Mechanism of action of Diuretics

Group members

• Amanda Powell
• Kevin Maskell
• Parimal Sura
• Paul Rotolo
• Ibrahim Habib
• Brian Lin

The function of nephron portions (Amanda)

Each kidney contains about 1 million nephrons, the funtional unit of the kidney. A nephron is a hollow tube, closed at one end at the location of the renal corpuscle. The nephron is a single layer of epithelium throughout its length, resting on a basement membrane. Each section of the nephron is specially designed to selectively filter or reaborb solutes or water. This complex system of filtering and reaborbing allows the kidney to regulate the water and electrolyte balance of the body, among other functions. Below is a more detailed description of the various portions of the nephron, including the renal corpuscle, the proximal tubule (convoluted and straight segment), the Loop of Henle (thin descending, thin ascending and thick ascending limb), the distal convoluted tubule and the collecting system. The diagrams below summerize the main purpose of each section of the nephron (left) as well as the general characteristics of the epithelial cells in each major section (right). These can be refered back to throughout the discussion.

The renal corpuscle is made up of a glomerular capillary bed surrounded by a Bowman’s capsule. Blood moves into the capillary bed via the afferent arteriole and out via the efferent arteriole. The endothelial cells which line the capillaries are fenestrated and a basement membrane lies between them and the epithelial cells of the Bowman’s capsule, podocytes. These three layers are the filtration barrier through which plasma is filtered out of blood and into the tubule of the nephron (EM below).

This barrier is designed to limit filtration based on size and charge (due to the negative charge depostied on the membrane). Anout 180 liters of filtrate is formed this way per day. Only about 1.4 liters of urine is produced, however, suggesting that more than 99% of filtered water is reaborbed into the capillaries surrounding the nephron (peritubular capillaries). This is the job of the remainder of the nephron structures.

The filtrate immediately enters the proximal convoluted tubule in the cortex of the kidney (PCT in figure above, left). Cells of the proximal tubule are low columnar. They have a brush border of microvilli on the apical, lumenal surface, which serves to greatly increase their surface area. This is important as 2/3 of filtered water, sodium and chloride is reaborbed here along with other filtered nutrients, such as glucose, which rarely appear in the urine (they are entirely reabosorbed). Bicarbonate is also reaborbed in large quantities along with water and sodium. Additionally, endocytic vescicles are found at the bases or the microvilli which take in small proteins from the filtrate. These go on to merge with lysosomes which digest these small poroteins so they can be reaborbed into the peritubullar capillaries.

(Above, Simple columnar epithelium lined tubule with visible microvilli brush border, Renal corpuscle with urinary pole visible, 20X)

As filtrate exits the proximal tubule it enters the thin descending limb of the Loop of Henle and then up the ascending thin limb. These two tubules are histologically identical. The cells in this region appear thin and flat in comparison to the other epithelia of the tubules in the medullary rays. The final stretch of the Loop is the thick ascending limb. This portion has a simple cuboidal cell structure and is very different physiologically from the thin limbs. The epithelium of the thin descending limb is freely permeable to water, but not to any solutes. It engages in little, if any, active transport across its epithelium, simply allowing water to move out via osmosis. This serves to concentrate solute in the tubule as filtrate passes through this portion of the nephron. The thick ascending limb, on the other hand, is impermeable to water and solute permeable via primary and secondary active transport. This functions to reabsorb salt and leave water behind, diluting the filtrate. These different permeabilities in the Loop of Henle are vital to kidney funtion because they set up the medullary countercurrent system which allows the kidenys to selectively reaborb solutes, nutrients and water that the body needs and produce conentrated urine containing only waste products.

The distal tubule is the next portion of nephron through which the filtrate passes (labeled DCT on the schemtic at the top of this section). Similarly to the proximal tubule, it is composed of a convoluted and straight portion. Functionally and histologically, the distal tubule is much like the thick ascending tubule; sodium is transported into the interstium and water does not follow in equal proportions. This serves to further dilute the filtrate in the lumen of the tubule. Beginning in the distal tubule and continuing into the collecting tubules and ducts, the nephron begins to have receptors in it's basolateral membrane for the hormone aldosterone. Aldosterone stimulates sodium reaborption into the interstium in exchange for potassium. In addition, hydrgen ions are secreted, which helps to regulate the acid-base balance of the body.

