Structure and function of the kidney 4717 Steve Ha

Structure and function of the kidney 4717 Steve Harper and Robert Unwin

ESSENTIALS The kidneys are the archetypal organs of homeostasis. Their pri- mary function is filtration and segregation. Through these processes they rebalance the continual electrolyte and chemical disturbances that derive from physiological and pathological metabolic pro- cesses. Electrolytes, hydrogen (H+) ions from metabolic acids, amino acids, fatty acids, plasma proteins, immunoglobulins, and metabolic products are either retained, reabsorbed, and recycled; or they are filtered, secreted, concentrated, and excreted. These processes con- vert approximately 120 ml/​min of primary glomerular filtrate into
1 ml/​min of urine. In addition, the kidneys play crucial roles in bone mineralization, the maintenance of cardiovascular tone, systemic blood pressure, and bone marrow synthetic function via their contri- bution to vitamin D production, the renin–​angiotensin system, and erythropoiesis. Introduction The kidneys control the milieu intérieur by rebalancing the con- tinual electrolyte and chemical disturbances that derive from physiological and pathological metabolic processes. Their mech- anism of action involves the conversion of about 120 ml/​min of plasma filtrate into 1 ml/​min of urine, with incredible precision in the control of excretion of an enormous variety of substances. Electrolytes, hydrogen (H+) ions from metabolic acids, amino acids, fatty acids, plasma proteins, immunoglobulins, and meta- bolic products are either retained, reabsorbed, and recycled, or they are filtered, secreted, concentrated, and excreted. To illustrate the vital homeostatic role of the kidney, we need to look no further than sodium, which with its main anions chloride and bicarbonate constitutes the major osmotically active solute in the extracellular fluid (ECF), and hence Na+ concentration is the chief determinant of ECF osmolality and fluid shifts between the ECF and intracellular fluid compartments. If the Na+ concentration changes, osmoreceptors in the hypothalamus detect the change and affect the sensation of thirst and modulate the release of vasopressin. In this way, the volume of the ECF, which is intimately related to blood pressure, is determined largely by its Na+ content, which in turn is controlled by renal Na+ excretion. The kidney is therefore inevitably involved in any condition where blood pressure or ECF volume is abnormal. The concept of effective circulating volume relates to the body’s perception of the ‘fullness’ of the vascular tree. Changes in effective circu- lating volume are detected by both high-​pressure baroreceptors in the aortic arch and carotid sinus, and low-​pressure recep- tors in the cardiac atria. Their stimulation produces corrective changes in Na+ excretion by the kidneys, mediated by appropriate changes in activity of the renin–​angiotensin–​aldosterone system, renal sympathetic nerves, and natriuretic peptides. In congestive heart failure, cirrhosis of the liver, and the nephrotic syndrome, the effective circulating volume is reduced even though the ECF volume is not, resulting in enhanced renal Na+ (and water) reten- tion causing oedema. The kidneys play a crucial role in the homeostasis of electrolytes other than sodium, as evidenced by the dangers of hyperkalaemia in renal failure. They also have critical actions in regard to bone mineralization and bone marrow synthetic function via their con- tribution to vitamin D production and erythropoiesis. This chapter describes how the structure and function of the kidney enables it to perform such remarkable feats. Ultrastructure The functional unit linking ‘blood in’ to ‘urine out’ is the nephron (Fig. 21.1.1). The blood supply to the nephron is eventually arrived at after sequential division of the renal arteries and subsequent vessels to reach specialized portal exchange microvascular beds. These are the glomerular capillaries, which occur in grape-​like clusters in the outer cortex of the kidneys and, in total, constitute 19 km of capillary filtra- tion area. The structure of the nephron is that the glomerulus, including the postfiltration barrier Bowman’s space (which in life is a potential space like the pleural and pericardial spaces), leads on to the proximal convoluted tubule (PCT), loop of Henle, and distal convoluted tubule (DCT) before reaching the collecting duct. The mammalian nephron has evolved to include systems for dynamic feedback from distal to proximal parts of the nephron (tubuloglomerular feedback) in that the 21.1 Structure and function of the kidney Steve Harper and Robert Unwin

SECTION 21 Disorder s of the ki dne y and u rinary trac 4718 (b) (c) (d) AVC AVC AVC Afferent Arteriole Conduit Conduit Efferent Arteriole Conduit (a) Fig. 21.1.2  Collagen supported Glomerular Vascular Chambers identified in human kidneys perfusion fixed under physiological hydrostatic and osmotic pressures. (a) Scale diagram of the Afferent (Red) and Efferent (Blue) ends of the glomerular vasculature within the glomerular stalk. Diagram shows size and branch relationships between arterioles, vascular chambers and 1st order conduit vessels (few branches); mesangium close to vascular pole—(grey). Scale bar 100 µm. (b) & (c) Still images from 3D-video reconstruction of physiologically fixed human glomerulus demonstrating elongated afferent vascular chamber (AVC) (red network) and right angle approach of incident arteriole. (d) Collapsed Vascular chambers are occasionally visible on human renal biopsy tissue. Source data from Neal, C., Arkill, K., Bell, J., et al., Novel hemodynamic structures in the human glomerulus, Renal Physiology, American Journal of Physiology, Volume 315 Issue 5, November 2018, Pages F1370–F1384. Copyright © 2018 the American Physiological Society. tubular structures, after descending into the medulla, wend their way back to the renal cortex so that the DCT lies adjacent to its own glom- erular stalk (the juxtaglomerular apparatus) (Fig. 21.1.1). Glomerulus The microvascular ultrastructure The glomerular capillary bed is composed of five to seven glomerular lobules fed by conduit capillaries that diverge from the afferent ar- teriole in the glomerular stalk. These initial lobular capillaries have few branches until they reach the periphery of the glomerular tuft, at which point multiple division results in approximately twice the number of efferent lobular vessels converging on the efferent arteriole. The efferent arterioles subsequently diverge again into the peritubular and medullary capillary plexi, which finally converge to the renal venous system via the returning medullary vasa recta. The glomerular capillaries are lined by a nondiaphragmatic highly fenestrated endo- thelium, with each fenestration being 15 times the diameter of an al- bumin molecule. The afferent arteriole enters the glomerular tuft at an acute right angle (Fig. 21.1.2). This, in conjunction with a subsequent vascular dilatation immediately within the glomerular stalk (the Afferent Vascular Chamber – AVC), is predicted to result in complex, even rotational flow, to promote a more even distribution of blood into the conduit vessels of the glomerular lobules which originate from the AVC at a variety of disparate positions, orientations and angles (Fig. 21.1.2). Both afferent and efferent arterioles are vasoactive, responding to—​among other agents—​prostaglandins (afferent vessel AA JG MD www.people.upei.ca Glomerulus Afferent arteriole Loop of Henle Distal convoluted tubule (DCT) Straight collecting tubule Urine Proximal convoluted tubule (PCT) Efferent arteriole Fig. 21.1.1  The nephron. Specialized cells of the thick ascending limb of the DCT (macula densa (MD)) approximate to the juxtaglomerular cells (JG) and afferent arteriole (AA) of its own glomerulus. This facilitates fine and rapid modification of SNGFR and Na+, water, and blood pressure homeostasis via the renin–​angiotensin and other systems.

