed Scopus (43) Google Scholar It has been estimated that about 20% of total Na entry may be mediated by Na/H exchange. Immunocytochemical data indicate that the exchanger isoform in the apical membrane is NHE2, whereas NHE4 is the isoform of the basolateral membrane.25.Peti-Peterdi J. Chambrey R. Bebok Z. et al.Macula densa Na(+)/H(+) exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms.Am J Physiol Renal Physiol. 2000; 278: F452-F463PubMed Google Scholar MD cells are thus distinct from TAL cells, where NHE3 is the dominant variant of the exchanger.26.Amemiya M. Loffing J. Lotscher M. et al.Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb.Kidney Int. 1995; 48: 1206-1215Abstract Full Text PDF PubMed Scopus (331) Google Scholar How Na/H exchange in MD cells could affect TGF has been unclear. One possibility is that the intracellular pH determines the magnitude of the response by disinhibiting MD neuronal nitric oxide synthase (nNOS) and thereby augmenting the generation of NO. In fact, it has been reported that Na/H exchange inhibition with amiloride augments TGF responses by preventing cell alkalinization and thereby causing relative inhibition of nNOS.27.Wang H. Carretero O.A. Garvin J.L. Inhibition of apical Na+/H+ exchangers on the macula densa cells augments tubuloglomerular feedback.Hypertension. 2003; 41: 688-691Crossref PubMed Scopus (25) Google Scholar,28.Liu R. Carretero O.A. Ren Y. et al.Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback.Kidney Int. 2005; 67: 1837-1843Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar In contrast to these observations in the isolated JGA preparation, there is no in vivo evidence that would support a major role of Na/H exchange activity in TGF responsiveness. Micropuncture studies in NHE2-deficient mice failed to show changes in TGF responses compared to wild-type animals,29.Lorenz J.N. Dostanic-Larson I. Shull G.E. et al.Ouabain inhibits tubuloglomerular feedback in mutant mice with ouabain-sensitive alpha1 Na,K-ATPase.J Am Soc Nephrol. 2006; 17: 2457-2463Crossref PubMed Scopus (17) Google Scholar and loop of Henle perfusion with amiloride or N-(isopropyl)-amiloride (EIPA) did not elicit measurable alterations of TGF response magnitude.4.Wright F.S. Schnermann J. Interference with feedback control of glomerular filtration rate by furosemide, triflocin, and cyanide.J Clin Invest. 1974; 53: 1695-1708Crossref PubMed Scopus (176) Google Scholar Thus, a major TGF-modulating role of Na/H exchange-dependent variations of cytosolic pH in TAL or MD cells is not supported by in vivo observations currently available. TGF responses have also been found to be well maintained in NHE3−/− mice,7.Lorenz J.N. Schultheis P.J. Traynor T. et al.Micropuncture analysis of single-nephron function in NHE3-deficient mice.Am J Physiol Renal Physiol. 1999; 277: F447-F453PubMed Google Scholar and autoregulation of GFR and renal blood flow was not altered in NHE3−/− mice with transgenic expression of NHE3 in the intestinal tract.30.Noonan W.T. Woo A.L. Nieman M.L. et al.Blood pressure maintenance in NHE3-deficient mice with transgenic expression of NHE3 in small intestine.Am J Physiol Regul Integr Comp Physiol. 2005; 288: R685-R691Crossref PubMed Scopus (51) Google Scholar Although an effect of apical transport inhibition on TGF is generally accepted, there is still considerable uncertainty about the subsequent steps in the juxtaglomerular signaling cascade. One possibility is that one of the intracellular consequences of NKCC2-dependent NaCl uptake is directly coupled to the mediating step. Detailed and sophisticated studies in the isolated perfused rabbit JGA have identified depolarization, alkalinization, and various ionic compositional changes as results of an increased NaCl uptake, and it is therefore conceivable that one or more of these changes trigger the signaling events directly.3.Schlatter E. Salomonsson M. Persson A.E. et al.Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl-K+ cotransport.Pflugers Arch. 1989; 414: 286-290Crossref PubMed Scopus (127) Google Scholar,31.Bell P.D. Lapointe J.Y. Peti-Peterdi J. Macula densa cell signaling.Annu Rev Physiol. 2003; 65: 481-500Crossref PubMed Scopus (93) Google Scholar A second possibility is that signal propagation is the consequence of transcellular NaCl transport, with apical uptake being the first step in the sequence. Transcellular NaCl transport across the renal tubular epithelium universally requires the support of Na,K-ATPase-dependent energy supply. Until recently, the available data about Na,K-ATPase in the MD made the transcellular transport hypothesis look less likely. First, cytochemical and immunological localization studies32.Beeuwkes R. Rosen S. Renal Na+-K+-ATPase: localization and quantitation by means of its K+-dependent phosphatase activity.in: EL B. Current Topics in Membranes and Transport. 13. Academic Press, New York1980: 343-354Google Scholar,33.Kashgarian M. Biemesderfer D. Caplan M. et al.Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments.Kidney Int. 1985; 28: 899-913Abstract Full Text PDF PubMed Scopus (215) Google Scholar suggested that the expression of Na,K-ATPase in the basolateral membrane of MD cells was quite low. In addition, a microenzymatic determination of Na,K-ATPase activity in microdissected rabbit MD cells arrived at the conclusion that the activity levels were about 50-fold lower than those in neighboring TAL cells.34.Schnermann J. Marver D. ATPase activity in macula densa cells of the rabbit kidney.Pflugers Arch. 1986; 407: 82-86Crossref PubMed Scopus (35) Google Scholar However, the interpretation of these studies failed to take into account the important impact of basolateral membrane folding on enzyme density. For example, in the enzyme activity studies, Na,K-ATPase was normalized to unit of cell volume rather than to membrane surface area, an approach that would underestimate a membrane-bound molecule by the membrane-folding factor. Although the basolateral membrane of MD cells is typically non-folded, extensive infolding in the neighboring TAL cells may increase its basolateral membrane by a factor of 10–20.35.Welling L.W. Welling D.J. Surface areas of brush border and lateral cell walls in the rabbit proximal nephron.Kidney Int. 1975; 8: 343-348Abstract Full Text PDF PubMed Scopus (64) Google Scholar Thus, the difference in enzyme activity per unit cell-surface area between MD and TAL cells may actually be at least 10-fold less than assumed. Species-specific differences may also play a role.25.Peti-Peterdi J. Chambrey R. Bebok Z. et al.Macula densa Na(+)/H(+) exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms.Am J Physiol Renal Physiol. 2000; 278: F452-F463PubMed Google Scholar,36.Peti-Peterdi J. Bebok Z. Lapointe J.Y. et al.Novel regulation of cell [Na(+)] in macula densa cells: apical Na(+) recycling by H-K-ATPase.Am J Physiol Renal Physiol. 2002; 282: F324-F329Crossref PubMed Scopus (45) Google Scholar Extensive studies in the rat using a well-defined antibody against α1 Na,K-ATPase and its colocalization with nNOS, an MD cell marker, have clearly established a robust presence of the enzyme in the basolateral membrane of MD cells.37.Wetzel R.K. Sweadner K.J. Immunocytochemical localization of Na-K-ATPase alpha- and gamma-subunits in rat kidney.Am J Physiol Renal Physiol. 2001; 281: F531-F545PubMed Google Scholar,38.Wetzel R.K. Sweadner K.J. Phospholemman expression in extraglomerular mesangium and afferent arteriole of the juxtaglomerular apparatus.Am J Physiol Renal Physiol. 2003; 285: F121-F129Crossref PubMed Scopus (23) Google Scholar In addition, MD cells also express the β1 subunit of the enzyme and a γ subunit that may be either γa or both γa and γb.37.Wetzel R.K. Sweadner K.J. Immunocytochemical localization of Na-K-ATPase alpha- and gamma-subunits in rat kidney.Am J Physiol Renal Physiol. 2001; 281: F531-F545PubMed Google Scholar,39.Arystarkhova E. Wetzel R.K. Sweadner K.J. Distribution and oligomeric association of splice forms of Na(+)-K(+)-ATPase regulatory gamma-subunit in rat kidney.Am J Physiol Renal Physiol. 2002; 282: F393-F407Crossref PubMed Scopus (70) Google Scholar,40.Farman N. Fay M. Cluzeaud F. Cell-specific expression of three members of the FXYD family along the renal tubule.Ann N Y Acad Sci. 2003; 986: 428-436Crossref PubMed Scopus (19) Google Scholar,41.Pu H.X. Cluzeaud F. Goldshleger R. et al.Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit.J Biol Chem. 2001; 276: 20370-20378Crossref PubMed Scopus (110) Google Scholar Again, the staining of MD cells appeared somewhat less intense than that in neighboring TAL cells, no doubt because of the absence of basolateral infoldings in MD cells. Pharmacological studies examining the effect of Na,K-ATPase inhibition on TGF were also inconclusive. In rats, Na,K-ATPase inhibition by ouabain, administered by luminal or peritubular microinfusion, did not elicit clear reductions of TGF responses (own unpublished data). However, the α1 subunit of Na,K-ATPase, by far the predominant isoform of the enzyme in the kidney, is largely resistant to ouabain in rats and mice. Mice homozygous for a complete α1 Na,K-ATPase-null mutation are not viable, whereas heterozygotes are essentially normal with respect to renal function.23.James P.F. Grupp I.L. Grupp G. et al.Identification of a specific role for the Na,K-ATPase alpha 2 isoform as a regulator of calcium in the heart.Mol Cell. 1999; 3: 555-563Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar Recently, however, the ingenious utilization of the possibilities of gene manipulation has brought important new information, suggesting an important role of Na,K-ATPase in supporting TGF.29.Lorenz J.N. Dostanic-Larson I. Shull G.E. et al.Ouabain inhibits tubuloglomerular feedback in mutant mice with ouabain-sensitive alpha1 Na,K-ATPase.J Am Soc Nephrol. 2006; 17: 2457-2463Crossref PubMed Scopus (17) Google Scholar TGF responses were determined in double-mutant mice in whom the normal ouabain-resistance pattern was reversed with α1 Na,K-ATPase mutated to be ouabain sensitive and α2 Na,K-ATPase to be ouabain resistant.42.Dostanic I. Lorenz J.N. Schultz Jel J. et al.The alpha2 isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice.J Biol Chem. 2003; 278: 53026-53034Crossref PubMed
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