Finally, the dilute filtrate enters the collecting duct system. It is the job of the collecting duct system to concentrate urine finally and adjust the urea content of the final urine. This process is regulated by the sensitivity of the collecting duct epithelial cells to ADH.

Multiple nephrons' collecting ducts empty into a collecting tubule system. These tubules gradually merge and grow larger as they move from the cortex into the medulla of the kidney and eventually enter papillary ducts and eventually the ureters to exit the kidney as urine. The small collecting tubules in the cortex are cuboidal epithelium and as they progress deeper into the medulla they become taller columnar epithial cells. The two main types of cells in the collecting tubules are principal cells, involved in water reabsorption, and intercalated cells, which regulate acid-base balance via the active transport of hydrogen ions. Intercalated cells fade out as the collecting tubules move into the medulla.

Osmotic diuretics (Kevin)

Osmotic diuretics are drugs which are filtered from the blood at the glomerulus, but do not completely reabsorb later in the nephron. The presence of additional osmotically active particles leads to increased diuresis. Given the action of these drugs, there is no transporter affected per se, it is simply a matter that the body’s ability to reabsorb or transport the drug has been exceeded. According to Goodman and Gilman’s The Pharmacological Basis of Therapeutics 11th edition, there are 4 currently available osmotic diuretics: glycerin, isosorbide, mannitol, and urea. Other sources also list glucose as an example of an osmotic diuretic, but only if it is present in sufficient quantities to exceed the kidney’s transport maximum.

As mentioned before, osmotic diuretics work by increasing the osmotic pressure within the nephron, forcing excretion of greater quantities of water and sodium. There has been some debate about the specific site of action. The original theory was that osmotic diuretics act in the proximal tubule, limiting the osmosis of water into the interstitial space and reducing Na+ concentration to the point that net Na+ reabsorption would cease. However recent studies have shown this to be a secondary mechanism, with the main location of action being the loop of Henle. There, they act by increasing renal blood flow by expanding the extracellular fluid volume and inhibiting renin release, which results in reduced medulary tonicity, causing less water to be extracted from the descending limb. This limits the concentration of NaCl which enters the thin ascending limb, diminishing the ability of the kidney to passively reabsorb NaCl there.

The overall effect of osmotic diuretics is to increase excretion of not just water, but almost all of the electrolytes, including Na+, K+, Ca2+, Mg2+, Cl-, HCO3 -, and phosphate. The increase in secretion of some of these electrolytes, such as Mg2+, is used as evidence that osmotic diuretics may have additional effects on transporters throughout the ascending limb, but these effects remain unknown. Their effect on renal blood flow, as noted above, is to increase it by a variety of mechanisms, though glomerular filtration rate is only changed to a very minimal extent.

Carbonic anhydrase (Ibrahim)

Normal Mechanism

Carbonic Anhydrase is an enzyme that catalyzes the interconversion of carbon dioxide and water into carbonic acid, which usually disassociates into a proton and bicarbonate. It is utilized almost everywhere in the circulatory system as it is essential to the transport of CO2. It is also found in the nephron in the kidney, and specifically acts on the proximal tubule. Carbonic Anhydrase is located intracellularly where it converts the reabsorbed water and CO2 into bicarbonate and protons. The protons are then pumped into the lumen to acidify the urine and in exchange for sodium ions. The bicarbonate is pumped in the opposite direction into the interstitial space in exchange for chloride ions. Consequently, for every hydrogen ion that is pumped into the lumen there is a bicarbonate ion reabsorbed into the circulatory system. The hydrogen ion in the lumen combines with HCO3- using a membrane bound Carbonic Anhydrase protein. The newly formed H2CO3 disassociates to form H2O and CO2 , which is reabsorbed. This cyclic process is how Carbonic Anhydrase is utilized to reabsorb water in the proximal tubule. Refer to the graphic below.