21.1  Structure and function of the kidney 4719 dilatation) and angiotensin (predominant efferent vessel constriction). This allows dynamic manipulation of flow through the glomerular capillary network, influencing intraglomerular pressure, ultrafiltra- tion, and single-​nephron glomerular filtration rate (SNGFR). Even modest alterations in vessel diameter can have a profound influence on vessel function since flow is proportional to the fourth power of the vessel radius—​hence the clinically relevant change in glomerular filtration rate (GFR) that can result from the use of prostaglandin and angiotensin-​converting enzyme (ACE) inhibitors. The barrier The glomerular filtration barrier (GFB) is a complex, integrated, multilayered structure that behaves in many ways like a simple an- ionic sieve. It appears to segregate primarily according to size, but molecular charge, shape, orientation, and solubility all contribute to the tendency for a molecule to be retained (or not) within the vas- cular lumen. The resulting differential restriction ensures poor per- meability to large, lipid insoluble or anionic molecules, for example, only 1 in 1600 albumin molecules traverse the barrier (Fig. 21.1.3). In contrast, the GFB is highly permeable to water and small water-​ soluble molecules (Fig. 21.1.4). In sequential order, from the glomerular capillary lumen out- wards, the fused layers of the barrier include the endothelial glycocalyx, the fenestrated endothelium, the glomerular basement membrane (GBM), the 40-​nm podocyte foot process/​slit diaphragm (between interdigitating foot processes of adjacent podocytes—​the slit diaphragms are composed of molecules that appear to have signalling as well as structural functions), the glycocalyx of the (b) (a) Fig. 21.1.3  (a) Isolated rabbit glomerulus, cannulated and perfused with 60-​kDa rhodamine-​labelled dextran. Dilute solution in afferent arteriole (AA) gives a signal comparable to background. Free water is ultrafiltrated out, [rhodamine-​dextran] rises to reveal intense fluorescence in the efferent arteriole (EA). (b) Mouse glomerulus. Red fluorescence = mouse albumin (atto-​565 labelled). Glomerular albumin sieving coefficient (Θalb): Urinary space [albuminatto565]/​glomerular capillary [albuminatto565] = 0.0006
(i.e. approximately 1 in 1600 albumin molecules penetrates the GFB). (a) Reproduced with permission from Salmon A, et al. (2009). New aspects of glomerular filtration barrier structure and function: five layers (at least) not three. Current Opinion in Nephrology and Hypertension, 18(3), 197–​205. Copyright © 2009 Lippincott Williams. (a) (b) Fig. 21.1.4  Two stills from a video showing the glomerular filtration barrier is highly permeable to water and small water-​soluble molecules (here Lucifer yellow). In this in vivo preparation, the time difference of the fluorescence intensity peak of glomerular filtrate at two regions of interest (ROI) can be used to visualize and investigate the SNGFR. G, glomerulus; PT, proximal tubule. Scale = 50 μm. http://​ ajprenal.physiology.org/​content/​ajprenal/​suppl/​2006/​04/​12/​00521.2005.DC1/​40XSNGFR_​short.avi. Reproduced with permission from Kang et al. Am J Physiol Renal Physiol 2006;291: F495–​F502. Copyright © 2006 the American Physiological Society.

