The Juxtaglomerular Apparatus: From Anatomical Peculiarity ...€¦ · 99 per cent must be...

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HOMER W. SMITH AWARD LECTURE The Juxtaglomerular Apparatus: From Anatomical Peculiarity to Physiological Relevance JURGEN SCHNERMANN National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland. Homer Smith died in 1962, the year before I started my career in the Department of Physiology of the University of Goettingen. At that time, his original work was still well known and widely quoted. Several decades later, this has of course changed, and the newer generation of renal physiologists and nephrologists is often unaware of who Homer Smith was and what he did. This is not totally surprising given the time elapsed and the short half-life of scientific fame and the fact that the electronic library, which we more and more rely on exclusively, does not go back beyond 1965 or so. I would therefore like to begin my presentation by honoring the man who is commemorated by this award. Born in 1895 in Denver, he received an A.B. from the University of Denver in 1917. He joined the armed forces and was transferred to the Chemical Warfare Station in Washington D.C., where he studied the biologic effects of gases, specifically of mustard gas, under the supervision of E.K. Marshall, who later provided proof for tubular secretion of organic compounds and its saturable nature. After the end of the war, Homer Smith enrolled in graduate studies at Johns Hopkins University and received the D.Sc. degree in chemistry in 1921. His early scientific work revolved around the chemothera- peutic value of arsenic compounds and the chemistry of secondary valence. After 2 years in the laboratory of Walter Cannon at Harvard University (1923–1925), he turned to studies of marine biology and of the physiology of fish. Around 1930, he began to develop and apply quantitative methods to studying the function of the mammalian kidney. He was appointed chairman of the Department of Physiology of the University of Virginia at the age of 30 yr. In 1928, he became chairman of the Physiological Laboratories at New York University College of Medicine, a position that he held until 1961. In the late 1920s, when Homer Smith started his work on the kidney, renal physiology as a field was not particularly advanced; in fact, it was considerably underdeveloped. This is reflected, for example, in the following statements of Alfred Richards, one of the other greats of kidney physiology in that same time: The literature of investigation of the kidney, since Bowman, contains no great illuminating discoveries comparable to those which are so conspicuous in other fields. Many of the questions of kidney function which are now being actively debated by physiologists are practically identical with those which were subjects of controversy seventy-five years ago. . . . Is the Malpighian body with its contained glomerulus a filter or is it a “secretory” structure? Is the epithelium which composes the tubule walls capable of secreting substances from blood into lumen of tubule, or is its task that of permitting or enabling restoration to the blood of substances lost to it in passage through the glomerulus? (41). These were very basic questions indeed! Thirty years later, by the end of the “Homer Smith era,” the function of the kidney at the organ level was adequately described in quanti- tative terms of filtration, perfusion, absorption, and secretion, through his work and through the work of others that he had stimulated. One could argue, as Robert Berliner has done, that his summary and interpretation of the available knowledge may be his most important contribution to the field (5). He wrote three textbooks on renal physiology and pathophysiol- ogy of which the second, The Kidney, was enormously influ- ential, because it was the first modern and correct summary of how the kidney works in quantitative terms (58,61,63). His scientific work is summarized in a little over 100 orig- inal publications — papers used to be fewer and longer then. His major scientific contribution was to co-invent, simulta- neously with Richards (42), the clearance methodology for measuring GFR and RBF, and then apply it in a wide array of conditions, mostly in clinical conditions, since Homer Smith had long-standing and intense collaborations with his clinical colleagues at New York University (14,23,54,64). Among his other contributions are the development of the concept of free water clearance with urinary dilution being a consequence of NaCl absorption across a water impermeable segment, the distal nephron, the discovery that urea back-diffusion accounts for its relatively low clearance and its dependence on urine flow (10), the notion that bulk absorption of the majority of filtered water occurs in the proximal tubule by active absorp- tion of Na followed by passive absorption of water, the concept of “obligatory” reabsorption in the proximal tubule and “fac- ultative” reabsorption in the distal tubule, and the introduction of the concept of glomerulotubular balance. Homer Smith was a much sought-after speaker as judged Correspondence to Dr. Jurgen Schnermann, Building 10, Room 4 D51, 10 Center Drive MSC 1370, Bethesda, MD 20892. Phone: 301-435-6580; Fax: 301-435-6587; E-mail: [email protected] 1046-6673/1406-1681 Journal of the American Society of Nephrology Copyright © 2003 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000069221.69551.30 J Am Soc Nephrol 14: 1681–1694, 2003

Transcript of The Juxtaglomerular Apparatus: From Anatomical Peculiarity ...€¦ · 99 per cent must be...

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HOMER W. SMITH AWARD LECTURE

The Juxtaglomerular Apparatus: From Anatomical Peculiarityto Physiological Relevance

JURGEN SCHNERMANNNational Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,Maryland.

