MPharm Programme Microbial Identification Quiz 5 PDF

MPharm Programme Microbial Identification Dr Callum Cooper [email protected] Learning Objectives • Describe different microbiological techniques to identify bacterial contaminants • Classical identification Vs molecular identification • Highlight limitations and advantages • Link l...

MPharm Programme Microbial Identification Dr Callum Cooper [email protected] Learning Objectives • Describe different microbiological techniques to identify bacterial contaminants • Classical identification Vs molecular identification • Highlight limitations and advantages • Link lecture together with laboratory work What is the contaminant? OR What is causing the infection? What is Diagnostic Microbiology? •Identification • USUALLY to species level •Typing • Distinguishing between strains within a species •Antimicrobial susceptibility testing •Pathogenicity profiling •Clinical applications, but also… • • • • Veterinary microbiology Plant (crop) pathology Food microbiology Pharmaceutical microbiology (characterising contaminants) Microbial Identification • Culture-based methods – Colony morphology – Selective media e.g. Mannitol salt agar – Chromogenic media e.g. MacConkey agar • Microscopic examination – Gram’s staining – Electron microscopy • Biochemical profiling • Molecular analysis – Immunologic – Genomic – Proteomic Colony morphology • Microbes are nearly everywhere and they often live in complex communities NOT as individual species • Highly inaccurate to identify species by colony morphology Colony morphology • To increase accuracy, individual colonies have to be purified • Done by sub culturing them What does this tell us about the microorganism? NOTHING • Selective and Differential media Selective media: Allows for the growth of particular species • • • Suppresses other species Antibiotic /nutrient supplements MacConkey Agar; inhibits G+ organisms due to bile salts and crystal violet • • • Neutral red dye turns pink in acid Sabouraud Dextrose Agar; selective for certain fungi due to low pH(5.6) and high glucose concentration (3-4%) Baird-Parker Agar: selective for Staphylococci, • • Pyruvate and glycine promote Staph growth Lithium chloride and tellurite inhibit nonStaph growth • Tellurite reduced by S. aureus • S. aureus shows shiny black colonies with clear zone around them • Selective and Differential media Differential media: enables bacterial species to be distinguished from one another on the same medium • • • • • • • • Mannitol salt agar (also selective); Inhibits G- organisms (and some G+) due to high salt content (7.5-10%) Mannitol content and phenol red differentiate between Staphylococci Eosin methylene blue (EMB) agar Selective for G- (e.g. E. coli) organisms Contains 2 stains, eosin and methylene blue • Toxic to G+ Lactose fermentation causes dye uptake and stains G- purple E. coli develops a metallic green sheen Microscopy Microscopy Light Fluorescence Electron Cryo Transmission Dark field Phase contrast Scanning Transmission Visible light Radiation source Electron beam Air Medium High Vacuum >1000 x Magnification >100000 x 0.2µm Resolution 0.5 nm Microscopy Light Microscopy- Sample preparation • • In order to see microorganisms more clearly they are often stained Common stains include; • • Bacteria • Gram Stain (Gram Positive Vs Gram Negative) • Ziehl–Neelsen stain (Acid fast bacteria; mainly Mycobacteria) • Malachite Green Stain (Endospore staining) Fungi • Periodic acid-Schiff stain • • • • Used to detect polysaccharides Only works on living fungi • India ink staining (also used for bacterial capsules) • Calcofluor White binds chitin and cellulose Protozoa • Direct microscopy (e.g Trypanosoma brucei in blood) • Giemsa stain Most viruses cannot be visualised with light microscopy Light Microscopy- Gram Staining • 1st step in identifying pure bacterial cultures • Differentiates between Gram positive and Gram negative bacteria based on cell wall composition Electron Microscopy Preparation method will vary by type of Electron microscope used Sample Spread over a support grid Chemical Fixation TEM Dehydration Embedding Critical Drying Ultramicrotomy SEM Metal Coating Immunolabelling Contrast staining Negative staining Biochemical Profiling • Can identify organisms that have been isolated in pure culture • • Requires Gram state to be known Individual tests