Urine Formation

The physiological events involved in urine formation can be categorised into three stages: glomerular filtration, tubular reabsorption and tubular secretion. The urine is formed and excreted due to the combining effect of the three stages.

Urine excreted = (Quantity of glomerular filtrate

— substances absorbed by the tubule) + secretions of the tubule.

9.4.1 Glomerular Filtration

Glomerular filtration is a passive, non-selective process where fluids and electrolytes are filtered through the three layers of the glomerular membrane into Bowman’s space under the influence of specific physical forces. It is called non-selective and passive because filtration is indiscriminate without expending energy. The fluid collected in the Bowman’s space is called glomerular filtrate. The amount of glomerular filtrate formed per minute is known as the glomerular filtration rate (GFR). The composition of this filtrate is similar to blood except for the presence of blood cells and plasma proteins. The plasma flow rate through both the kidneys per minute is called renal plasma flow (RPF). Usually, about 20% of plasma that enters the glomeruli is filtered, producing 170 L of glomerular filtrate with an aver­age GFR of 125 mL/min or 180 L/24 h by both kidneys in humans. The fraction of renal plasma flow that is filtered is called the filtration fraction:

Filtration fraction = GFR/renal plasma flow

The amount of any substance present in plasma reaching the kidneys per minute is the plasma load for that substance. The plasma load that filters into the capsular space is known as the tubular load of the substance.

The physical forces involved in glomerular filtration are (1) glomerular capillary hydrostatic pressure that favours filtration, (2) colloid osmotic pressure (COP) of plasma proteins that oppose filtration, (3) hydrostatic pressure of fluid in the Bowman’s capsule which opposes the filtration and (4) Bowman's capsule osmotic pressure that favours the filtration.

Besides the net filtration pressure, GFR also depends on the glomerular capillary surface area and the permeability or the hydraulic conductivity of the capillary membrane. The product of these two factors is known as the filtration coeffi­cient (K_f). The value of K_f cannot be estimated directly but can be calculated as

So, from the values mentioned above, K_f can be calculated as 125/10 = 12.5 mL/min/mm of Hg of filtration pressure.

Chronic uncontrolled hypertension and diabetes mellitus affect the glomerulus’s permeability characteristics, reducing the K_f value and thereby reducing GFR.

9.4.2 Physiologic Control of GFR and Renal Blood Flow

Among the physical forces controlling filtration, plasma col­loid osmotic pressure and hydrostatic pressure of capsular fluid are usually not regulated for controlling glomerular filtration.

In acute conditions, like severe haemorrhage and ischae­mia, sympathetic stimulation causes vasoconstriction of renal arterioles resulting in reduced GFR. Apart from that, it can also contract the mesangial cells, reducing the surface area of the glomerulus participating in filtration resulting in the reduction of K_f value and GFR. Under normal resting conditions, the role played by these nerves in the regulation of GFR is meagre.

Humoral factors, like epinephrine, norepinephrine and endothelin, cause vasoconstriction and reduction in GFR, whereas prostaglandins (PGE₂ and PGI₂) and bradykinin cause vasodilatation, an increased renal blood flow and increase in GFR. Angiotensin II is a peptide showing prefer­ential vasoconstrictor property with the efferent arteriole of the kidney. This peptide is produced from angiotensinogen, a plasma protein. Angiotensinogen is converted to angiotensin I on proteolytic cleavage with renin of the JG apparatus. Angiotensin I in the lungs is converted to angiotensin II by the angiotensin-converting enzyme.

Autoregulation: Autoregulation is a process involving vari­ous intrinsic mechanisms by which the kidneys maintain relatively constant renal blood flow and GFR within a wide range of mean systemic arterial pressure. Two mechanisms are mainly involved, myogenic response and tubuloglomerular feedback.

Myogenic response: When the blood enters the afferent arteriole, the stretch receptors of smooth muscles on the walls of the blood vessels experience increased or decreased tension depending on the hydrostatic pressure exerted by blood on the walls. The increased pressure within the vessel causes the vessel wall to stretch which inherently contracts the vessel wall to restrict the blood flow. At the same time, the inherent relaxation of an unstretched afferent arteriole increases blood flow when the pressure decreases.

