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Renal Physiology (4th Sem CC4-9-TH)

Structural Organization of :

The kidneys are a pair of bean-shaped structures that are located just below and posterior to the liver in the peritoneal cavity. The adrenal glands sit on top of each kidney and are also called the suprarenal glands. Kidneys filter and purify it. All the blood in the human body is filtered many times a day by the kidneys; these organs use up almost 25 percent of the oxygen absorbed through the lungs to perform this function. Oxygen allows the kidney cells to efficiently manufacture chemical energy in the form of ATP through aerobic respiration. The filtrate coming out of the kidneys is called .

Externally, the kidneys are surrounded by three layers. The outermost layer is a tough connective tissue layer called the renal fascia. The second layer is called the perirenal fat capsule, which helps anchor the kidneys in place. The third and innermost layer is the renal capsule. Internally, the kidney has three regions—an outer cortex, a medulla in the middle, and the renal pelvis in the region called the hilum of the kidney. The hilum is the concave part of the bean-shape where blood vessels and nerves enter and exit the kidney; it is also the point of exit for the ureters. The is granular due to the presence of —the functional unit of the kidney. The medulla consists of multiple pyramidal tissue masses, called the renal pyramids. In between the pyramids are spaces called renal columns through which the blood vessels pass. The tips of the pyramids, called renal papillae, point toward the renal pelvis. There are, on average, eight renal pyramids in each kidney. The renal pyramids along with the adjoining cortical region are called the lobes of the kidney. The renal pelvis leads to the ureter on the outside of the kidney. On the inside of the kidney, the renal pelvis branches out into two or three extensions called the major calyces, which further branch into the minor calyces. The ureters are urine-bearing tubes that exit the kidney and empty into the urinary bladder.

The kidney tissue (parenchyma) contains about one million little filter units, the so-called Nephrons. These nephrons filter the blood and produce the urine. Each consists of a so-called renal corpuscule followed by a tubule. Together, corpuscule and tubule form a functional unit: the corpuscules filter the blood, thereby producing about 100 litres of urine every day. To reduce this large volume, the tubules regain most of the filtered and fluids (about 99 %) and return it to the blood circulation. As a result, only about a tenth of the initially produced urine (called primary urine), for example 1 - 1.5 liters, is finally released. The volume of the primary and final urine produced by the corpuscules per day mainly depends on the fluid intake as well as on the total blood volume, which is higher in adults than in children. While the renal corpuscules are found in the outer renal cortex, the tubules are located within the inner and drain – via special collecting ducts – into the so-called renal calyces. This is where the urine is collected and transported to the renal pelvis, ureter and urinary bladder.

Figure1. Kidneys filter the blood, producing urine that is stored in the bladder prior to elimination through the urethra.

Microstructural organization of kidney :

Because the kidney filters blood, its network of blood vessels is an important component of its structure and function. The arteries, veins, and nerves that supply the kidney enter and exit at the renal hilum. Renal blood supply starts with the branching of the aorta into the renal arteries (which are each named based on the region of the kidney they pass through) and ends with the exiting of the renal veins to join the inferior vena cava. The renal arteries split into several segmental arteries upon entering the kidneys. Each segmental artery splits further into several interlobar arteries and enters the renal columns, which supply the renal lobes. The interlobar arteries split at the junction of the renal cortex and medulla to form the arcuate arteries. The arcuate “bow shaped” arteries form arcs along the base of the medullary pyramids. Cortical radiate arteries, as the name suggests, radiate out from the arcuate arteries. The cortical radiate arteries branch into numerous afferent arterioles, and then enter the supplying the nephrons. Veins trace the path of the arteries and have similar names, except there are no segmental veins.

As mentioned previously, the functional unit of the kidney is the nephron. Each kidney is made up of over one million nephrons that dot the renal cortex, giving it a granular appearance when sectioned sagittally. There are two types of nephrons—cortical nephrons (85 percent), which are deep in the renal cortex, and juxtamedullary nephrons (15 percent), which lie in the renal cortex close to the renal medulla. A nephron consists of three parts—a , a renal tubule, and the associated network, which originates from the cortical radiate arteries.

Figure 2. The internal structure of the kidney is shown.

Renal Corpuscle

The renal corpuscle, located in the renal cortex, is made up of a network of capillaries known as the and the capsule, a cup-shaped chamber that surrounds it, called the glomerular or Bowman’s capsule.

Renal Tubule

The renal tubule is a long and convoluted structure that emerges from the glomerulus and can be divided into three parts based on function. The first part is called the proximal convoluted tubule (PCT) due to its proximity to the glomerulus; it stays in the renal cortex. The second part is called the , or nephritic loop, because it forms a loop (with descending and ascending limbs) that goes through the renal medulla. The third part of the renal tubule is called the distal convoluted tubule (DCT) and this part is also restricted to the renal cortex. The DCT, which is the last part of the nephron, connects and empties its contents into collecting ducts that line the medullary pyramids. The collecting ducts amass contents from multiple nephrons and fuse together as they enter the papillae of the renal medulla.

Capillary Network within the Nephron

The capillary network that originates from the renal arteries supplies the nephron with blood that needs to be filtered. The branch that enters the glomerulus is called the afferent arteriole. The branch that exits the glomerulus is called the efferent arteriole. Within the glomerulus, the network of capillaries is called the glomerular capillary bed. Once the efferent arteriole exits the glomerulus, it forms the peritubular capillary network, which surrounds and interacts with parts of the renal tubule. In cortical nephrons, the peritubular capillary network surrounds the PCT and DCT. In juxtamedullary nephrons, the peritubular capillary network forms a network around the loop of Henle and is called the vasa recta.

