Toxic responses of liver & kidney

Question 1

The liver has numerous functions, mention three.

Answer 1

• Metabolic homeostasis: Control of synthesis and utilization of carbohydrates, lipids and proteins. Strategically placed between the intestines and rest of the body, venous blood from the intestines goes through the liver via the portal vein and then out into the systemic circulation, so the liver act as a first “filter” for everything from the stomach and intestines.
• Metabolism of xenobiotics and bioactivation
• Vascular functions such as formation of lymph and the hepatic
  phagocytic functions.
• Secretory and excretory functions, particularly with respect to the
  synthesis and secretion of bile.

Question 2

What are the sources of hepatic blood and how much do they each account for?

Answer 2

About 70% of hepatic blood flow is from the portal vein (from the stomach and intestine) and about 30% from the hepatic artery (from the heart via the aorta).

Xenobiotics may be extensively metabolized by the liver so that little of the parent xenobiotic enters the systemic circulation (first pass effect)

Question 3

There are two concepts for classifying the basic functioning unit of the liver, which? Explain them.

Answer 3

• Lobule (classic): Hexagonal lobules with a clear boundary surrounding a central vein, with portal triads in each corner consisting of a portal vein, a hepatic arteriole, and a bile duct. Divided into three zones; centrilobular, midzonal, and periportal. In this classification the middle is where blood flows out.
• Acinus: (preferred): The acinus extends over two lobules, with adjacent portal triads in the base and top, and the central vein of each lobule on the sides. In an acinus, the entry of blood from the portal vein and hepatic arteriole is central, zone 1; then comes the intermediate zone (zone 2) and the blood flows out in the terminal hepatic vein (central vein) and this part is zone 3. This means that the concentration of oxygen, hormones, nutrients, and xenobiotics is highest in zone one and lowest in zone 3 and the hepatocytes in these zones also contain different constituents because of the different environments. In this classification the blood flows in in the middle.

Question 4

Explain how the blood flows in the liver.

Answer 4

The blood from the portal triads goes into sinusoids (unique capillaries in the liver with pores/fenestrations) and pass sheets of hepatocytes (parenchymal cells) in which biotransformation happens. The blood from all the sinusoids then collect in the central veins and go to the heart.

Question 5

What types of cells are found in the liver? explain their function.

Answer 5

• Hepatocytes: parenchymal cells that are rich in enzymes that facilitate biotransformation of substances. (78% of liver volume)
• Sinusoidal cells: (6% of the liver volume)
• Endothelial cells that build up the sinusoids (capillaries)
• Kupffer cells: Fixed macrophages situated in the sinusoidal lumen of the liver (80% of the body’s fixed macrophages are kupffer cells!!) that ingest and degrade particulate matter. They are also antigen presenting cells that can be recognized by T-cells in the immune system.
• Stellate cells: (Ito cells; fat-storing cells) are found in the perisinusoidal space (space of Disse). They store vitamin A and fat and produce extracellular matrix and collagen. (The stellate cells (Ito cell) is the major cell type involved in liver fibrosis, which is the formation of scar tissue in response to liver damage.)

The remaining 16% of volume of the liver is just space, sinusoidal and ECM.

Question 6

The regional expression (zone 1-3) of xenobiotic-metabolizing enzymes determines the zone-specific localization of damage by reactive toxicants. which two enzymes are notably expressed in zone 1 and 3 of the acinus? What consequence of zone specific damage does this have?

Answer 6

• High levels of glutathione in zone 1: antioxidant, so it protects from oxidative stress.
• high levels of CYP450 in zone 3: Produce reactive electrophile metabolites.

The combination of high reactive electrophile metabolites and low antioxidants result in zone 3 being more vulnerable/susceptible to oxidative stress.

Question 7

Bile formation is a specialized function of the liver. How is bile formed?

Answer 7

Bile consist of bile salts, bilirubin, cholesterol, phospholipids, water and soluble xenobiotic metabolites and other waste products and is formed in the hepatocytes from enzymatic reactions. The bile components are actively transported out of the hepatocytes into canaliculi (small channels in between the hepatocytes) that merge into the common hepatic duct leading to the gallbladder, where the bile is stored and gets concentrated. When needed, the bile goes through the common bile duct to the small intestine where it facilitates the digestion and absorption of fats.

