Renal Physiology

Homeostasis, the Milieu Interieur, and the Wisdom ofthe Nephron

Melanie P. Hoenig and Mark L. Zeidel

AbstractThe concept of homeostasis has been inextricably linked to the function of the kidneys for more than a centurywhen it was recognized that the kidneys had the ability to maintain the “internal milieu” and allow organisms the“physiologic freedom” to move into varying environments and take in varying diets and fluids. Early ingenious,albeit rudimentary, experiments unlocked a wealth of secrets on the mechanisms involved in the formation ofurine and renal handling of the gamut of electrolytes, as well as that of water, acid, and protein. Recent scientificadvances have confirmed these prescient postulates such that the modern clinician is the beneficiary of a richunderstanding of the nephron and the kidney’s critical role in homeostasis down to the molecular level. Thisreview summarizes those early achievements and provides a framework and introduction for the new CJASNseries on renal physiology.

Clin J Am Soc Nephrol 9: ccc–ccc, 2014. doi: 10.2215/CJN.08860813

IntroductionCritical advances in our understanding of renal phys-iology are unfolding at a rapid pace. Yet, remarkably,the lessons learned from early crude measurements andcareful study still hold true; indeed, classic articles stillserve as the basis for introductory textbooks on renalphysiology and provide a solid working knowledge toclinicians. Drawings with just a handful of transportersat each nephron segment, known for more than halfa century, are sufficient to understand basic mecha-nisms of autoregulation, clearance, and the effects ofdiuretics—the tools needed to care for patients. Yet weclinicians also benefit from a treasure trove of subse-quent scientific advances, which have given us a de-tailed and comprehensive understanding of how thekidney maintains stable body chemistries and volumebalance.

The layers of complexity and the mysteries thatcontinue to unravel make it difficult to stay abreastof current research. Still, the modern nephrologist isin good company. In 1959, a medical student wroteto Homer Smith, the uncontested patriarch of modernnephrology at the time, to inquire about his recti-linear depiction of the nephron (Figure 1) and whyhe failed to mention the counter current theory inhis famous 1956 textbook, the Principles of RenalPhysiology (1,2). Indeed, the structure of the loopof Henle had been well known since the mid-1800s, but the importance of that eponymous struc-ture, the gradient that it generated, and its role inthe final product urine was only just elucidated atthe time of the student’s correspondence. Beforethis, Homer Smith felt that the hairpin turn wasjust a vestige of embryology. This student’s mis-sive was a curiosity, rather than a criticism. Care-fully framed questions have always served to

advance our understanding. In this overview, wewill describe, all too briefly, the ingenious methodsused by early investigators and the secrets theyunlocked to help create the in-depth understandingof renal physiology and pathophysiology that weenjoy today. Additional details will follow in thenew CJASN series of review articles on the physiologyof the kidney.

The Milieu Interieur and the Kidney’s EssentialRoleIn the early 1800s, Darwin’s Theory of Evolution

combined with the recognition that the body chemis-tries of many disparate species were remarkablysimilar led Claude Bernard to develop his theoryof the Milieu Intérieur: “The constancy of the inter-nal environment is the condition of a free and in-dependent existence” (3). By this, he meant that theability of our ancestor organisms to leave the oceansrequired that they develop the ability to “carry theocean with them” in the form of an internal ocean,bathing their cells constantly in fluids that resemblethe very seas from which they evolved. This concept,although reminiscent of the notion of bodily humors(4,5), marked an enormous advance because Bernarddescribed both the features of bodily fluids and theneed to maintain that internal milieu. Maintenance ofthe internal milieu was first called the “wisdom ofthe body” by Starling (6), who recognized that or-ganisms must maintain the constancy of this internalocean despite great fluctuations in diet, fluid intake,and other environmental conditions. The term homeo-stasis was later coined by behaviorist Walter Cannonto describe the physiologic processes that, in ag-gregate, maintain the constancy of the internal

Division ofNephrology, BethIsrael DeaconessMedical Center,Harvard MedicalSchool, Boston,Massachusetts

Correspondence:Dr. Melanie P. Hoenig,Division ofNephrology,Department ofMedicine, Beth IsraelDeaconess MedicalCenter Clinic, Fa 8/185 Pilgrim Road,Boston, MA 02215.Email: MHoenig@bidmc.harvard.edu

www.cjasn.org Vol 9 July, 2014 Copyright © 2014 by the American Society of Nephrology 1

. Published on May 1, 2014 as doi: 10.2215/CJN.08860813CJASN ePress

chemistries, as well as BP, body temperature, and energybalance (7).The earliest insights into renal physiology came from the

assiduous study of anatomy because, to a large degree, renalfunction follows structure. Meticulous drawings and histo-logic study of the animal and human kidney from WilliamBowman (8,9), Jacob Henle (10), and others, complete withcapsule, capillaries, and convoluted tubules were availablein Bernard’s time, yet the mechanisms for the formation ofurine and the kidney’s role in homeostasis were not em-braced until the next century. Until the 1920s, a debate ragedon the mechanism of urine formation. Some researcherschampioned a filtration doctrine and others ascribed secre-tory power to both the glomerulus and tubules (11,12). Thesecretory theory was more popular, however, because thesheer volume of blood that would first need to be filteredand then reabsorbed by the kidney was enormous. HomerSmith noted that the filter-reabsorption strategy “seemedextravagant and physiologically complicated” (2).

