Chapter 303: Cellular and Molecular Biology of the Kidney

The kidney is one of the most highly differentiated organs in the body. At the conclusion of embryologic development, nearly 30 different cell types form a multitude of filtering capillaries and segmented nephrons enveloped by a dynamic interstitium. This cellular diversity modulates a variety of complex physiologic processes. Endocrine functions, the regulation of blood pressure and intraglomerular hemodynamics, solute and water transport, acid-base balance, and removal of drug metabolites are all accomplished by intricate mechanisms of renal response. This breadth of physiology hinges on the clever ingenuity of nephron architecture that evolved as complex organisms came out of water to live on land.

Kidneys develop from intermediate mesoderm under the timed or sequential control of a growing number of genes, described in Fig. 303-1. The transcription of these genes is guided by morphogenic cues that invite two ureteric buds to each penetrate bilateral metanephric blastema, where they induce primary mesenchymal cells to form early nephrons. The two ureteric buds emerge from posterior nephric ducts and mature into separate collecting systems that eventually form a renal pelvis and ureter. Induced mesenchyme undergoes mesenchymal epithelial transitions to form comma-shaped bodies at the proximal end of each ureteric bud leading to the formation of S-shaped nephrons that cleft and enjoin with penetrating endothelial cells derived from sprouting angioblasts. Under the influence of vascular endothelial growth factor A (VEGF-A), these penetrating cells form capillaries with surrounding mesangial cells that differentiate into a glomerular filter for plasma water and solute. The ureteric buds branch and each branch produce a new set of nephrons. The number of branching events ultimately determines the total number of nephrons in each kidney. There are ~900,000 glomeruli in each kidney in normal birth weight adults and as few as 225,000 in low-birth-weight adults, with the latter producing numerous comorbid risks.

Glomeruli evolve as complex capillary filters with fenestrated endothelia under the guiding influence of VEGF-A and angiopoietin-1 secreted by adjacently developing podocytes. Epithelial podocytes facing the urinary space envelop the exterior basement membrane supporting these emerging endothelial capillaries. Podocytes are partially polarized and periodically slough into the urinary space by epithelial-mesenchymal transition, and to a lesser extent apoptosis, only to be replenished by migrating parietal epithelia from Bowman capsule. Impaired replenishment results in heavy proteinuria. Podocytes attach to the basement membrane by special foot processes and share a slit-pore membrane with their neighbor. The slit-pore membrane forms a filter for plasma water and solute by the synthetic interaction of nephrin, annexin-4, CD2AP, FAT, ZO-1, P-cadherin, podocin, TRPC6, PLCE1, and Neph 1-3 proteins. Mutations in many of these proteins also result in heavy proteinuria. The glomerular capillaries are embedded in a mesangial matrix shrouded by parietal and proximal tubular epithelia forming Bowman capsule. Mesangial cells have an embryonic lineage consistent with arteriolar or juxtaglomerular cells and contain contractile actin-myosin fibers. These mesangial cells make contact with glomerular capillary loops, and their local matrix holds them in condensed arrangement.

Between nephrons lies the renal interstitium. This region forms a functional space surrounding glomeruli and their downstream tubules, which are home to resident and trafficking cells such as fibroblasts, dendritic cells, occasional lymphocytes, and lipid-laden macrophages. The cortical and medullary peritubular capillaries, which siphon off solute and water following tubular reclamation of glomerular filtrate, are also part of the interstitial fabric as well as a web of connective tissue that supports the kidney’s emblematic architecture of folding tubules. The relational precision of these structures determines the unique physiology of the kidney.

Each nephron is partitioned during embryologic development into a proximal tubule, descending and ascending limbs of the loop of Henle, distal tubule, and the collecting duct. These classic tubular segments build from subsegments lined by highly unique epithelia serving regional physiology. All nephrons have the same structural components, but there are two types whose structures depend on their location within the kidney. The majority of nephrons are cortical, with glomeruli located in the mid-to-outer cortex. Fewer nephrons are juxtamedullary, with glomeruli at the boundary of the cortex and outer medulla. Cortical nephrons have short loops of Henle, whereas juxtamedullary nephrons have long loops of Henle. There are critical differences in blood supply as well. The peritubular capillaries surrounding cortical nephrons are shared among adjacent nephrons. By contrast, juxtamedullary nephrons depend on individual capillaries called vasa recta that run alongside the long loops of Henle. Cortical nephrons perform most of the glomerular filtration because there are more of them and because their afferent arterioles are larger than their respective efferent arterioles. The juxtamedullary nephrons, with longer loops of Henle, create an osmotic gradient for concentrating urine. How developmental instructions specify the differentiation of all these unique epithelia among various tubular segments is still unknown.

