1 Thomas Visalli, PhD, DABT Adjunct Assistant Professor, Pharmacology and Physiology Rutgers, GSBS Toxic Responses of the Liver and Kidney

2 Toxic Responses of the Liver

3 Facts About the Liver  Strategic location between intestinal tract and the rest of the body facilitates its task of metabolic homeostasis in the body  Extraction of ingested nutrients, vitamins, metals, drugs, toxicants, etc. from the blood for catabolism, storage, and/or excretion into bile

4 Facts About the Liver  Bile formation is essential for uptake of lipid nutrients, protection from oxidative insult, and excretion of endogenous xenobiotic compounds

5 The Liver is a Dominant Site of Specific Toxins  To understand why, the following must be considered:  Major functions of the liver  Structural organization of the liver  Processes involved in the excretory function of the liver (bile formation, etc.)

6 I. Hepatic Functions  Venous blood from the stomach and intestines flows via the portal vein through the liver before entering the systemic circulation  Liver is the first organ to encounter ingested substances  Scavenging or uptake processes extract these minerals for catabolism, storage, or excretion into bile

7 I. Hepatic Functions Liver GB Small Intestine Hepatic Portal Vein

8 Functions of Liver and Consequences of Impaired Hepatic Functions  All of these functions may be dramatically altered by acute or chronic exposure to certain toxicants

9 II. Structural Organization of the Liver Fig. 13-2, Casarett & Doull’s

10 II. Structural Organization of the Liver  Blood flow to the liver:  70%: Oxygen-depleted blood from portal vein  30%: Oxygenated blood from hepatic artery  Hepatic Artery feeds liver with oxygen  Portal Vein brings blood from digestive tract  Sinusoids: channels between hepatocytes where blood travels on its way to CV  CV (HV) drains blood from liver to systemic circulation

11 II. Structural Organization of the Liver  Many gradients exist:  Oxygen  Zone 1 = oxygen rich; Zone 3 = hypoxic  Bile Salts  Bilirubin  Many organic ions

12 II. Structural Organization of the Liver  Gradients of enzymes involved in detoxification also exist:  Zone 1: Glutathione  Zone 3: Cytochrome p450 proteins

13 II. Structural Organization of the Liver  Sinusoids: Contain 3 major cell types  Endothelial Cells  Line sinusoids; very porous to allow for transfer of necessary proteins to hepatocytes  Kupffer Cells  Resident macrophages of liver  Ito Cells  Fat storage cells (stellate cells); synthesize collagen and store vitamin A.

14 II. Structural Organization of the Liver Fig. 13-4 Casarett & Doull’s

15 III. Bile Formation  Bile: yellow fluid containing bile salts, glutathione, phospholipids, cholesterol, bilirubin, organic anions, proteins, metals, ions, xenobiotics  Bile formation essential for:  Uptake of lipid nutrients from small intestine  Protection of small intestine from oxidative insult  Excretion of endogenous and xenobiotic compounds

16 III. Bile Formation  Hepatocytes transport bile salts, glutathione, and other solutes into canalicular lumen  Canaliculi: Channels between hepatocytes that drain into a common bile duct  Lumen is sealed with tight junctions  Bile concentrated and stored in gall bladder before its release into duodenum

17 III. Bile Formation  Major driving force for bile formation is active transport of bile salts and other osmolytes into canalicular lumen Fig. 13-5 Casarett & Doull’s MDR: (Multi-drug resistance glycoprotein): Exports lipids and lipophilic drugs CMOAT: (Canalicular Multiple Organic Anion Transporter): Exports conjugates of glutathione and glucuronides

18 III. Bile Formation  Bilary excretion is very important in the homeostasis of metals  Metals excreted into bile via:  Facilitated uptake across sinusoidal membranes via facilitated diffusion or receptor-mediated endocytosis  Specific canalicular membrane transporters

19 III. Bile Formation  Bile is modified along its route to gallbladder  Epithelial cells lining bile ducts contain phase I and phase II enzymes to detoxify toxicants present in bile BileIntestine Elimination of Toxicants (?)

20 Major Types of Hepatic Injury  Hepatic response to chemical insult depends upon:  Intensity of insult  Cell population affected  Length of exposure (acute, chronic, etc.)