Homer Smith died in 1962, the year before I started my career inthe Department of Physiology of the University of Goettingen. Atthat time, his original work was still well known and widelyquoted. Several decades later, this has of course changed, and thenewer generation of renal physiologists and nephrologists is oftenunaware of who Homer Smith was and what he did. This is nottotally surprising given the time elapsed and the short half-life ofscientific fame and the fact that the electronic library, which wemore and more rely on exclusively, does not go back beyond 1965or so. I would therefore like to begin my presentation by honoringthe man who is commemorated by this award. Born in 1895 inDenver, he received an A.B. from the University of Denver in1917. He joined the armed forces and was transferred to theChemical Warfare Station in Washington D.C., where he studiedthe biologic effects of gases, specifically of mustard gas, under thesupervision of E.K. Marshall, who later provided proof for tubularsecretion of organic compounds and its saturable nature. After theend of the war, Homer Smith enrolled in graduate studies at JohnsHopkins University and received the D.Sc. degree in chemistry in1921. His early scientific work revolved around the chemothera-peutic value of arsenic compounds and the chemistry of secondaryvalence. After 2 years in the laboratory of Walter Cannon atHarvard University (1923–1925), he turned to studies of marinebiology and of the physiology of fish. Around 1930, he began todevelop and apply quantitative methods to studying the functionof the mammalian kidney. He was appointed chairman of theDepartment of Physiology of the University of Virginia at the ageof 30 yr. In 1928, he became chairman of the PhysiologicalLaboratories at New York University College of Medicine, aposition that he held until 1961.

In the late 1920s, when Homer Smith started his work on thekidney, renal physiology as a field was not particularly advanced;in fact, it was considerably underdeveloped. This is reflected, forexample, in the following statements of Alfred Richards, one ofthe other greats of kidney physiology in that same time:

The literature of investigation of the kidney, since Bowman,contains no great illuminating discoveries comparable tothose which are so conspicuous in other fields. Many of thequestions of kidney function which are now being activelydebated by physiologists are practically identical with thosewhich were subjects of controversy seventy-five years ago.. . . Is the Malpighian body with its contained glomerulus afilter or is it a “secretory” structure? Is the epithelium whichcomposes the tubule walls capable of secreting substancesfrom blood into lumen of tubule, or is its task that ofpermitting or enabling restoration to the blood of substanceslost to it in passage through the glomerulus? (41).

These were very basic questions indeed! Thirty years later,by the end of the “Homer Smith era,” the function of thekidney at the organ level was adequately described in quanti-tative terms of filtration, perfusion, absorption, and secretion,through his work and through the work of others that he hadstimulated. One could argue, as Robert Berliner has done, thathis summary and interpretation of the available knowledgemay be his most important contribution to the field (5). Hewrote three textbooks on renal physiology and pathophysiol-ogy of which the second, The Kidney, was enormously influ-ential, because it was the first modern and correct summary ofhow the kidney works in quantitative terms (58,61,63).

His scientific work is summarized in a little over 100 orig-inal publications — papers used to be fewer and longer then.His major scientific contribution was to co-invent, simulta-neously with Richards (42), the clearance methodology formeasuring GFR and RBF, and then apply it in a wide array ofconditions, mostly in clinical conditions, since Homer Smithhad long-standing and intense collaborations with his clinicalcolleagues at New York University (14,23,54,64). Among hisother contributions are the development of the concept of freewater clearance with urinary dilution being a consequence ofNaCl absorption across a water impermeable segment, thedistal nephron, the discovery that urea back-diffusion accountsfor its relatively low clearance and its dependence on urineflow (10), the notion that bulk absorption of the majority offiltered water occurs in the proximal tubule by active absorp-tion of Na followed by passive absorption of water, the conceptof “obligatory” reabsorption in the proximal tubule and “fac-ultative” reabsorption in the distal tubule, and the introductionof the concept of glomerulotubular balance.

Homer Smith was a much sought-after speaker as judged

Correspondence to Dr. Jurgen Schnermann, Building 10, Room 4 D51, 10Center Drive MSC 1370, Bethesda, MD 20892. Phone: 301-435-6580; Fax:301-435-6587; E-mail: [email protected]

1046-6673/1406-1681Journal of the American Society of NephrologyCopyright © 2003 by the American Society of Nephrology

DOI: 10.1097/01.ASN.0000069221.69551.30

J Am Soc Nephrol 14: 1681–1694, 2003

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from the number of talks he was invited to give, and it is fromthe transcripts of these talks that one can glean a more personalview of who Homer Smith was. The titles of some of thesepresentations indicate his interests, titles such as “Error inPhysiology” (1936), “Plato and Clementine” (1947), “Objec-tives and Objectivity in Science” (1949), “Agnosticism versusAtheism” (1956), “De Urina” (1958), and “The Biology ofConsciousness” (1959) among others. It is apparent that he wasbroadly interested; he was a humanist, an evolutionary biolo-gist, a paleontologist, a philosopher, and a musician, somewhatof a renaissance man. Throughout his life, he struggled with theplace of man in nature and therefore with the interface ofscience, philosophy, and religious beliefs. He did not believe inthe supernatural or in creationism, and was skeptical about allestablished religions. In his words:

All human history reveals that transcendental metaphysics isnot only futile but dangerous. Those who have, frequently bydishonest means, foisted upon the naıve and gullible theirown unsupported speculations have served to retard man’sself realization more than any other misfortune that has everbefallen him. History also reveals that man does not needany brand of transcendental metaphysics — his lasting con-tentments and achievements he has found wholly within theframe of reference that takes things as they are in the hereand now (62).

In addition to his textbooks, he wrote four other books,Kamongo, or The Lungfish and the Padre (Viking Press,1932), The End of Illusion (Harper, 1935), Man and His Gods(Little Brown, 1952), and From Fish to Philosopher (LittleBrown, 1953). Particularly pleasant to read is From Fish toPhilosopher. His enthusiasm and understanding for compara-tive physiology has substantial resonance in this era in whichwe learn more and more about the extent of evolutionaryconservation both at the genomic and functional level.