available; • • • Oxidase test (tests is bacteria is able to produce certain cytochrome oxidase or indophenol oxidase) TMPD or DMPD as redox indicators • Ox + Pseudomonadaceae • Ox - Enterobacteriacae Commercially available kits; • • Miniaturised biochemical tests • API strips (bioMerieux, 1970) • BD BBL Crystal Produces a numerical code which identifies bacterium from a database What about in practice: Classical Non-sterile Pharmaceutical Micro TVC Sample TYMC Streak to purity Gram Stain Biochemical Assay Definitive ID Biochemical Profiling: ATP determination • • Adenosine Triphosphate (ATP) one of the main sources of energy in bacterial cells ATP quantified by the amount of light released following breakdown by firefly luciferase • • • Provides a direct measure of bacterial number Does not require/provide any other information about contaminant Susceptible to contamination Molecular Identification • Immunological Identification • • Genomic Identification • • • • ELISA PCR Whole Genome Sequencing 16s rRNA Proteomic Identification • MALDI TOF Molecular Identification: ELISA • Enzyme-linked immunosorbent assay (ELISA) • Used in medical diagnostics (e.g. HIV, TB etc) • 2 main types used in diagnostics • Direct ELISA (non specific coating of antigens) • Sandwich ELISA (allows for capture of specific antigens) • Requires specific monoclonal antibodies to work Molecular Identification: Genomic • PCR: Amplifies a short sequence of DNA • Sequences amplified determined by primers • • • • Efficacy dependant on how selective primers are Provides positive/negative results Used for identification of antibiotic resistance e.g. mecA Need to know what you are looking for Molecular Identification: Genomic • DNA Sequencing: can sequence whole genome or targeted regions (e.g 16s rRNA) • Can identify multiple changes at once (presence/absence of genes, SNPs etc) • Multi locus sequence typing (MLST): • Amplify housekeeping genes, then sequence • 7 genes used in S. aureus; carbamate kinase (arcC) shikimate dehydrogenase (aroE) glycerol kinase (glpF) guanylate kinase (gmk) phosphate acetyltransferase (pta) triosephosphate isomerase (tpi) acetyl coenzyme A acetyltransferase (yqiL) • Databases exist for different bacteria species • http://www.mlst.net/ • https://pubmlst.org/ • Need for balance between turnaround time and sensitivity • Used less frequently as WGS costs decreases Molecular Identification: MALDI TOF • • • Increasingly used due to speed of identification and lower cost compared to conventional methods Identifies samples based on ionized samples Spectra peaks compared to reference database • Can be used to identify bacterial species from selective media • Still needs specialist oversight • Can also provide information about resistance • Only in certain cases E. coli Summary • Identified different ways of identifying microbial contaminants • • • • • Morphology Biochemistry Immunologic Genetic The ideal diagnostic should be; • • • • ACCURATE SENSITIVE COST EFFECTIVE FAST Extra Reading • • Hugo and Russell’s Pharmaceutical Microbiology: Chapter 3 Section 8; Chapter 25 Section 4.1.2 Singhal et al 2015. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Frontiers in microbiology MPharm Programme Disease and Pathogenesis Dr Callum Cooper [email protected] Learning Objectives • Sources of disease • Look at different stages of disease process • Pathogenic virulence factors • Examples of toxin producers • Examples of biofilms How did this person get infected? How can we stop this disease spreading? Clincal Microbial infections are a major public health problem • Infectious disease is still a major killer of people globally; • Lower respiratory diseases killed 3 million in 2016 • Diarrhoeal disease killed 1.4 million in 2016 • In developed countries, 5-12% of hospital patients encounter nosocomial infection • i.e. one they were not admitted with • In EU each year, ca.400,000 nosocomial infections are resistant to antimicrobial agents; ca. 25,000 of these patients die as a result1 • Risk is determined by individual susceptibility and is multifactorial; • Chronic illness • Genes • Previous exposure • Age 1 WHO • Medicines • Surgery • Malnourishment data: http://www.euro.who.int/en/health-topics/disease-prevention/antimicrobial-resistance Scale of Disease • Endemic: an infection present in a population which is maintained constantly with no external