Renal blood flow normally drains ~20% of the cardiac output, or 1000 mL/min. Blood reaches each nephron through the afferent arteriole leading into a glomerular capillary where large amounts of fluid and solutes are filtered to form the tubular fluid. The distal ends of the glomerular capillaries coalesce to form an efferent arteriole leading to the first segment of a second capillary network (cortical peritubular capillaries or medullary vasa recta) surrounding the tubules (Fig. 303-2A). Thus, nephrons have two capillary beds arranged in a series separated by the efferent arteriole that regulates the hydrostatic pressure in both capillary beds. The distal capillaries empty into small venous branches that coalesce into larger veins to eventually form the renal vein.

The hydrostatic pressure gradient across the glomerular capillary wall is the primary driving force for glomerular filtration. Oncotic pressure within the capillary lumen, determined by the concentration of unfiltered plasma proteins, partially offsets the hydrostatic pressure gradient and opposes filtration. As the oncotic pressure rises along the length of the glomerular capillary, the driving force for filtration falls to zero en route to the efferent arteriole. Approximately 20% of the renal plasma flow is filtered into Bowman space, and the ratio of glomerular filtration rate (GFR) to renal plasma flow determines the filtration fraction. Several factors, mostly hemodynamic, contribute to the regulation of filtration under physiologic conditions.

Although glomerular filtration is affected by renal artery pressure, this relationship is not linear across the range of physiologic blood pressures due to autoregulation of GFR. Autoregulation of glomerular filtration is the result of three major factors that modulate either afferent or efferent arteriolar tone: these include an autonomous vasoreactive (myogenic) reflex in the afferent arteriole, tubuloglomerular feedback (TGF), and angiotensin II-mediated vasoconstriction of the efferent arteriole. The myogenic reflex is a first line of defense against fluctuations in renal blood flow. Acute changes in renal perfusion pressure evoke reflex constriction or dilatation of the afferent arteriole in response to increased or decreased pressure, respectively. This phenomenon helps protect the glomerular capillary from sudden changes in systolic pressure.

TGF changes the rate of filtration and tubular flow by reflex vasoconstriction or dilatation of the afferent arteriole. TGF is mediated by specialized cells in the thick ascending limb of the loop of Henle called the macula densa that act as sensors of solute concentration and tubular fluid flow rate. With high tubular flow rates, a proxy for an inappropriately high filtration rate, there is increased solute delivery to the macula densa (Fig. 303-2B) that evokes vasoconstriction of the afferent arteriole causing GFR to return toward normal. One component of the soluble signal from the macula densa is adenosine triphosphate (ATP) released by the cells during increased NaCl reabsorption. ATP is metabolized in the extracellular space to generate adenosine, a potent vasoconstrictor of the afferent arteriole. During conditions associated with a fall in filtration rate, reduced solute delivery to the macula densa attenuates TGF, allowing afferent arteriolar dilatation and restoring GFR to normal levels. Angiotensin II and reactive oxygen species enhance, while nitric oxide (NO) blunts TGF.

The third component underlying autoregulation of GFR involves angiotensin II. During states of reduced renal blood flow, renin is released from granular cells within the wall of the afferent arteriole near the macula densa in a region called the juxtaglomerular apparatus (Fig. 303-2B). Renin, a proteolytic enzyme, catalyzes the conversion of angiotensinogen to angiotensin I, which is subsequently converted to angiotensin II by angiotensin-converting enzyme (ACE) (Fig. 303-2C). Angiotensin II evokes vasoconstriction of the efferent arteriole, and the resulting increased glomerular hydrostatic pressure elevates GFR to normal levels.

The renal tubules are composed of highly differentiated epithelia that vary dramatically in morphology and function along the nephron (Fig. 303-3). The cells lining the various tubular segments form monolayers connected to one another by a specialized region of the adjacent lateral membranes called the tight junction. Tight junctions form an occlusive barrier that separates the lumen of the tubule from the interstitial spaces surrounding the tubule and also apportions the cell membrane into discrete domains: the apical membrane facing the tubular lumen and the basolateral membrane facing the interstitium. This regionalization allows cells to allocate membrane proteins and lipids asymmetrically. Owing to this feature, renal epithelial cells are said to be polarized. The asymmetric assignment of membrane proteins, especially proteins mediating transport processes, provides the machinery for directional movement of fluid and solutes by the nephron.

EPITHELIAL SOLUTE TRANSPORT

There are two types of epithelial transport. Movement of fluid and solutes sequentially across the apical and basolateral cell membranes (or vice versa) mediated by transporters, channels, or pumps is called cellular transport. By contrast, movement of fluid and solutes through the narrow passageway between adjacent cells is called paracellular transport. Paracellular transport occurs through tight junctions, indicating that they are not completely “tight” or occlusive. Indeed, some epithelial cell layers allow rather robust paracellular transport to occur (leaky epithelia), whereas other epithelia have more restrictive tight junctions (tight epithelia). In addition, because the ability of ions to flow through the paracellular pathway determines the electrical resistance across the epithelial monolayer, leaky and tight epithelia are also referred to as low- or high-resistance epithelia, respectively. The proximal tubule contains leaky epithelia, whereas distal nephron segments, such as the collecting duct, contain tight epithelia. Leaky epithelia are most well suited for bulk fluid reabsorption, whereas tight epithelia allow for more refined control and regulation of transport.