SECTION 21 Disorder s of the ki dne y and u rinary trac 4720 podocyte, and the space under the podocyte (the sub-​podocyte space) (Fig. 21.1.5). It has been shown in in vivo models, and in clin- ical disease, that faults in any one of these layers can lead to a failure of normal permselectivity (Fig. 21.1.5). The wide acceptance of the pore theory of permeability (see ‘Theories of permeability’) and the high density of fenestrations both initially lent credence to the view of the GFB as a glorified sieve (i.e. a continuous barrier with pores of regular size and spacing). However, the GFB is now regarded as a dynamic structure, with all cellular and acellular (matrix or space) components subject to dynamic modifi- cation: for example, the hydraulic resistance of the GBM increases as hydrostatic pressure rises; the cell surface layers (glycocalyx) can present formidable barriers to protein flux in their own right and can be altered by permeability modifying growth factors such as vascular endothelial growth factor and angiopoietin I (produced by podocytes); and even podocytes themselves appear to be able to con- tract and migrate. Barrier permeability The permeability of a capillary barrier is defined as the volume of fluid traversing the barrier per unit area, per unit time, per unit driving force (units: m.s−1.pascal−1). The factors governing this phenomenon were first described by Starling in 1896. Hence the Starling equation: Flow Area Driving Force (ΔP − Δπ) Permeability (to water) A Jv = Lp[(Pc−Pi)−σ(πp−πi)] Pod GCL 2 5 3 4ii 4i 1 Glomerular Capillary Lumen (GCL) 6. Sub-podocyte space 6. Sub-pod ocyte space (a) (b) Fig. 21.1.5  (a) A two-​photon in vivo image of the subpodocyte space between podocyte and glomerular endothelial cell. (b) Transmission electron microscopy of a perfusion fixed human glomerulus with addition of Lanthanum Dysprosium GlycosaminoGlycan Adhesion (or LaDy GAGa) stain (section 80 nm thick) which highlights the multilayered GFB including glycocalyx. Scale = 200 nm. http://​www.ncbi.nlm.nih.gov/​pmc/​articles/​PMC3922634/​bin/​1471-​2369-​15-​24-​ S15.mov .Acquired or genetic abnormalities in multiple layers of the GFB can result in abnormal glomerular permselectivity and/​or glomerular disease: 1. Endothelial glycocalyx: thickness reduced by enzymatic degradation or pathological hyperglycaemia (causes proteinuria). Restored experimentally by angiopoietin I or VEGF165b with a reduction in protein leak. 2. Fenestrated endothelium: fenestration density modified by podocyte-​derived pro-​ and antipermeability isoforms of vascular endothelial growth factor. .3. GBM: target for Goodpasture’s antibody; mutations of GBM components cause proteinuria (e.g. Alport’s syndrome). Increased GBM thickness and altered GBM composition seen in diabetic nephropathy. 4. Podocyte: slit
diaphragm proteins—​mutations cause steroid-​resistant (e.g. Finnish) nephrotic syndrome.
Nonslit diaphragm podocyte gene mutations cause proteinuria and glomerular injury, for example, WT1 (Denys–​Drash syndrome). 5. Glycocalyx of the podocyte (yet to be investigated). 6. The subpodocyte space: endows as much resistance as GBM itself. Alters in foot process effacement and as podocytes migrate.

21.1  Structure and function of the kidney 4721 where ΔP is the hydrostatic pressure gradient, Δπ is the functional oncotic pressure, and σ is the reflection coefficient. The tendency of a barrier to resist macromolecular flux: if, for example, 50% of the macromolecular solute penetrated the barrier, then Δπ would be functionally only 50% of that measured. Effectively flow depends on the balance of two opposing forces: driving force and resistance (permeability−1), thus the scenario is very similar to Ohm’s law governing the flow of current around an electrical circuit which is dependent on voltage (driving force) and electrical resistance (conductance−1). Theories of permeability How the observed structure of the barrier (or that of any capillary) equates to its function has long been debated. Early work suggested endothelia possessed ‘size-​limiting structures’ to the movement of solutes and water, leading to the pore theory of permeability. However, structural pores of the correct size and number could not be identified. The subsequent identification of the molecular mesh or network structure of the glycocalyx and basement membrane as well as the podocyte glycocalyx and glomerular slit diaphragm led to the ‘fibre-​matrix junction-​break’ theory of permeability, which pos- tulates that the complex molecular network of the above-​mentioned structures behave functionally as one might expect pores to func- tion, but there are no discrete ‘holes’, that is, there are pathways through endothelial barriers, not pores. Glomerular filtration rate Total GFR is the sum of all SNGFRs. From Starling’s formula this would therefore be ( ) [ ( ) ] SNGFR Lp P A ∑ ∑

− × ∆ ∆π . Permeability and filtration area can be influenced by glomerular cell function, and glom- erular structure and number (and adversely by damage and sclerosis). The predominant cause of loss of GFR as we age, for example, is numer- ical loss (6500 glomeruli per year) reducing the available ‘A’. Reduced hydraulic permeability has been implicated in declining GFR of early pre-​ eclampsia and in some forms of glomerular disease (e.g. membranous nephropathy) in which a fall in both area and Lp has been cited. Although SNGFR does increase with falling Δπ, increased capillary flow and in- creased afferent arteriolar pressure, the predominant factor influencing the driving force is intraglomerular pressure. This in turn is determined by the balance of afferent and efferent arteriolar tone, which are influenced by many molecules including prostaglandins, angiotensin II, and natri- uretic peptides (atrial and brain). These changes can happen rapidly, sup- porting the argument that the glomerulus is dynamic and responsive (Figs. 21.1.6 and 21.1.7). Tubuloglomerular feedback The proximity of the DCT to the afferent arteriole of its own glom- erulus facilitates a unique fine tuning of single nephron function (GFR and sodium handing) via mechanisms controlled by the juxta- glomerular apparatus. This involves some specialized cells of the DCT that sense NaCl delivery to the distal nephron, unique cells at the glomerular stalk, and renin-​producing granular cells of the af- ferent arteriole (sometimes called juxtaglomerular cells). A decrease in DCT NaCl content is interpreted as a decrease in GFR or systemic blood pressure. This induces the release of renin from preformed granules prompting the indirect production of angiotensin II, re- sulting in systemic vasoconstriction and preferential efferent glom- erular arteriolar vasoconstriction, raised intraglomerular pressure, and maintained or increased GFR. Conversely, an increase in DCT NaCl content prompts a parallel system which leads to adenosine-​ mediated afferent arteriolar vasoconstriction and reduced GFR (Figs. 21.1.6 and 21.1.7). Clinical relevance Glomerular selectivity dysfunction and glomerular disease Derangement of the differential permeability of the glomeruli is a characteristic sign of the various types of glomerulonephritis (GN), as well as other glomerular diseases of which the most prevalent is diabetic nephropathy. The glomerulonephritides are a spectrum of conditions associated with abnormal glomerular cell number, injury, type, or morphology, with or without the deposition of immune-​ or nonimmune-​related proteins, resulting in injury and destruction of (a) (b) Fig. 21.1.6  Dynamic SNGFR and tubuloglomerular feedback. Rapid changes in glomerular physiology, SNGFR, and downstream nephron flow are a normal feature in health. (a) and (b) show a rapid in vivo degranulation of renin stores (green quinacrine stain) in the glomerulus (G) and afferent arteriole (AA) (R18, red) from juxtaglomerular cell (JG) apparatus in an isolated rabbit kidney, in this instance in response to hyperglycaemia with resultant AA dilatation:
http://​www.jci.org/​articles/​view/​33293/​sd/​2. Reproduced with permission from Toma I, Kang JJ, Sipos A, Vargas S, Bansal E, Hanner F, et al. Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney. J Clin Invest 118:2526–​2534 (2008). Copyright © 2018 American Society for Clinical Investigation.