Question 8

What is meant by “hepatotoxicity”?

Answer 8

Hepatotoxicity is chemical-derived damage to the liver. Can be drugs or toxins.

Question 9

There are many sorts of hepatotoxicity, name three.

Answer 9

• Steatosis (fatty liver)
• Cholestasis
• Fibrosis
• Hepatocyte necrosis
• Tumors

Question 10

What is fatty liver (steatosis) caused by?

Answer 10

Fatty liver or steatosis is a condition caused by accumulation of lipids (triglycerides or phospholipids), and is diagnosed when lipids make up greater than 5% of the total liver weight. The accumulation occurs due to an imbalance in the uptake of fatty acids and their secretion as very low density lipoproteins (VLDL), typically associated with acute exposure to many hepatotoxins.

Question 11

Name two hepatotoxins that can cause fatty liver.

Answer 11

Ethanol, carbon tetrachloride, tributyl tin (TBT).

Question 12

Can steatosis (fatty liver) be treated?

Answer 12

Yes, in early stages it’s fully reversible. If alcohol abuse is the cause, stopping drinking can make you fully recover.

Prolonged exposure can lead to cell death, apoptosis or necrosis, which can be focal (random spots of dead hepatocytes), zonal (death in certain functional regions) or panacinar (widespread). in order from least to most detrimental.

Question 13

What condition do you have if you have a decreased bile flow? How is it clinically defined?

Answer 13

Cholestasis.

The clinical definition of cholestasis is any condition in which substances normally excreted into bile are retained.

Question 14

What three causes can lead to cholestasis?

Answer 14

• Impaired secretion by hepatocytes (can either be impaired influx into them, impaired transport inside the cell, or impaired efflux out in canaliculi)
• Obstruction of bile flow through intra- or extrahepatic bile ducts (can be due to impaired contractility of the canaliculi or leaky paracellular junctions)
• Decreased bile formation

Toxicant-induced cholestasis can be transient or chronic; when substantial, it is associated with cell swelling, cell death, and inflammation.

Question 15

What substance is commonly measured in plasma to diagnose cholestasis?

Answer 15

Conjugated bilirubin (bilirubin+glucuronic acid). Bilirubin is a byproduct of the breakdown of red blood cells, and is normally excreted in bile. If something is causing bile to be retained, the serum levels of bilirubin is heightened.

Question 16

Name two hepatotoxins that can cause cholestasis.

Answer 16

Chlorpromazine (antipsycotic medicine), estrogens, phalloidins (toxin in death cap mushroom) and penicillin/antifungals such as erythromcyin, ampicillin.

Question 17

What symptom is caused by excess bilirubin levels? How does it manifest?

Answer 17

Jaundice. Jaundice is characterized by eyes and skin turning yellow and urine becoming bright yellow or brown due to the yellow pigment of bilirubin. Liver and inside of mouth and nose also turns yellow.

Question 18

What is liver fibrosis/cirrhosis?

Answer 18

Liver fibrosis is the accumulation of extracellular matrix (ECM) proteins, mostly collagens Type I and Type III, in response to liver injury (either hepatotoxic or cholestatic) that result in scarring. The stellate cells play a critical role in the progression of fibrosis. This is initially a response to protect the liver, and in early stages it can be reversed, but can become very dangerous.

After an extended time this can cause cirrhosis; When the scarring gets so extensive it takes over from normal cells and divides the liver into nodules, which leads to impaired liver function. Cirrhosis is not reversible and has a poor prognosis or survival.

Question 19

What xenobiotics can cause fibrosis/cirrhosis?

Answer 19

The leading cause is viral hepatitis, but can also be caused by chronic exposure to ethanol and heavy metals.

Question 20

Explain the mechanism of acetaminophen (paracetamol) toxicity in stort.

Answer 20

Metabolism: Liver breaks down acetaminophen into many metabolites, one of which can be toxic: NAPQI (produced by CYP2E1).
Detoxification: NAPQI is neutralized by glutathione when taking a normal dose.
Overdose: Excess acetaminophen depletes glutathione.
Accumulation: Unneutralized NAPQI damages liver cells.