MEMBRANE TRANSPORT

Cell membranes are composed of hydrophobic lipids that repel water and aqueous solutes. The movement of solutes and water across cell membranes is made possible by discrete classes of integral membrane proteins, including channels, pumps, and transporters. These different mechanisms mediate specific types of transport activities, including active transport (pumps), passive transport (channels), facilitated diffusion (transporters), and secondary active transport (cotransporters). Active transport requires metabolic energy generated by the hydrolysis of ATP. Active transport pumps are ion-translocating ATPases, including the ubiquitous Na⁺/K⁺-ATPase, the H⁺-ATPases, and Ca²⁺-ATPases. Active transport creates asymmetric ion concentrations across a cell membrane and can move ions against a chemical gradient. The potential energy stored in a concentration gradient of an ion such as Na⁺ can be used to drive transport through other mechanisms (secondary active transport). Pumps are often electrogenic, meaning they can create an asymmetric distribution of electrostatic charges across the membrane and establish a voltage or membrane potential. The movement of solutes through a membrane protein by simple diffusion is called passive transport. This activity is mediated by channels created by selectively permeable membrane proteins, and it allows solute or water to move across a membrane driven by favorable concentration gradients or electrochemical potential. Facilitated diffusion is a specialized type of passive transport mediated by simple transporters called carriers or uniporters. For example, hexose transporters such as GLUT2 mediate glucose transport by tubular cells. These transporters are driven by the concentration gradient for glucose that is highest in extracellular fluids and lowest in the cytoplasm due to rapid metabolism. Many other transporters operate by translocating two or more ions/solutes in concert either in the same direction (symporters or cotransporters) or in opposite directions (antiporters or exchangers) across the cell membrane. The movement of two or more ions/solutes may produce no net change in the balance of electrostatic charges across the membrane (electroneutral), or a transport event may alter the balance of charges (electrogenic). Several inherited disorders of renal tubular solute and water transport occur as a consequence of mutations in genes encoding a variety of channels, transporter proteins, and their regulators (Table 303-1).

Each anatomic segment of the nephron has unique characteristics and specialized functions enabling selective transport of solutes and water (Fig. 303-3). Through sequential events of reabsorption and secretion along the nephron, tubular fluid is progressively conditioned into urine. Knowledge of the major tubular mechanisms responsible for solute and water transport is critical for understanding hormonal regulation of kidney function and the pharmacologic manipulation of renal excretion.

The proximal tubule is responsible for reabsorbing ~60% of filtered NaCl and water, as well as ~90% of filtered bicarbonate and most critical nutrients such as glucose and amino acids. The proximal tubule uses both cellular and paracellular transport mechanisms. The apical membrane of proximal tubular cells has an expanded surface area available for reabsorptive work created by a dense array of microvilli called the brush border, and leaky tight junctions enable high-capacity fluid reabsorption.

Solute and water pass through these tight junctions to enter the lateral intercellular space where absorption by the peritubular capillaries occurs. Bulk fluid reabsorption by the proximal tubule is driven by high oncotic pressure and low hydrostatic pressure within the peritubular capillaries. Cellular transport of most solutes by the proximal tubule is coupled to the Na⁺ concentration gradient established by the activity of a basolateral Na⁺/K⁺-ATPase (Fig. 303-3A). This active transport mechanism maintains a steep Na⁺ gradient by keeping intracellular Na⁺ concentrations low. Solute reabsorption from the tubular lumen is coupled to the Na⁺ gradient by Na⁺-dependent transporters such as Na⁺-glucose and Na⁺-phosphate cotransporters present in apical membranes. In addition to the paracellular route, water reabsorption also occurs through the cellular pathway enabled by constitutively active water channels (aquaporin-1) present on both apical and basolateral membranes.

Proximal tubular cells reclaim nearly all filtered bicarbonate by a mechanism dependent on carbonic anhydrases. Filtered bicarbonate is first titrated by protons delivered to the lumen mainly by Na⁺/H⁺ exchange. The resulting carbonic acid (H₂CO₃) is metabolized by brush border carbonic anhydrase to water and carbon dioxide. Dissolved carbon dioxide then diffuses into the cell, where it is enzymatically hydrated by cytoplasmic carbonic anhydrase to re-form carbonic acid. Finally, intracellular carbonic acid dissociates into free protons and bicarbonate anions, and bicarbonate exits the cell through a basolateral Na⁺/HCO₃⁻ cotransporter. This process is saturable, which can result in urinary bicarbonate excretion when plasma levels exceed the physiologically normal range (24–26 meq/L). Carbonic anhydrase inhibitors such as acetazolamide, a class of weak diuretic agents, block proximal tubule bicarbonate reabsorption and are useful for alkalinizing the urine.