Tubuloglomerular feedback: Tubuloglomerular feedback is another mechanism of autoregulation to maintain opti­mum filtration pressure when the kidney experiences altered perfusion pressure. When GFR increases due to elevated glomerular hydrostatic pressure, macula densa cells sense an increase in the concentration of Na⁺ and Cl^- in the tubular fluid. This sensitisation of macula densa cells, by some unknown means, constricts the afferent arteriole lowering hydrostatic pressure and also contracts the mesangium lowering the filtration surface area. Both these effects reduce GFR. Macula densa cells can sense the reduced concentration of Na⁺ and Cl^- in the tubular fluid when GFR decreases due to lower hydrostatic pres­sure. Then renin is released from the JG cells, ultimately causing the formation of angiotensin II. Angiotensin II selectively constricts the efferent tubule offering more resistance to blood flow, and thus GFR is increased.

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Transmembrane Proteins in Excretion

Polycystin 1 and polycystin 2 are the two transmem­brane proteins coded by PKD1 and PKD2 genes expressed in different locations of renal epithelial cells, including the primary cilia present on the apical membrane of these cells. These cilia protrude from the cells to the lumen and act as tubular fluid flow sensors. They transduce the alterations in the tubular flow into a cellular response regulating fluid and electrolyte trans­port. Whenever increased tubular fluid flow occurs, there will be bending of cilia, which activates PKD1/ PKD2-dependent calcium ion influx into the cell and results in potassium ion secretion. It is also reported that functional loss of polycystins results in renal cyst formation.

9.4.3 Tubular Reabsorption

The water and solutes of the tubular fluids when transported to the peritubular capillaries throughout the nephron, includ­ing the collecting duct, are called tubular reabsorption. The substances reabsorbed across the tubular epithelial layer reach the renal interstitial, are then absorbed into the peritubular capillaries and finally get into the systemic circu­lation.

Along with water, soluble solutes like potassium, magne­sium and chloride ions and organic solvents are also taken to the interstitium through a process known as solvent drag. About 65% of filtered sodium, chloride, bicarbonate, magne­sium and potassium and almost all filtered amino acids and glucose are absorbed through PCT. The epithelial cells of PCT are provided with numerous mitochondria for meeting the increased metabolic demand associated with transport mechanisms. The microvilli’s extensive surface area provided on the luminal surface also favours bulk reabsorption.

Capillary dynamics in the peritubular capillaries favours reabsorption by bulk flow. In the peritubular capillaries, hydrostatic pressure and COP are 17 and 30 mm of Hg, respectively, whereas in the interstitial fluid, the values are 6 and 10 mm of Hg, respectively. Hence, the reabsorption pressure (30 + 6 = 36 mmHg) exceeds filtration pressure (17 + 10 = 27 mmHg) by 9 mmHg (36 - 27 = 9 mmHg), favouring reabsorption to peritubular capillaries. The tubular fluid remains isosmotic with plasma as both solutes and water are reabsorbed.

The amount of a substance filtered through the glomerular filtrate and presented to the tubule per minute is known as the tubular load of that particular substance. The maximum rate at which the substance is reabsorbed from the tubular lumen to the peritubular fluid is the tubular transport maximum (Tm). The renal threshold is the plasma concentration of a substance at which it first appears in the urine. The property of tubules to increase reabsorption by the increased tubular load due to increased GFR is known as glomerulotubular balance.

Na⁺-K⁺ ATPase present on the basolateral side of the tubular epithelial cells hydrolyses ATP. The released energy is used to transport sodium ions from the tubular cells to the interstitium. Potassium ions are taken in return into the inte­rior of the cells from the interstitial space. This is known as primary active transport. The increased transport of sodium ions out of the cell creates an intracellular potential of - 70 mV. This negative potential and decreased intracellular sodium concentration favour sodium diffusion into the cell from the tubular fluid in a concentration gradient. The energy released during primary active transport is used to transport another substance known as secondary active transport. For example, glucose or amino acid is transported along with sodium. Hence, secondary active transport is known as co-transport. If a secondary secretion occurs along with pri­mary active sodium transport, that is known as counter­transport. The inward influx of sodium ions accompanied by the outflow of hydrogen ions is an example of counter­transport.