Early Investigations Form the FrameworkAn interest in comparative physiology and the advent of

the marine biologic laboratories that studded the Atlanticcoast at the turn of the century helped frame an earlyunderstanding of the kidney and its role in evolution.Remarkably, increasingly complex fish with salty interiorsadapted to fresh water, whereas amphibians rose from thesea to face the challenges mandated by scarce water. In hisfamed opus, Smith summarized the observations to dateand waxed poetic (and philosophic), declaring, “Superfi-cially, it might be said that the function of the kidneys is tomake urine; but in a more considered view, one can saythat the kidneys make the stuff of philosophy itself” (13).This impression, the sense of wonder at the intricate deal-ings of the kidney, permeates the scientific writings fromthat time to this day.

Studies performed in frogs, rabbits, and dogs in the early1900s showed that the constituents of blood and urinediffered because urine contained urea, potassium, andsodium salts, whereas blood contains protein, glucose, andvery little urea. Furthermore, balance studies suggestedthat the volume and constituents of urine changed depend-ing on changing intake or experimental infusions. In his“The Secretion of Urine” monograph published in 1917,Cushny summarized the available literature to date anddescribed the brisk diuresis that followed sodium chlorideinfusions. He also reported findings that showed that thekidney produced acid urine in humans and the carnivora,whereas the herbivora had alkaline urine unless fed a pro-tein diet (14). Despite this careful review and his presen-tation of the “modern view” that acknowledged that bothfiltration and secretion could exist, Cushny struggled withthe data and felt that the theories were “diametrically op-posed” because secretion and reabsorption would result inopposing currents along the renal epithelium. Clearly,methods were needed that could allow direct measure-ment of the filtrate and its modification along the nephron.When Wearn and Richards introduced the micropunc-

ture technique to the study of the kidney (first in amphibia,which had large renal structures amenable to manipula-tion), the debate on the formation of urine was resolved(11,15). By sampling the fluid elaborated from the glomer-ular capsule of a frog, the team demonstrated a protein-free filtrate that was otherwise similar to blood. By contrast,the frog bladder urine had a different composition fromthe blood and was free of glucose. These findings, in lightof earlier data, supported the notion that urine is formedby glomerular filtration, and the urine is then modified inthe tubules, by a combination of reabsorption and secre-tion. Subsequent studies by Walker and others inserted oil“plugs” or “blocks” in various segments of the nephronand distal to the sampling pipette so that the investigatorscould avoid contamination but still study urine from

Figure 1. | Study of the kidney requires consideration of both the role of each segment and the three-dimensional architecture. (A) HomerSmith’s rectilinear nephron mimics the straight trajectory of the fish nephron. (B) The human nephron is more intricate; the distal nephron greets itsown glomerulus before it returns to the environs established by the loop of Henle. TX, treatment. A is modified from reference 1, with permission.

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different segments of the nephron and, therefore, charac-terize each segment’s function, the ions absorbed, and theosmolarity of the fluid (16). These investigators then de-veloped the “stop flow” technique in which theyplaced a pipette distal to the oil droplet, infused fluidinto that segment, and then sampled the fluid at the endof that segment to determine how the fluid had beenaltered (Figure 2A) (17).This meticulous work was confirmed in mammals by

extension of the micropuncture technique to rat and guineapig kidneys (Figure 2B). However, despite the ingenioususe of oil blocks to prevent upstream tubular fluid fromreaching downstream segments and then substituting ar-tificial perfusates, micropuncture studies did not permitcontrol of the composition of fluids on both sides of thetubular epithelium. This limitation was remedied afterWorld War II, when Hans Ussing developed his famedchamber methods (Figure 3). With this strategy, transportacross isolated epithelia could be studied quantitatively bysystematically altering the ionic composition and voltages

of the solutions on either side of the epithelium (18). Care-ful transport studies using model epithelia from nonmam-mals, including the toad bladder, the turtle bladder, andthe flounder bladder, which anatomically and functionallymodel collecting duct principal cells, collecting duct inter-calated cells, and distal tubule cells, respectively, gaveimportant insights into transport mechanisms in thesesegments. In the late 1960s, Burg and colleagues devel-oped methods for isolating and perfusing individualmammalian nephron segments, first from rabbits andthen from mice. These preparations, along with the abilityto measure minute quantities of ions and volumes fromthese tubules with ion-specific electrodes, including thepicapnotherm (which measures minute quantities of carbondioxide), permitted investigators to examine in detail themechanisms, driving forces, and regulation of transportacross individual nephron segments (19). With painstakingeffort, investigators dissected tubules, perfused the segmentwith fluid of specific ion concentrations, and collected the“waste” fluid from the other end of the segment (Figure 4).

Figure 2. | Micropunctureand“stopflow”techniqueswereused tohelpdefine the roleof each segmentof thenephron. (A)Theproximal tubule fromthe kidney of the aquatic salamander is illustrated here. A micropipette removes the filtrate at a point just proximal to a “plug” of mineral oil. To de-termine the role of the tubule in handling of individual constituents (reabsorption, secretion, or diffusion), fluid was injected into the tubule at differentlocations and then collected distally. This “artificial” fluid could be altered to differ from the normal filtrate by one or more constituents. (B) A sketch ofa camara lucida drawing of a guinea pig nephron after microdissection (these drawings were created with the aid of a light projector because pho-tomicrographs were not readily available at the time). Oil or mercury blocks could be inserted at various points along the nephron and fluid from thelumen could be collected and studied. A is modified from reference 17, with permission; B is modified from reference 16, with permission.