input • E.g. Chickenpox • Epidemic: An infection which rapidly spreads in a short time period (usually ≤2 weeks or less) • E.g. Ebola outbreaks from 2014 • Pandemic: An epidemic which has spread across a large region (e.g. multiple continents/globally etc) • Historically very important e.g. Black death or Spanish Flu • E.g. 2009 H1N1 Influenza pandemic Terminology • Pathogenicity: Ability of a pathogen to produce an infectious disease in an organism • Virulence:Relative degree of damage done by a pathogen, or the degree of pathology caused by a pathogen • Virulence / pathogenicity factor: Microbial product or strategy that contributes to virulence or pathogenicity • Colonization of a niche in the host (this includes attachment to cells) • Immunoevasion, evasion of the host's immune response • Immunosuppression, inhibition of the host's immune response • Entry into and exit out of cells (if the pathogen is an intracellular one) • Obtain nutrition from the host Sources of Disease • People – Major source, particularly of highly infectious disease: HIV, smallpox, diphtheria etc. • Obligate pathogen: A microorganism that has to cause disease to be passed between hosts and must also infect a host in order to survive e.g. Mycobacterium tuberculosis WHAT ABOUT COMMENSAL ORGANISMS? • Opportunistic pathogen: Normally a commensal or non harmful microorganism that can cause disease when the host's resistance is low e.g. Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa. Commensal microorganisms in disease • Commensal microorgansims are part of the normal flora • Usually non-pathogenic • Some may be pathogenic but unable to enter disease process • Lack ability to attach to suitable surface • Held in check by other organisms • Prevent disease by; • Blocking attachment sites • Producing antimicrobial products as part of normal metabolism • Can cause disease in humans when; • Microbial balance is upset (e.g. following antibiotic treatment) • Microbes get places where they shouldn’t (e.g. blood stream infections) • Host immunity is compromised Sources of Disease • Environment – – – – Waterborne: cholera, typhoid, Legionella Food borne: Salmonella, Campylobacter, E. coli Surfaces: Soil: Clostridium botulinum and C. tetani • Animals (Zoonoses) – Major source of disease with most new human diseases are believed to have an animal origin; – Bacterial: anthrax, E. coli, plague – Parasites: Toxoplasmosis – Viruses: avian influenza, CCHF, Ebola and Rift Valley fever – Other: BSE • The role of animals in infectious disease Zoonoses are infectious diseases that can be naturally transmitted between non-humans (usually vertebrates) and humans • Animals can act as a reservoir (place where agents normally reside) • Contribute to development of pandemics • Exposure to infected animals or contaminated environments is a major risk factor The role of occupation in infectious disease exposure • Traditionally occupations which come in close contact with animals or animal products • Farmers, tanners, slaughterhouse workers etc • Rules implemented to reduce risk • These days some occupations at higher risk of exposure to infectious agents; • Laboratory • Healthcare • Still occasionally get cases https://www.theguardian.com/p olitics/2019/jun/14/call-fortattooists-and-body-piercers-toqualify-in-infection-control The role of animals in infectious disease • Humans can contract animal viruses “Bird Flu” H5N1 & "swine flu” H1N1 • Viruses can recombine to increase pathogenicity • Antigenic shift: Combination of two or more different strains • Produces new form of virus which shows a mixture of surface antigens • Antigenic drift: Accumulation of mutations within antibody binding regions • Reduces the effectiveness of the immune system Koch’s postulates 1st described in 1890 Proof available for: • Plague, anthrax, diphtheria, cholera, typhoid etc. But less evident when… • Condition is chronic or minor • Condition has multiple causes or pathogen is non-culturable • No suitable animal model of infection • Agents causing pneumonia, wound infection, UTIs, septicaemia Updated Koch: Molecular Postulates • Identify gene (or gene product) responsible • Show that gene is present in disease causing strains of bacteria • Not present in avirulent strains • Show that disrupting the gene reduces virulence • Introduce a cloned gene into avirulent strain • Should confer virulence • The gene is expressed in vivo • Specific immune response to gene protects The Disease Process Disease can be broadly split into 3 stages; • Transmission & exposure Pathogen Exposure Skin/mucosa adherence Further exposure Epithelia invasion • Attachment and Invasion • Colonisation and Damage Further exposure at local sites Colonisation and growth Invasiveness – further growth at Original/othersites Tissue damage and disease Local/systemic toxicity Transmission and Exposure • Transmission is the passing of a communicable disease from an infected host to a particular individual or group, regardless of whether the other individual was previously infected. • Vertical transmission: Mother to child e.g. HIV • Horizontal transmission: Person to person transmission e.g. Chlamydia • Vehicle borne transmission: Transmission through an inanimate object. • Vector borne transmission: Transmission through another organism e.g. Malaria • Diseases can be transmitted via different routes • HIV can be vertical, horizontal or vehicle borne Transmission and Exposure • Horizontal exposure to disease tends to be one of three routes; • Inhalation • Ingestion • Contact • Successful exposure depends on multiple factors • • • • Microbe count: more = better Airborne: size, density, surface features. Waterborne: density, surface features, hydrophobicity Contact: environmental persistence. • Particularly important in hospital acquired infections • Distribution on host: into cuts, digestive system, GU system, lungs etc. • Vectors Transmission and Exposure Utilities Facilities Process Contaminated Product Materials Equipment Personnel Transmission and Exposure Attachment & Invasion • Bind to receptors on host cells to stick to cells and avoid host defenses • Depends on surface break or on active microbial procedures • Virulence factors play an important role; • Pili or adhesins (aid attachment to cells) • Capsules (resist phagocytosis) • Enzyme production Biofilms • A biofilm comprises any group of microorganisms in which cells stick to each other and often also to a surface • Naturally occurring, but clinically important • Estimated 80% of infections involve biofilms • Increases average hospital stay • Increases resistance to treatment • Especially important in Cystic fibrosis sufferers • Biofilms form within lung Ref: D. Monroe. "Looking for Chinks in the Armor of Bacterial Biofilms". PLoS Biology 5 (11, e307) Colonisation and Damage: Consequences • Growth in tissues requires cells to resist host responses • Potentially neutralise – e.g. enzymes, capsules etc. • Growth also requires appropriate nutrients • Some tissues have limited (e.g. iron) or specific nutrients which slow or enhance microbial growth • Host cell death as a result of; • Damage • Toxin accumulation • Enzymatic digestion • Pathogenicity factors play an important role; • Bacterial toxins • Exotoxins • Endotoxins (LPS) • Capsule • Bacterial enzymes Microbial toxins • Microbial toxins promote infection by damaging tissue • Endotoxin (LPS) • Exotoxin (secreted toxin) • Endotoxins part of the bacterial cell membrane • Can lead to septic shock • Highly controlled in sterile pharmaceutical products • Clostridium difficile produces two main toxins; • Toxin A (enterotoxin) & Toxin B (cytotoxin) • Play important role in disrupting gut epithelia • Some toxins have found other uses; • Botulinum toxin among the most powerful • Estimated LD50 of 1.3–2.1 ng/kg IV or IM Microbial Exotoxins • Three general types of microbial exotoxin based on effect; • Cytotoxin (kills cells) • Neurotoxins (interfere with normal nerve impulses) • Enterotoxins (affects cells lining the GI tract) • Can be further divided; • Type I • Superantigens; Cause intense immune response due to nonspecific cytokine release from host cells • Produced by some S. aureus strains • Type II • Membrane disrupting toxins; Disrupt phospholipid bilayer or make protein channels in PM e.g Cholera toxin • Type III • Protein modification; e.g Diphtheria and Shiga toxin inhibit synthesis • Tetanus toxin cleaves protein involved neurotransmitter release Microbial Endotoxins • LPS (Lipopolysaccharides) • O Antigen • Lipid A • Heat Stable (c.f. exotoxins – typically heat liable [proteins / peptides]) • Quite difficult to remove from pharmaceutical preparations • Lipid A - Toxin portion of the LPS • Responsible for fever that is associated with