Figure 3. The nephron is the functional unit of the kidney. The glomerulus and convoluted tubules are located in the kidney cortex, while collecting ducts are located in the pyramids of the medulla.

Mechanism of Urine formation in Kidney :

Every one of us, including plants and animals, depend on the process for the removal of certain waste products from our body. During the process of excretion, both the kidneys play an important role in filtering the blood cells. Excretion is a biological process, which plays a vital role by eliminating toxins and other waste products from the body. In plants and animals including humans, as the part of metabolism, lot of waste products are produced. Plants usually excrete through the process of transpiration and animals excrete the wastes in different forms such as by urine, sweat, faeces, and tears. Among all these, the usual and the main form of excretion is the urine.

Urine formation begins with the delivery of blood to the glomerulus followed by its past the glomerular barrier. The filtered portion of plasma continues through the nephron whereas the unfiltered portion passes into the peritubular capillaries. As the filtered portion travels through the nephron, and certain solutes are resorbed back into the peritubular capillaries whilst other solutes are secreted from the peritubular capillaries into the nephron. Whatever fluid is remains at the end of the nephron is discarded as urine.

Urine Formation - Formation of urine is a process important for the whole organism. Not only acid-base balance is modulated by it, but also blood osmolarity, plasma composition and fluid volume, and thus it influences all cells in our body. A healthy adult person produces 1.5-2 liters of urine per day and this process involves three basic mechanisms: 1) Glomerular filtration

2) Tubular

3) Tubular secretion

Functional histology - Glomerulus consists of fenestrated capillaries without diaphragm that form important part of a renal filtration barrier. Blood flow and in afferent and efferent arteriole is strictly regulated, which allows glomerular filtration into Bowman’s capsule. Visceral layer of Bowman’s capsule consists of podocytes and their pedicels that tightly fit to the basement membrane of capillaries. Parietal layer is formed by a single layer of simple squamous epithelium. The renal filtration barrier is composed of the fenestrated capillary endothelium, the basement membrane and the pedicles of podocytes. Pedicels interdigitate with one another forming filtration slits that are spanned by slit diaphragms (formed by protein nephrin). Due to its negative charge it prevents the filtration of plasma protei

In the glomerulus we can find mesangium that provides mechanical support, has the phagocytic activity and secrets . Mesangial cells outside the glomerulus together with the macula densa cells (distal segment of the ascending limb of loop of Henle) and the granular cells (modified smooth muscle cells of the afferent arteriole) from juxtaglomerular apparatus. This is the place where renal corpuscle gets into contact with the renal tubular system.

The is lined by simple cuboidal epithelium with a well- developed brush border on the luminal side, the thin portion of Henle’s loop is lined by a simple squamous epithelium (poor in organelles). The distal tubule cells are smaller than those of proximal tubule and lack the brush border. Collecting ducts consist of principal intercalated cells.

Glomerular filtration - The volume of liquid filtered per unit time in all glomeruli can be expressed as the glomerular filtration rate (GFR). Its physiological value is 120 ml/min/1,73m2 body surface area, thus 180 l/day. About 99 % of the filtrate gets reabsorbed by the tubular resorption to the (back into the body), leaving only 1.5-2 l of urine per day. Movement of the fluid through the filtration membrane is controlled and determined by the ratio of the hydrostatic pressure in the capillaries and oncotic pressure of plasma proteins (less by the hydrostatic pressure of the interstitial fluid and oncotic pressure in the filtrate). These forces are called Starling´s forces and there are a few differences from the general principles: 1) Fluid is not exchanged between the capillary and the interstitium, but between the capillary and the fluid of Bowman’s capsule

2) Hydrostatic pressure in the capillaries is different, the movement is thus only one-sided (in the direction of filtration) 3) Filtration barrier (see above) has a unique structure and properties which do not allow passage of proteins into the filtrate (primary urine)

GFR is therefore dependent on the renal blood flow, the filtration pressure, the plasma oncotic pressure, and the size of the filtration area.

The principles governing the GFR are in reality a specialized application of the principles discussed in microcirculatory physiology and are thus ultimately governed by Starling Forces. However, because the glomerular capillaries are surrounded by the fluid in the Bowman's Capsule, the hydrostatic and oncotic pressure of Bowman's Space is used instead of those of the 'Interstitial Fluid'. Importantly, because no plasma proteins can cross the glomerular barrier during glomerular filtration, the oncotic pressure of fluid in Bowman's Space is essentially zero and is thus removed from the .

Formally:

GFR = Kf [(Pc-Pb)-(Πc)]

GFR = Glomerular Filtration Rate (ml/min). Equivalent of Jv in Starling Forces Kf = Permeability Constant of glomerular capillaries Pc = Glomerular Capillary Hydrostatic Pressure Pb = Bowman's Space Hydrostatic Pressure Πc = Glomerular Capillary Oncotic Pressure

Glomerular Capillary Hydrostatic Pressure Modulation

Overview

The GFR can be modulated by changing any of the variables mentioned above. However, the primary physiological mechanism by which GFR is modulated is through modification of the glomerular capillary hydrostatic pressure. The glomerular capillary pressure is itself determined by three variables as discussed below.