The proximal tubule contributes to acid secretion by two mechanisms involving the titration of the urinary buffers ammonia (NH₃) and phosphate. Renal NH₃ is produced by glutamine metabolism in the proximal tubule. Subsequent diffusion of NH₃ out of the proximal tubular cell enables trapping of H⁺, which is secreted by Na⁺/H⁺ exchange, in the lumen as ammonium ion (NH₄⁺). Cellular K⁺ levels inversely modulate proximal tubular ammoniagenesis, and in the setting of high serum K⁺ from hypoaldosteronism, reduced ammoniagenesis promotes type IV renal tubular acidosis. Filtered hydrogen phosphate ion (HPO₄²⁻) is also titrated in the proximal tubule by secreted H⁺ to form H₂PO₄⁻, and this reaction constitutes a major component of the urinary buffer referred to as titratable acid. Most filtered phosphate ion is reabsorbed by the proximal tubule through a sodium-coupled cotransport process that is regulated by parathyroid hormone (PTH).

Chloride is poorly reabsorbed throughout the first segment of the proximal tubule, and a rise in Cl⁻ concentration counterbalances the removal of bicarbonate anion from tubular fluid. In later proximal tubular segments, cellular Cl⁻ reabsorption is initiated by apical exchange of cellular formate for higher luminal concentrations of Cl⁻. Once in the lumen, formate anions are titrated by H⁺ (provided by Na⁺/H⁺ exchange) to generate neutral formic acid, which can diffuse passively across the apical membrane back into the cell where it dissociates a proton and is recycled. Basolateral Cl⁻ exit is mediated by a K⁺/Cl⁻ cotransporter.

Reabsorption of glucose is nearly complete by the end of the proximal tubule. Cellular transport of glucose is mediated by apical Na⁺-glucose cotransport coupled with basolateral, facilitated diffusion by a glucose transporter. This process is also saturable, leading to glycosuria when plasma levels exceed 180–200 mg/dL, as seen in untreated diabetes mellitus.

The proximal tubule possesses specific transporters capable of secreting a variety of organic acids (carboxylate anions) and bases (mostly primary amine cations). Organic anions transported by these systems include urate, dicarboxylic acid anions (succinate), ketoacid anions, and several protein-bound drugs not filtered at the glomerulus (penicillins, cephalosporins, and salicylates). Probenecid inhibits renal organic anion secretion and can be clinically useful for raising plasma concentrations of certain drugs like penicillin and oseltamivir. Organic cations secreted by the proximal tubule include various biogenic amine neurotransmitters (dopamine, acetylcholine, epinephrine, norepinephrine, and histamine) and creatinine. The ATP-dependent transporter P-glycoprotein is highly expressed in brush border membranes and secretes several medically important drugs, including cyclosporine, digoxin, tacrolimus, and various cancer chemotherapeutic agents. Certain drugs like cimetidine and trimethoprim compete with endogenous compounds for transport by the organic cation pathways. Although these drugs elevate serum creatinine levels, there is no actual change in GFR in this setting.

Calcium and phosphorus homeostasis depends upon normal functioning of the proximal tubule. Approximately 60–70% of filtered calcium and ~85% of filtered phosphorus (in the form of inorganic phosphate) are reabsorbed by the proximal tubule. Whereas calcium reabsorption is mostly by passive diffusion through the paracellular route, phosphate reabsorption is mediated by sodium-coupled cotransport. In addition to direct reabsorption, the proximal tubule contributes to systemic mineral balance by participating in specific endocrine pathways. Circulating 25-hydroxy vitamin D (calcidiol) is bioactivated by proximal tubular 1α-hydroxylase to produce 1,25-di-hydroxy vitamin D (calcitriol), the most active form of the hormone, that acts on the small intestine to promote calcium absorption. Phosphate balance is affected by circulating fibroblast growth hormone 23 (FGF23), a bone-derived hormone that interacts with its receptor (FGFR1) and co-receptor (Klotho) on proximal tubular cells to suppress sodium-phosphate cotransport and promote renal phosphate excretion. PTH stimulates proximal tubular 1α-hydroxylation of vitamin D while it suppresses sodium-phosphate cotransport. Derangements in PTH and FGF23 account for abnormal calcium and phosphate balance in chronic kidney disease.

