Introduction to Renal Structure and Function Renal Genital 2019

INTRODUCTION TO RENAL STRUCTURE AND FUNCTION

Orson W. Moe, M.D. Office: H05122; Phone: 82754 Email: [email protected]

LEARNING OBJECTIVES:

• Concept: The is a homeostatic organ that maintains constancy of the composition and amount of internal environment. Deviations from normal are sensed and then rectified. Sensing is largely extrarenal but some mechanisms can be intrarenal. • The kidneys achieve homeostasis by executing three functions as an excretory, metabolic, and endocrine organ. • Anatomically identify the , , renal calyces, medullary pyramids, , renal , and . Trace the from the main renal artery to the glomerular blood vessels, , the , and . • Appreciate the unique features of the renal circulation. • Learn the structural components of the and the juxtaglomerular apparatus. Describe the three layers of glomerular filtration barrier separating the blood from urine. • Walk axially down the and learn the different tubular segments traversed sequentially by the ultrafiltrate from Bowman’s space and finally reaching the . • Understand the difference between filtration, reabsorption, and secretion and how the three processes work in concert to achieve excretion. • Learn the generic design of a polarized transporting epithelium. Understand paracellular vs. transcellular transport, the model of an epithelial cell with polarized location of transporting proteins, and how the currency of energy is translated from metabolic substrates to ATP, and finally, to electrochemical gradients. • In addition to excretion, the kidney also has metabolic and endocrine functions. The kidney is a major site of gluconeogenesis. Certain endocrine hormones are secreted by the kidney into the circulation such as renin, vitamin D, and erythropoietin.

I. Introduction

Multicellular organisms evolved to house their cells in a constant organism world internal environment. The kidneys are regulatory organs poised at an interface between the organism and the external world that maintain a constant optimal internal environment in both composition and amount (Figure 1). Deviations of any parameter beyond the normal physiologic range are sensed, and corrective Maintain constancy measures are deployed to correct the parameter back to normal limits (Figure 2). Failure of correction sets the stage for disease. Composition Quantity Figure 1: Kidney: Interface between the organism and the world Introduction to Renal Structure and Function Renal Genital Urinary System 2019

There are mechanisms that monitor the physiologic parameter of interest and hence constitute the afferent arm of the loop (Figure 2). Effectors are the rectifying mechanisms that constitute the efferent arm of the homeostatic loop. Depending on the variable Physiologic sensed, the afferent sensing arms can be extra-renal or renal. Afferent parameter Efferent Sensor Effector One principal function of the kidney is excretion. The substances excreted can be exogenous toxins or drugs that are not normally present, or they can be endogenous substances that normally reside in the body but are present in excess. When the body is

Figure 2: Renal homeostatic loop deficient of an endogenous substance, the kidney conserves it by decreasing its excretion. In addition to excretion, the kidney also achieves homeostasis by providing metabolic substrates such as glucose to the body. The kidney also acts an endocrine organ, secreting circulating hormones to act on other organs.

The importance of renal function is easily appreciated in patients with either acute or chronic kidney disease where secondary failure of multiple organs ensues because of the disruption of the maintenance of the internal environment.

Figure 3: Genitourinary system and gross anatomy of kidney II. Renal Anatomy The kidneys are seated against the posterior wall of the abdomen in the retroperitoneal space (Figure 3). The kidney is surrounded by a capsule. The bisected surface of the kidney has a lighter colored outer region called the cortex and the darker inner region called the medulla (Figure 3). The medulla is divided radially into outer and inner regions with the outer medulla subdivided into outer and inner stripes. The medulla has multiple conical contours called renal pyramids with their apices abutting on the renal pelvis to form the papillae. The contact points of the renal pelvis with the renal parenchyma are cup-like structures called calyces. Interpolated between the pyramids are centripetal extensions of cortical tissue into the medulla called columns of Bertin. In contrast to the human kidney, the rodent kidney (which is used extensively as an experimental model) has only one pyramid and papilla. Introduction to Renal Structure and Function Renal Genital Urinary System 2019

Each kidney usually receives blood from a single renal artery (although there can be two or more renal arteries per kidney). Just before or after the renal artery enters the kidney, it divides into a series of smaller branches. These branches give rise to smaller interlobar arteries that pass between the pyramids of the kidney radially up the Columns of Bertin (Figure 4).