Systemic Arterial Pressure

Naturally, increased or decreased incoming systemic arterial blood pressures will increase or decrease the hydrostatic pressures within the glomerular capillaries. However, due to autoregulatory mechanisms discussed in Autoregulation of GFR and RBF incoming arterial pressures into the glomerulus are largely constant over a wide range of systemic arterial pressures and thus are not a major source of regulation. Renal Afferent Arteriolar Resistance

Whatever the incoming arterial pressure, the resistance offered by the renal afferent arteriole is a major determinant of the ultimate hydrostatic pressure within the glomerulus. If afferent arteriolar resistance is increased, the hydrostatic pressure within the glomerulus declines and so too does the glomerular filtration rate. Conversely, if the afferent arteriolar resistance is decreased, the hydrostatic pressure within the glomerulus increases and so too does the glomerular filtration rate. Consequently, regulation of renal afferent arteriolar resistance is a major source of modulation of the GFR.

Renal Efferent Arteriolar Resistance

The relationship between the resistance of the renal efferent arteriole with GFR is more complex and not well-understood. A hand-waving explanation is that increased renal efferent arteriolar resistance causes backup of blood into the glomerulus and consequently increases glomerular hydrostatic pressures and thus GFR. However, as efferent arteriolar resistance increases, the renal blood flow also declines, resulting in reduced GFR. Empirically, these two antagonistic phenomenon result in a biphasic relationship to increased efferent arteriolar resistance and GFR. Initial increments in efferent arteriolar resistance increase GFR; however, larger increments in efferent resistance result in decreased GFR.

Modulation of other variables

Overview

Although modulation of any of the other variables mentioned under "Physical Determinants" can change the GFR, these are not major sources of physiological GFR regulation. However, as mentioned below, some of these variables are affected in certain disease states and thus can result in pathological changes in GFR.

Permeability Coefficient

The permeability of the glomerular barrier can decline due to thickening of the glomerular basement membrane as occurs in . Additionally, total glomerular permeability can decline due to reductions in the total number of functional glomeruli, thus reducing the total glomerular surface area as occurs in hypertension.

Bowman's Space Hydrostatic Pressure In cases of urinary tract obstruction resulting in hydronephrosis, fluid backup into Bowman's Space can increase its hydrostatic pressure and thus reduce GFR.

Control of glomerular filtration - Its main determinant is the renal blood flow that is directly proportional to the pressure difference between renal artery and and inversely proportional to the peripheral resistance of the afferent and efferent arteriole and the interlobular artery. We distinguish local and central regulatory mechanisms. Local regulatory mechanisms - Local regulatory mechanisms consist mainly of myogenic autoregulation and tubuloglomerular feedback.

Myogenic autoregulation Elevated blood pressure leads to the contraction of renal blood vessels, thereby increasing peripheral resistance. The reverse process occurs when the blood pressure decreases. Thanks to this regulatory mechanism remains the renal blood flow (and thus the GFR) relatively unchanged during normal fluctuations of the mean arterial blood pressure (80-180 mmHg).

Tubuloglomerular feedback A decrease in GFR is registers by macula densa (part of the juxtaglomerular apparatus). As an answer to the detection of a low flow of or a reduced amount of it sends paracrine chemical signal that causes vasodilation of the afferent arteriole, leading to an increase in a hydrostatic pressure and to a restoration of normal GFR.

Central regulatory mechanisms - The central regulatory mechanisms are less important. They are represented by the sympathetic nervous system, epinephrine, - II, prostaglandins and adenosine. Postganglionic neurotransmitter of the sympathetic nervous system norepinephrine causes particularly in the afferent arteriole vasoconstriction, thereby reducing the renal blood flow (and thus the GFR) It is important especially in stressful situations, including pain and bleeding. Epinephrine has a similar effect. Angiotensin II (via angiotensin receptor AT1) acts on both the afferent arteriole and the efferent arteriole in similar way as sympathetic nervous system and epinephrine. Locally produced prostaglandins (especially E2 and I2) reduce the effects of sympathetic nervous system and angiotensin II on both the afferent arteriole and the efferent arteriole. Adenosine is generally effective vasodilator, in afferent arteriole but acting as vasoconstrictor. Furthermore, the renal blood flow is increased by atrial natriuretic peptide (ANP), , nitric oxide or kinins,whereas antidiuretichormone (ADH), ATP and endothelin cause a reduction in the renal blood flow.

Assessment of the glomerular filtration rate - If we want to determine GFR, which is one of the basic function of our kidneys, we have to use a substance that is excreted from the body only by glomerular filtration (, ) and is not affected by tubular processes. As an example we can mention the calculation of the (plasma volume that is per unit time completely cleaned of marker substances) of endogenous creatinine, whose formula has the following form:

U – urine creatinine concentration in mmol/l V – volume of urine () in ml/s

P – plasma creatinine concentration in mmol/l

In clinical practice, we use more complex calculations, corrected for body surface area (and other physical parameters) – e.g. equation by Cockroft and Gault, equation MDRD etc.

Tubular reabsorption and secretion - As it is mentioned above, about 99 % of the filtrate gets reabsorbed by the tubular resorption to the extracellular fluid (back into the body), leaving only 1.5-2 l of urine per day. The main task for renal tubules is therefore an isosmotic tubular reabsorption of primary urine. They absorb water, ions (sodium, , , , , or ), urea, and amino acids. All of this is independent on the extracellular fluid volume in the body – we speak about the obligatory resorption. Its primary role is to maintain fluid volume in the body under normal conditions.

Transport can be carried by passive (in the direction of the concentration or electrical gradient), primary against gradient (needs energy – ATP) or secondary active transport (transport protein uses the concentration gradient created by a primary active transport realized by other transport protein). Substances can be transported by paracellular or transcellular routes. Transport of water is always passive. Na+/K+-ATPase located on the basolateral membrane plays important role in the secondary active transport. It creates a concentration gradient for Na+. Transport proteins act as symporters (transport of compound is coupled to the transport of Na+ in the same direction) or antiporters (transport of compound is coupled to the transport of Na+ in the opposite direction). To understand the processes in the tubular system, we must imagine tubular epithelial cells, their apical membrane facing the tubular fluid (primary urine), basolateral membrane, on the other hand, is in contact with the peritubular fluid (here is located the Na+/K+-ATPase).