The proximal tubule, through distinct classes of Na⁺-dependent and Na⁺-independent transport systems, reabsorbs amino acids efficiently. These transporters are specific for different groups of amino acids. For example, cystine, lysine, arginine, and ornithine are transported by a system comprising two proteins encoded by the SLC3A1 and SLC7A9 genes. Mutations in either SLC3A1 or SLC7A9 impair reabsorption of these amino acids and cause the disease cystinuria. Peptide hormones, such as insulin and growth hormone, β₂-microglobulin, albumin, and other small proteins, are taken up by the proximal tubule through a process of absorptive endocytosis and are degraded in acidified endocytic lysosomes. Acidification of these vesicles depends on a vacuolar H⁺-ATPase and Cl⁻ channel. Impaired acidification of endocytic vesicles because of mutations in a Cl⁻ channel gene (CLCN5) causes low-molecular-weight proteinuria in Dent disease.

The loop of Henle consists of three major segments: descending thin limb, ascending thin limb, and ascending thick limb. These divisions are based on cellular morphology and anatomic location, but also correlate with specialization of function. Approximately 15–25% of filtered NaCl is reabsorbed in the loop of Henle, mainly by the thick ascending limb. The loop of Henle has an important role in urinary concentration by contributing to the generation of a hypertonic medullary interstitium in a process called countercurrent multiplication. The loop of Henle is the site of action for the most potent class of diuretic agents (loop diuretics) and also contributes to reabsorption of calcium and magnesium ions.

The descending thin limb is highly water permeable owing to dense expression of constitutively active aquaporin-1 water channels. By contrast, water permeability is negligible in the ascending limb. In the thick ascending limb, there is a high level of secondary active NaCl transport enabled by the Na⁺/K⁺/2Cl⁻ cotransporter on the apical membrane in series with basolateral Cl⁻ channels and Na⁺/K⁺-ATPase (Fig. 303-3B). The Na⁺/K⁺/2Cl⁻ cotransporter is the primary target for loop diuretics. Tubular fluid K⁺ is the limiting substrate for this cotransporter (tubular concentration of K⁺ is similar to plasma, about 4 meq/L), but transporter activity is maintained by K⁺ recycling through an apical potassium channel. The cotransporter also enables reabsorption of NH₄⁺ in lieu of K⁺, and this leads to accumulation of both NH₄⁺ and NH₃ in the medullary interstitium. An inherited disorder of the thick ascending limb, Bartter’s syndrome, also results in a salt-wasting renal disease associated with hypokalemia and metabolic alkalosis. Loss-of-function mutations in one of five distinct genes encoding components of the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC2), apical K⁺ channel (KCNJ1), basolateral Cl⁻ channel (CLCNKB, BSND), or calcium-sensing receptor (CASR) can cause Bartter’s syndrome.

Potassium recycling also contributes to a positive electrostatic charge in the lumen relative to the interstitium that promotes divalent cation (Mg²⁺ and Ca²⁺) reabsorption through a paracellular pathway. A Ca²⁺-sensing, G-protein-coupled receptor (CaSR) on basolateral membranes regulates NaCl reabsorption in the thick ascending limb through dual signaling mechanisms using either cyclic AMP or eicosanoids. This receptor enables a steep relationship between plasma Ca²⁺ levels and renal Ca²⁺ excretion. Loss-of-function mutations in CaSR cause familial hypercalcemic hypocalciuria because of a blunted response of the thick ascending limb to extracellular Ca²⁺. Mutations in CLDN16 encoding paracellin-1, a transmembrane protein located within the tight junction complex, leads to familial hypomagnesemia with hypercalciuria and nephrocalcinosis, suggesting that the ion conductance of the paracellular pathway in the thick limb is regulated.

The loop of Henle contributes to urine-concentrating ability by establishing a hypertonic medullary interstitium that promotes water reabsorption by the downstream inner medullary collecting duct. Countercurrent multiplication produces a hypertonic medullary interstitium using two countercurrent systems: the loop of Henle (opposing descending and ascending limbs) and the vasa recta (medullary peritubular capillaries enveloping the loop). The countercurrent flow in these two systems helps maintain the hypertonic environment of the inner medulla, but NaCl reabsorption by the thick ascending limb is the primary initiating event. Reabsorption of NaCl without water dilutes the tubular fluid and adds new osmoles to medullary interstitial fluid. Because the descending thin limb is highly water permeable, osmotic equilibrium occurs between the descending limb tubular fluid and the interstitial space, leading to progressive solute trapping in the inner medulla. Maximum medullary interstitial osmolality also requires partial recycling of urea from the collecting duct.