Mechanism of Action

Carbonic Anhydrase inhibitors do exactly just that. These agents are sulfonamide in structure and the diuretic effect was actually found as a side effect of sulfanilamide, which was used as an antibiotic in the past. Some notable clinically used carbonic anhydrase inhibitors are Acetazolamide, dichlorphenamide and methazolamide. These proteins block the enzyme carbonic anhydrase and thus they prevent the reabsorption of NaHCO3- in the proximal tubule resulting in diuresis. As stated above, bicarbonate is absorbed into the interstitial space and if there is no bicarbonate being formed as a result of a decrease in activity of the catalyst carbonic anhydrase, less water is reabsorbed, and thus, more water is ultimate excreted.

The following are the structures of Sulfonamide, Acetazolamide, and dichlorphenamide respectively.

Effect on Kidney Function:

Overall, the diuretic increases water excretion as well as the excretion of sodium and numerous other electrolytes. Also, as a result of HCO3- loss, there is metabolic acidosis. Sodium loss is not as profound because the ascending loop of Henle and the distal tubule are efficient in maintaining proper sodium concentration. In addition to the above effects, Carbonic Anhydrase inhibitors also decrease renal blood flow and glomerular filtration rate via feedback regulation resulting from the increased solute delivery to the distal tubule, which results in an increase in afferent arteriolar resistance.Though carbonic anhydrase inhibitors are diuretics, they are currently not used for diuretic purposes. Instead, these agents are used to reduce intraocular pressure in the treatment of glaucoma. This occurs because carbonic anhydrase inhibitors inhibit intraocular carbonic anhydrase, which facilitates the formation of aqueous humor. These agents are also used to treat epilepsy and motion sickness.

Loop Diuretics (Paul)

Types of Loop Diuretics

This class of diuretics refers to drugs that affect the Na+-K+-2Cl- symport in the Thick Ascending Limb of the Loop of Henle(referred to as TAL), hence the "loop" descriptor. Examples of loop diuretics include furosemide (LASIX), bumetanide (BUMEX), ethacrynic acid (EDECRIN), and torsemide (DEMADEX)

Normal Mechanism

The Na+-K+-2Cl- symporter takes advantage of the electrochemical gradient of sodium in the thick ascending limb(TAL). The gradient is created by the Na+ pump located on the basolateral side of the loop's epithelial cells. This pump continually pumps sodium out of the cell into other tissue, such as blood vessels, creating a low concentration in the cell. This allows the higher concentration of sodium in the lumen of the Loop to move down the gradient into the epithelial cell. The symporter uses that Na+ gradient to move K+ and Cl- ions into the cell, "uphill", or against their electrochemical gradients.

The symporter is located on the lumen side of the epithelial cell. Transporting the Na+ ion along its gradient into the cell allows the symporter to use that gradient to capture a K+ ion and 2 Cl- ions and move them into the cell simultaneously with the Na+. This symporter allows the TAL to greatly dilute the urine. Water follows the ions, so the water in the filtrate will follow. The action of the symporter is very effective and can remove much of the ions, leaving a more dilute solution behind.

Loop Diuretic Mechanism

The Loop Diuretic class of drugs are inhibitors of the Na+-K+-2Cl- symporter in the TAL. The drug binds to the symporter, preventing the symporter from binding and moving the ions into the cell. There is evidence that shows the drug binds to the chloride ion binding site of the protein. The movement of ions across the membrane of the epithelial cell stops, and the ions stay in the lumen of the TAL. This creates a highly concentrated solution in the lumen, and water cannot escape. The concentration can be so high that it actually pulls water in from interstitial fluid and the blood vessels that travel through the kidney. This becomes the key for loop diuretic treatments.

Effect on Kidney

The blocking of the symporter leads to increased excretion of Na+ and Cl- in the urine, up to about 25% of the filitered load of Na+. The inability of the ions to move across the membrane also disrupts the transepithelial potential difference, which in turn leads to a higher excretion of Ca++ and Mg++ in the urine. Some of the loop diuretics also have a weak carbonic anhydrase inhibitor effect. Furosamide is an example of such a loop diuretic. As can be read in another section here, this leads to increased HCO3- and phosphate. All symport inhibitors will also lead to increased K+ excretion, due to a larger amount of Na+ reaching the distal tubule.