Chronic alcohol abuse increases the risk (Ethanol induces CYP2E1).

Question 21

Explain the mechanism of ethanol toxicity in short.

Answer 21

Ethanol is bioactivated by alcohol dehydrogenase (ADH) to acetaldehyde, a reactive aldehyde, which is subsequently detoxifed to acetate by aldehyde dehydrogenase (ALDH2) in mitochondria. Overconsuming ethanol leads to accumulation of acetaldehyde which is reactive and can generate ROS and free radicals, which in turn can have indirect effects such as:

• Influence on membrane fluidity
• Formation of toxic fatty acid ethyl esters
• Damage mitochondrial inner membranes

Genetic polymorphisms relevant here, as 50% of Asians have a “slow” ALDH2 which results in accumulation of acetaldehyde.

Question 22

List three consequences acetaldehyde accumulation in mitochondria.

Answer 22

Production of Reactive Oxygen Species (ROS): Acetaldehyde can lead to an increase in the production of reactive oxygen species (ROS) within mitochondria. ROS are highly reactive molecules that can cause damage to mitochondrial proteins, lipids, and DNA.

Oxidative Stress: The increased production of ROS results in oxidative stress within the mitochondria. This oxidative stress can lead to the modification and damage of mitochondrial proteins and other macromolecules, disrupting their normal functions.

Mitochondrial DNA (mtDNA) Damage: Mitochondria have their own DNA (mtDNA), and alcohol abuse has been associated with increased mutations and damage to mtDNA. This can affect the mitochondrial respiratory chain and energy production.

Altered Electron Transport Chain: Mitochondria play a central role in the electron transport chain, which is essential for energy production through oxidative phosphorylation. Alcohol abuse can disrupt this chain, leading to decreased ATP (adenosine triphosphate) production, the primary energy currency of cells.

Mitochondrial Dysfunction: As mitochondria become damaged, their ability to produce ATP and regulate cellular energy decreases. This can affect various tissues and organs, leading to symptoms like muscle weakness, fatigue, and a decreased ability to handle oxidative stress.

Impaired Autophagy and Mitophagy: Autophagy is a cellular process that helps remove damaged or dysfunctional mitochondria. Chronic alcohol abuse can impair this process, leading to the accumulation of dysfunctional mitochondria within cells.

Cellular Damage and Apoptosis: Mitochondrial dysfunction can lead to the release of pro-apoptotic factors, triggering programmed cell death (apoptosis). This can contribute to tissue damage and organ dysfunction.

Organelle Swelling: Chronic alcohol consumption can lead to mitochondrial swelling, which further impairs their function and structure.

Organelle Fragmentation: Alcohol can disrupt the dynamic process of mitochondrial fusion and fission, leading to fragmentation of the organelles, which can compromise their function.

Question 23

Explain carbon tetrachloride toxicity in short.

Answer 23

Carbon tetrachloride (CCl4) is metabolized to CCl3´ (radical) by CYP450 and that can react with lipids –> fatty acid radical.

1. Initiation: Production of a fatty acid radical
2. Propagation: Fatty acid radical further reacts with oxygen forming peroxyl fatty acid radicals. Cyclical radical chain reaction.
3. Termination: Two radicals react to form non-radical species

Results in reactive aldehydes causing cell membrane damage

Question 24

What are the core functions of the kidney?

Answer 24

Excretion (metabolic waste and xenobiotics) and Homeostasis:

– Electrolyte/water composition
– Renin production (blood pressure regulation)
– Erythropoietin production (Hb synthesis)
– Metabolism and activation of vitamin D
– Metabolism of parathyroid hormone (Ca2+) regulation
– Acid/base balance

Question 25

Why is the kidney so vulnerable to toxicant accumulation?

Answer 25

Since the kidneys main function is to concentrate substances to conserve water, it's also susceptible to concentrate toxicants! Non toxic concentrations in plasma can become toxic in the kidney.

Question 26

The kidney can be divided into three parts, which? The three parts also receive a very uneven distribution of the blood, how much does each part get and what consequences does this get for toxicity?