The proximal tubule - Reabsorption of sodium ions is in the first half of the proximal tubule coupled with the reabsorption of bicarbonate, glucose, amino acids, lactate, urea and phosphate. Absorbed compounds are osmotically active, thereby draining water from tubules. This leads to an increased concentration of ions in the tubular fluid that is very important for a resorption in other parts of the proximal tubule.

Reabsorption of bicarbonate ions in the proximal tubule Movement of bicarbonate and ions depends on the transport sodium ions. This process is catalyzed by (located in the apical membrane and in the intracellular part of the epithelial cells). The first step is the secretion of H+ into the tubular fluid through the Na+/H+ antiport, located at the luminal (apical) membrane of proximal tubule cells. Transferred H+ may in the tubular fluid react with filtered bicarbonate ions to form carbonic acid. Carbonic anhydrase facilitates the decomposition of carbonic acid in the tubular fluid to water and . Both compounds can freely diffuse into the tubule epithelial cells, where carbonic acid is restored by the carbonic anhydrase. Molecules of carbonic acid dissociates into hydrogen and bicarbonate ions. Bicarbonate ions then pass through the basolateral + – membrane into the interstitial fluid through Na /3HCO3 - – – + cotransporter or anion exchanger (Cl /HCO3 ). H returns via antiport with + + + – Na into the tubular fluid. For each secreted H , Na and HCO3 is absorbed (Na+ is returned to the blood by active transport in exchange for K+ – Na+/K+- ATPase).

Renal (tubular) threshold Glucose, and many other organic compounds are in this part of the tubule completely resorbed under physiological conditions. This transport has some maximum value – so-called renal/tubular threshold. As an example we can mention the renal threshold for glucose. When this renal threshold is exceeded (due to too high plasma concentration – such as 10 mmol/l for glucose), glucose reabsorption in the proximal tubule is incomplete and some amount of glucose remains in the final urine. Unabsorbed osmotically active molecules drain water molecules to renal tubules, thereby increasing diuresis (osmotic polyuria).

Reabsorption of sodium ions is in the second half of the proximal tubule coupled with the transport of chloride ions, used are both transcellular (on basolateral membrane helps K+/Cl–-symport) and paracellular routes. Relatively abundant positively charged ions (sodium, potassium, calcium, magnesium) in the tubular fluid accompany chloride ions in . Transport of ions is followed by passive reabsorption of water. Loop of Henle Henle’s loop absorbs about 25 % of the solutes (thick segment of the ascending limb), but only about 15 % water (descending limb). Its proper function (thick part of the ascending limb is impermeable to water and has active transport of Na+ and Cl–) is essential for the formation of a high osmotic pressure (hyperosmolarity) in the renal medulla that ensures a production of highly concentrated urine. Some mechanisms of reabsorption of ions are similar to those in the proximal tubule. Very important is the specific symport of Na+, K+ and 2 Cl– across the apical membrane. This symport uses energy derived from the transport of sodium and chloride ions in the direction of their concentration gradient for the transport of potassium ions into the (against their concentration gradient). Some of these ions leave cells on the basolateral membrane (together with Cl–), some return back into the tubular fluid, thereby creating an electrical imbalance. Due to this, positively charged ions (Na+, K+, Ca2+, Mg2+) are resorbed by paracellular route (very important mechanism for resorption of solutes). This is especially significant for formation of a hypertonic renal medulla. Hypotonic fluid leaves the loop of Henle and enters the distal tubule.

Clinical correlation: Substances that block the symport (e.g. furosemide) are used as very effective drugs – loop .

Distal convoluted tubule and collecting duct Distal convoluted tubule and collecting duct resorbe about 7 % of solutes (mainly Na+ and Cl–) and approximately 17 % water. Their resorption is affected by (e.g. ADH) – facultative resorption. Hydrogen and potassium ions are secreted here. The distal convoluted tubule and the collecting duct thus play an important role in the formation of the final urine and in the regulation of osmolarity and pH. Sodium and chloride ions are absorbed in the first part of the distal convoluted tubule. The distal part of the distal convoluted tubule and the collecting duct consist of two cell types: 1) Principal cells responsible for the resorption of sodium ions and water (dependent on ADH) and secretion of K+ ions 2) Intercalated cells containing carbonic anhydrase. They are involved in acid- base balance, because they can secrete both hydrogen and bicarbonate ions

Calcium and phosphate reabsorption and secretion Plasma concentration of total calcium is 2.25-2.75 mmol/l and for ionized calcium 1.1-1.4 mmol/l. Only ionized calcium (about 48 % of total) is filterable by kidneys. Resorption takes place by both active (15-20 %) and passive paracellular (80 %) mechanisms. It is localized in the proximal tubule, the ascending part of Henle’s loop and partially in the distal convoluted tubule. Parathyroid stimulates the reabsorption by transcellular route in this segment. acts the same way, just mostly in the distal convoluted tubule. In contrast, calcitonin increases the excretion of calcium ions by inhibition of tubular reabsorption.

Serum phosphate concentration is 0.7-1.5 mmol/l, urine concentration is 15-90 mmol/l. are also influenced by the (inhibits the resorption of phosphates) and by the calcitonin (also reduces the resorption of phosphates).

Control of tubular processes We can distinguish local and central regulatory mechanisms.