SECTION 21 Disorder s of the ki dne y and u rinary trac 4722 the filtering mechanism and subsequent glomerulosclerosis. A clin- ical hallmark of GN is proteinuria due to loss of the usual molecular segregation of glomerular filtration. The abnormalities provoking these conditions can be inherent to the host, with some types of GN recurring after kidney transplantation. The search for circulating causative factors and underlying immune abnormalities continues in different patterns of GN, but with variable success. Clinically GN is a wide spectrum of disease. At one end of the morphological spectrum of injury is minimal change disease, char- acterized by morphological changes only in the podocytes (foot process effacement or flattening), and only observable on electron microscopy, yet provoking such marked proteinuria that nephrotic syndrome is the clinical presentation. At the other extreme are se- vere, destructive inflammatory lesions causing necrosis and forma- tion of extracapillary crescents, typically presenting with rapidly progressive renal failure and seen, for example, in renal vasculitis, Goodpasture’s disease, and systemic lupus. Other common patterns of GN of intermediate morphological and functional severity include IgA nephropathy, membranous nephropathy, mesangiocapillary GN, and focal segmental glomerulosclerosis. Diabetic nephrop- athy is a particularly complex form of glomerular injury involving every facet of glomerular physiology, from resistance, pressure, and glomerular flow rate to endothelial, GBM, and podocyte defects, as well as altered communication between layers and between nephron segments. Proteinuria, as well as being a marker of glomerular injury, is also an independent risk factor for cardiovascular and all-​cause mor- tality in people with kidney disease. The explanation is unknown, but abnormalities in the glycocalyx have been implicated since these are consistent features despite the ultrastructural changes in capil- laries and macrovessels being widely different. Abnormal flattening of the glycocalyx (Fig. 21.1.8) in the endothelial cells of the glom- erular capillary is a feature of diabetic nephropathy, and emerging data suggest that similar abnormalities are simultaneously present in systemic vessels. Furthermore sodium and hyperaldosteronism have also been associated with a reduction in glycocalyx thickness in in vivo models. Tubule Figure 21.1.1 is a reasonably natural representation of the nephron, with the tubule folding back on itself, but it is easier to consider renal tubular function along its course by representing the nephron diagram- matically in a linearized ‘opened up’ form (Fig. 21.1.9). However, both Figs. 21.1.1 and 21.1.9 are oversimplified: in particular they do not illus- trate that the nephron is up to 1000 times as long (3–​5 cm) as it is wide (50–​60 µm). A mathematically similar garden hose would be 20 m long.   0 50 100 150 Time (s) CCD PT Glom Fluorescence intensity 200 250 300 Fig. 21.1.7  An in vivo rat kidney perfused with rhodamine-​labelled dextran. Modest amounts of dextran can be detected in filtrate and downstream flow detected in distal parts of the nephron PCT and cortical collecting duct (CCD). The apparent oscillations in flow are rapid and affected by multiple parameters including those of tubuloglomerular feedback. (a) (b) (c) (d) (e) Fig. 21.1.8  Confocal images. Glycocalyx (GLX) disruption is associated with increased glomerular permeability and proteinuria. This can be reproduced experimentally by enzymatic disruption, for example, with neuraminidase (a, b). Glomerular endothelial cells (EC) of rats stained green (Lucifer yellow), GLX coloured red (Alexa-​594-​wheat germ agglutinin (WGA) lectin). GCL, glomerular capillary lumen. (c–​e) GLX disruption seen in diabetes. Rat kidneys perfused in vivo with cell membrane labelled (R18, red) and glycocalyx labelled (alexa-​ 488-​WGA lectin; green) then imaged with confocal microscopy. In (c) to (e), the GCL is on the right of each panel and the EC to the left. The healthy control in (c) has normal GLX depth (solid arrows). (d) Absent glomerular endothelial GLX lining the luminal surface of EC in a diabetic proteinuric animal. (e) GLX restored after VEGF-​A165b treatment with reduction of proteinuria.