Clin J Am Soc Nephrol 9: ccc–ccc, July, 2014 Homeostasis and the Nephron, Hoenig and Zeidel 3

This arrangement allowed investigation of individualsegments of the nephron to better characterize the featuresof transport, electrochemical gradients, coupling with otherions, active versus passive transport, the threshold for reab-sorption, and the permeability to water (1). The resultingflurry of studies, spanning nearly 2 decades, defined thephenomenology and regulation of transport, and identified,at least functionally, the transporter proteins responsible forhomeostasis (20).Meanwhile, in the clinical realm, the flame photometer

became available in the late 1940s and this innovation made itpossible to measure more than a dozen samples of blood forboth sodium and potassium in under an hour. Before thistime, electrolyte measurements were onerous and involvedboth chemical extractions and precipitations (21). Studies ofelectrolytes and the metabolic derangements were now pos-sible; when this process was linked to an autoanalyzer thatalso provided chloride and total CO2, interest in acid-basedisorders soared and the concept of “Gamblegrams” flour-ished (22). Dr. Gamble, a disciple of Henderson, studied arange of different insults from gastrointestinal losses to ad-vanced CKD and their effect on electrolytes, and describedthe kidney as the “remarkable organ of regulation, the kidneysustains the chemical structure of extracellular fluid” (23).Later, availability of the automatic analyzers also spurred a

large literature using metabolic balance studies to character-ize everything from bed rest orwater immersion to the effects

of pharmacologic agents like chlorothiazide (24,25). In thesedetailed studies, investigators characterized vital signs,weight, intake, excretion, electrolytes, clearance, plasma vol-ume, and hormonal levels. These data helped solidify theconcepts of the steady state in homeostasis (26).The stage was now set to identify specific renal trans-

porters, describe how they function, and characterize howthey are regulated. The remarkable reabsorptive task of thenephron tubules requires energy and active transport. Itwas not long after Nobel laureate Jens Skou’s 1957 discov-ery of the Na-K-ATPase in crab nerve microsomes (27,28)that this critical transporter was identified in the kidney.Because of its abundance, the enzyme was identified incrude homogenates of the renal cortex and medulla andNa-K-ATPase activity was later measured in individualsegments of the nephron. The highest activity was in thethick ascending limb and distal convoluted tubule (DCT)and considerable activity was also observed in the proxi-mal tubule. Further study helped identify the polarity ofthe renal epithelial cells with the Na-K-ATPase at the baso-lateral membrane. This finding helped solidify the conceptthat energy generated from this housekeeping enzyme,which maintains the normal cellular ion concentration, isharnessed by the kidney to reabsorb the bulk of the filteredsodium along with a host of other substances (29).With a map in place for the role of each segment of the

nephron and a solid understanding of factors that influence

Figure 3. | TheUssingChamber can be used tomeasure ion transport between the two sides of an epithelial cellmembrane by polarized cells.Here, a monolayer of epithelial cells separates two compartments. Fluid in the two compartments is identical to eliminate the contribution ofpassive paracellular diffusion driven by differences in concentration, osmotic pressure, or hydrostatic pressure. Voltage electrodes placed nearthe epithelial membranemaintain the potential difference at zero so that the current measured by the current electrodes reflects the movementof ions by active transport through the epithelial cells.

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the actions in those segments, research efforts by a multitudeof investigators have set out to characterize the wide arrayof transporters in the nephron, their molecular structure,their distribution in the nephron, and their role in normalphysiology and disease. This effort has been aided bysophisticated and rapidly evolving techniques with PCR,cloning and amplification of cDNAs, and expression inXenopus oocytes, knockout mice, genome-wide associationstudies, and other models.

The Ultrastructure of the Glomerulus, the Concept ofClearance, and AutoregulationOnce the debate on the formation of urine was settled

and it was clear that urine was formed by filtration,reabsorption, and secretion, mysteries of the elegant filtra-tion design were explored and the concept of clearance wasdeveloped. Use of scanning electron microscopy allowedresearchers to appreciate the three-dimensional structure ofthe cells and the ingenious design of the glomerulus.Podocytes were visualized extending their primary, sec-ondary, and tertiary projections to interdigitate with neigh-boring “feet” and form the filtration slit diaphragm.Meanwhile, within the capillary bed, the delicate fenes-trated endothelium drapes the basement membrane. Aseries of investigations into the dynamics of glomerularultrafiltration, first in a unique strain of Wistar rats withsurface glomeruli and later in primates, helped definefactors that create the net driving force for filtrationand provided the mathematical framework for our currentunderstanding of these forces (30). In addition, studies onthe permselectivity of the glomerular capillary wall usingferritin molecules of various sizes that were neutral, anionic,or cationic revealed that particles were restricted based onboth charge and size; this explained the limited clearance ofalbumin at 39 A°, which is smaller than the 42 A° poresobserved in the glomerular endothelial cell (31).More recently, new techniques have helped further

characterize these observations. For example, models ofnephron development, such as the zebrafish, transparentand rapidly growing fish with a single pair of nephrons,have been indispensable to determining the effects of singledefects on kidney function and development (32). Myriad

proteins that form the filtration slit diaphragm are charac-terized and defects in several of these proteins can predict-ably cause the nephrotic syndrome. Details of the complexmeshwork of the basement membrane, a joint effort by theendothelial and epithelial cells, can result in thin basementmembrane disease or hereditary syndromes, such as Alportsyndrome. The elixir, vascular endothelial growth factor, ap-pears to support the endothelium; when it is compromised,endothelial cells cannot sustain the regular challenges re-quired to support normal structure and function of thisunique capillary bed and, thus, thrombosis and endothelialinjury can occur. In addition, direct micropuncture of glo-meruli allowed a detailed description of normal glomerularhemodynamics and identified the derangements in glomer-ular function that occur with disease. Application of glo-merular micropuncture in animals with glomerulardamage caused by hypertension, diabetes, or loss of renalmass led directly to current therapies, including dietaryprotein restriction and use of angiotensin-convertingenzyme inhibition.The role of the glomerulus in “clearance” has provoked