The Juxtaglomerular ApparatusI would like to turn to the topic of this overview, the

mechanisms of juxtaglomerular cell communication, withthese quotes of Homer Smith that are as true and puzzling nowas they were then:

Examining the pattern of the human kidney, we must not besurprised to find that it is far from a perfect organ. In fact, itis in many respects grossly inefficient. It begins its task bypouring some 125 cc of water into the tubules each minute,demanding for this extravagant filtration one quarter of allthe blood put out by the heart. Out of this stream of water,99 per cent must be reabsorbed again. This circuitousmethod of operation is peculiar, to say the least. . . . Inconsequence of the circuitous pattern of the filtration andreabsorption of water, nearly half a pound of glucose andover three pounds of sodium chloride per day, not to men-tion quantities of phosphate, amino acids and other sub-stances, must be saved from being lost in the urine by beingreabsorbed from the tubular stream. There is enough waste

motion here to bankrupt any economic system — other thana natural one. . . (59).

The astounding conclusion that over three pounds of NaClmust be filtered and absorbed per day was a derivative of therecognition of the nature of glomerular filtrate formation as anultrafiltration process and the determination of the GFR mag-nitude by the inulin clearance. Avid absorption takes placealong the proximal part of the nephron by an array of trans-porters, and their activity reduces the amount of Na to anapparently trivial number by the time the collecting duct isreached. There is good evidence that most or all of the regu-lation of Na absorption that is necessary to match salt intakeand excretion over the physiologic range occurs along thecollecting duct through variations in Na absorption across thehighly regulated epithelial Na channel ENaC. The critical roleof ENaC-dependent Na absorption is highlighted by observa-tions in patients and mice, in which ENaC deficiency is asso-ciated with severe Na loss and volume depletion, that in thecase of the ENaC knockout mice, regardless of ENaC subunit,lead to rapid postnatal death, at least to a substantial extent byvolume loss and circulatory collapse (1,20,33). The same out-come is seen in mice that are deficient in the mineralocorticoidreceptor, and therefore do not respond to aldosterone, the mostimportant regulator of ENaC (4). Conversely, ENaC overac-tivity as in Liddle syndrome causes severe volume-dependentarterial hypertension with all its dire consequences (17,55).Because the capacity of the collecting duct is limited, onecould envisage unwanted Na loss by collecting duct overload-ing with excess Na when there is an increase in GFR or whenthere is a decrease in Na absorption in the proximal nephron. Itis now clear that such changes in NaCl delivery are sensed inthe juxtaglomerular region of the nephron and are used as asignal that feeds back to the glomerulus and tends to returndistal Na delivery to its original state.

This feedback system is anatomically represented in thejuxtaglomerular apparatus (JGA), the complex of epithelial,mesangial, and vascular cells formed as the result of the returnof the thick ascending limb to its origin, a fact well appreciatedand illustrated in Homer Smith’s textbook (Figure 1). In es-sence, specialized epithelial cells called macula densa (MD)cells make contact with a mesangial cell type, and throughthem with the smooth muscle and renin-producing granularcells of the glomerular arterioles. It is now clear that thisstructure serves two functions, both related to Na homeostasis.One is the vascular feedback control called tubuloglomerularfeedback or TGF for short; the other is NaCl-dependent controlof renin secretion and renin synthesis. Although both of theseeffector systems respond to the same input, they differ in theirtemporal characteristics.

Macula densa control of vascular tone has a fast responsetime with full activation or deactivation occurring within 20 to60 s, it compensates for random and high frequency variationsof the MD signal in the vicinity of its operating point, it acts asa minute-to-minute controller of salt delivery to the distalnephron, and prolonged out-of-range deviations of the maculadensa signal cause resetting of the response curve. Macula

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densa control of renin secretion has a slow response time anda slow frequency response (about 2 to 3 mHz or cycle timesbetween 5 and 8 min), it responds to prolonged deviations ofthe MD signal, those associated with changes in EC volume forexample, it acts as a long-term controller of body salt balancethrough changes in systemic angiotensin II levels, and it isinexhaustible because the MD signal can also affect renintranscription.

Characteristics of Tubuloglomerular FeedbackExperimental studies of tubuloglomerular feedback began

with the microinjection experiments that Klaus Thurau de-signed and initiated (69). After outlining the proximal seg-ments that belong to the same nephron, its distal tubule waspunctured and fluid was manually injected. When this fluidwas an isotonic solution of either NaCl or NaBr, the previouslyidentified proximal tubule would usually completely collapse,

Figure 1. Schematic diagram of a nephron demonstrating the return of the thick ascending limb to the vascular pole of its own glomerulus. Ahistological view of the juxtaglomerular apparatus is shown in the upper left hand corner. The diagram is from Smith H, The Kidney (Plate I)(61).