21.1  Structure and function of the kidney 4723 Proximal convoluted tubule The PCT, extending from Bowman’s space, is divided in func- tional terms, and to a lesser extent histologically, into three parts (Fig. 21.1.9). As we have shown previously, the passage of water and solutes across the GFB is primarily dependent on Starling’s forces, and has been regarded as a passive process. The proximal tubular epithelium is highly permeable (of low resistance—​often termed ‘leaky’), meaning that water (fluid) and some solutes can pass with relative ease between cells (a paracellular pathway as determined by Starling’s forces—​Fig. 21.1.10), and that a small osmotic (δπ) and/​or hydrostatic pressure gradient (δP) between blood (in the peritubular capillaries; Fig. 21.1.1) and tubular fluid (glomerular filtrate) can have a big effect on fluid reabsorption. The phenomenon of glomerulotubular balance reflects this, and is another way in which glomerular filtrate delivery to the tubule and tubular reabsorption are normally kept in balance. In addition, however, and in contrast to mechanisms at the GFB, in the tubule, water and solutes can be transported transcellularly (often against concentration gradients) by three active transport mechanisms involving (1)  molecule-​specific channels (acting via electrochemical gradients), (2)  cotransporters, or (3)  exchange ‘pumps’. Both cotransporters and exchange pumps are described as secondarily active in that they are driven by the ATP-​generated low intracellular Na+ concentration resulting from action of the sodium–​ potassium ATPase (Na+,K+-​ATPase) pump sited in the basolateral membrane (Fig. 21.1.10). The movement of solutes transcellularly is driven in all tubular cells by this sodium pump which keeps the Na+ concentration inside the cell low (20–​30 mM), creating a fa- vourable concentration gradient for Na+ entry from the tubular fluid on the apical (luminal) side, where its concentration (at the start) is similar to plasma (140–​145 mM). Other solutes are reabsorbed by a coupling or exchange mechanism that can operate in parallel (cotransport), as for glucose, phosphate, and amino acids, or in ex- change (counter-​transport), as with protons (H+) for bicarbonate re- absorption, and for ammonium (NH4+) secretion. In addition to its reabsorptive function, the PCT has a metabolic role in converting 25-​OH vitamin D to active 1,25-​(OH)2 vitamin D, and also an enzyme that can inactivate vitamin D (24-​hydroxylase). The proximal tubule also synthesizes glucose (and ammonium), as well as several paracrine factors such as angiotensin II and prostaglandins. The PCT is where the bulk (about 65%) of filtered sodium and water is reabsorbed, but unlike in the collecting duct (see ‘Late distal convoluted tubule and collecting duct’), the amount reabsorbed is relatively fixed (obligate) (Fig. 21.1.11). Table 21.1.1 lists the main transport functions of the PCT. Early proximal convoluted tubule (S1) S1 reabsorbs several selected filtered solutes, including glucose and amino acids, and significant amounts of phosphate and bicar- bonate, and—​importantly—​small or low molecular weight proteins (LMWPs), including any filtered albumin (Figs. 21.1.12 and 21.1.13). Reabsorption Filtration Excretion ~150 litres/day ~20,000 mmol Na+/day S3 S2 S1 Thick ascending Loop of Henle CD DCT Secretion PCT ~1.5 litres/day ~150 mmol Na+/day Reabsorption when solute clearance < creatinine clearance Secretion when solute clearance > creatinine clearance descTL ascTL Fig. 21.1.9  Linearized representation of the nephron contrasting input load to output excretion. DCT, distal convoluted tubule; PCT, proximal convoluted tubule. Cotransporters Exchangers Channels Low [Na+] Tight junctions (variable claudin content) Na+ Na+ Na+ Na+ X K+ K+ Basolateral membrane ∆π and ∆P gradients affect paracellular net transport Apical membrane Y+ Lumen Interstitium Sodium pump Fig. 21.1.10  A prototypical polarized renal tubular cell membrane. The intracell [Na+] is kept actively low (20–​30 mM) by the Na+,K+-​ATPase pump in the basolateral membrane (red fill). Three types of transporter reside in apical membrane: (1) channels; these (Na+ entry and K+ exit) are electrochemical gradient dependent (for Na+, both concentration and voltage favour entry; for K+, concentration favours exit, limited by voltage). (2) Cotransporters; X = for example, glucose, PO4 3-​, and amino acids. (3) Exchangers; Y = H+ or NH4 +. Tight junctions vary in composition (different claudins being a key component) along the tubule. High permeability (leaky) in the PCT and low permeability (tight) in the TALH and beyond, which is why a transepithelial potential difference can be established across the distal nephron. Sodium and Water reabsorption <1% excreted 19% 25% 15% 50% 50% 5% ~0 15% 15% ~0

4% 1% excreted Fig. 21.1.11  Sites of relative reabsorption of Na+ and water along the nephron.