A loop diuretic's inhibition of the reabsorption of Na+ and Cl- and other ions in the TAL leads to an inability of the kidney to concentrate urine. A watery urine, as well as increased amounts of urine, are the result.

There are some adverse effects, although those not related to the diuretic mechanism are rare. Loop diuretics can be "abused", leading to a depletion of sodium ions in the body. This can lead to serious concerns, such as hyponatremia, hypotension, circulatory collapse and more. If the patient does not have a large enough intake of K+, the continual loss of K+ from the use of loops diuretics could lead to hypokalemia. This can induce cardiac arrhythmias. A 2003 study by Rejnmark indicated that use of loop diuretics in "postmenopausal osteopenic women" should be avoided, as the increased loss of Ca++ could have severe effects on bone metabolism.

Loop diuretics have also been linked to ototoxicity, causing such sometimes reversible symptoms as tinnitus, vertigo, and deafness. This seems to occur most frequently with loop diuretics delivered quickly through intravenous measures. When administered via oral doses, these effects are less frequent.

Treatments

Why would loop diuretics be used? What is the clinical value of these drugs?

The major use of loop diuretics is to treat acute pulmonary edema. The increased diuresis decreases the fluid going to the heart, and therefore the filling pressures in the heart, relieving the edema.

Another use for loop diuretics is in the treatment of chronic congestive heart failure. The diuretic helps to decrease the extracelular fluid volume.

Loop diuretics are not as useful as other diuretics in regards to treating hypertension. While loop diuretics do decrease blood pressure, they are not as effective due to their short elimination half-lives.

Thiazide diuretics (Brian)

www.toxlab.co.uk/images/thiazide.gif

Thiazide diuretics are a class of drugs that reduce plasma volume and often used to treat a variety of pathophysiological conditions associated with edema or hypertension. Drugs in this family include bendroflumethiazide, benzthiazide, chlorothiazide, chlortalidone, hydrochlorothiazide, hydroflumethiazide, indapamide, metolazone, polythiazide and many others. Depending on their formulation characteristics, thiazide diuretics are variably absorbed at different rates and mainly metabolized in the kidney or liver.

www.clinic-clinic.com/images/phrmclgy/drtcs2.gif

Thiazide diuretics are secreted into the tubular fluid by organic anion transporters in the straight segment of the proximal tubule. While some thiazides have weak carbonic anhydrase activity, their principal effects are the inhibition of Na+/Cl- symporter on the luminal membrane of distal convoluted tubule. This inhibition results in increased excretion of sodium, potassium, and magnesium as well as an overall increase in urine volume. The decreased excretion of calcium is observed but its mechanism is poorly understood at this point.

The use of thiazide diuretics causes a significant and constant loss of potassium. Thus, potassium supplements are frequently prescribed and its levels monitored. In severe cases of hypokalemia, ventricular rhythms are disturbed and muscle cramps and pain are observed. With increased dosage of thiazides consumed, the occurrence and severity of the other side effects have been reported.

Uses of diuretics (Parimal)

Diuretics have many uses for clinicians. They are versatile drugs used in numerous cases. Doctors prescribe diuretics to treat a wide variety of disorders in which include high blood pressure, heart failure, kidney stone prevention, renal failure, cirrhosis, and diabetes insipidus. These are few of the diseases or symptoms that call for diuretics.

The reason why diuretics are an effective treating agent against hypertension is because of their great ability to regulate blood pressure. Diuretic alone have controlled blood pressure in over 50% of patients and when used in combination with other drugs, treat 80% of patients(Papademetriou, V). Some diuretics lower blood pressure by increasing water and sodium filtration and thus lowering blood volume and pressure. Others inhibit vasopressin release, or inhibit reabsorption, or increase glomerular filtration rate. Each of these mechanism help in lowering blood pressure.

Diuretics are effective in treating kidney stones. Kidney stones come in many forms that include calcium, uric acid, and cystine. These stones form because of their insolubility and when they form, they can produce extreme flank pain. Often times, stones are small enough to pass through in the urine. Drinking more fluids is one treatment to help to dissolve these stones, but when that is not enough, diuretics or surgical methods may be used. Diuretics help by increasing the water content in the urine and thus making kidney stones more soluble.