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and this was interpreted as indicating a severe reduction inGFR. In retrospect, it would appear that the choice of theexperimental conditions was fortunate because complete ces-sation of GFR is not the typical response to distal saturationwith NaCl. A subsequent approach permitted an evaluation ofthe TGF response in a more quantitative way (50). Afteridentifying the segments of a proximal tubule by dye injection,loops of Henle were microperfused from the last superficialproximal segment. Flow in an upstream segment was inter-rupted by injecting an immobile block, and fluid collectionswere made in a segment even further upstream for measure-ment of GFR, and in the distal tubule for measurement of Naor Cl concentration. The results of such experiments showedthat as perfusion rate increased there was an increase in Naconcentration and a parallel decrease in single nephron GFR(SNGFR). A decrease of SNGFR was not seen when theperfusate was an isotonic mannitol of Na sulfate solution (50).A further refinement (Figure 2, left) consisted of performingnine repeat measurements of SNGFR at nine different flowrates, and this design revealed a sigmoidal relationship be-tween SNGFR and end-proximal flow rate, with SNGFR fall-ing by about 50%, and the flow causing a half-maximal re-sponse being precisely in the range of normal end-proximalflows (7). In a variant approach, first performed in collabora-tion with Erik Persson, stop flow pressure, a close correlate ofglomerular capillary pressure, was measured instead of

SNGFR; this technique is technically somewhat easier and hasproven useful as a screening method (48). Stop flow pressureshows the same sigmoidal relationship to perfusion rate, withthe exception that responses are less easily triggered in thelow-flow range, and the midpoint is therefore shifted to theright (Figure 2, right). Thomson and Blantz (68) developed atechnique in which the tubule is not blocked, and in which loopflow is perturbed by the addition or withdrawal of fluid at lowrates. Tubular flow is measured upstream from the perturbationby a videometric quasi-continuous method, permitting an as-sessment of the compensatory power of the entire feedbackloop. A compensation of one would be optimal because itwould indicate that an addition of 1 nl/min had caused a fall inupstream flow of 1 nl/min. Maximal error compensation ofbetween 0.8 and 0.9 was observed in response to small pertur-bations around normal flows.

An important characteristic feature of TGF is its tendency toreset by shifting its operational range into a higher or lowerflow range and by changing its sensitivity by either decreasingor increasing the slope of the TGF function. This typicallyoccurs when the signal is forced out of operating range for toolong. Often, left shifts are situations of increased angiotensinII, states of volume depletion for example, and right shifts arestates of reduced angiotensin II and perhaps increased nitricoxide, volume expansion for example.

In summary, the TGF control system describes an inverse

Figure 2. (Left) Relationship between the rate of loop of Henle perfusion and single nephron GFR (SNGFR) determined as the product of earlyproximal tubular flow rate (VEP) and the tubular fluid to plasma inulin ratio. Data are from Briggs et al. (7). (Right) Relationship between therate of loop of Henle perfusion and the associated change of stop flow pressure (PSF). Data are from Schnermann et al. (48).

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and sigmoidal relationship between SNGFR and VLP; its op-erating point is at the function midpoint, its optimal errorcompensation is around the operating point, it can reset itsoperational range and its sensitivity to flow perturbations, ithas a tendency for stable oscillations at 35 to 50 mHz (28), itis a single nephron mechanism although TGF-induced smoothmuscle activation can spread by electrotonic coupling (24), andits vascular effect is long-lasting.

Mechanisms of JGA-Mediated Regulation of VascularTone and Renin Secretion

The information pathway linking the tubular lumen with thevascular effector cells sequentially involves the different celltypes of the JGA. It begins with a signaling event in the tubularlumen that affects two end points, contractility and renin se-cretion, through initiating an epithelial cell response, mostlikely of macula densa cells, followed by an extracellulartransmission mechanism that bridges the JGA interstitium.

Luminal SignalTubuloglomerular Feedback. To identify the nature of the

tubular signal that is required to alter the vascular endpoint, amodified technique was used in which test solutions wereperfused into the distal tubule, a site that is much closer to theJG region so that one can assume that the test solutions reachthe sensing site largely unmodified (49). Pure NaCl solutionselicited the full response at a constant flow rate, and responses

were seen in the hypotonic range with saturation reached atabout 60 mM (Figure 3). Half-maximum concentration wasabout 35 mM, which we estimate to be close to the normalNaCl concentration in the macula densa region. Studies in anisolated rabbit preparation show that half-maximum and satu-ration concentrations may be even lower in that species (40).Using the same technique of retrograde perfusion, normalresponses were seen when Na was substituted with smallunivalent cations, but not when Cl was substituted with anumber of anions, the exception being a Br substitution (49).We concluded from this that variations in Cl concentrations areresponsible for causing the TGF response, whereas variationsin Na concentration or osmolarity are not a mandatory require-ment for the vascular reaction. To summarize, the luminalsignal for TGF is dependent on graded changes of Cl andpossibly K, the Cl concentration causing half-maximum re-sponse is about 35 mM, substitution of Na by other monovalentcations has no effect on the response, and signaling is inde-pendent of flow, osmolarity, and luminal calcium concentra-tion (47).

Renin Secretory Response. Macula densa–dependent re-nin secretion has been proposed and studied in vivo by Vander(74), but renin secretion is under the control of importantdrives in addition to that of the macula densa; it was thereforedifficult to decide whether and to what extent these other driveswere involved. To study MD-dependent renin secretion in

Figure 3. Relationship between the NaCl concentration in solutions perfused into the distal tubule at a rate of 15 nl/min and the associatedpercent change in SNGFR. C1/2 indicates the NaCl concentration causing the half-maximum decrease of SNGFR. Data are from Schnermannet al. (49).