Sodium reabsorption mainly (65%) occurs at the proximal PCT as co-transport and counter-transport. But chloride- driven Na⁺ transport takes place from the distal portions of the PCT. In the first two modes of transportation, sodium- coupled carrier molecules are involved, whereas in the chloride-driven Na⁺ transport, both Cl^- and Na⁺ ions are transported through the leaky tight junctions. About 25% of Na⁺ present in the tubular fluid is absorbed in the thick ascending limb of both cortical and medullary segments of the loop of Henle. Sodium is transported by co-transport using Na⁺-K⁺-2Cl^- carriers present on the luminal surface of the loop of Henle. Nearly 5% tubular Na⁺ is absorbed from the proximal segment of the distal tubule along with Cl^- co-transport. The second half of the distal tubule has principal cells and intercalated cells.

The principal cells reabsorb sodium and water from the lumen and secrete potassium ions into the lumen. The intercalated cells absorb potassium ions and secrete hydrogen ions. The principal cells are the site of action of the adrenal cortical hormone, aldosterone. The proteolytic enzyme renin is released from JG cells, and it affects the production of angiotensin II, which stimulates the adrenal cortex to release aldosterone. Aldosterone favours sodium reabsorption by increasing the number of Na⁺-K⁺ ATPase on the basolateral membrane of tubular epithelial cells. Aldosterone also increases potassium secretion by principal cells. The remaining 5% of sodium ions in the tubular fluid are absorbed under the control of aldosterone, depending on the body’s requirement. Atrial natriuretic peptide (ANP) inhibits the renal absorption of sodium by exerting its influence on the principal cells.

The excess positive charge generated due to absorption of sodium ions is neutralised up to 75% by chloride ion trans­port. Chloride transport occurs through tight junctions, and chloride can also be passively absorbed in a concentration gradient at the time of solvent drag. From the thick limb of the loop of Henle and the proximal segment of the distal tubule, chloride ions are absorbed by way of secondary active transport or co-transport with sodium.

Glucose and amino acids are reabsorbed by co-transport with sodium ions. They are released from the carrier molecules and transported to the peritubular space by facilitated diffusion inside the cells. In human beings, if GFR is 125 mL and plasma concentration of glucose is 1 mg/mL (100 mg/dL), tubular load of glucose will be 125 ? 1 = 125 mg/min. The transport maximum or tubular maximum (Tm) value for glucose is estimated to be an average of 375 mg/min for an adult human being. It is the maximum milligrams per minute at which a substance is transported from the tubular lumen to the interstitial fluid. Beyond the Tm value, the same increment in the serum level of a substance will be excreted through urine. When the blood glucose level increases to 2 mg/mL from 1 mg/mL, the tubular load becomes 250 mg/min. In this stage, a trace amount of glucose may appear in urine because some indi­vidual nephrons may have lower Tm values, and some other nephrons may not absorb to their maximum capacity. At a tubular load of 375 mg, both kidneys’ nephrons absorb glucose at their maximum capacity. The increased urinary concentration of any substance will reflect the increased plasma level of that substance. The Tm value for amino acids is 1.5 mM/min (Table 9.1).

Table 9.1 Renal threshold for glucose in different domestic species

Lower thresholds may occur in diabetes in cats, but stress causes hyperglycaemia and glycosuria.

Proteins of molecular weight less than 69,000 will be entirely absorbed from the PCT by active pinocytosis. Inside the cells, they will be degraded by cellular lysozyme to amino acids and these amino acids are transported through the basolateral membrane to the peritubular space. The peptides are hydrolysed at the luminal brush border, and the amino acids formed are transported by co-transport.

When water is reabsorbed osmotically, that will facilitate urea transport to the peritubular space in a concentration gradient. In the inner medullary collecting ducts, facilitated diffusion occurs through urea transporters. Half of the amount filtered will be reabsorbed, and the remaining half is excreted through urine. Since the tubular membrane is impermeable to creatinine, the amount filtered will be excreted as a whole through urine.