Cirrhosis is the seventh leading cause of disease-related death in the United States. It is characterized by chronic degeneration of normal liver cells. Liver tissue gets converted into scar tissue. Ascities, or abdominal fluid build-up, is the most common complication that comes from cirrhosis. The liver is vital organ and thus many other complications may arise from cirrhosis including liver cancer, fever, muscle weakness, anemia, edema, nerve pain, slurred speech, tremors, brain damage, hemorrhage, impotence, sepsis, liver failure (inability to synthesize and metabolize necessary chemicals), and death. Diuretics with salt and water restrictions in the diet help get rid of excessive abdominal fluid (ascities) that builds up in patients with cirrhosis and better the quality of patients’ lives.

"In the early stages of cirrhosis, the liver expands and takes on a yellowish hue due to an increase in the presence of adipose tissue, which may also be accompanied by an increase in fibrous scar tissue and bile ducts. Over time, the liver develops a granular consistency due to an even greater proportion of fibrous tissue, and the blood vessels passing through the organ thicken, often hindering blood flow. In the final stages of liver degeneration, the organ substantially reduces in size and completely loses it typical lobular organization. At this point all fat in the organ has disappeared and all that remains is greatly damaged liver tissue." (Nikon Microscopy U)

Diabetes insipidus is a disease that is characterized by excreting large amounts of dilute urine. It occurs because patients are not responsive to ADH or do not produce ADH, and the kidneys can no longer concentrate the urine. The main symptoms are polyuria and nocturia as well as polydipsia and constant thirst. Nephrogenic diabetes insipidus, where the kidneys are no longer responsive to vasopressin, can be treated by monitoring electrolyte and water intake and content and also administering Thiazide diuretics. This may seem counterintuitive to use a diuretic in a patient that is excreting large quantities of water, but thiazide diuretics have an ability to concentrate the urine. This somehow helps reduce the urine volumes of individuals.

Diuretics have contributed to better the lives of so many. Their role aids in many diseases that include cardiovascular, hepatic, and renal. The versatile role of diuretics in medicine make them doctors and patients good friend.

Sources

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Loop Diuretics:

Goodman & Gilman's The Pharmacological Basis of Therapeutics - 11th Ed. (2006) (via RFUMS Library STAT!Ref)

Rejnmark, L, Vestergaard, P., Pedersen, A.R., et al. Dose-effect relations of loop and thiazide diuretics on calcium homeostasis: A randomized, double-blinded, Latin-square, multiple cross-over study in postmenopausal osteopenic women. Eur. J. Clin. Invest., 2003, 33:41-50.

Papademetriou, V. Diuretics in hypertension: clinical experiences. European Heart Journal. 1992; 13 Suppl G:92-5. http://www.medscape.com/medline/abstract/1486913?src=emed_ckb_ref_0

Human Digital Pathology Image Gallery, Liver Cirrhosis. Nikon Microscopy U. 2006. http://www.microscopyu.com/galleries/pathology/livercirrhosislarge.html

Libby M. Morimoto, Polly A. Newcomb, Cornelia M. Urlich, Roberd M. Bostick, Cynthia J. Lais, and John D. Potter. Risk Factors for Hyperplastic and Adenomatous Polyps: Evidence for Malignant Potenttial. Cancer Epidemiology, Biomarkers and Prevention. Vol 11, 1012-1018, October 2002.

Wielondek M, Wilczak W, Bielicki D, Krygier-Stojaloska A, and Marlicz K. Flow cytometric DNA ploidy and cell proliferative activity in colorectal adenomatous polyps. Pol J Pathol. 1998; 49(3):135-9

Toshio Iinuma, Sadamu Homma, Tetsuo Noda, Donald Kufe, Tsuneya Ohno, and Gotaro Toda. Prevention of gastrointestinal tumors based on adenomatous polyposis coli gene mutation by dendritic cell vaccine. J Clin Invest. 2004 May 1; 113(9): 1307-1317.