Answer 26

1. Renal cortex (outer layer): receives about 90% of blood flow 2. Renal medulla (middle layer: receives about 6-10% of BF 3. Renal pelvis/papilla (center): about 1-2% of BF. Since the cortex receives most, it's the most susceptible to toxicants. However, the residence time in the medulla and pelvis is higher due to the more sluggish blood flow, so that can be problematic too.

Question 27

Which part of the nephron is most vulnerable to toxicity? What three things make it so susceptible?

Answer 27

The proximal tubule, since that's where the first stage of filtrate passes. - First, The proximal tubule have a leaky epithelium favoring influx of compounds into proximal tubule cells that can accumulate and cause site specific toxicity. - Second, we have a lot of active transport for reabsorption here, it's packed with mitochondria to provide ATP, and if they are compromised, the homeostasis in the body is threatened and things go bad FAST! - There is also biotransformation of xenobiotics in the proximal tubule, CYP450 among others, that can cause accumulation of reactive metabolites/ROS that can damage the cells here.

Question 28

The afferent and efferent arteriole differ in diameter, how?

Answer 28

The afferent arteriole receives blood from the heart and leads it to the glomerulus, where the initial filtration happens. It has a larger diameter than the efferent (which returns filtered blood to the systemic circulation) and this forces solutes out of capillary into Bowmans capsule.

Question 29

What is filtered out in the glomerulus?

Answer 29

The glomerulus is the site of filtration, where visceral epithelial cells (podocytes) filter the blood from capillaries into ultrafiltrate in Bowmans space and to the proximal tubule. Size and charge matter for what is filtered out: – Size: smaller molecules under 60kDa, molecule needs to be smaller than albumin – Charge: anionic molecules are not filtered but cationic and neutral are.

Question 30

Toxicity in the kidney can be intrarenal or extrarenal, what does this mean?

Answer 30

Intrarenal toxicity: toxin affects the the nephron directly. Extrarenal toxicity: toxin affect other parts in the body that affects nephron function, for example toxins that have an effect of lowering cardiac output, which lowers GFR (glomerular filtration rate).

Question 31

What determines the GFR (glomerular filtration rate)?

Answer 31

Glomerular filtration rate (GFR, ml per minute) depends on: – hydraulic permeability (surface area available for filtration) – Colloid osmotic pressure (30 mmHg) <-- – Capsular hydrostatic pressure (15 mmHg) <-- – Glomerular blood hydrostatic pressure (55 mmHg) --> Net force = 10 mmHg --> The osmotic pressure is often affected by toxicants.

Question 32

How much blood is filtered through glomeruli a day? How much of a drop in GFR is needed to stop filtration entirely?

Answer 32

GFR is about 120 mL/min, 180 L/day. (the sum of filtration from roughly 2 million glomeruli!) A drop of 20 % in glomerular blood hydrostatic pressure will completely stop filtration!

Question 33

How can GFR be increased/decreased?

Answer 33

Vasodilation of the afferent arteriole and vasoconstriction of the efferent arteriole increase GFR. Vasodilation of the efferent arteriole and vasoconstriction of the efferent arteriole decrease GFR, so vice versa!

Question 34

What hormones are involved and how does it work when GFR needs to be increased in the body?

Answer 34

1. When filtrate flow or NaCl concentration is decreased the cells of macula densa synthesize and secrete Prostaglandin E2 (vasodialator) 2. Prostaglandin causes afferent arteriole vasodilatation 2. Juxtaglomerular cells release renin, which results in angiotensin II, leading to efferent arteriole constriction --> GFR increases

Question 35

There is another mechanism that can increase GFR, what and how?

Answer 35

Compensatory renal hypertrophy (size increase of the kidney (common when one kidney is lost) which restores normal GFR again. More blood can be filtered --> increase in GFR.

Question 36

What is acute renal injury/failure characterized by?

Answer 36

ARF/ARI is characterized by an acute drop in GFR and azotemia (= increase in blood urea nitrogen (BUN) and creatinine). Acute kidney injury may have maaaany different causes upon exposure to a nephrotoxicant. Very complex and often interconnected mechanisms.

Question 37

Name three common mechanisms for acute renal injury/failure.