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isolation, Skott and Briggs (56) developed a technique inwhich the thick ascending limb and its adherent glomeruluswere dissected from rabbit kidneys and perfused with theBurg technique through the ascending limb. The perfusedpreparation was retracted into an outer pipette that functionsas a superfusion chamber. After immersion of this wholesetup in oil, the superfusate carrying the released reninwould form droplets that could be collected in defined timeintervals for measurement of renin activity (Figure 4, A andB). The change in renin release in response to changing theluminal perfusate composition is macula densa-mediated,because this preparation does not consist of much more thanthe tubuloglomerular contact area and because it obviouslyhas no baroreceptor or regulated sympathetic input. Asshown in Figure 4C, an increase in NaCl concentrationcaused a reversible inhibition and a decrease in NaCl areversible stimulation of renin release with maximal deflec-tions being reached after about 20 min (29). Resolution ofthe concentration dependency of renin secretion showed thatthe renin response, like TGF, covers the hypotonic concen-tration range below 60 mM, with a half-maximum Cl con-centration of about 25 to 30 mM (19). To summarize, theluminal signal for the renin secretory response is dependent

on graded changes of Cl, the Cl concentration causing thehalf-maximum response is about 25 to 30 mM, substitutionof Na by Rb or choline has no effect on the response,substitution of Cl by isethionate or acetate eliminates theresponse, and the response is largely determined by NaClconcentration, not NaCl load.

Epithelial Cell Response Elicited by a Luminal NaClConcentration Change. Loop diuretics such as furosemide,bumetanide, and others have been observed to completelyblock TGF responses and to stimulate renin secretory re-sponses (30,79). In a study in which we determined the dosedependence of TGF and transport inhibition, we found thatbumetanide inhibited TGF at exactly those concentrations atwhich a change in distal Cl concentration indicated inhibitionof transport, indicating that these two events were causallylinked. Recent studies with the K channel blocker U37883A, aswell as earlier experiments with Ba from our laboratory,showed that K channel blockade largely blocked TGF re-sponses (45,73). Furthermore, Lorenz et al. (32) recentlyshowed that ROMK knockout mice have very small or no TGFresponses. The identical effect of inhibitors of two entirelydifferent transporters indicates that their common role in de-termining NaCl transport activity rather than any other effects

Figure 4. (A) Preparation of a glomerulus (Glom) with attached thick ascending limb (TAL) isolated from rabbit kidney. The pipette perfusingthe thick ascending limb is visible on the left. In the rabbit, the macula densa cells (MD) form a plaque that bulges into the tubular lumen (56).(B) The specimen attached to the perfusion pipette has been withdrawn into an outer glass pipette that serves as a superfusion chamber. Afterestablishing a superfusion flow, the superfusion pipette is immersed in mineral oil. The superfusate containing the released renin forms dropletsthat are withdrawn in time intervals for renin analysis (57). (C) Increasing perfusate NaCl concentration from Cl/Na 7/26 mM to 122/141 mMcauses a reversible decrease in renin secretion rate (top); decreasing it from Cl/Na 122/141 mM to 7/26 mM causes a reversible increase in reninsecretion. Data are from Weihprecht et al. (29) and Lorenz et al. (77).

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of NKCC2 or ROMK is an early epithelial event in the maculadensa pathway.

Studies by Rainer Greger (16) in cortical TAL segments hadshown that the NaCl cotransport activity is modified by Cl withan affinity constant of about 50 mM, whereas the affinities forNa or K are so high that the cotransporter is essentially alwayssaturated. Together with the inhibitory effect of loop diuretics,we believe that this finding may explain why, as alluded toearlier, the TGF response is regulated by changes in Cl con-centration and why it is apparently Na independent. Implicit inthis argument is the notion that affinity constants of NKCC2 inthe thick ascending limb are comparable to those in the MD.This notion has been challenged by the recent findings ofGimenez and Forbush, who have observed that the Cl affinityof the NKCC2 B isoform, the isoform most likely expressed inmacula densa cells, had a much higher affinity, only 9 mM,when expressed in oocytes (13).

It is still unclear which of the epithelial cell changes that aredependent on increased activity of NKCC2 are part of the

juxtaglomerular signaling pathway. NaCl cotransport activa-tion has been shown to cause increases of cytosolic Na and Kconcentration, an increase of cytosolic Cl concentration, celldepolarization, probably an increase in cytosolic Ca concen-tration, cell swelling, and cell alkalinization (12,25–27,43,44).All of these changes could be involved in initiating down-stream events, perhaps with the exception of cell alkalinization,because loop diuretics were found to increase cell pH withouteliciting a TGF response (12). A related problem is to deter-mine which of the transporters and receptors that have beenidentified by the work of several laboratories, most notablythose of Bell/Lapointe (3) and Persson (38), to be present inapical or basolateral membranes of macula densa cells play arole in juxtaglomerular regulatory pathways. Up until now,with the exception of NKCC2 and ROMK, none of the othermembrane proteins have been unequivocally implicated in theTGF or renin pathway (Figure 5). Ongoing studies will hope-fully shed some light on the roles of the luminal NHE, H/KATPase, angiotensin II receptors, and others in determining the

Figure 5. Summary of membrane transporters and surface receptors found in apical and basolateral membranes of macula densa cells. Thepresence of NOS 1 and COX-2 in macula densa cells is indicated. For proteins outlined in red, a connection to the specific function of themacula densa cells to affect vascular tone or renin secretion has been established. See text for references.

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vascular response. A role for NO generated by macula densanNOS has been established that on the basis of inhibitor studiesconsists of an attenuation of the full vasoconstrictor response(39,78).