significant inquiry as well. Early clinical investigators ex-plored the clearance of urea and creatinine after ingestionor determined the clearance with the infusion equilibriummethods, first with inulin and later with a host of radio-active markers, such as 125I-iothalamate. When it becamepossible to measure the low concentrations of creatinine inserum, use of endogenous creatinine to calculate and laterestimate clearance became possible. The pitfalls of thisstrategy (e.g., contribution from secretion, variability basedon muscle mass, and the changes in serum levels related todiet and volume status, all of which make it less precisethan the measured GFR) are more than balanced by theconvenience (33).It was soon evident that massive daily glomerular

filtration could translate into life-threatening losses if therewere no mechanisms in place to limit them in the event oflow perfusion. Some researchers heralded these strategiesas “acute renal success” (34); however, further study of theintegrated response that maintains the GFR over a widerange of perfusion pressures provided an understandingof the roles of the juxtaglomerular apparatus in autoregu-lation and tubuloglomerular feedback (35).

Figure 4. | Study of isolated perfused tubular segments allowed study of each of the different nephron segments independently. (A) Aphotomicrograph of a portion of a rabbit proximal convoluted tubule during perfusion. (B) A schematic diagram of the technique. One end ofthe dissected tubule was connected to a micropipette, which was used to perfuse the lumen, and the other end was connected to a collectionmicropipette. Both the luminal fluid and the peritubular fluid could be controlled to assess tubular transport characteristics. A is reprinted withpermission from Burg MB: Perfusion of isolated renal tubules. Yale J Biol Med 45: 321–326, 1972.

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Sodium and Water HomeostasisSodium, the major extracellular cation, plays a pivotal

role in the maintenance of extracellular fluid volume andperfusion of vital organs and capillary beds. The kidneyhas an elaborate array of sodium transporters throughoutthe nephron (36). In the proximal tubule, it is linked to anelegant mechanism to reabsorb the filtered bicarbonateload by excreting H1 ions with the electroneutral antipor-ters or Na1/H1 exchangers. Reabsorption of the amplefiltered sodium also plays an important role in the reab-sorption of glucose, sulfate, phosphate, and several aminoacids. The remaining fraction of filtered sodium is reabsorb-ed with unique transporters in each of the subsequentnephron segments in which apical reabsorption of sodiumis rate limiting. These transporters include the furosemide-sensitive channel in the loop of Henle, the thiazide-sensitivesodium chloride cotransporter that is primarily in the DCT,and the epithelial sodium channel transporter that is locatedprimarily in the collecting tubules (Figure 5).Our understanding of the mechanisms of sodium trans-

port along the nephron comes from disparate sources. Theadvent of sulfonamides, investigated initially as much-needed antibiotics after World War II, were promptlyrecognized for their saluretic effects and soon revealed awealth of secrets regarding the transport of sodiumthroughout the nephron (37). Genetic disorders also pro-vided important clues. Endocrinologist Frederick Bartterand others described a set of youths afflicted with growthand mental retardation, muscle cramps, salt craving,

polyuria, and polydipsia. Bartter initially attributed hiseponymous syndrome to a state of aldosterone excesswith angiotensin resistance but when three-fourths adre-nalectomy did not resolve the defect, he focused on theloop of Henle. Subsequent contributions from physiolo-gists helped distinguish this disorder from Gitelman’s syn-drome and helped define the interplay of transporters inthe loop of Henle and the DCT. However, it was the stun-ning characterizations by geneticists that helped identifymutations in several genes; these discoveries explained thesubtle differences in the phenotype of these disorders andwill be carefully considered within this series (38,39)An endocrinopathy was also the initial theory that Dr.

Liddle invoked to describe a family with early onset severehypertension and hypokalemia that was notable for sup-pressed renin and aldosterone. As soon as the epithelialsodium channel was characterized, investigators demon-strated complete linkage in affected individuals with adefect in this transporter that resulted in a constitutivelyactive sodium channel (40). Insights into the molecular bi-ology, structure, function, and regulation of each of thesesodium transporters has clearly enriched our grasp of re-nal physiology and complemented earlier predictions.Hormonal and sympathetic nervous input can greatly

augment sodium reabsorption, particularly by angiotensin IIin the proximal tubule and aldosterone in the distal nephron,whereas the effect of atrial natriuretic peptide in themedullarycollecting duct was found to be the opposite (41). Knowledgeof these transporters is critical to the understanding of the

Figure 5. | The unique transporters and cell structure of each segment of the nephron work in concert to maintain homeostasis. ENaC,epithelial sodium channel; NKCC2, Na+-K+-2Cl cotransporter; ROMK, renal outer medullary potassium.

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clinical use of diuretics and the care of patients with a widevariety of issues, from the patient with essential hypertensionto the complex patient with cirrhosis.In the 1960s, Guyton proposed that all hypertension,

ultimately, is a result of the failure of the kidney to excretethe excess of total body sodium with a normal pressurenatriuresis (42). Although this theory has been disputedover the years, it is notable that monogenic defects thatlead to hypertension or hypotension are found exclusivelyin genes that encode either renal transporter proteins orproteins that regulate the function of renal transporter pro-teins and ultimately renal sodium handling.Despite mounting evidence on the importance of sodium