The distal convoluted tubule reabsorbs ~5% of the filtered NaCl. This segment is composed of a tight epithelium with little water permeability. The major NaCl-transporting pathway uses an apical membrane, electroneutral thiazide-sensitive Na⁺/Cl⁻ cotransporter in tandem with basolateral Na⁺/K⁺-ATPase and Cl⁻ channels (Fig. 303-3C). Apical Ca²⁺-selective channels (TRPV5) and basolateral Na⁺/Ca²⁺ exchange mediate calcium reabsorption in the distal convoluted tubule. Ca²⁺ reabsorption is inversely related to Na⁺ reabsorption and is stimulated by PTH. Blocking apical Na⁺/Cl⁻ cotransport will reduce intracellular Na⁺, favoring increased basolateral Na⁺/Ca²⁺ exchange and passive apical Ca²⁺ entry. Loss-of-function mutations of SLC12A3 encoding the apical Na⁺/Cl⁻ cotransporter cause Gitelman syndrome, a salt-wasting disorder associated with hypokalemic alkalosis and hypocalciuria. Mutations in genes encoding WNK kinases, WNK-1 and WNK-4, cause pseudohypoaldosteronism type II (Gordon syndrome) characterized by familial hypertension with hyperkalemia. WNK kinases influence the activity of several tubular ion transporters. Mutations in this disorder lead to overactivity of the apical Na⁺/Cl⁻ cotransporter in the distal convoluted tubule as the primary stimulus for increased salt reabsorption, extracellular volume expansion, and hypertension. Hyperkalemia may be caused by diminished activity of apical K⁺ channels in the collecting duct, a primary route for K⁺ secretion. Mutations in TRPM6 encoding Mg²⁺ permeable ion channels also cause familial hypomagnesemia with hypocalcemia. A molecular complex of TRPM6 and TRPM7 proteins is critical for Mg²⁺ reabsorption in the distal convoluted tubule.

The collecting duct modulates the final composition of urine. The two major divisions, the cortical collecting duct and inner medullary collecting duct, contribute to reabsorbing ~4–5% of filtered Na⁺ and are important for hormonal regulation of salt and water balance. Cells in both segments of the collecting duct express vasopressin-regulated water channels (aquaporin-2 on the apical membrane, aquaporin-3 and -4 on the basolateral membrane). The antidiuretic hormone vasopressin binds to the V2 receptor on the basolateral membrane and triggers an intracellular signaling cascade through G-protein-mediated activation of adenylyl cyclase, resulting in an increase in the cellular levels of cyclic AMP. This signaling cascade stimulates the insertion of water channels into the apical membrane of collecting duct cells to promote increased water permeability. This increase in permeability enables water reabsorption and production of concentrated urine. In the absence of vasopressin, collecting duct cells are water impermeable, and urine remains dilute.

The cortical collecting duct contains high-resistance epithelia with two cell types. Principal cells are the main water, Na⁺-reabsorbing, and K⁺-secreting cells, and the site of action of aldosterone, K⁺-sparing diuretics, and mineralocorticoid receptor antagonists such as spironolactone and eplerenone. The other cells are type A and B intercalated cells. Type A intercalated cells mediate acid secretion and bicarbonate reabsorption also under the influence of aldosterone. Type B intercalated cells mediate bicarbonate secretion and acid reabsorption.

Virtually all transport is mediated through the cellular pathway for both principal cells and intercalated cells. In principal cells, passive apical Na⁺ entry occurs through an amiloride-sensitive, epithelial Na⁺ channel (ENaC) with basolateral exit mediated by the Na⁺/K⁺-ATPase (Fig. 303-3D). This Na⁺ reabsorptive process is tightly regulated by aldosterone and is physiologically activated by a variety of proteolytic enzymes that cleave extracellular domains of ENaC; plasmin in the tubular fluid of nephrotic patients, for example, activates ENaC, leading to sodium retention. Aldosterone enters the cell across the basolateral membrane, binds to a cytoplasmic mineralocorticoid receptor, and then translocates into the nucleus, where it modulates gene transcription, resulting in increased Na⁺ reabsorption and K⁺ secretion. Activating mutations in ENaC increase Na⁺ reclamation and produce hypokalemia, hypertension, and metabolic alkalosis (Liddle’s syndrome). The potassium-sparing diuretics amiloride and triamterene block ENaC, causing reduced Na⁺ reabsorption.

Principal cells secrete K⁺ through an apical membrane potassium channel. Several forces govern the secretion of K⁺. Most importantly, the high intracellular K⁺ concentration generated by Na⁺/K⁺-ATPase creates a favorable concentration gradient for K⁺ secretion into tubular fluid. With reabsorption of Na⁺ without an accompanying anion, the tubular lumen becomes negative relative to the cell interior, creating a favorable electrical gradient for secretion of potassium. When Na⁺ reabsorption is blocked, the electrical component of the driving force for K⁺ secretion is blunted, and this explains lack of excess urinary K⁺ loss during treatment with potassium-sparing diuretics or mineralocorticoid receptor antagonists. K⁺ secretion is also promoted by aldosterone actions that increase regional Na⁺ transport, which favor more lumen electronegativity, and by increasing the number and activity of potassium channels. Fast tubular fluid flow rates that occur during volume expansion or diuretics acting “upstream” of the cortical collecting duct also increase K⁺ secretion, as does the presence of relatively nonreabsorbable anions (including bicarbonate and semisynthetic penicillins) that contribute to the lumen-negative potential. Off-target effects of certain antibiotics, such as trimethoprim and pentamidine, block ENaCs and predispose to hyperkalemia, especially when renal K⁺ handling is impaired for other reasons. Principal cells, as described below, also participate in water reabsorption by increased water permeability in response to vasopressin.