Mechanisms Mediating between the Macula Densa andEffector Cells

Tubuloglomerular Feedback. It is widely assumed that anincrease in luminal NaCl concentration causes changes in theinterstitium of the JGA that alter the function of granular andsmooth muscle cells. Because of the cellular discontinuity, themost common notion is that these changes are changes in theappearance and interstitial concentration of a paracrine medi-ator. A number of such mediators have been invoked on thebasis of pharmacologic inhibitor studies, but conclusive evi-dence and therefore a consensus has been difficult to reach. Itis in this area of research that the use of knockout mice hasbeen and will continue to be quite helpful. For example, micewith knockout mutations in the thromboxane receptor haveperfectly normal TGF responsiveness, suggesting that throm-boxane is not a major player in this response under normalconditions (66).

Rather solid pharmacologic evidence in various in vivo andin vitro preparations has implicated adenosine 1 receptors(A1AR) as participating in the TGF response. For example, theA1AR antagonist FK 838 fully prevented the vascular responseto an elevation in luminal NaCl concentration in an in vitroperfused rabbit preparation in which the thick ascending limbwas perfused and in which diameter changes of the perfusedafferent arterioles served as TGF end point (40). Thus, on theassumption that the endogenous A1AR ligand, adenosine, maytrigger the TGF response through this receptor subtype, asoriginally suggested by Osswald (37), Daqing Sun in our labwith the help of Linda Samuelson (65) generated mice with aknockout mutation in this receptor subtype. In fact, TGF re-sponses of stop flow pressure or single nephron GFR werefound to be entirely absent in A1AR�/� mice (65). Thedependence of TGF responsiveness on the presence of intactA1 adenosine receptors was confirmed in another strain ofA1AR knockout mice that was independently generated byFredholm and studied in the laboratory of Erik Persson (8).Thus, we conclude that an increase in luminal NaCl causes theappearance in the JGA interstitium of adenosine and thatadenosine through A1AR activation causes TGF vasoconstric-tion. The exact source of adenosine still remains to be identi-fied. Adenosine is presumably generated inside macula densacells by ATP degradation, and it could be exported by one ofthe so-called equilibrative nucleoside transporters that arefound in many membranes. Alternatively, adenosine may begenerated extracellularly from released ATP. Bell et al. (2)have provided some evidence that ATP may in fact be releasedin a NaCl concentration-dependent fashion, perhaps through anonspecific anion channel, and an attenuation of TGF re-sponses by an inhibitor of ecto-5'nucleotidase has been ob-served by Thomson et al. (67).

Renin Secretory Response. While adenosine is probablyresponsible for high NaCl-induced vasoconstriction, it is lesscertain that it plays a major role in the stimulation of renin

secretion by low luminal NaCl. For example, the stimulation ofrenin secretion caused by furosemide, likely mediated to amajor extent by the macula densa pathway, was found to beunaltered in isolated perfused kidneys of A1AR knockout mice(52). The conclusion that inhibition of adenosine production isnot the primary cause for the stimulation of renin release bylow NaCl agrees with earlier studies in the isolated JGApreparation in which the addition of adenosine to the bath didnot induce a major inhibition of renin secretion (31). On theother hand, prostaglandins have been invoked in the maculadensa pathway of renin secretion for a while. An involvementof prostaglandins in macula densa–mediated renin secretionwas supported by the observation in the isolated rabbit JGApreparation that two nonsteroidal cyclooxygenase (COX) in-hibitors essentially entirely blocked the renin stimulationcaused by low NaCl (15). The interpretation of these findingswas not entirely clear because macula densa cells were notbelieved to express COX-1, the only known COX enzyme atthe time. A breakthrough development was the demonstrationby Harris et al. (18) that macula densa cells as well as sur-rounding cTAL cells express COX-2, the second isoform ofCOX, usually called the inducible isoform. We subsequentlyshowed that inhibition of COX-2 with the specific inhibitor NS398 eliminated the renin secretory response in the isolatedrabbit JGA preparation, whereas valeryl salicylate used as aCOX-1 inhibitor had no such effect (72). A role for COX-2 inthe renin secretory pathway is also suggested by studies fromour laboratory in which the stimulatory effect of chronic bu-metanide administration on plasma renin activity was found tobe largely absent in COX-2 knockout mice, whereas it wasessentially normal in the absence of COX-1 (R. Topaloglu,unpublished observations). The strength of this argument de-pends on the extent to which the administration of loop diuret-ics stimulates renin through the macula densa pathway. Webelieve that it is this prostaglandin-dependent pathway thatcauses the marked stimulation of the renin-angiotensin systemin patients with Bartter syndrome, a state characterized byincreased PGE2 production (53).

To study the cellular mechanisms that link a low luminalNaCl concentration with an activation of COX-2, we utilized acell line derived in our lab from macula densa cells, theso-called MMDD1 cells (80). Lowering medium NaCl concen-tration caused a prompt increase in the release of PGE2 fromthese cells, and a delayed induction of COX-2 expression.Substitution experiments showed that these effects were elic-ited by a low medium Cl concentration, whereas a selectivelowering of Na had no stimulatory effect. A stimulation ofCOX-2 expression was also caused by furosemide and bumet-anide. The release of PGE2 was inhibited by the COX-2inhibitor NS 398 with the same efficacy as by the nonspecificCOX inhibitor indomethacin, suggesting that it was derivedfrom COX-2. A lowering of medium Cl concentration wasfollowed by rapid phosphorylation of p44/42 and p38 MAPkinases, and the presence of p44/42 and p38 inhibitors pre-vented the stimulation of COX-2 expression by low chloride.Lowering of Cl did not cause transient or steady-state changesof cytosolic calcium concentration (80). It should be pointed

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out that this cellular response to lowering NaCl does not appearto be restricted to macula densa cells, but it appears to beshared by thick ascending limb cells (11). In summary, adecrease in luminal NaCl concentration activates and transcrip-tionally induces COX-2, causing PGE2 release, and EP4-me-diated stimulation of renin secretion and renin synthesis. Thus,the vascular and renin secretory responses, while initiated bythe same luminal signal, appear to be elicited by largely dif-ferent extracellular mediators.