Figure 4: Renal vasculature hierarchy (left). Latex casts of renal vessels (right)

Interlobar arteries divide to form smaller vessels, arcuate arteries, which run along the junction of the medulla and cortex parallel to the surface of the kidney (Figures 4). Arcuate arteries give rise to still smaller arteries, cortical ascending arteries, which carry blood from the deep part of the cortex to the superficial cortex. As glomeruli are located exclusively in the cortex, it is the cortical ascending arteries that bring arterial blood in proximity to glomeruli. then distribute blood from cortical ascending arteries to individual glomeruli. In each glomerulus, the incoming afferent arteriole forms a highly branched network of specialized glomerular capillaries. There are four unique features of the renal circulation (Table 1).

Feature Implication Little or no anastomoses Very prone to regional disruption of blood flow High blood flow per gram tissue Lowest oxygen extraction (lowest A-VO2 difference) Functional arteriovenous shunts Solutes and gases (e.g., O2) can diffuse directly from artery to vein without passing through the capillaries Multiple capillaries in tandem The two capillaries serve difference functions in sequence Table 1: Four salient feature of the renal circulation

Introduction to Renal Structure and Function Renal Genital Urinary System 2019

Figure 5: The glomerular and peritubular capillaries are in tandem (left). The intrarenal vasculature is shown with arteries and shown under red and blue shaded areas, respectively (right).

The glomerular capillaries provide the site for glomerular ultrafiltration, the first step in making urine (Figure 5). The plasma that is not filtered at the end of each glomerular capillary network enters the that conveys blood out of the glomerulus. Note that even though this “arteriole” is situated after a capillary system (glomerular capillary), it is not called a venule. It is an arteriole because it is in front of a second capillary system- the peritubular capillaries. surrounding the tubular elements in both the cortex and medulla. The peritubular capillaries provide oxygen and nutrients for the bulk of the kidney. They work with the tubules by collecting the fluid and solutes reabsorbed by tubules to return to the circulation. They also deliver solutes that are secreted by tubules from the blood into urine. Blood in the peritubular capillaries is returned to the circulation by a venous system that follows the architectural structure of the arterial supply: interlobular vein, , interlobar vein, and renal vein (Figure 5).

IV. Renal Excretion Excretion of a substance can be mediated by one or a combination of three processes: 1) Filtration, 2) Secretion, and 3) Reabsorption. Figure 6 compares two systems – pure filtration vs. filtration-reabsorption – and shows what they imply in Figure 6: Functional implications of filtration, secretion, and reabsorption. terms of demands on regulation. Consider the excretion of 1L/day by purely filtration. A 5% error (reduction in filtration) will mean that only 0.95 L/day will be excreted - a 50 ml error. Now compare this to a filtration-reabsorption model where 170 L/day is filtered and 169 L/day is reabsorbed resulting in the same 1 L/day excretion. A 5% error (reduction in reabsorption) will mean 160L/day of reabsorption resulting in excretion of 10L/day and an absolute error of 9L. A clear consequence of a filtration-reabsorption design is that regulation has to be exquisitely tight and even small errors are not tolerated. Introduction to Renal Structure and Function Renal Genital Urinary System 2019

V. Microscopic Anatomy

A. The Nephron The kidney is composed of a large number of functional units called (Figure 7). Each human kidney has approximately 1 million nephrons. The nephron is composed of multiple different types of cells. Approximately 30% of nephrons have their glomeruli situated deep in the cortex near the medulla; these are called juxtamedullary nephrons. The nephrons whose glomeruli are located in the mid or superficial cortex are called superficial or cortical nephrons. The different cell types are summarized in Table 2.

Figure 7: Overview of nephrons- One juxtamedullary and one cortical nephron are shown Introduction to Renal Structure and Function Renal Genital Urinary System 2019