SECTION 21 Disorder s of the ki dne y and u rinary trac 4724 The mechanism for LMWP reabsorption is highly specialized, involving receptor-​mediated endocytosis and internalization, with eventual protein degradation in lysosomes. The apical surface re- ceptors responsible for binding LMWPs are megalin and cubilin (Fig. 21.1.14). Late proximal convoluted tubule (S2) The S2 segment reabsorbs more than 50% of the filtered Na+ and K+ ions, in addition to Cl− and residual glucose and amino acids (Fig. 21.1.15). Bicarbonate is reabsorbed as a result of H+ ion se- cretion generated by intracellular carbonic anhydrase inside (type II) and at their apical membranes (type IV) by conversion of bicarbonate to CO2 for rapid cell entry to form bicarbonate for basolateral extrusion. Carbonic anhydrase inhibitors (still used orally for treating glaucoma) prevent bicarbonate reabsorption, increasing urinary bicarbonate (and potassium) excretion and causing a form of proximal renal tubular acidosis. This mimics the carbonic anhydrase (type II) mutations of osteopetrosis. Secretory transport mechanisms handle endogenous (dietary) organic anions and cations, also xenobiotics, drug metabolites, and drugs (e.g. diuretics) that require secretion for their action. Urate is secreted and reabsorbed, the overall effect being that around 10% of filtered urate is excreted. Increased urate reabsorption occurs when the ΔP across the PCT favours paracellular reabsorption (see ‘Proximal convoluted tubule’), for example, with hypovolaemia or diuretic therapy. In chronic kidney or liver disease, weak organic acids (e.g. uric and lactic acids) and conjugates of glucuronic acid increase and interfere with the secretion (and excretion) of drugs such as chlorothiazide, penicillin, and probenecid (and vice versa). Potassium reabsorption is mainly paracellular, driven by the small concentration (and electrical) gradients and reabsorption of water (convection—​solvent drag). Chloride reabsorption is both paracellular, depending on electrical (S1) and concentration (S2) gradients and convection, and transcellular via specialized car- riers (S2). S2 also reabsorbs (paracellularly) some calcium and magnesium. Straight proximal tubule (S3) This segment forms the beginning of the descending limb of the loop of Henle and will be discussed in this context later. Its proper- ties and function are similar to S2. Clinical relevance PCT abnormalities can be acquired (usually drugs, e.g. amino­ glycosides and cytotoxics) or genetic. Chronic damage from drugs (e.g. ifosfamide), myeloma, and amyloidosis can cause a renal Fanconi’s syndrome with urinary glucose, phosphate, and bicar- bonate wasting, and tubular (LMWP) proteinuria. Genetic forms of renal Fanconi’s syndrome include Dent’s disease (CLCN5) and Lowe’s syndrome (OCRL) (both X-​linked). Selective genetic trans- port defects include renal glycosuria (SGLT2; SCL5A2) and a Table 21.1.1  Key transport functions of the proximal tubule Reabsorption Secretion • c.65% of filtered Na+, H2O, K+, and Cl− • Organic anions (e.g. salicylate and furosemide) • All filtered glucose and amino acids • Organic cations (e.g. creatinine, histamine, cimetidine,a and amiloride) • All filtered LMWPs (megalin/​ cubilin receptor dependent) • H+ • Most filtered bicarbonate (coupled to H+ secretion) • NH4 + • Most filtered phosphate (inhibited by parathyroid hormone) • Most filtered urate a Cimetidine can be used to reduce creatinine secretion, which increases in chronic kidney disease, and to estimate GFR more accurately from creatinine clearance. Lumen S1 Na+ Cotransporter Cotransporter Cotransporter Exchanger Na+ Na+ Na+ H+ H2O AQP1 AQP1 K+ K+ Na+ Interstitium glucose Low molecular weight proteins (LWMP - RBP/albumin) → amino acids phosphate Fig. 21.1.12  S1 contains AQP1 diffusive water channels, so there is some transcellular diffusive water movement driven osmotically.

21.1  Structure and function of the kidney 4725 form of isolated proximal renal tubular acidosis (SLC4A4). See Chapter 21.16 for further discussion. Loop of Henle The loop of Henle is defined as the segment between S2 and the DCT and includes S3, the thin descending (present in deep, long-​ looped juxtamedullary nephrons) and ascending limbs, and the thick ascending limb of Henle (TALH). The loop reabsorbs 40% of Na+ and 30% of filtered water. In the thin descending limb, water is reabsorbed osmotically into the hypertonic medullary interstitium. In contrast, the thin and thick ascending limbs are impermeable to water, although significant quantities of Na+ are reabsorbed passively in the thin ascending limb and actively in the TALH (Fig. 21.1.16). Na+ reabsorption in the water-​impermeable ascending limb, to- gether with osmotic equilibration in the descending limb, and coun- tercurrent flow (due to its U shape) generates an osmotic gradient (from cortex to medulla) in the medullary interstitium, which can reach approximately 1200 mOsm/​kg in the papillae (countercurrent multiplication). This gradient ensures concentrated urine in the col- lecting ducts. Countercurrent multiplication is driven by active trans- port of Na+ along the TALH. Na+ enters via the triple cotransporter (Na+,K+,2Cl−; NKCC2), which is the target of ‘loop diuretics’ such 2.0 1.5 1.0 0.5 0 25 20 % of proximal tubular length HCO3- Na+ CI- Inulin TF/P ratio Amino acids Glucose 75 10 0 Fig. 21.1.13  Relative concentration of molecules along the PCT. Results based on micropuncture data using the nonreabsorbed, but freely filtered, marker inulin. As water is reabsorbed along the proximal tubule, the inulin concentration in tubular fluid relative to plasma (TF/​P) rises, but that for Na+ stays the same (TF/​P = 1), because water is reabsorbed in proportion to Na+. Phosphate and bicarbonate decrease, since they are reabsorbed; chloride (Cl−) increases slightly, because reabsorption is delayed and slower. LMW proteins Albumin PTH DBP - 25(OH)D3 Lumen RBP/albumin Megalin/ cubilin Recycling endosome Early endosome Late endosome Lysosome Ligand receptor Proximal tubule cell Coated vesicle Fig. 21.1.14  The mechanism for low molecular weight (LMW) protein reabsorption involving receptor-​mediated endocytosis and internalization, with eventual protein degradation in lysosomes. Albumin and LMW proteins, including parathyroid hormone (PTH) and vitamin D-​binding protein (DBP) with 25(OH)D3, are filtered into the primary urine and endocytosed by PT cells via the megalin-​ cubilin receptor pathway. Following internalization in coated vesicles, the receptor-​ligand complexes progress along the endocytic pathway. The endosomes undergo a progressive, ATP-​dependent acidification that results in the dissociation of the receptor–​ligand complexes, with megalin and cubilin being recycled in the apical membrane, whereas the ligand is directed to lysosomes for degradation. In the case of 25(OH)D3-​DBP, DBP is degraded in lysosomes, whereas 25(OH)D3 is released in the cytosol and metabolized to active 1,25(OH)2D3 in mitochondria before being released into the circulation. Adapted with permission from Devuyst O and Pirson Y (2007). Genetics of hypercalciuric stone forming diseases. Kidney International, 72(9), 1065–​72. Copyright © 2007 International Society of Nephrology.