21 Major Types of Hepatic Injury  Fatty Liver (Steatosis)  Results from disruptions in lipid metabolism  Lipids accumulate in hepatocytes  Commonly a response to acute exposure  Usually reversible  Caused by cycloheximide, ethanol

22 Major Types of Hepatic Injury  Cell death  Can occur by two modes  Apoptosis (lack of inflammation)  Necrosis (inflammation occurs)  ALT/AST  Can be:  Focal (random)  Zonal (death in certain functional region)  Panacinar (widespread, massive cellular death)  Acetaminophen, Ecstasy, Cocaine (oral exposure)

23  Canalicular Cholestasis  Results from:  Decrease in functional integrity of sinusoidal and canalicular transporters  Diminished transcytosis  Diminished contractility of canaliculus  Weakened junctions between blood and canalicular lumen  Solutes leak out of lumen  Loss of charge and size gradient between canalicular lumen and blood Major Types of Hepatic Injury

24  Canalicular Cholestasis  Decrease in bile formation  Bile pigments often accumulate in skin and eyes when excretion of these pigments into bile is impaired – Jaundice  Can result in cell swelling, cell death and inflammation  Cyclosporine can cause canalicular cholestasis

25 Fig. 13-11 Casarett & Doull’s Mechanisms of Cholestasis

26 Major Types of Hepatic Injury  Bile Duct Damage  Damage to ducts that carry bile from liver to GI tract  Can result in loss of bile ducts (vanishing bile duct syndrome)  Similar to symptoms seen with canalicular cholestasis  Amoxicillin

27 Major Types of Hepatic Injury  Sinusoidal damage  Occurs from:  Dilation of lumen  Blockade of lumen  Progressive endothelial destruction of endothelial cell wall of lumen  Extensive sinusoidal blockade or cell wall destruction results in liver becoming engorged with blood cells causing shock  Anabolic steroids, Acetaminophen

28 Major Types of Hepatic Injury  Cirrhosis  Accumulation of extensive amounts of collagen fibers in response to injury or inflammation  Following repeated chemical insult, destroyed hepatic cells are replaced by fibrotic scars  Architecture of the liver is disrupted  Decreases liver’s capacity to perform its essential functions  Not reversible!  Repeated exposure to ethanol

29 Major Types of Hepatic Injury  Tumors  Can arise from hepatocytes, bile duct cells, or cells of the sinusoidal lining (rare)  Aflatoxin  Thorotrast (radioactive thorium dioxide)  Accumulates in Kupffer cells  Emits radioactivity throughout its long half-life

30 Factors in Liver Injury  Why is the liver the target site for so many toxins of diverse structure?  Why do many hepatotoxins preferentially damage one type of liver cell?

31 Factors in Liver Injury  Why is the liver the target site for so many toxins of diverse structure?  Specialized uptake processes result in higher exposure in the liver versus other tissues  Abundant capacity for bioactivation reactions

32 Factors in Liver Injury  Why do many hepatotoxins preferentially damage one type of liver cell?  Specialized processes are located in the liver  Example: Cocaine an acetaminophen cause Zone 3 hepatocellular necrosis  Zone 3 is site of high levels of cytochrome p450  P450 enzymes produce harmful metabolites of these two drugs

33 Factors in Liver Injury  Hepatocytes have fenestrated epithelial layers  Liver is membrane-rich and has the ability to concentrate lipophilic compounds  Liver contains many sinusoidal transporters which toxins may be substrates for  Example: Vitamin A hepatotoxicity initially affects sinusoidal Ito cells, which extract the vitamin

34 Factors in Liver Injury  Cytochrome p450 enzymes may bioactivate many toxins to free radicals  Conditions in which cytochrome p450 is depleted has been shown to decrease liver damage during exposure to certain hepatotoxins

35 Factors in Liver Injury  Example:  Therapeutic doses of acetaminophen are not hepatotoxic  However, fasting or other conditions that deplete glutathione may enhance acetaminophen hepatotoxicity  Ethanol may increase Cytochrome P4502E1 causing increased acetaminophen hepatotoxicity

36 Schematic of Bioactivation and Hepatotoxicity of Acetaminophen Fig. 13-7, Casarett & Doull’s

37 Factors in Liver Injury  Activation of Kupffer cells increases ROS and reactive nitrogen species in the liver  Example: LPS  In addition, migration (infiltration) of neutrophils, lymphocytes, and other inflammatory cells may occur to combat infection but also may add to damage by depleting glutathione, etc., through release of excessive amounts of ROS and proteases, etc.