Intercalated cells do not participate in Na⁺ reabsorption but, instead, mediate acid-base secretion. These cells perform two types of transport: active H⁺ transport mediated by H⁺-ATPase (proton pump), and Cl⁻/HCO₃⁻ exchange. Intercalated cells arrange the two transport mechanisms on opposite membranes to enable either acid or base secretion. Type A intercalated cells have an apical proton pump that mediates acid secretion and a basolateral Cl⁻/HCO₃⁻ anion exchanger for bicarbonate reabsorption (Fig. 303-3E). Aldosterone increases the number of H⁺-ATPase pumps, sometimes contributing to the development of metabolic alkalosis. Secreted H⁺ is buffered by NH₃ that has diffused into the collecting duct lumen from the surrounding interstitium. By contrast, type B intercalated cells have the Cl⁻/HCO₃⁻ exchanger on the apical membrane to mediate bicarbonate secretion while the proton pump resides on the basolateral membrane to enable acid reabsorption. Under conditions of acidemia, the kidney preferentially uses type A intercalated cells to secrete the excess H⁺ and generate more HCO₃⁻. The opposite is true in states of bicarbonate excess with alkalemia where the type B intercalated cells predominate. An extracellular protein called hensin mediates this adaptation.

Inner medullary collecting duct cells share many similarities with principal cells of the cortical collecting duct. They have apical Na⁺ and K⁺ channels that mediate Na⁺ reabsorption and K⁺ secretion, respectively (Fig. 303-3F). Sodium reabsorption by inner medullary collecting duct cells is also inhibited by the natriuretic peptides called atrial natriuretic peptide or renal natriuretic peptide (urodilatin); the same gene encodes both peptides but uses different posttranslational processing of a common preprohormone to generate different proteins. Atrial natriuretic peptides are secreted by atrial myocytes in response to volume expansion, whereas urodilatin is secreted by renal tubular epithelia. Natriuretic peptides interact with either apical (urodilatin) or basolateral (atrial natriuretic peptides) receptors on inner medullary collecting duct cells to stimulate guanylyl cyclase and increase levels of cytoplasmic cGMP. This effect in turn reduces the activity of the apical Na⁺ channel in these cells and attenuates net Na⁺ reabsorption, producing natriuresis.

The inner medullary collecting duct transports urea out of the lumen, returning urea to the interstitium, where it contributes to the hypertonicity of the medullary interstitium. Urea is recycled by diffusing from the interstitium into the descending and ascending limbs of the loop of Henle.

The balance of solute and water in the body is determined by the amounts ingested, distributed to various fluid compartments, and excreted by skin, bowel, and kidneys. Tonicity, the osmolar state determining the volume behavior of cells in a solution, is regulated by water balance (Fig. 303-4A), and extracellular blood volume is regulated by Na⁺ balance (Fig. 303-4B). The kidney is a critical modulator of both physiologic processes.

WATER BALANCE

Tonicity depends on the variable concentration of effective osmoles inside and outside the cell causing water to move in either direction across its membrane. Classic effective osmoles, like Na⁺, K⁺, and their anions, are solutes trapped on either side of a cell membrane, where they collectively partition and obligate water to move and find equilibrium in proportion to retained solute; Na⁺/K⁺-ATPase keeps most K⁺ inside cells and most Na⁺ outside. Normal tonicity (~280 mosmol/L) is rigorously defended by osmoregulatory mechanisms that control water balance to protect tissues from inadvertent dehydration (cell shrinkage) or water intoxication (cell swelling), both of which are deleterious to cell function (Fig. 303-4A).

The mechanisms that control osmoregulation are distinct from those governing extracellular volume, although there is some shared physiology in both processes. While cellular concentrations of K⁺ have a determinant role in any level of tonicity, the routine surrogate marker for assessing clinical tonicity is the concentration of serum Na⁺. Any reduction in total body water, which raises the Na⁺ concentration, triggers a brisk sense of thirst and conservation of water by decreasing renal water excretion mediated by release of vasopressin from the posterior pituitary. Conversely, a decrease in plasma Na⁺ concentration triggers an increase in renal water excretion by suppressing the secretion of vasopressin. Whereas all cells expressing mechanosensitive TRPV1, 2, or 4 channels, among potentially other sensors, respond to changes in tonicity by altering their volume and Ca²⁺ concentration, only TRPV⁺ neuronal cells connected to the organum vasculosum of the lamina terminalis are osmoreceptive. Only these cells, because of their neural connectivity and adjacency to a minimal blood-brain barrier, modulate the downstream release of vasopressin by the posterior lobe of the pituitary gland. Secretion is stimulated primarily by changing tonicity and secondarily by other nonosmotic signals such as variable blood volume, stress, pain, nausea, and some drugs. The release of vasopressin by the posterior pituitary increases linearly as plasma tonicity rises above normal, although this varies, depending on the perception of extracellular volume (one form of cross-talk between mechanisms that adjudicate blood volume and osmoregulation). Changing the intake or excretion of water provides a means for adjusting plasma tonicity; thus, osmoregulation governs water balance.