in the kidney’s role in homeostasis, investigators in the1950s soon recognized that the measured serum sodiumcorrelated poorly with the total body sodium by compar-ing these values in heterogeneous patients with a varietyof chronic conditions. Instead, the serum sodium corre-lated well with the serum osmolality (particularly whencorrections were made for the osmotic contributions ofglucose and nonprotein nitrogen) (43). (Of note, the sameinvestigators also recognized that the Na21-K1/total bodywater ratio correlated closely with “corrected” serum so-dium and explained the importance for accounting of po-tassium repletion during the treatment of hyponatremia.)Maintenance of the plasma osmolality was noted to betightly regulated by both the release of vasopressin andthe kidneys’ response (44). This interplay between the twois essential for water homeostasis, a critical factor in themaintenance of cell volume. Although cells have devel-oped strategies to deal with excess or insufficient water,these volume regulatory changes require extrusion or in-clusion of electrolytes, which alters the cellular interiormilieu and wreaks havoc on normal cellular function.Later adaptations allow cells to return toward normalcybut only within a small range. Water reabsorption requiresthe ability to both establish an osmotic gradient in thekidney and to reabsorb water from the urinary filtrate.The kidney has an elegant strategy to concentrate or dilutethe urine by its response to vasopressin and the ability todeploy aquaporins to the luminal membrane (45). At leastseven aquaporin isoforms are expressed in the kidney andplay important roles at different sites. In the proximal tu-bule and thin descending limb, aquaporin 1 appears toserve as the dominant gateway for water reabsorption,whereas trafficking of aquaporin 2 along cytoskeletal ele-ments in the collecting duct cells allows reabsorption ofwater and urine concentration in the principal cells ofthe collecting ducts (46). Detailed study of the molecularstructure and cell physiology of these transporters has al-lowed insight into the rare genetic diseases that affectaquaporins, such as congenital nephrogenic diabetes insipidusand the common acquired defects related to lithium, cal-cium, and even urinary obstruction. Similarly, study of thevasopressin receptor has resulted in new strategies andpharmacologic agents for the treatment of states of excessantidiuretic hormone and polycystic kidney disease.

Acid-Base HomeostasisMaintenance of pH is a critical activity of the kidney and

is essential for normal cellular function because the pH

dictates the charged state of proteins that affects confor-mational shape, enzymatic activity, binding, and cellulartransport and, thus, allows proteins to perform essentialmetabolic functions. Although some acid-base enthusiastsenjoy consideration of the “strong ion difference” to rec-oncile data, the normal kidney’s remarkable response tosubtle differences in pH or, more likely, intracellular CO2

does not take these differences into account. Although theexact mechanisms used to sense pH are still not yet un-derstood, it is well known that the kidney plays a domi-nant role in the regulation of the acid-base balance (47).Indeed, with acidosis, a complex intracellular cascade en-sues, including activation of the electroneutral sodium-coupled amino acid transporter for glutamine, an increasein glutamine metabolism and ammoniagenesis, as well asan increase in the expression of the sodium hydrogen anti-porter (48,49). Additional orchestrated responses in thedistal nephron with the help of the machinery in the in-tercalated cells and neighboring principal cells contributeto the kidneys’ response to the challenges of acidemia (50).By contrast, metabolic alkalosis can be easily and rapidly

handled by the kidney by excretion of excess filtered bi-carbonate or may be perpetuated by the kidney because of aresponse to volume depletion with secondary activation ofthe renin-angiotensin and aldosterone axis or from primaryhyperaldosteronism. These processes are intimately linked tothe regulation of potassium as well. Finally, the kidneyresponds to the challenges of primary respiratory disordersto offset the effects of these disturbances in a predictablefashion (51,52). In chronic respiratory alkalosis, the kidneycan decrease acid excretion by decreasing ammonia produc-tion and bicarbonate retention; in respiratory acidosis, activ-ity of the Na-H antiporter is augmented so that morebicarbonate is reabsorbed (53). Understanding of these com-plex processes helps in the care of actual patients who de-velop acid-base disorders from a wide variety of insults.

Potassium HomeostasisExcretion of potassium in excess of the amount filtered is

another triumph of the kidney in electrolyte homeostasis andits mechanism, another important milestone in the under-standing of renal pathophysiology (54–56). Potassium, themajor cation in the body, must be maintained in high con-centrations in the intracellular space and a low concentrationin the extracellular fluid to allow both normal cellular func-tion and the considerable gradient required for excitation ofnerves and contractions of muscles. The kidney plays its roleby reabsorption of nearly all of the filtered load proximallyand variable secretion in the distal nephron. Along the way,potassium is secreted into the lumen with the help of therenal outer medullary potassium transporter, which providessufficient substrate to the NaKC2 transporter, and by the“big potassium,” “maxi,” or high conductance transporter(57). Both appear to play an important role in the secretionof potassium in the distal nephron dictated by the influenceof aldosterone and magnitude of distal flow (58).

Divalent Cations and Phosphate HomeostasisThe kidney plays a critical role in maintaining both normal

extracellular calcium ion levels and the vast repositories of

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calcium needed for normal intracellular function and main-tenance of the skeleton. Integrated control by parathyroidhormone and 1,25-dihydroxyvitamin D helps achieve thisend (59). Calcium is reabsorbed through a paracellular routein the proximal tubule and the thick ascending limb,whereas there are unique, well characterized transient recep-tor potential ion channels in the DCT. Each of these regionsis controlled by local effects prescribed by the calcium sens-ing receptor and modulated by the pH (60). There is also aninteresting pas de deux between sodium and calcium han-dling. Volume depletion or decreased sodium delivery de-creases urinary calcium either by increasing proximalreabsorption or by promoting reabsorption in the DCTwith changes in the activity of the basolateral Na1/Ca1