Responses of the Effector CellsSmooth Muscle Cells. The first determination of the

changes in the hemodynamic determinants of filtration causedby high luminal NaCl was performed by Briggs and Wright(6). In their study, increasing the macula densa signal fromnormal to maximum, caused a 35% reduction in SNGFR, a24% reduction in single nephron filtration fraction, a 20%reduction in stop flow pressure, and a 20% reduction innephron plasma flow. Using standard filtration models, theseauthors calculated that this data set necessitated an increase in

afferent arteriolar resistance, depending on conditions between25 and 50%, while efferent resistance could have slightly fallenor slightly increased (6). It has also been suggested that TGFactivation may reduce the filtration coefficient (21). Preglo-merular vasoconstriction, the dominant response elicited byTGF engagement, has now been observed directly in two invitro preparations, in the isolated perfused rabbit preparation,and in the juxtamedullary nephron preparation (9,22).

Because adenosine has been identified as being required forTGF responses, one would expect that the effects of exogenousadenosine in afferent arterioles are consistent with the knowncharacteristics of the TGF response. In fact, adenosine added tothe medium bathing isolated perfused afferent arterioles frommouse kidneys caused a vasoconstriction at concentrations of10�8 M and higher that was particularly pronounced in theglomerular entrance segment of the arteriole (P. Hansen, un-published observations). Vasoconstriction was not seen in af-ferent arterioles from A1AR knockout mice, indicating that itwas A1AR-mediated. These observations confirm earlier ob-

Figure 6. Perfused afferent arteriole with attached glomerulus under control conditions (top) and during perfusion with a solution containingadenosine at 10�7 M (bottom). In this particular preparation, the region of maximal contraction was observed to be located inside theglomerulus after the first division of the arteriole. Black arrows point to the region of the first branching, and white arrows indicate the diameterof the lower branch before and during adenosine.

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servations by Weihprecht and Lorenz (75) in a similar vesselpreparation from the rabbit kidney. In preparations in whichthe optical resolution and orientation were ideal, the mostpronounced constrictor effect of adenosine was observed in thevery terminal segment of the arteriole adjacent to the maculadensa (Figure 6). Thus, exogenous adenosine constricts exactlywhere the TGF mechanism is likely to constrict, stronglysupporting the identity of the two. This glomerulus-near por-tion of the afferent arteriole is also the segment that is partic-ularly responsive to angiotensin II and is therefore probably thesite where adenosine and angiotensin II synergistically interact.Two additional observations in isolated perfused afferent arte-rioles, inhibition of adenosine-induced vasoconstriction by ni-fedipine and long duration of the effect of adenosine, furtheremphasize the notion that the vasoconstriction caused by ex-ogenous adenosine and by TGF share the same characteristics.Inhibition of TGF responses by blockers of voltage-activatedCa channels has been well documented (35,36), and an unal-tered vasoconstrictor effect elicited through the TGF pathwayhas been observed for at least 30 min (7). Finally, the effec-tiveness with which adenosine constricts afferent arterioles ismarkedly affected by the degree of simultaneous activation ofAT1A angiotensin II receptors (76). This effect is mirrored bynumerous observations showing that the magnitude of the TGFresponse depends on ambient levels of angiotensin II and

AT1A receptor engagement. Thus, in AT1A receptor or ACEknockout mice or during treatment with ACE inhibitors orangiotensin receptor blockers, a given stimulus to the maculadensa is much less effective than normally, and the glomerularvascular response to an A1AR agonist is markedly attenuated(51,70,71). Conversely, an increase in angiotensin II levels, byangiotensin infusion for example, appears to enhance the re-sponse to adenosine, and this interaction may to some extent bethe underlying cause for the resetting of the TGF responsealluded to earlier (34,46). The mechanism of the interactionbetween angiotensin II and adenosine is still unclear. Evidencefrom nonrenal tissue would suggest that the simultaneous ac-tivation of PLC through Gq- and Gi-dependent receptorscauses an enhanced release of calcium from intracellular storesthat may include a synergistic component.

SummaryChanges of NaCl and perhaps KCl concentrations at the

macula densa serve as the luminal signal that ultimatelyresults in activation of vascular smooth muscle or granularcells. An increase in luminal NaCl concentration initiates anepithelial cell response that consists of NKCC2-mediatedcell swelling, depolarization, and an increase of cytosoliccalcium. These or some other consequences of NKCC2activation cause the appearance in the juxtaglomerular in-

Figure 7. The effect of elevating NaCl at the macula densa is shown schematically to consist of an increase in the levels of adenosine in thejuxtaglomerular apparatus (JGA) interstitium and adenosine 1 receptors (A1AR)–mediated constriction of the afferent arteriolar smooth musclecells.