38 Factors in Liver Injury  Liver cells are vulnerable to same types of insult that injure other tissues  Preferential liver damage occurs due to the location of the liver and due to its high capacity for converting chemicals to reactive entities

39 Other Mechanisms of Liver Injury  Cytoskeleton disruption  Mitochondrial damage

40 Other Mechanisms of Liver Injury  Cytoskeleton disruption  Phalloidin (mushroom):  Upon uptake into hepatocytes, prevents disassembly of actin filaments, affecting dynamic nature and integrity of the hepatocyte cytoskeleton  Leads to accentuated “actin web” resulting in dilation of the canalicular lumen Amanita phalloides

41 Other Mechanisms of Liver Injury  Mitochondrial Damage  Mitochondrial DNA codes for several proteins in the mitochondrial electron transport chain  Certain toxins affect mitochondrial DNA  Mitochondrial DNA has limited capacity for repair!

42  Liver is susceptible to toxicological insult because of:  The liver’s proximity and involvement with the GI tract  The liver’s diverse and vital functions  Bile formation  Detoxification reactions  Extraction of diverse substances Summary

43 Toxic Responses of the Kidney

44 Vital Role of the Kidney  Kidney contributes to total body homeostasis  Excretion of metabolic waste  Synthesis of renin and erythropoietin  Regulation of extracellular volume  Acid/base balance  Kidney receives relatively large levels of xenobiotics

45 Schematic of Human Kidney  Divided into 3 major areas:  Cortex  Medulla  Papilla  Nephron: Functional unit of the kidney  Vascular element  Glomerulus  Tubular element (reabsorption/excretion throughout) Tremendous osmotic gradient exists in the kidney!

46 Nephron and Renal Vasculature  Afferent Arteriole  Blood to glomerulus  Efferent Arteriole  Blood leaving glomerulus  Surrounds entire nephron for continual reabsorption and excretion  Flow rate to glomerulus is highly controlled and responds to nerve stimulation, hormones, signaling molecules, etc.

47 The Nephron  Glomerulus  Specialized capillary bed that filters a portion of the blood to an ultrafiltrate which enters the proximal tubule  Glomerular filtration is highly dependent on transcapillary hydrostatic pressure, oncotic pressure, and permeability of the glomerular capillary wall.  Glomerular capillary wall  Permits high rate of fluid filtration  Provides a barrier to the transglomerular passage of macromolecules

48 The Nephron  Proximal Tubule  Reabsorbs approximately 60-80% solutes, small proteins, and water filtered at the glomerulus  Numerous transport systems  Specific endocytotic protein reabsorption processes

49 The Nephron  Loop of Henle  Reabsorbs Na + /K + and water  Possesses Na + /K + /2Cl - co-transporters  Water is freely permeable in descending limb  Ascending limb is impermeable to water

50 The Nephron  Distal Tubule/Collecting Duct  Sensitive to physiologic triggers that may cause decrease glomerular filtration rate (GFR)  To prevent massive loss of fluid/electrolytes if impaired tubular reabsorption occurs  Collecting duct performs final adjustments to urinary volume and composition  Responsive to ADH (increased ADH = increased permeability of collecting duct to water = increased water reuptake)

51 Acute Renal Failure  Characterized by low GFR and azotemia (buildup of nitrogenous wastes in the blood)  Drug may precipitate within kidney causing obstruction  Drug may cause vasoconstriction

52 Acute Renal Failure  Impaired Tubular Integrity  Chemical may compromise cell to cell adhesion in kidney tubules  Results in gaps in cell lining causing back-leak of filtrate and decreased GFR  Detached cells may cause obstruction of tubules

53 Mechanisms of GFR Reduction Fig. 14-4 Casarett & Doull’s

54 Acute Renal Failure Fig. 14-6 Casarett & Doull’s

55 Adaptation Following Toxic Insult  Kidney has a remarkable ability to compensate for loss in functional renal mass  Example:  Following unilateral nephrectomy, GFR of the remaining kidney increases 40-60%!  In addition, compensatory increases in all other functions of the nephron occur (reabsorption, etc.)  Good and Bad: If a chemical induced changes in renal function…problem may not be detected until compensatory mechanisms are overwhelmed

56 Fig. 14-7 Casarett & Doull’s Response to Nephrotoxic Insult

57 Chronic Renal Failure  May occur from long-term exposure to various chemicals  Adaptation following nephron loss causes increased GFR in functional neurons  Whole kidney GFR is maintained

58 Chronic Renal Failure  With time, adaptations can be maladaptive  Glomerulosclerosis eventually develops leading to tubular atrophy and interstitial fibrosis  Mechanical damage occurs as a result of chronically increased GFR