Local mechanisms Local mechanisms are represented mainly by Starling´s forces (increased plasma oncotic pressure leads to an increased reabsorption of water and solutes from the interstitium into the capillaries, thereby supporting the tubular resorption) and glomerulotubular balance (increased GFR leads to an increase in glucose, amino acids and sodium ions resorption, these are followed by water – volume of resorbed fluid increases proportionally with increased GFR).

Central mechanisms Central mechanisms are represented by many hormones – such as ADH, , angiotensin II, epinephrine, natriuretic peptides (ANP and BNP) or parathyroid hormone. Sympathetic nervous system has a role also.

ADH (antidiuretic hormone, ) is produced in the and secreted by the posterior pituitary gland in a response to an increased osmolarity of extracellular fluid (to a lesser extent as an answer to a decrease of extracellular fluid volume). ADH binds to the V2-receptor located on collecting duct cells (partly on distal tubule cells). Its effect increases the number of in cell membranes and water molecules can pass along the osmotic gradient into peritubular fluid (ECF). ADH acts also on a transport of urea in the collecting duct and on a transport of Na+ and Cl– in the thick segment of the ascending limb of the loop of Henle. Aldosterone is secreted by the zona glomerulosa of the adrenal cortex in response to increasing plasma concentrations of angiotensin II and potassium ions. It plays therefore an important role in maintaining of constant level of potassium ions (accelerates secretion of potassium ions in the thick segment of the loop of Henle and in the distal tubule) and in regulation of volume of ECF. As the part of the -angiotensin-aldosterone system, it stimulates reabsorption of sodium ions, accompanied by passive water resorption (distal tubule and collecting ducts). This system is activated by decrease in the plasma volume.

Angiotensin II stimulates aldosterone secretion and resorption of sodium ions (and consequently resorption of water molecules) in the proximal tubule.

Sympathetic nervous system and epinephrine stimulate reabsorption of sodium ions and water molecules in the proximal tubule and in the thick segment of the loop of Henle.

As the name suggests, natriuretic peptides (ANP – atrial natriuretic peptide and BNP – brain natriuretic peptide) increase natriuresis. They inhibit Na+ reabsorption in the distal tubule, thereby increasing its loss in urine. Sodium ions drain water molecules, result is increased diuresis. Both peptides are secreted by our heart. ANP is secreted by atrial cardiomyocytes, the stimulus for its secretion is an increased wall stress (increased venous return causes dilation of heart). BNP is secreted by ventricular cardiomyocytes, the signal is increased tension in the ventricular wall. Natriuretic peptides thus mediate response of our organism to an excess of Na+ and increased blood volume. Only natriuretic peptides (together with dopamine) increase diuresis. Parathyroid hormone reduces Ca2+ excretion (stimulates reabsorption of Ca2+ from the primary urine) and increases excretion of phosphates in our kidneys. In result, it increases calcaemia and decreases phosphatemia. Control of urine osmolarity There are several processes controlling the urine osmolarity. Excretion of excess water leads to a formation of hypotonic urine, excretion of excess solutes results in a formation of hypertonic urine.

1) Dilution of urine a) The loop of Henle creates an osmotic gradient from the cortex to the hypertonic medulla (due to impermeability of the thick segment to water molecules and high reabsorption of solutes) b) Production of ADH is reduced c) Urea passes from the medulla into the tubular system, thereby reducing hypertonicity of the medulla

2) Production of hypertonic urine a) The loop of Henle creates an osmotic gradient (hypertonic medulla); Na+, Cl– (see above) and urea play an important role – hypertonicity of the renal medulla reaches its maximum b) Production of ADH is increased c) Urea circulates in the renal medulla – increased hypertonicity of the medulla

Final urine - Final urine is characteristically malodorous, clear, golden yellow liquid. Its specific gravity varies between 1 003-1 038 kg/m3 and its pH between 4.4-8.0. It contains Na+ (100-250 mmol/l), K+ (25-100 mmol/l), Cl– (about 135 mmol/l), Ca2+, creatinine, vanillylmandelic acid (degradation product of ), uric acid, urea, etc. Healthy kidneys do not allow a significant amount of proteins and glucose to reach the final urine (they are almost completely reabsorbed). Presence of high amount of proteins and glucose in the final urine is a pathological finding. Normal diuresis is 1.5-2 l/day. Polyuria is diuresis higher than 2 l/day, oliguria lower than 0.5 l/day and anuria lower than 0.1 l/day

Acid Base balance by kidney –

Acid-base and pH regulation are critical for both normal physiology and cell metabolism and function. The importance of this regulation is evidenced by a variety of physiologic derangements that occur when plasma pH is either high or low. The kidneys have the predominant role in regulating the systemic bicarbonate concentration and hence, the metabolic component of acid-base balance. This function of the kidneys has two components: − reabsorption of virtually all of the filtered HCO3 and production of new bicarbonate to replace that consumed by normal or pathologic acids. This − production or generation of new HCO3 is done by net acid excretion. Under normal conditions, approximately one-third to one-half of net acid excretion by the kidneys is in the form of titratable acid. The other one-half to two-thirds is the excretion of ammonium. The capacity to excrete ammonium under conditions of acid loads is quantitatively much greater than the capacity to increase titratable acid. Multiple, often redundant pathways and processes exist to regulate these renal functions. Derangements in acid-base homeostasis, however, are common in clinical medicine and can often be related to the systems involved in acid-base transport in the kidneys. Acid-base homeostasis and pH regulation are critical for both normal physiology and cell metabolism and function. The importance of this regulation is evidenced by a variety of physiologic derangements that occur when plasma pH is either high or low. The kidneys have the predominant role in regulating the systemic bicarbonate concentration and hence, the metabolic component of acid-base balance. This function of the kidneys has two components: reabsorption of virtually all of the − filtered HCO3 and production of new bicarbonate to replace that consumed by − normal or pathologic acids. This production or generation of new HCO3 is done by net acid excretion. Under normal conditions, approximately one-third to one-half of net acid excretion by the kidneys is in the form of titratable acid. The other one-half to two-thirds is the excretion of ammonium. The capacity to excrete ammonium under conditions of acid loads is quantitatively much greater than the capacity to increase titratable acid. Multiple, often redundant pathways and processes exist to regulate these renal functions. Derangements in acid-base homeostasis, however, are common in clinical medicine and can often be related to the systems involved in acid-base transport in the kidneys