SECTION 21 Disorder s of the ki dne y and u rinary trac 4726 as furosemide, and is transported across the basolateral membrane by the sodium pump (Na+,K+-​ATPase) (Fig. 21.1.16). There is also a small contribution from Na+/​H+ exchanger as in the PCT. TALH ion transport produces a lumen-​positive transepithelial potential differ- ence (10–​15 mV) that drives cation reabsorption (Ca2+ and Mg2+; and some Na+ and K+) through a selective paracellular (paracellin/​ claudin) pathway. By blocking the triple cotransporter in the TALH, loop diuretics dissipate the medullary osmotic gradient and positive transepithelial potential difference, leading to natriuresis and diur- esis, and increased K+, Ca2+, and Mg2+ excretion. At the ‘top’ of the TALH are the specialized macula densa cells that ‘sense’ NaCl delivery and can adjust the rate of NaCl delivery to match the reabsorptive capacity of the TALH through the mech- anism of tubular glomerular feedback (mentioned previously), which vasoactively (via local release of angiotensin II and adeno- sine) reduces glomerular filtration. Loop diuretics block the sensing mechanism and can inhibit tubular glomerular feedback, thus aug- menting their natriuretic and diuretic effect. Clinical relevance The autosomal recessive Bartter’s syndrome has a phenotype similar to chronic loop diuretic administration, and its various clinical sub- types are the result of mutations in transporters involved in Na+ re- absorption along the TALH (Fig. 21.1.16): Bartter type 1, NKCC2 (apical Na+ entry; SLC12A2); Bartter type 2, K+ channel (apical K+ recycling; ROMK/​KCNJ1); Bartter type 3, Cl− channel (basolateral Cl− exit; CLCNKB); and Bartter type 4, Cl− channel regulatory sub- unit (basolateral Cl− exit; BSND). These are examples of ‘loss-​of-​ function’ mutations; however, there is also a type 5 variant due to an activating mutation of a basolateral calcium sensing receptor (CASR) that inhibits TALH Na+ reabsorption. See Chapter 21.16 for further discussion. Early distal convoluted tubule The early DCT (Fig. 21.1.17) is part of the so-​called diluting seg- ment because it is still relatively impermeable to water. Sodium reabsorption is via the thiazide-​sensitive Na+-​Cl− cotransporter (NCC). Unlike loop diuretics, thiazides reduce Ca2+ excretion, in part as a result of a lower intracellular Na+ concentration, leading to an increase in Na+/​Ca2+ exchange and basolateral Ca2+ extrusion (Fig. 21.1.17), as well as a mild degree of diuretic-​induced hypovol- aemia increasing proximal reabsorption (see earlier). Clinical relevance Thiazides are used to reduce hypercalciuria in renal stone formers, and are modest antihypertensive drugs with few side effects, apart from hypokalaemia and hyponatraemia that are especially prevalent in elderly women. The autosomal recessive Gitelman’s Lumen Urate Na+ Citrate2- CI- Na+ Na+ H+ K+ K+ Na+ NaDC-1 URAT1 ABCG2, NPT1,4 S2 K+ H2O AQP1 AQP1 CA-II CA-IV Interstitium Fig. 21.1.15  Late proximal convoluted tubule. Lumen Interstitium Loop diuretics work here K+ K+ K+ K+ CI- CI- Ca2+ CaSR CIC-Ka CIC-Kb Barttin regulatory subunits CLDN16/19 TALH (Paracellin) 10–15 mV lumen positive potential difference Encourages reabsorption K+ Ca2+ Mg2+ 2CI- Na+ Na+ Na+ δ+ δ– * Fig. 21.1.16  Thick ascending limb of Henle.