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terstitium of adenosine, and the suppression of the release ofPGE2. Adenosine activates A1 adenosine receptors on vas-cular smooth muscle and/or extraglomerular mesangialcells, and this activation results in G�i-dependent activationof phospholipase C, depolarization, activation of voltage-dependent Ca channels, and contraction (Figure 7). A de-crease in luminal NaCl concentration initiates an epithelialresponse that consists of activation of multiple MAP kinasesand stimulation of COX-2 activity and induction of COX-2expression. This is followed by the appearance in the jux-taglomerular interstitium of PGE2 and the suppression ofadenosine. PGE2 activates EP4 receptors on granular cells,and this results in G�s-mediated adenylate cyclase activa-tion and PKA-mediated renin secretory and transcriptionalactivation (Figure 8). Or, maybe more provocatively, onecould envisage two extreme states in which the maculadensa cells exist: one when the luminal NaCl is extremelylow, or when NaCl uptake is pharmacologically inhibited. Inthis situation, the MD cell is a PGE2-producing cell throughthe activation of COX-2. On the other hand, when the NaClconcentration and therefore NKCC2-mediated uptake ismaximal, the MD cell is an ATP/adenosine-releasing cell.

In his book The Kidney, Homer Smith notes about thejuxtaglomerular apparatus that:

Goormaghtigh has called the modified arteriolar wall and itsassociated extravascular cells the juxtaglomerular apparatus;

he believes that it has an important endocrine function, butsome writers have proposed that this apparatus functions inthe control of the renal blood flow, in diuresis, etc. (61).

He himself had not made up his mind about the role of thisanatomical structure. We now can be certain that it serves atleast the two functions that were the topic of this presentation;it controls vascular tone and renin secretion. Nevertheless, inthe words of Homer Smith,

The first lesson of science is that every man must be free toexplore the cosmos in his own way, which is to say that hemust avoid all a priori certitudes. The second lesson ofscience is that no certitudes have been discovered a poste-riori. Scientific truth itself is spelled with a lower case ”t“;it is to be defined as a body of belief which has beenrepeatedly tested and found to be verifiable, but which isnone the less always held subject to re-examination andre-interpretation . . . (60).

Re-examination and re-interpretation indeed!

AcknowledgmentsOver the years, major financial support came from the Deutsche

Forschungsgemeinschaft, the National Institutes of Health, and theAmerican Heart Association.

The partnership with Josie Briggs has been a source of much joy inall aspects of my life. I am grateful for the guidance provided by Hans

Figure 8. The effect of decreasing NaCl concentration at the macula densa is schematically shown to cause an increase of PGE2 in the JGAinterstitium and an EP4-mediated release of renin from granular cells.

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Sarre, Kurt Kramer, Klaus Thurau, and Gerhard Giebisch, and Iacknowledge with gratitude the collaboration with numerous col-leagues: Walter S. Wilde, Wolfram Nagel, Marshall Cortney, DouglasLandwehr, David Z. Levine, Michael Horster, Heinz Valtin, Fred S.Wright, Wilhelm Kriz, Hans-Jurgen Hoehling, Hermann Koepsell,Frank C. Carone, Erik G. Persson, Hans Ulfendahl, Mats Wolgast,Herbert Dahlheim, John M. Bauman, Hartmut Osswald, GiovanniGambaro, Peter C. Weber, Burkhard Scherer, June Mason, DieterHaeberle, Diana Marver, Heino Velasquez, Ole Skott, Joan Keiser,Paul D. Killen, Peter Mundel, Sebastian Bachmann, Evelyne Fischer,Geza Fejes-Toth, Volker Vallon, David E. Smith, Frank Brosius,Richard L. Malvin, Thomas Coffman, Michael Oliverio, John H.Krege, Alan S. Verkman, Tonghui Ma, Mark Knepper, Chung-LinChou, Gary E. Shull, Patrick Schultheis, Kenneth Spring, Helle Prae-torius, Tomas Berl, Luciano Barajas, Linda C. Samuelson, ThomSaunders, John N. Forrest, Ganes Sen, Kenneth Bernstein, FrankSchweda, and Armin Kurtz.

Particular thanks are due to my present and former students, post-doctoral fellows, and chief technicians: Peter Wunderlich, MichaelWahl, Heide Fischbach, Gerd Liebau, John M. Davis, Wolfhart v.Stackelberg, Gunther Grill, Boris Steipe, Bengt Agerup, WolfgangSchutz, Bhomittra Dev, V.N. Puri, Nicholas T. Stowe, David W.Ploth, Christian Drescher, Leon C. Moore, Shiroh Yarimizu, BrendanColgan, Marcos Marin-Grez, Connie L. Davis, Pichian Angchanpen,Hidehisa Soejima, Karyn Todd-Turla, Min Chen, Horst Weihprecht,Peter Sawaya, Aris Urbanes, Bharvi Trivedi, John Lorenz, SuzanneGreenberg, Xiao-Rui He, Wei-Hua Wang, Matthias Kretzler, MichaelP. Harris, Demian Rose, Kimberley Hill, Tingxi Yu, Xiaoquan Shu,Daqing Sun, Timothy Traynor, Zhaohui Wang, Inderjit Singh, SohailA. Hassan, Andrew Pham, Samir Sayegh, Tianxin Yang, John M.Park, Lois J. Arend, Daniel E. Michele, Yuning Huang, YoshimiEndo, Hans Pohl, Rezzan Topaloglu, Anita Pasumarthy, Seiji Hashi-moto, Alexander Paliege, Angelika Luedtke, Pernille Hansen, HayoCastrop, Monika Hermle, Gisela Schubert, Ann Smart, Jo-Ann Davis,Xiulian L. Zhu, and Diane Mizel.

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