− Renal Control of Plasma HCO3

The kidneys have the predominant role of regulating the systemic − HCO3 concentration and hence, the metabolic component of acid-base balance. This function of the kidneys has two components: reabsorption of virtually all − − of the filtered HCO3 and production of new HCO3 to replace that consumed − by normal or pathologic acids. This production or generation of new HCO3 is − done by net acid excretion. In other words, the kidneys make new HCO3 by − excreting acid. Because HCO3 is freely filtered at the glomerulus, − approximately 4.5 mol HCO3 is normally filtered per day − (HCO3 concentration of 25 mM/L ×GFR of 0.120 L/min ×1440 min/d). − Virtually all of this filtered HCO3 is reabsorbed, with the urine normally − − essentially free of HCO3 . Seventy to eighty percent of this filtered HCO3 is reabsorbed in the proximal tubule; the rest is reabsorbed along more distal − segments of the nephron . In addition to reabsorption of filtered HCO3 , the − kidneys also produce additional HCO3 beyond that which has been filtered at the glomerulus. This process occurs by the excretion of acid into urine. (As indicated above, the excretion of acid is equivalent to the production of alkali.) The net acid excretion of the kidneys is quantitatively equivalent to the amount − − of HCO3 generation by the kidneys. Generation of new HCO3 by the kidneys is usually approximately 1 mEq/kg body wt per day (or about 70 mEq/d) and − replaces that HCO3 that has been consumed by usual endogenous acid production (also about 70 mEq/d) as discussed above. During additional acid loads and in certain pathologic conditions, the kidneys increase the amount of − acid excretion and the resulting new HCO3 generation. Net acid excretion by the kidneys occurs by two processes: the excretion of titratable acid and the + excretion of ammonium (NH4 ). Titratable acid refers to the excretion of protons with urinary buffers. The capacity of the nephron to excrete acid as free protons is limited as illustrated by the fact that the concentration of protons (H+), even at a urine pH 4.5, is <0.1 mEq. However, the availability of urine buffers (chiefly phosphate) results in the excretion of acid coupled to these urine buffers . Under normal conditions, approximately one-third to one-half of net acid excretion by the kidneys is in the form of titratable acid. The other one-half + to two-thirds is the excretion of NH4 . The mechanism whereby the excretion of + NH4 results in net acid excretion will be discussed below. The capacity to + excrete NH4 under conditions of acid loads is quantitatively much greater than the capacity to increase titratable acid. Net acid excretion in the urine is, therefore, calculated as Loss of alkali in − the urine in the form of HCO3 decreases the amount of net acid excretion or − new HCO3 generation. Loss of organic anions, such as citrate, in the urine − represents the loss of potential alkali or HCO3 . However, in humans, the loss of these organic anions is not usually quantitatively significant in whole–body acid-base balance.

− Proximal Tubule HCO3 Reabsorption The proximal tubule reabsorbs at least 70%–80% of the approximately 4500 − mEq/d filtered HCO3 . The proximal tubule, therefore, has a high capacity for − − HCO3 reabsorption. Most (probably >70%) of this HCO3 reabsorption occurs by proton secretion at the apical membrane by sodium-hydrogen exchanger NHE3 . This protein exchanges one Na+ for one H+ ion, driven by the lumen to cell Na+ gradient (approximately 140 mEq/L in the lumen and 15–20 mEq/L in the cell). The low intracellular Na+ is maintained by the basolateral Na+/K+- ATPase. An apical H+-ATPase and possibly, NHE8 under some circumstances account for the remaining portion of proximal tubule H+ secretion and − + HCO3 reabsorption. This H -ATPase shares most subunits with the distal tubule H+-ATPase. In the lumen of the proximal tubule, the secreted H+ reacts − with luminal HCO3 to generate CO2 and H2O, which for the purposes here, can be considered to be freely permeable across the proximal tubule and reabsorbed. This reaction in the lumen is relatively slow unless catalyzed, which occurs normally, by carbonic anhydrase (CA; in this case, membrane-bound CAIV tethered in the lumen). There are multiple isoforms of CA, but membrane- bound CAIV and cytosolic CAII are most important in renal acid-base transport . Mutations in CAII are known to cause a form of mixed proximal and distal with osteopetrosis (22). + + − The source of the H in the cells is the generation of H and HCO3 from CO2 and H2O catalyzed in the cell by intracellular CA, predominantly cytosolic − CAII. The HCO3 generated within the proximal tubule cell by apical + − H secretion exits across the basolateral membrane. Most of this HCO3 exit − occurs by sodium HCO3 cotransport, NBCe1-A, or SLC4A4. The stoichiometry and transported ionic species have been active areas of − − investigation, but studies suggest that 3 HCO3 Eq or 1 HCO3 Eq and 1 −2 + CO3 Eq are transported with each Na ; this electrogenic stoichiometry results + − in Na and HCO3 being driven out of the cell by the cell–negative membrane voltage. Mutations in this (NBC-e1) protein cause proximal renal tubular acidosis (24). Chloride-bicarbonate exchange may also be present on the basolateral membrane of the proximal tubule but is not the main mechanism of − HCO3 reabsorption. The proximal tubule is a leaky epithelium on the basis of its particular tight junction proteins and therefore, unable to generate large transepithelial solute or − electrical gradients; the minimal luminal pH and HCO3 obtained at the end of the proximal tubule are approximately pH 6.5–6.8 and 5 mM, respectively. The inability of the proximal tubule to lower pH further may also result from the dependence on the Na+ gradient driving the H+ (pH) gradient. In the late proximal tubule, ongoing reabsorption of the low concentrations of luminal − HCO3 may be countered and balanced by passive backleak of plasma or − + + peritubular HCO3 into the lumen; however, continued Na /H exchange activity will result in continued Na+ reabsorption.