Structure Cell types Main function Epithelia have parietal and visceral cells, Filtration of protein-poor and cell- Glomerulus Capillaries comprise endothelial cells, free fluid [150-180 liters/day]. Between the capillaries reside mesangial cells. Proximal Extensive brush border which are finger-like Bulk reabsorption of filtered Na+, + - - 3- 2+ convoluted tubule projections of apical membrane that greatly K , Cl , HCO3 , PO4 , Ca , glucose, (PCT) expands the surface area. This is the only cell in and organic solutes; ammonia the kidney with such apical membrane structure. production; and secretion. The basolateral membrane is also highly Gluconeogenesis. invaginated to increase surface area. The cell has Production of 1,25-dihydroxy- lots of mitochondria, many of them located near vitamin D. the basal membrane. Proximal straight Simpler than the proximal convoluted tubule. Very similar to PCT. Usually lesser tubule (PST) Fewer microvilli on the apical surface and in magnitude. invaginations on the basolateral side. Mitochondria are smaller and randomly scattered throughout the cytoplasm. + - + 2+ 2+ - Thick ascending Moderately well-developed apical surfaces; Na , Cl , K , Ca , Mg , and HCO3 limb (TAL) basolateral surfaces are highly invaginated. Lots reabsorption; maintenance of of mitochondria located in the basal invaginations. medullary tonicity; Medullary (mTAL) and cortical portion (cTAL) ammonia/ammonium transport. Macula densa cells are highly specialized tubule Sensing filtrate composition, epithelial cells of the TAL. osmolality, and volume; critical for tubuloglomerular feedback. + - - 2+ Distal convoluted Similar to the thick ascending limb cell: Na , Cl , HCO3 , and Mg tubule (DCT) moderately developed apical surface, invaginated reabsorption. Ca2+ reabsorption is basolateral membrane; lots of mitochondria, many transcellular of them located in the basal invaginations. Connecting tubule Similar to DCT Similar to DCT and cortical (CNT) collecting tubule: features of both Cortical collecting Principal cells: Few short, stubby apical Na+ reabsorption. K+ secretion. tubule (CCT) microvilli; moderate amount of basolateral ADH-sensitive H2O reabsorption. invaginations; few mitochondria scattered Acid-base balance: throughout the cytoplasm. -intercalated cells: H+ secretion; K+ Intercalated cells: -intercalated cells all along the reabsorption. - CT. -intercalated cells: Only in cortical CT. Both - intercalated cells: HCO3 secretion cell types have poorly developed apical and basolateral Medullary Inner medullary collecting tubule (IMCD) cells; Fine tune regulation of NaCl collecting tubule less extensively developed apical and basolateral reabsorption, K+ reabsorption, H+ - (MCT) surfaces and fewer mitochondria compared to secretion (HCO3 regeneration), intercalated cells. ADH-sensitive H2O reabsorption, urea reabsorption. Table 2: Summary of characteristics of individual nephron segments

Introduction to Renal Structure and Function Renal Genital Urinary System 2019

B. Structure of the Glomerulus The glomerulus is composed of vascular, epithelial, and mesangial components. In the human the glomerulus is about 200 nm in diameter (Figure 8). Afferent arterioles lose their smooth muscle upon entering the glomerular tuft, and ramify into the glomerular capillary. The capillary is housed inside an epithelial structure call the Bowman’s capsule which has a visceral layer and a parietal layer (Figure 8). Between the parietal and visceral layer of Bowman’s capsule is Bowman’s space where urine is first formed.

Figure 8: (Top left) A micrograph of the glomerulus and (Top right) the glomerular structure. A “punched-out” balloon example will be given in class to aid in your understanding of the glomerular structure. (Bottom) Structures within the glomerulus. The visceral cell of the Bowman’s capsule facing the urine side is the (Figure 9); so named because of the extensive “foot processes”. The smooth muscle layers of the afferent and efferent arterioles are critical in determining arteriolar tone. The glomerular capillary is suspended in close contact to the mesangium on one side and separated from the foot processes of the visceral epithelium of Bowman’s capsule (podocyte) on the opposite side by a characteristic basement membrane. The capillaries themselves have large lumina and possess fenestrations (50-100 nm in diameter). Introduction to Renal Structure and Function Renal Genital Urinary System 2019

Afferent arterioles lose their smooth muscle upon entering the glomerular tuft, and ramify into the glomerular capillary. The capillary is housed inside an epithelial structure call the Bowman’s capsule which has a visceral layer and a parietal layer. Between the parietal and visceral layer of Bowman’s capsule is Bowman’s space where urine is first formed.

A. C.

B. D.

Figure 9: A. Scanning electron micrograph of glomerular capillary wrapped by foot processes of . B. Cartoon and corresponding transmission electron micrograph of a glomerular capillary and a podocyte. C. and D. Close up of the filtration barrier of endothelium, basement membrane and podocyte.

C. Glomerular Filtration Barrier The glomerulus filters large volumes of water and solutes while retaining most of the proteins and all of the cellular elements of blood. The barrier is a three-layered structure (Figure 10):

I. Fenestrated endothelial cells. The innermost part of the barrier. The fenestrations are not just “holes” but repels negatively charged large molecules in the blood.

II. Glomerular basement membrane. The middle layer of the barrier. It has an electron dense central layer flanked by two thinner layers. The glomerular basement membrane (GBM) restricts movement of large molecules (e.g., large proteins) into Bowman’s space. There is also negatively charged glycoproteins with sialic acid residues which repels negatively charged plasma proteins.