The kidneys play a vital role in maintaining water balance through the regulation of renal water excretion. The ability to concentrate urine to an osmolality exceeding that of plasma enables water conservation, whereas the ability to produce urine more dilute than plasma promotes excretion of excess water. For water to enter or exit a cell, the cell membrane must express aquaporins. In the kidney, aquaporin-1 is constitutively active in all water-permeable segments (e.g., proximal tubule, descending thin limb of the loop of Henle), whereas aquaporin-2, -3, and -4 in the collecting duct promote vasopressin-regulated water permeability. Net water reabsorption is ultimately driven by the osmotic gradient between dilute tubular fluid and a hypertonic medullary interstitium.

SODIUM BALANCE

The perception of extracellular blood volume is determined, in part, by the integration of arterial tone, cardiac stroke volume, heart rate, and the water and solute content of extracellular fluid. Na⁺ and accompanying anions are the most abundant extracellular effective osmoles and together support a blood volume around which pressure is generated. Under normal conditions, this volume is regulated by sodium balance (Fig. 303-4B), and the balance between daily Na⁺ intake and excretion is under the influence of baroreceptors in regional blood vessels and vascular hormone sensors modulated by atrial natriuretic peptides, the renin-angiotensin-aldosterone system, Ca²⁺ signaling, adenosine, vasopressin, and the neural adrenergic axis. If Na⁺ intake exceeds Na⁺ excretion (positive Na⁺ balance), then an increase in blood volume will trigger a proportional increase in urinary Na⁺ excretion. Conversely, when Na⁺ intake is less than urinary excretion (negative Na⁺ balance), blood volume will decrease and trigger enhanced renal Na⁺ reabsorption, leading to decreased urinary Na⁺ excretion.

The renin-angiotensin-aldosterone system is the best-understood hormonal system modulating renal Na⁺ excretion. Renin is synthesized and secreted by granular cells in the wall of the afferent arteriole. Its secretion is controlled by several factors, including β₁-adrenergic stimulation to the afferent arteriole, input from the macula densa, and prostaglandins. Renin and ACE activity eventually produce angiotensin II that directly and indirectly promotes renal Na⁺ and water reabsorption. Stimulation of proximal tubular Na⁺/H⁺ exchange by angiotensin II directly increases Na⁺ reabsorption. Angiotensin II also promotes Na⁺ reabsorption along the collecting duct by stimulating aldosterone secretion by the adrenal cortex. Constriction of the efferent glomerular arteriole by angiotensin II indirectly increases the filtration fraction and raises peritubular capillary oncotic pressure to promote tubular Na⁺ reabsorption. Finally, angiotensin II inhibits renin secretion through a negative feedback loop. Alternative metabolism of angiotensin by ACE2 generates the vasodilatory peptide angiotensin 1-7 that acts through Mas receptors to counterbalance several actions of angiotensin II on blood pressure and renal function (Fig. 303-2C).

Aldosterone is synthesized and secreted by granulosa cells in the adrenal cortex. It binds to cytoplasmic mineralocorticoid receptors in the collecting duct principal cells that increase activity of ENaC, apical membrane K⁺ channel, and basolateral Na⁺/K⁺-ATPase. These effects are mediated in part by aldosterone-stimulated transcription of the gene encoding serum/glucocorticoid-induced kinase 1 (SGK1). The activity of ENaC is increased by SGK1-mediated phosphorylation of Nedd4-2, a protein that promotes recycling of the Na⁺ channel from the plasma membrane. Phosphorylated Nedd4-2 has impaired interactions with ENaC, leading to increased channel density at the plasma membrane and increased capacity for Na⁺ reabsorption by the collecting duct.

Chronic exposure to aldosterone causes a decrease in urinary Na⁺ excretion lasting only a few days, after which Na⁺ excretion returns to previous levels. This phenomenon, called aldosterone escape, is explained by decreased proximal tubular Na⁺ reabsorption following blood volume expansion. Excess Na⁺ that is not reabsorbed by the proximal tubule overwhelms the reabsorptive capacity of more distal nephron segments. This escape may be facilitated by atrial natriuretic peptides that lose their effectiveness in the clinical settings of heart failure, nephrotic syndrome, and cirrhosis, leading to severe Na⁺ retention and volume overload.