Distal Tubule Acidification Several segments after the proximal tubule contribute substantially to acid-base homeostasis. First, the thick ascending limb (TAL) reabsorbs a significant − amount of HCO3 , approximately 15% of the filtered load, predominantly through an apical Na+/H+ exchanger. TAL acid-base transport is regulated by a variety of factors (e.g., Toll-like receptors, dietary salt, aldosterone, angiotensin II, etc., but the integrated role of the thick limb acid-base transport in various acid-base disorders has not been well clarified. Beyond the TAL, the distal tubule compared with the proximal tubule has a + − limited capacity to secrete H and thereby, reabsorb HCO3 . During inhibition − − of proximal tubule HCO3 reabsorption, increased distal delivery of HCO3 can overwhelm this limited reabsorptive capacity. However, the collecting tubule is able to generate a large transepithelial pH gradient (urine pH <5 with blood pH approximately 7.4). This large pH gradient is achievable because of the primary active pumps responsible for distal nephron H+ secretion (discussed below) and because of the relative impermeability of the distal tubule to ions (i.e., a tight epithelial membrane). The large pH gradient ensures the generation of titratable + − acid and the entrapment of NH4 . The limitation in HCO3 reabsorption is likely, in part, caused by the absence of luminal CA discussed below, but there may be other factors as well, such as a limited number of H+ pumps. The distal tubule beyond the TAL consists of several distinct morphologic and functional segments, including the distal convoluted tubule, the connecting segment, and several distinct collecting duct segments, each with several cell types. Although several of these cell types can secrete H+, the CA–containing (and mitochondrial–rich) intercalated cells (ICs) are chiefly responsible for acid-base transport. There are at least three types of ICs: type A or α ICs, which + − secrete H ; type B or β ICs, which secrete HCO3 ; and non–A, non–B ICs, with a range of function that remains under investigation. Conceptually, H+ secretion − in the type A ICs will result in HCO3 reabsorption in the presence of luminal − − HCO3 but will acidify the urine and generate new HCO3 in the absence of − − luminal HCO3 (i.e., if virtually all of the filtered HCO3 has been reabsorbed upstream). The mechanism of this H+ secretion involves primarily an apical H+- ATPase as illustrated in. The apical H+-ATPase is an electrogenic active pump (vacuolar ATPase) able to secrete H+ down to a urine pH of approximately 4.5. This is a multisubunit ATPase resembling that in intracellular organelles. The subunits are in two domains: a V0 transmembrane domain and a V1 cytosolic domain. Mutations in certain subunits are a known cause of distal renal tubular acidosis. Regulation of this pump is primarily by recycling between subapical vesicles and the plasma membrane involving the actin cytoskeleton and microtubules . Regulation may also be accomplished, in certain situations by assembly or disassembly of the two domains and phosphorylation of subunits. H+ secretion also occurs through another set of pump(s): apical H+/K+-ATPases. H+/K+-ATPases in the collecting duct include both the gastric form of H+/K+- ATPase and the colonic form of H+/K+-ATPase. On the basis of inhibitor studies, there may also be a third type of H+/K+-ATPase in the collecting duct. These transporters likely contribute to urine acidification under normal conditions but particularly during states of potassium depletion. Conceptually, − − HCO3 reabsorption (or new HCO3 generation) as in the proximal tubule is a + − two-part process: secretion of H into the lumen and HCO3 exit from the cell across the basolateral membrane. Therefore, to complete the process of − − − HCO3 reabsorption or new HCO3 generation, HCO3 produced in the cell from CO2 and H2O exits across the basolateral membrane. In other words, the − HCO3 generated intracellularly by any of these pumps exits the basolateral membrane. This exit into the blood occurs through a chloride-bicarbonate exchanger, a truncated version of the anion exchanger 1 (AE1; band 3 protein), − − which is the Cl / HCO3 exchanger in red blood cells that facilitates CO2 transport. This is an electroneutral exchanger driven by the ion − concentrations of chloride and HCO3 . Mutations in AE1 can also cause distal renal tubular acidosis. Two other transporters SLC26a7 (a − − C1 /HCO3 exchanger) and KCC4 (a KCl cotransporter) are also in the basolateral membrane of some acid secreting cells in the collecting duct and likely contribute to urine acidification in certain conditions. − In the cortical collecting duct, in addition to HCO3 reabsorption by type A ICs, − simultaneous HCO3 secretion occurs in separate cells, the type B ICs. This process involves an apical chloride-bicarbonate exchanger and a basolateral H+- − ATPase . In animal studies, HCO3 secretion is stimulated by alkali loading and inhibited by low luminal chloride. The basolateral H+-ATPase is the same pump − − as in the apical membrane of type A IC, but the apical Cl / HCO3 exchanger has been identified as , a protein first identified as being abnormal in some patients with hereditary deafness (39,52,53). The importance of − HCO3 secretion in human pathophysiologic circumstances is not well characterized but may be important in the maintenance and/or repair of metabolic alkalosis. The type B ICs, non–A, non–B ICs, and pendrin have now also been implicated in NaCl transport/balance and hypertension . This involves transport of Cl− and − − HCO3 on pendrin and also, a sodium–driven chloride/HCO3 exchanger (NDCBE or SLC4A8); Cl− exits the cell on AE4. Cytosolic CA, CAII, is present in all ICs of the collecting tubule, and it + − facilitates provision of H to be secreted into the lumen and HCO3 to be released into the basolateral aspect and blood. However, several segments of the distal nephron do not have luminal CA (i.e., CAIV). The implications of this are that, as H+ is secreted into the lumen, luminal pH may be below equilibrium pH, − with pH, CO2, and HCO3 only coming to equilibrium later downstream in the + final urine. Final urine PCO2 may, therefore, rise above blood PCO2 (as H and − HCO3 react and come to equilibrium with CO2) and be an index of distal nephron H+ secretion.