exchanger or hyperpolarization of the luminal plasmamembrane (61,62). The fate of magnesium homeostasis isintimately linked to that of other cations. In parallel tocalcium, the majority of filtered magnesium is reabsorbedin the proximal tubule and thick ascending limb by para-cellular movement mediated, in part, by the claudin pro-teins, which govern ion movement through the otherwisetight junctions in those regions (63). In addition, the actionof the renal outer medullary potassium transporter to sup-ply more potassium in the lumen for the NaKC2 trans-porter is thought to create a lumen-positive transepithelialpotential difference that can favor cation reabsorption inthis segment (64). By contrast, in the DCT, movement ofMg21 is an active transcellular process. At this segment,cation channels from the melastin transient receptor po-tential subfamily play a major role in this endeavor, evi-denced by Mg21 wasting seen in the rare genetic disorderswith defects in this channel (65).Emerging details on phosphate metabolism have

identified a family of sodium phosphate transporters thathelp reabsorb the bulk of the filtered phosphate in theproximal tubule (66). These transporters appear to be affectedby a series of factors, including the recently characterizedfibroblast growth factor 23 and its obligate coreceptor Klotho,which together, via the fibroblast growth factor receptor,inhibit the reabsorption of sodium-dependent phosphatereabsorption and lower vitamin D levels by downregulat-ing the gene for 1a-hydroxylase (67). Phosphate that es-capes proximal reabsorption and is delivered distally isavailable to bind H1 as an important source of “titratableacid” (68).

Protein MetabolismEven Richard Bright (69) in the 1820s recognized that in

“dropsy” (or edema) of renal origin, urea was increased inthe blood and decreased in the urine such that urea couldserve as a marker of kidney failure. One hundred years later,Thomas Addis tried to assess renal function using ureaclearance and “rest” the kidneys from the “work” of clearingproteins by prescribing a low-protein diet (70). Landmarkstudies by Brenner’s group suggested that this strategy wascorrect because high-protein diets fed to laboratory animalscan be shown to increase renal blood flow and glomerularfiltration and subsequently contribute to the progression ofCKD (71). Nevertheless, the specific mediators that lead tohyperfiltration and contribute to the changes seen with ahigh-protein diet are yet to be determined.

Renal Physiology Has Significant Clinical RelevanceOne of the considerable gifts to the field of nephrology is

that there is a deep and growing understanding of theintricacies of kidney function and the ingeniousmethods thatthe kidney uses to govern homeostasis. These discoveriescomplement observations made with careful considerationand primitive measurements in the past. Predictions on themovement of ions have been translated by detailed charac-terization, on the molecular level, of ion transporters andprovide insight into the integrated responses of the kidney tothe maintenance of the internal milieu. Those of us who areprivileged enough to care for patients with disorders of thekidney can utilize knowledge gleaned in the laboratory tounderstand real clinical concerns.Because it is difficult for any practicing nephrologist to

stay abreast of the rapidly unfolding revelations, this newrenal physiology series will serve as an update to thecurrent understanding of the nephron. CJASN will providecareful reviews of the nephron, sequentially, from the glo-merulus to the collecting duct, followed by a review on thecontrol of urinary drainage and bladder function. Next,there will be a series of cohesive reviews that will addresshow the kidney factors in the integrated response to so-dium and water homeostasis, potassium handling, acid-base homeostasis, excretion of organic cations and anions,and divalent cations and phosphate homeostasis. Proteinmetabolism and control of renal nitrogen excretion as wellas sensory functions of the kidney will follow. Finally, therole of the interstitium and hormonal function of the kid-ney will be considered.In jingoistic banter that many nephrologists can echo

with sincerity, Homer Smith asserted, “The responsibilityfor maintaining the composition of [the internal milieu] . . .devolves to the kidneys. It is no exaggeration to say thatthe composition of the body fluids is determined not bywhat the mouth takes in but by what the kidneys keep;they are the master chemists of our internal environment”(13).With this series in hand, the CJASN reader will beprivy to the cutting-edge science of nephrology; knowl-edge of the dramatic advances in our understanding willlikely turn any nephrologist into a philosopher.

Authors’ NoteThe landmark works described in this article are freely

accessible on the Internet for those who would like toindulge in the primary sources. These texts and manu-scripts have been made available as part of Google Scholarand the Internet Archive, nonprofit digital libraries withthe mission to allow universal access to all knowledge.These archival texts include Homer Smith’s The Principalsof Renal Physiology and From Fish to Philosopher, Bowman’streatise On the Structure and Use of the Malpighian Bodies ofthe Kidney, and texts by Starling, Cushny, Cannon, andBernard.

DisclosuresNone.

References1. Smith HW: Principles of Renal Physiology, New York, Oxford

University Press, 1956

8 Clinical Journal of the American Society of Nephrology

2. SmithHW: The fate of sodium andwater in the renal tubules. BullN Y Acad Med 35: 293–316, 1959

3. Bernard C: Lecons sur les phenomenes de la vie communs auxanimaux et aux vegetaux, Paris, Bailliere JB, 1878

4. Bernard C: An Introduction to the Study of Experimental Medi-cine 1865, London, Macmillan & Co Ltd, 1927

5. Gross CG: Claude Bernard and the constancy of the internalenvironment. Neuroscientist 4: 380–385, 1998

6. Starling EH: The Harvreian Oration, delivered before The RoyalCollege of Physicians of London on St. Luke’s Day, 1923. BMJ 2:685–690, 1923

7. CannonWB:Wisdom of the Body, New York,WWNorton, 19398. Bowman W: On the structure and use of the malphighian bodies

of the kidney, with observations on the circulation through thatgland. Philos Trans R Soc Lond 132: 57–80, 1942