Water is reabsorbed extensively from the PCT. Although the descending thin segment of the loop of Henle is perme­able to water, it is highly impermeable to solutes. The ascend­ing segments (including both thin and thick) are highly impermeable to water. In the presence of vasopressin or antidiuretic hormone (ADH), the late distal tubule and the collecting ducts are made permeable to water (Fig. 9.7). Whenever the extracellular fluid volume decreases or osmo­larity increases, ADH is released from the posterior pituitary. The water reabsorption by ADH is mediated through an intracellular protein called aquaporin-2 (AQP-2). When ADH binds to the plasma membrane receptors of late distal tubules, collecting tubules and collecting ducts, there will be increased formation of cAMP, activation of protein kinases and translocation of intracellular AQP-2 proteins from the interior to the plasma membrane. Fusion of AQP-2 to the luminal plasma membrane results in the opening of water channels in these regions, resulting in water entry to the interior of the cell. Water exits the cell through a different water channel (either AQP-3 or AQP-4) permanently posi­tioned at the basolateral border and then enters the blood, in this way being reabsorbed.

About 50% of the plasma calcium is ionised, and the remainder binds to the plasma proteins or exists in combina­tion with anions such as phosphate. So, the glomerulus can filter only about 50% of the plasma calcium. Usually, about 99% of the filtered calcium is reabsorbed by the tubules, and only about 1% of the filtered calcium is excreted. About 65% of the filtered calcium is reabsorbed in the proximal tubule, about 25-30% is reabsorbed in the loop of Henle and 4-9% is reabsorbed in the distal and collecting tubules. Parathyroid hormone increases calcium reabsorption, especially from the distal tubules, and magnesium reabsorption from the loop of Henle and decreases phosphate reabsorption by PCT.

Fig. 9.7 ADH-mediated water reabsorption from renal tubules. Dehydration causes the release of vasopressin (ADH) from the posterior pituitary, which attaches to the basolateral plasma membrane of epithelial cells of the late distal tubule, collecting tubule and collecting duct. This attachment causes the translocation of intracellular water channels AQP-2 to the luminal membrane increasing water permeability

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Aquaporins

Aquaporins (AQPs) constitute a family of proteins located in the plasma membrane and mediate water transport. A total of 13 proteins (AQP1-12Α, Β) are included in the AQP family in humans. Though the majority of AQPs facilitate water reabsorption, AQPs (like AQP3, AQP7 and AQP9) also play a significant role in glycerol transportation, thus being referred to as aquaglyceroporins. Because of AQPs' fundamental roles in essential water homeostasis, the distribution of AQPs was initially regarded as ubiquitous from prokaryotes to eukaryotes.

Renal excretion of magnesium is increased markedly dur­ing increased magnesium ion levels but decreases to almost nil during times of its depletion in the blood. Regulation of magnesium excretion is achieved significantly by changing tubular reabsorption. The proximal tubule usually reabsorbs only about 25% of the filtered magnesium. The loop of Henle is the primary site of reabsorption, where about 65% of the filtered load of magnesium is reabsorbed. Only a minimal amount (less than 5%) of the filtered magnesium is reabsorbed in the distal and collecting tubules. Increased extracellular fluid magnesium concentration, extracellular volume expansion and increased extracellular fluid calcium concentration may increase magnesium excretion.

9.4.4 Tubular Secretion

Hydrogen ions are secreted to the PCT in return to sodium ions, produced by the dissociation of H₂CO₃ formed by the hydration of CO₂ inside the cell. The intercalated cells of the late distal tubule and cortical and medullary collecting ducts secrete hydrogen ions by an active hydrogen-ATPase mech­anism. The principal cells of distal and cortical collecting tubule segments of nephrons secrete potassium under the influence of aldosterone whenever dietary potassium intake is high. Organic acids and bases, like bile salts, urates and catecholamines, are secreted from the proximal tubule. In addition, some waste products of metabolism, harmful drugs and toxins are also secreted to the tubular fluid of PCT.

9.5