III. Epithelium. There are visceral and parietal layers of cells of the Bowman’s capsule. The visceral epithelium consists of the podocytes and contributes to the filtration barrier; the parietal layer does not. The podocytes have a highly interdigitating system of epithelial foot processes that rest against the basement membrane. The podocyte cell bodies lie within the extracellular matrix. The spaces between foot processes are called filtration slits diaphragms which are negatively charged and contributes to the restriction of middle-size negatively charged particles. Introduction to Renal Structure and Function Renal Genital Urinary System 2019

There have been momentous advances in the identification of the components of slit diaphragm complex and in understanding their functions, although the current model is far from complete. Some of the proteins on the growing list are listed in Figure 10.

Urine

Slit diaphragm Foot process of Filtration Podocyte

Proteins of the complex 300 nM Nephrin Podocin CD2AP Foot P-cadherin Neph1 Densin-180 Glomerular Basement Membrane process FAT1

50-100 nm Proteins of the slit diaphragm Fenestrated Endothelium Blood Figure 10: (Left) Three layers of the filtration barrier. (Right) Molecular composition of the slit diaphragm. Interposed between the glomerular capillary loops is the mesangium. The mesangium is composed of mesangial cells embedded in the mesangial matrix. Mesangial cells have contractile functions and can alter intraglomerular renal blood flow as well as the single nephron glomerular filtration rate. Mesangial cells also produce mesangial matrix, have phagocytic capabilities, and perform paracrine functions such as synthesis of prostaglandins. Part of the mesangium extends beyond the glomerular corpuscle forming the extraglomerular mesangium, which is part of the juxtaglomerular apparatus. The parietal epithelium is flat and squamous with few organelles. At the vascular pole, the parietal epithelium is contiguous with a different epithelium – the proximal convoluted tubule.

VI. Juxtaglomerular Apparatus The juxtaglomerular apparatus (JGA) is a specialized tripartite structure in the vascular pole of the glomerulus (Figure 11) consisting of 1) the afferent arteriole and efferent arteriole, 2) extraglomerular mesangium and mesangial cells, and 3) a specialized part of the thick ascending limb that physically contacts the glomerulus called the macula densa.

The granular cells in the vascular wall are contains renin. The unique juxtaposition of the glomerular arterioles, mesangium, free access to systemic circulation, and Figure 11 Juxtaglomerular complex consisting of the afferent Na+-transporting tubular epithelium arteriole with granular cells in its wall, mesangial cells, macula densa cells at the end of the thick ascending , and allows the JGA to fulfill its critical role in efferent arteriole. There are nerve fibers innervating the afferent + systemic regulation of Na balance and arteriole blood pressure. This is achieved via the endocrine renin-angiotensin system and the intrarenal regulation of glomerular filtration rate via tubular glomerular feedback.

Introduction to Renal Structure and Function Renal Genital Urinary System 2019

VII. Structure of the Renal Tubule The epithelial cell is the key player of the renal Brush border tubule. The renal tubule is a prototypical polarized Protrusions epithelium. The salient characteristics are summarized in Figure 12. There are structures Tight designed to amplify the surface area available for junctions transport. In the luminal side (the apical membrane), this is achieved by either protrusions or a special extensive form of protrusions called the brush border. Intercellular space Between cells are structures called tight junctions. Although they are called tight junctions, some are truly tight (high resistance to solute and charge Infoldings Interdigitation movement), whereas others can be quite leaky (low Figure 12: Characteristics of a renal tubule. Two resistance to solute and charge movement). adjacent cells are depicted in white and grey.

In addition to resistance, the junctional complexes also regulate whether the junction is more permeable to one ion vs. another (relative and selective permeability). For example, one anion such as chloride may be more permeant than another one such as bicarbonate. Behind the tight junctions is the intercellular space which is contiguous with the interstitial space between the tubule and the peritubular capillary. On the interstitial-capillary side (the basolateral Active Transport membrane), surface area is amplified by either infoldings into the cell or interdigitations between cells. Currency exchange