Answer 37

Tubular toxicity: (Aminoglycosides, cisplatin, vancomycin, radiocontrast agents, heavy metals, haloalkane-cysteine conjugates) - Changes in specific reabsorptive or secretory mechanisms - Altered general permeability of the tubular epithelium - Tubular necrosis Glomerular toxicity: (NSAIDs, gold, penicillamine) - Altered permeability --> compromised filtration Vasoconstriction: (NSAIDs, radiocontrast agents, Cyclosporine, Tacrolimus, Amphotericin B) - Reduced renal blood flow -> reduced GFR Crystalluria: (Sulfonamides, methotrexate, acyclovir, ethylene glycol) - Crystals may obstruct renal tubule-> reduced GFR

Question 38

What is the common target for site specific toxicity of the glomerulus? Name one intra- and one extrarenal factor.

Answer 38

Permeability is a common target of nephrotoxicants affecting the glomerulus and GFR. Intrarenal factors: - local vasoconstriction - vascular damage and altered permeability, by altering the size- and charge-selective functions. - endothelial cell damage Extrarenal factors: - Circulating immune cells can get trapped in glomerulus, and associated ROS and cytokines can damage glomeruli (heavy metals and hydrocarbons for example). - Antibody/immune responses to some chemicals can deposit in the glomerulus, leading to glomerular injury.

Question 39

Name two types of toxicants which cause site specific injury to the proximal tubule, and explain how in short.

Answer 39

Heavy metals and halogenated compounds. Heavy metals: bind to sulfhydride groups of copmounds needed in the cells, which make them lose their function and highjack the transport system into the cells and cause issues. – Hg (mercury/kvicksilver): conjugated to glutathione or cysteine and co- transported into cells. Early signals = mitochondrial dysfunction which results in necrosis. – Cd (cadmium): Targets cell-cell adhesion signaling pathways, dysfunction of tubule and consequently interstitial nephritis (inflammation) and necrosis. - Lead Halogenated compounds: – Chloroform: bioactivated by P450 into unstable trichloromethanol, forms phosgene (COCl 2 ) with HCl which reacts with proteins and results in necrosis. - carbon tetrachloride (CCl4): ROS

Question 40

Name the seven mechanisms for renal cell injury.

Answer 40

- Apoptosis (mitochondria driven) - ROS – damage proteins, lipids, DNA - Loss of ATP and therefore ion transport and ion balance - Loss of polarity and therefore effective cell function - Lysosomal overload and enzyme release - Alterations to Ca 2+ dynamics within the cell - Impaired membrane function This is the pattern of toxicity in most of the body too.

Question 41

What is a problematic consequence about toxic injury to individual cells?

Answer 41

When cells are so toxically injured that they die (apoptosis/necrosis), the remaining cells lose connections to each other, and can lose their grip and form obstructions in the tubes, leading to decreased GFR and ARF/ARI.

Question 42

Explain the nephrotoxicity mechanism of NSAIDs (nonsteroidal anti-inflammatory drugs) like Ibuprofen, aspirin, Voltaren.

Answer 42

Most NSAIDs inhibit cyclooxygenase-1 (COX-1) and -2 (COX-2) -> inhibited prostaglandin synthesis -> inhibited vasodilation -> constriction of the afferent arteriole -> decreased GFR. This can cause kidney toxicity: - acute tubular necrosis - acute interstitial nephritis - papillary necrosis (ischemic (no oxygen))

Question 43

Explain the nephrotoxicity mechanism of ACE inhibitors (drugs that lower blood pressure).

Answer 43

ACE inhibitors lower blood pressure by inhibiting the synthesis of angiotensin (vasoconstrictor) and this leads to blocked vasoconstriction of the efferent arteriole to --> reduced GFR.

Question 44

Is acute kidney injury/failure treatable?

Answer 44

Yes! Luckily, most acute kidney injuries are reversible! The kidney is very adaptable, and can compensate, regenerate and repair most damage. - Removal of kidney results in GFR increase from 40 to 60% in remaining kidney. BUT...compensation can mask underlying damage... Chronic renal failure (irreversible) needs to be treated with dialysis or kidney transplant. If untreated, it can be lethal.