9. Eknoyan G: Sir William Bowman: His contributions to physiol-ogy and nephrology. Kidney Int 50: 2120–2128, 1996

10. Kinne-Saffran E, Kinne RK: Jacob Henle: The kidney and beyond.Am J Nephrol 14: 355–360, 1994

11. Wearn HT, Richards AN: Observations on the composition ofglomerular urine, with particular reference to the problem ofreabsorption in the renal tubules. Am J Phys 71: 209–227, 1924

12. Gottschalk CW: A history of renal physiology to 1950. in: TheKidney: Physiology and Pathophysiology, edited by Seldin DW,Giebisch G , 2nd Ed., New York, Raven Press, Ltd, 1992

13. Smith HW: From Fish to Philosopher, Garden City, NY, Dou-bleday and Company, Inc, 1961

14. Cushny AR: The secretion of urine. In: Monographs on Physiol-ogy, edited by Starling EH, London, Longmans, Green and Co.,1917

15. Sands JM: Micropuncture: Unlocking the secrets of renal func-tion. Am J Physiol Renal Physiol 287: F866–F867, 2004

16. Walker AM, Bott PA, Oliver J, MacDowell MD: The collectionand analysis of fluid from single nephrons of the mammaliankidney. Am J Physiol 134: 580–595, 1941

17. Richards AN, Walker AM: Methods of collecting fluid fromknown regions of the renal tubules of amphibia and of perfusingthe lumen of a single tubule. Am J Physiol 118: 111–120, 1937

18. Ussing HH, Zerahn K: Active transport of sodium as the source ofelectric current in the short-circuited isolated frog skin. ActaPhysiol Scand 23: 110–127, 1951

19. Burg MB, Grantham J, Abramow M, Orloff J, Schafer JA: Prepa-ration and study of fragments of single rabbit nephrons. J Am SocNephrol 8: 675–683, 1997

20. Burg MB, Knepper MA: Single tubule perfusion techniques.Kidney Int 30: 166–170, 1986

21. Peitzman SJ: The flame photometer as engine of nephrology: Abiography. Am J Kidney Dis 56: 379–386, 2010

22. Gamble JL: Chemical Anatomy, Physiology and Pathology ofExtracellular Fluid, A Lecture Syllabus, Cambridge, HarvardUniversity Press, 1942

23. Harvey AM: Classics in clinical science: James L. Gamble and“Gamblegrams”. Am J Med 66: 904–906, 1979

24. HollanderW, Chobanian AV,Wilkins RW: Relationship betweendiuretic and antihypertensive effects of chlorothiazide and mer-curial diuretics. Circulation 19: 827–838, 1959

25. Chobanian AV, Lille RD, Tercyak A, Blevins P: Themetabolic andhemodynamic effects of prolonged bed rest in normal subjects.Circulation 49: 551–559, 1974

26. Bonventre JV, Leaf A: Sodium homeostasis: Steady stateswithout a set point. Kidney Int 21: 880–883, 1982

27. Skou JC: The influence of some cations on an adenosine tri-phosphatase from peripheral nerves. Biochim Biophys Acta 23:394–401, 1957

28. Skou JC: Nobel Lecture. The identification of the sodium pump.Biosci Rep 18: 155–169, 1998

29. Katz AI: Renal Na-K-ATPase: Its role in tubular sodium and po-tassium transport. Am J Physiol 242: F207–F219, 1982

30. Maddox DA, Deen WM, Brenner BM: Dynamics of glomerularultrafiltration. VI. Studies in the primate. Kidney Int 5: 271–278,1974

31. Bohrer MP, Baylis C, Humes HD, Glassock RJ, Robertson CR,Brenner BM: Permselectivity of the glomerular capillary wall.Facilitated filtration of circulating polycations. J Clin Invest 61:72–78, 1978

32. Drummond IA: Kidney development and disease in the zebrafish.J Am Soc Nephrol 16: 299–304, 2005

33. Narayanan S, Appleton HD: Creatinine: A review.Clin Chem 26:1119–1126, 1980

34. Thurau K, Boylan JW: Acute renal success. The unexpected logicof oliguria in acute renal failure. Am J Med 61: 308–315, 1976

35. Peti-Peterdi J, Harris RC: Macula densa sensing and signalingmechanisms of renin release. J Am Soc Nephrol 21: 1093–1096,2010

36. Feraille E, Doucet A: Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: Hormonal control.Physiol Rev 81: 345–418, 2001

37. Suki W, Rector FC Jr, Seldin DW: The site of action of furosemideand other sulfonamide diuretics in the dog. J Clin Invest 44:1458–1469, 1965

38. Bartter FC, Pronove P, Gill JR Jr, MacCardle RC: Hyperplasia ofthe juxtaglomerular complex with hyperaldosteronism and hy-pokalemic alkalosis. A new syndrome. Am J Med 33: 811–828,1962

39. Proesmans W: Threading through the mizmaze of Bartter syn-drome. Pediatr Nephrol 21: 896–902, 2006

40. Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C,Hansson JH, Schambelan M, Gill JR Jr, Ulick S, Milora RV,Findling JW, Canessa CM, Rossier BC, Lifton RP: Liddle’s syn-drome: Heritable human hypertension caused by mutations inthe beta subunit of the epithelial sodium channel. Cell 79: 407–414, 1994

41. Zeidel ML, Kikeri D, Silva P, Burrowes M, Brenner BM: Atrialnatriuretic peptides inhibit conductive sodium uptake by rabbitinner medullary collecting duct cells. J Clin Invest 82: 1067–1074, 1988

42. Montani J, Van Vliet BN: Understanding the contribution ofGuyton’s large circulatory model to long-term control of arterialblood pressure. Exp Physiol 94: 382–398, 2009