The movement of a solute can be through a cell, termed transcellular transport (Figure 12), or it can be around the High ATP Electrochemical energy gradient cell, which is called paracellular transport (Figure 12). substrates Solute transport is an energy-requiring process that requires metabolic fuels. In the kidney there is constant conversion of “fuel currency.” Urine Metabolic Interstitium substrates ATP Metabolic substrates such as amino acids, glucose, organic 3Na+ 2K+ anions, and fats are converted to ATP, the universal energy unit for all cells (Figure 13). ATP can be directly Low [Na+] Na+ hydrolyzed by transport proteins called ATPases that couple -60 mV the energy release from ATP directly to the uphill movement Glucose + + of ions. Figure 13 shows the example of how the Na /K - ATPase converts ATP to create a low intracellular Na+ and Figure 13: (Top) Switch in fuel utilization. a negative interior voltage. The Na+-glucose co-transporter (Bottom) Metabolic substrates generates ATP. on the apical membrane couples the movement of a Na+ to a The Na-K-ATPase translates ATP into + electrochemical gradients such as low cell Na glucose (carrying a net positive charge). The low cell Na and negative interior voltage, which in turn concentration and negative voltage will energize glucose provides the electrochemical driving force for uptake across the apical membrane. glucose uptake in the apical membrane.

There are different kinds of transport proteins shown in Figure 14. ATPases directly couple to ATP hydrolysis to transport. There are co-transporters (symporters) that move two solutes in the same direction. There are counter-transporters (antiporters) that move two different solutes in opposite directions. There are channels that are protein “holes” that allows ions or solutes to permeate. Introduction to Renal Structure and Function Renal Genital Urinary System 2019

Different transporters can also be coupled together to literally “create” a new transport system. Finally there are proteins that protrude outside the cell in the junctional area to provide a conduit for paracellular transport.

Figure 14: Types of transporter proteins

VII. The Kidney as an Endocrine Organ In addition to excretion of unwanted solute and water, the kidney is also an important endocrine organ. There are a number of hormones that are secreted by the kidney into the systemic circulation. Three of these are manipulated by clinicians every day. Renin is important for maintenance of the integrity of the circulation and it permits the kidney to have a constant glomerular filtration rate in the face of low and fluctuating slay intake. This is vital for terrestrial existence. Erythropoietin is produced in the kidney to act on the bone marrow to produce erythrocytes. The location of erythropoietin producing cells in the cortical interstitium is due to the unique ability of these cells when positioned here to sense the balance between oxygen delivery and consumption. The 1-alpha-hydroxylase is perfectly situated in the one segment of the nephron () where the whole body defense of phosphate is being defended. Lesser expression of the same enzyme is found in the rest of the nephron segments.

VIII. Glossary of terms Glomerular filtration: Net movement of water and solutes from the glomerular capillaries into Bowman’s space. Introduction to Renal Structure and Function Renal Genital Urinary System 2019

Ultrafiltration: Formation of fluid similar to plasma except most proteins and all cells are missing. This is called the ultrafiltrate because the filter prevents passage of molecules as small as 5-10 nm in diameter – 1000 times smaller than red blood cells. The fluid within Bowman's space contains most solutes in concentrations approximately equal to those in plasma.

Tubular reabsorption: Movement of a substance from the tubule lumen into the peritubular capillaries without Net specifying the nature or mechanism of the transport process.

Tubular secretion: Net movement of a substance from the peritubular capillaries into the tubule lumen without specifying the nature or mechanism of the transport process. Physiologically important solutes such as H+, K+, + NH4 , and urate are normally secreted into the tubular urine (Figure 15).

Urinary excretion: Refers to what actually appears in the urine. Excretion is usually expressed as mass per time. In general, Excretion = Filtration - Reabsorption + Secretion.

IX: Fractional excretion: Fractional excretion is abbreviated by FE. This is the Figure 15: Excretion is a net result of three processes: Glomerular filtration, tubular amount excreted in the urine divided by the amount filtered secretion and tubular reabsorption. Each at the glomerulus (Figure 15). A substance that is filtered substance is handled differently by a but neither reabsorbed not secreted will have its excretion combination of these three processes. rate exactly equal to its filtered rate. The FE will be 1. A substance that is filtered and reabsorbed will have an FE < 1. A substance that is filtered and secreted will have an FE > 1.

IX. Concept of Clearance Clearance is an artificial concept. The clearance of substance X is defined as the volume of body fluid that is void of X in unit time; hence clearance has the unit of volume/time.

Excretion rate of X Clearance: An imaginary concept: Clearance of any substance X = Theoretical volume of body fluid that is cleared of substance X Plasma [X] Clearance has the unit of volume/time Cr Excretion UCr V Creatinine Clearance ClCr = = Plasma [Cr] PCr Plasma concentration Urea Excretion Uurea V of X Urea Clearance Clurea = = Plasma [urea] P Production [X] Excretion Urea Mass Mass Mass Ucr = urine creatinine concentration U = urine urea concentration Time Volume Time urea V = urine in unit time PCr = plasma creatinine concentration P = plasma urea concentration urea Figure 16: Definition and calculation of clearance