21.1  Structure and function of the kidney 4727 syndrome has a similar phenotype to chronic thiazide diuretic use and is due to mutations in the gene encoding NCC (SLC12A3). See Chapter 21.16 for further discussion. Late distal convoluted tubule and collecting duct The DCT epithelial permeability is low, hence almost all transepithelial transport across the DCT is active and transcellular. DCT and collecting duct cells are of two main types: principal cells (most numerous) that reabsorb Na+ and water and secrete K+ (Fig. 21.1.18), and intercalated cells that secrete H+ (α-​cells) or bicarbonate (β-​cells) ions (Fig. 21.1.19), α-​cells outnumber β-​intercalated cells. Potassium-​sparing diuretics (e.g. amiloride) block the apical Na+ channel (ENaC) and decrease the transepithelial voltage, reducing K+ secretion and potentiating hyperkalaemia, especially in combin- ation with nonsteroidal anti-​inflammatory drugs or ACE inhibitors, and occasionally with trimethoprim. Principal cells respond to two key hormones:  aldosterone (Fig. 21.1.18) and vasopressin (anti- diuretic hormone), which activates adenylate cyclase via basolateral membrane V2-​receptors, increasing aquaporin water channel in- sertion into apical membrane. Collecting duct fluid then becomes osmotically equilibrated with the surrounding interstitial fluid, iso- tonic in the cortex but increasingly hypertonic in the medulla such that the osmolality of maximally concentrated urine can be approxi- mately 1200 mOsmol/​kg. In the absence of antidiuretic hormone, the TALH hypotonic fluid remains hypotonic and the urine dilute. Clinical relevance DCT and collecting duct defects in water handling lead to polyuria. Lithium interferes with vasopressin signalling causing a nephrogenic diabetes insipidus. Genetic mutations in the vasopressin V2 (AVPR2) receptor cause an X-​linked form of nephrogenic diabetes insipidus, and mutations in the gene encoding aquaporin 2 (AQP2) cause both reces- sive and dominant forms. A rare activating mutation of V2 receptor can be a cause of hyponatraemia. See Chapter 21.2.1 for further discussion. Mutations in ENaC can cause a form of pseudohypoaldosteronism (type IA), as can mutations of the mineralocorticoid receptor (type IB) (recessive). A gain-​of-​function mutation in ENaC is the cause of Gordon’s syndrome (dominant) with the expected clinical features of hypertension and hypokalaemia. Genetic mutations in the acid–​base transporters of the α-​intercalated cells cause distal renal tubular acidosis, which can be recessive (apical H+ secretion; ATP6V1B1, ATP6V0A4) or dominant and recessive (basolateral Cl−/​bicarbonate exchanger; SLC4A1). See Chapters 16.17.4 and 21.15 for further discussion. Interstitium The interstitium comprises the intertubular, extraglomerular, and extravascular space of the kidney, limited by the tubular and vas- cular basement membranes, and containing extracellular matrix and interstitial fluid as well as lymphatics. It also contains immune cells (dendritic cells, macrophages, and lymphocytes) and fibroblasts. The interstitium is not only a source of hormonal and paracrine fac- tors produced locally and secreted from other renal cells, including renin (from juxtamedullary cells), adenosine (transforming growth Na+ Na+ Lumen DT Interstitium Thiazide diuretics work here K+ K+ CI– CI– Ca2+ Ca2+ Ca2+ Mg2+ Na+ Fig. 21.1.17  Early DCT. Amiloride works here 20–25 mV lumen negative potential difference ENaC Na+ K+ K+ K+ Na+ H2O ADH AQP2 AQP3 or AQP4 Late DT and CD ROMK δ+ δ– + ++ Aldosterone enhances all three pathways Spironolactone acts here Fig. 21.1.18  The principal cell of DCT. Na+ enters via the apical epithelial Na+ channel (ENaC) and exits by the usual sodium pump. This process generates a lumen negative transepithelial potential difference (cf. TALH) that favours K+ secretion via apical K+ channels (ROMK). Aldosterone increases Na+ reabsorption and K+ secretion by stimulating the basolateral sodium pump and enhancing activity of the apical Na+ (ENaC) and K+ (ROMK) channels. ADH inserts AQP2 into the apical membrane. Spironolactone acts by competing with nuclear mineralocorticoid receptors in epithelial cell nuclei. Atrial and brain natriuretic peptides decrease sodium reabsorption in the DCT and cortical collecting duct via 3ʹ,5ʹ-​cyclic guanosine monophosphate (cGMP)-​dependent phosphorylation of ENaC. Lumen H+ H+ HCO3

H2O + CO2 Late DCT and CD β (beta) α (alpha) CA-II HCO3

HCO3

CI- CI- H+ CI- CI- Interstitiun Fig. 21.1.19  α-​Intercalated cells actively secrete H+ across their apical membrane via an H+-​ATPase pump (cf. the sodium pump) and with the help of carbonic anhydrase (type II—​cf. the proximal tubule), bicarbonate exits across the basolateral membrane via an exchanger with Cl−, similar to the one present in red blood cells. CD, collecting duct.

SECTION 21 Disorder s of the ki dne y and u rinary trac 4728 factor), and endothelin, but it also has a major role in the pathogen- esis of interstitial fibrosis, a key factor in the progression of chronic kidney disease. The renal fibroblast is the key cell with a complex lineage and several functions, including synthesis of extracellular matrix, and it is also the source of erythropoietin that is reduced in chronic kidney disease, resulting in anaemia. Some authorities have also speculated that the renin-​secreting juxtaglomerular cells may be fibroblast precursors, linking the two hormonal systems. ACE inhibitors and angiotensin receptor blockers are known to reduce the haematocrit, decrease postrenal transplant erythrocytosis, and to increase the clinical requirement for recombinant erythropoietin in chronic kidney disease. Acknowledgement In memoriam: the authors are grateful for the slides and teaching materials of the late Professor David Shirley on which Figs. 21.1.9 to 21.1.19 are based. The authors also wish to thank Miss Nia Harper for further de- tailed artwork. FURTHER READING Burford JL, et al. (2017). Combined use of electron microscopy and intravital imaging captures morphological and functional features of podocyte detachment. Pflugers Arch, 469(7–8), 965–74. Butler MJ, et al. (2019). Aldosterone induces albuminuria via matrix metalloproteinase-dependent damage of the endothelial glycocalyx. Kidney Int, 95(1), 94–107. Gyarmati G, et al. (2018). Advances in Renal Cell Imaging. Semin Nephrol, 38(1), 52–62. Mount DB (2014). Thick ascending limb of the loop of Henle. Clin J Am Soc Nephrol, 9, 1974–​86. Neal CR, et al. (2018). Novel hemodynamic structures in the human glomerulus. Am J Physiol Renal Physiol, 315(5), F1370–F1384. https://www.ncbi.nlm.nih.gov/pubmed/30388047. Palmer LG, Schnermann J (2015). Integrated control of Na transport along the nephron. Clin J Am Soc Nephrol, 10, 676–​87. Subramanya AR, Ellison DH (2014). Distal convoluted tubule. Clin J Am Soc Nephrol, 9, 2147–​63. Other material The following articles obtain videos that are very useful on this subject: http://​ajprenal.physiology.org/​content/​ajprenal/​suppl/​2006/​04/​12/​ 00521.2005.DC1/​40XSNGFR_​short.avi http://​www.ncbi.nlm.nih.gov/​pmc/​articles/​PMC3922634/​bin/​1471-​ 2369-​15-​24-​S15.mov http://​www.ncbi.nlm.nih.gov/​pmc/​articles/​PMC3884556/​bin/​ NIHMS530039-​supplement-​3.mov http://​www.ncbi.nlm.nih.gov/​pmc/​articles/​PMC3884556/​bin/​ NIHMS530039-​supplement-​4.mov http://​www.jci.org/​articles/​view/​33293/​sd/​2