Ammonia Excretion

+ NH4 excretion represents the other major mechanism whereby the kidneys − excrete acid. Of the net acid excreted (or new HCO3 generated) by the kidneys, approximately one-half to two-thirds (approximately 40–50 mmol/d) is because + of NH4 excretion in the urine. Additionally, during acid loading or acidosis, + NH4 excretion can increase several fold, in contrast to a more limited increase in titratable acidity. + Traditionally, NH4 excretion was viewed as the excretion of protons in conjunction with a buffer (ammonia [NH3]): However, + NH3/NH4 is not an effective physiologic buffer, because its pKa is too high (approximately 9.2), and most of total ammonia (>98%) at physiologic pH is + + NH4 on the basis of the pKa. Furthermore, NH4 , not NH3, is produced within the kidneys. However, total ammonia excretion in the urine does represent acid − excretion (or the production of new HCO3 ) when considered in conjunction with the metabolism of the deamidated carbon skeleton of . Glutamine is the predominant precursor of NH3 in the kidney (Figure 5). Conceptually, the metabolism of the carbon skeleton of glutamine can result in the formation of − + one HCO3 formed for every NH4 excreted. Other amino acids can also be used to some extent. The predominant enzymatic pathway for NH3 formation is mitochondrial phosphate–dependent glutaminase in the proximal tubule, which + produces one NH4 and glutamate. Glutamate dehydrogenase can then convert + glutamate into α-ketoglutarate and a second NH4 . The α-ketoglutarate produced − can be converted to glucose or completely metabolized to HCO3 . Other pathways for NH3 production also exist but are thought to be less important. Other nephron segments other than the proximal tubule can produce NH3 but not to the same extent, and the other segments do not have the same significant adaptive increases in ammoniagenesis with sustained acidosis as the proximal tubule. A variety of conditions affect the renal production of NH3, but the most important is that of acid-base status. Chronic can increase NH3 production several fold in the kidneys. Potassium balance also alters NH3 production, such that suppresses NH3 formation (and transport in both the proximal tubule and thick limb), and hypokalemia increases NH3 production by the kidneys. Each of these changes has clinical correlates in overall acid-base balance. A variety of hormones (angiotensin II, prostaglandins, etc.) have also been shown to alter NH3 production. The + transport of the produced NH4 along the nephron and into the urine is a + complex process that is still a topic of intense stud. Most of the NH4 produced is excreted into the urine rather than added to the venous blood; this is particularly true with acidosis—the proportions into urine and blood are almost even in normal conditions. Therefore, during acidosis, both total NH3 production and secretion into urine are increased. The overall picture of + + NH3/NH4 transport is summarized as follows. In the proximal tubular, NH4 is produced from glutamine as discussed above and then, secreted into the lumen. + + + + This secretion occurs by both Na /NH4 exchange on the Na /H exchanger and NH3 diffusion across the apical membrane. Interestingly, the secretion of + NH4 across the apical membrane is augmented by angiotensin II . Whether the diffusion of NH3 across the apical membrane of the proximal tubule is lipid- phase diffusion as long thought or facilitated by certain gas channels has not + been determined. In the loop of Henle, NH4 ion is reabsorbed into the + interstitium, where it accumulates in the medulla. In the TAL, NH4 is transported across the apical membrane predominantly on the Na+-K+-2Cl + + cotransporter, with perhaps some entry on K channels (72). NH4 has a similar hydrated size as K+ and therefore, can often be transported through many K+ pathways (73). Remarkably, the apical membrane of the TAL is virtually impermeable to NH3 in contrast to most cell membranes (74). NH3 probably leaves the TAL cell across the basolateral membrane by NH3 diffusion and + + NH4 movement by NHE4 (72). One fraction of this medullary NH4 is secreted + back into the late proximal tubule as NH3 diffusion coupled to H secretion, and + therefore, it is partially recycled. Another fraction of medullary NH4 enters the lumen of the cortical and medullary collecting ducts and as such, bypasses the superficial/cortical distal segment of the nephron. A small fraction of interstitial + NH4 is shunted to the systemic circulation for eventual detoxification by the liver.

Overview - Acid-base homeostasis is critical for normal physiology and health. Hence, multiple, often redundant pathways and processes exist to control systemic pH. Derangements in acid-base homeostasis, however, are common in clinical medicine and can often be related to the systems involved in acid-base transport in the kidneys.