43. Edelman IS, Leibman J, O’Meara MP, Birkenfeld LW: Inter-relations between serum sodium concentration, serum osmo-larity and total exchangeable sodium, total exchangeablepotassium and total body water. J Clin Invest 37: 1236–1256,1958

44. Verney EB: Renal excretion of water and salt. Lancet 273: 1237–1242, 1957

45. Knepper MA: Molecular physiology of urinary concentratingmechanism: Regulation of aquaporins water channels by vaso-pressin. Am J Physiol Renal Physiol 272: F3–F12, 1997

46. Nielsen S, Frøkiaer J, Marples D, Kwon TH, Agre P, Knepper MA:Aquaporins in the kidney: From molecules to medicine. PhysiolRev 82: 205–244, 2002

47. Pitts RF, Lotspeich WD, Schiess WA, Ayer JL, Miner P: The renalregulation of acid-base balance in man; the nature of themechanism for acidifying the urine. J Clin Invest 27: 48–56, 1948

48. Boron WF: Acid-base transport by the renal proximal tubule. JAm Soc Nephrol 17: 2368–2382, 2006

49. Curthoys NP, Gstraunthaler G: Mechanism of increased renalgene expression during metabolic acidosis. Am J Physiol RenalPhysiol 281: F381–F390, 2001

50. Koeppen BM: The kidney and acid-base regulation. Adv PhysiolEduc 33: 275–281, 2009

51. Arbus GS, Herbert LA, Levesque PR, Etsten BE, Schwartz WB:Characterization and clinical application of the “significanceband” for acute respiratory alkalosis.NEngl JMed 280: 117–123,1969

52. Brackett NC Jr, Wingo CF, Muren O, Solano JT: Acid-base re-sponse to chronic hypercapnia in man. N Engl J Med 280: 124–130, 1969

53. Krapf R, Pearce D, Lynch C, Xi XP, Reudelhuber TL, Pouyssegur J,Rector FC Jr: Expression of rat renal Na/H antiporter mRNA levelsin response to respiratory and metabolic acidosis. J Clin Invest87: 747–751, 1991

54. Berliner RW, Kennedy TJ Jr: Renal tubular secretion of potas-sium in the normal dog. J AmSocNephrol9: 1341–1344, discussion1344–1345, 1998

55. Malnic G, Klose RM, Giebisch G: Micropuncture study of renalpotassium excretion in the rat. Am J Physiol 206: 674–686, 1964

56. Giebisch G: Renal potassium transport: Mechanisms and regu-lation. Am J Physiol 274: F817–F833, 1998

Clin J Am Soc Nephrol 9: ccc–ccc, July, 2014 Homeostasis and the Nephron, Hoenig and Zeidel 9

57. Greger R, Schlatter E, Hebert SC: Milestones in nephrology:Presence of luminal K1, a prerequisite for active NaCl transportin the cortical thick ascending limb of Henle’s loop of rabbitkidney. J Am Soc Nephrol 12: 1788–1793, 2001

58. Sansom SC,Welling PA: Two channels for one job. Kidney Int 72:529–530, 2007

59. PeacockM: Calciummetabolism in health and disease.Clin J AmSoc Nephrol 5[Suppl 1]: S23–S30, 2010

60. Felsenfeld AJ, Levine BS: Milk alkali syndrome and the dynamicsof calcium homeostasis. Clin J Am Soc Nephrol 1: 641–654,2006

61. Nijenhuis T, Vallon V, van der Kemp AW, Loffing J, HoenderopJG, Bindels RJ: Enhanced passive Ca21 reabsorption and re-duced Mg21 channel abundance explains thiazide-inducedhypocalciuria and hypomagnesemia. J Clin Invest 115: 1651–1658, 2005

62. Gagnon KB, Delpire E: Physiology of SLC12 transporters: Lessonsfrom inherited human genetic mutations and genetically en-gineeredmouse knockouts.Am J Physiol Cell Physiol 304: C693–C714, 2013

63. Hou J, Rajagopal M, Yu ASL: Claudins and the kidney. Annu RevPhysiol 75: 479–501, 2013

64. Ferre S, Hoenderop JG, Bindels RJM: Sensing mechanisms in-volved in Ca21 and Mg21 homeostasis. Kidney Int 82: 1157–1166, 2012

65. Dimke H, Monnens L, Hoenderop JG, Bindels RJ: Evaluation ofhypomagnesemia: Lessons from disorders of tubular transport.Am J Kidney Dis 62: 377–383, 2013

66. Lederer E, Miyamoto K: Clinical consequences of mutations insodium phosphate cotransporters. Clin J Am Soc Nephrol 7:1179–1187, 2012

67. HuMC, Shiizaki K, Kuro-oM,MoeOW: Fibroblast growth factor 23and Klotho: Physiology and pathophysiology of an endocrine net-work of mineral metabolism. Annu Rev Physiol 75: 503–533, 2013

68. ScheissWA, Ayer JL, LotspeichWD, Pitts RF: The renal regulationof acid-base balance in man. Factors affecting the excretion oftitratable acid by the normal human subject. J Clin Invest 27: 57–64, 1948

69. Jay V: Richard Bright—physician extraordinaire. Arch Pathol LabMed 124: 1262–1263, 2000

70. Lemley KV, Pauling LS: Thomas Addis: 1881-1949, Washington,DC, National Academy of Sciences, 1994

71. Brenner BM,Meyer TW,Hostetter TH: Dietary protein intake andthe progressive nature of kidney disease: The role of hemody-namically mediated glomerular injury in the pathogenesis ofprogressive glomerular sclerosis in aging, renal ablation, andintrinsic renal disease. N Engl J Med 307: 652–659, 1982

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10 Clinical Journal of the American Society of Nephrology