59 Susceptibility of the Kidney to Toxic Injury  Kidneys constitute 0.5% total body weight, but receive 25% of resting cardiac output  Therefore, any drug or toxin in the systemic circulation will be delivered to the kidney in relatively high concentrations

60 Susceptibility of the Kidney to Toxic Injury  The kidney concentrates urine and may concentrate toxicants in tubular fluid, driving passive diffusion of toxicants into tubular cells  Further, the kidney is very sensitive to circulating vasoconstrictors and prostaglandins (vasodilators). Any interference with these substances = renal involvement

61 Sites of Renal Injury  Glomerular Injury  Initial site of chemical exposure in the nephron  Cyclosporine, Amphotericin B (antifungal)  Impair glomerular filtration by causing renal vasoconstriction and decreasing glomerular filtration  Injury may occur to glomerular cell walls (cyclosporine)

62 Sites of Renal Injury  Proximal Tubular Injury  Most common site of toxicant-induced renal injury  Proximal tubule has leaky epithelium that favors the flux of compounds into the tubule

63 Sites of Renal Injury  Loop of Henle/Distal Tubule/Collecting Duct Injury  Amphotericin B, cisplatin (chemotherapeutic)  Cause impaired concentrating ability

64 Sites of Renal Injury  Papillary Injury  Agents that inhibit vasodilatory prostaglandins compromise renal blood flow the the medulla/papilla and result in tissue ischemia

65 Assessment of Renal Function  Non-invasive:  Urine volume measurement  Osmolality  pH  Urinary composition  GFR determination (via measurement of creatinine clearance)

66 Biochemical Mechanisms of Renal Cell Injury  Cell death:  Apoptosis  Organized, usually affects scattered, individual cells  Oncosis  Affects many contiguous cells, cells rupture releasing cellular contents, inflammation follows  As toxicant concentration increases, process usually shifts from apoptosis to oncosis

67 Biochemical Mediators of Toxicity  A chemical can initiate cellular injury by a variety of mechanisms Fig. 14-12 Casarett & Doull’s

68  Cell Volume and Ion Homeostasis  Both tightly regulated and critical for reabsorptive properties of tubular epithelial cells  Toxicants can affect these parameters by increasing ion permeability and disrupting cell volume, or by disrupting ATP production Biochemical Mediators of Toxicity

69  Cytoskeleton and Cell Polarity  Toxicants may disrupt membrane integrity by:  Alteration of cytoskeletal components  Disruption of energy metabolism or calcium and phospholipid homeostasis Biochemical Mediators of Toxicity

70  Mitochondria  Nephrotoxins may compromise cellular respiration and ATP production causing mitochondrial dysfunction Biochemical Mediators of Toxicity

71  Lysosomes  Exposure to unleaded gasoline induces cellular injury through rupture and release of lysosomal enzymes Biochemical Mediators of Toxicity

72  Ca 2+ Homeostasis  Free cytosolic Ca 2+ (Ca 2+ pool) is critical in renal cells  Ca 2+ level is maintained by a series of pumps located on the endoplasmic reticulum  Certain nephrotoxins may disrupt these mechanisms  High calcium levels may cause activation of degradative calcium-dependent enzymes (phospholipases) which may degrade cellular components Biochemical Mediators of Toxicity

73 Specific Nephrotoxicants  Heavy Metals  Different metals have different primary targets in the kidney  Most metals bind to sulfhydryl groups of critical proteins, inhibiting their normal functions and causing renal cell injury  Mercury  Cadmium  Lead

74 Specific Nephrotoxicants HCL forms phosgene (chemical warfare agent) which injures cellular macromolecules  Halogenated Hydrocarbons Example: Chloroform  Targets proximal tubule Chloroform Trichloro- Ethanol (instable) HCL release

75 Specific Nephrotoxicants  Mycotoxins  Commonly found on corn  May affect lipid metabolism in the kidney

76 Specific Nephrotoxicants  Acetaminophen  May cause proximal tubular necrosis  Renal cytochrome p450 activates acetaminophen resulting in nephrotoxicity

77 Specific Nephrotoxicants  Advil (Ibuprofen)  Aleve (Naproxen) Inhibit vasodilatory prostaglandins  Non-steroidal Anti-Inflammatory Drugs Unopposed vasoconstriction Decreased renal blood flow Renal Ischemia Acute renal failure

78 Summary  Toxins in the systemic circulation are delivered to the kidney in relatively high amounts  Toxins may be concentrated in the kidney  Chemical disruption of the kidney’s vital functions may affect total body homeostasis