many G- bacterial infections • When G- cells are ‘digested’, endotoxins are released: causes fever • If immune system acting on high G- bacterial load, large release of endotoxins can trigger septic shock • Antibiotics can initially cause fever / shock due to release of endotoxins Haemolysins • Heamolysins cause red blood cell lysis by forming pores in the plasma membranes • Most are proteins but some are lipid biosurfactants • Not to be confused with haemolysis • Bacteria can be classified on basis of haemolysis • Alpha (Oxidises iron) • S. pneumoniae • Beta (ruptures RBC) • S. pyogenes • Gamma (non-haemolytic) • Can lead to haemolytic anaemia • Bone marrow cant produce RBC fast enough to replace loss By Y tambe - Y tambe's file, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=860707 Non-bacterial disease: Mycoses • Mycoses are diseases caused by fungi • • • • • Can be persistent (chronic) Can be localised or systemic Often from inhalation of fungal spores Most common in immunocompromised patients or those who have taken antibiotics Fungal diseases commonly include; • • • Athletes foot (irritation, dry and inflamed skin with localised pain) • Caused by species of Trichophyton, Epidermophyton, and Microsporum Thrush By James Heilman, MD - Own work, CC BY• Caused by Candida albicans (most SA 3.0, https://commons.wikimedia.org/w/index.p hp?curid=17770316 common) Aspergillosis • Generally caused by Aspergillus fumigatus • Can be invasive in immune compromised patients Non-bacterial disease: Viral • Viral diseases can cause both communicable and noncommunicable diseases • Communicable: • Herpes • HIV • Influenza • AND MANY MANY MORE • Non-communicable: • HPV → Cervical cancer1 • Human adenovirus Ad-36 → Obesity2 • Mouse mammary tumor virus → human breast cancer3 1: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC145302/ 2: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517116/ 3: https://www.ncbi.nlm.nih.gov/pubmed/16061645 Extra reading • Prescotts Microbiology:Part VIII; section 35, 36, 38 • Brock Microbiology: Part VI • Percival, S.L et al. 2015. Healthcare-associated infections, medical devices and biofilms: risk, tolerance and control. J Med Microbiol, 64(4), pp.323-334. WEEK 16 MPharm Programme Renal Dr Praveen Bhugra PHA112 Slide 1 of 41 PHA112 Renal WEEK Learning Objectives 16 From this lecture you should be able to:  Understand and explain briefly the organs and function of the urinary system  Understand and explain in detail the anatomy of kidney including location, internal and external structure, the blood and nerve supply and also the function of the kidney.  Understand and describe in detail the structure of the nephron and the organisation of its blood supply including its function  Understand and explain in detail the renal physiology including mechanism of urine formation, glomerular filtration, tubular reabsorption, tubular secretion and hormonal regulation and homeostasis Slide 2 of 41 PHA112 Renal WEEK 16 Slide 3 of 41 Urinary System PHA112 Renal WEEK 16 Slide 4 of 41 Urinary System PHA112 Renal WEEK 16 Urinary System •Are paired, bean-shaped organs •Located along the back body wall below the diaphragm and adjacent to the vertebral column • Right kidney lower than the left because liver occupies larger area above the kidney on the right side Slide 5 of 41 PHA112 Renal WEEK 16 Structure of Kidney 8-18 2-3 Slide 6 of 41 PHA112 Renal WEEK 16 Slide 7 of 41 Structure of Kidney MPharm PHA112 Renal WEEK 16 Blood and Nerve Supply • Renal arteries deliver ~ 1/4 (1200 ml) of cardiac output to the kidneys each minute • Arterial flow into and venous flow out of the kidneys follow similar paths • Nerve supply is via sympathetic fibers from the renal plexus Slide 8 of 41 PHA112 Renal WEEK 16 Slide 9 of 41 Blood Supply of Kidney PHA112 Renal WEEK 16 Slide 10 of 41 Renal Blood Supply PHA112 Renal WEEK 16 Functions of the Kidney • Removal of toxins, metabolic wastes, and excess ions from the blood • Regulation of blood volume, chemical composition, and pH • Gluconeogenesis during prolonged fasting • Endocrine functions • Renin: regulation of blood pressure and kidney function • Erythropoietin: regulation of RBC production • Activation of vitamin D Slide 11 of 41 MPharm PHA112 Renal WEEK 16 Functions of the Kidney 1,25 dihydroxycholecalciferol or 1,25-dihydrooxyvitamin D3 or calcitriol Slide 12 of 41 PHA112 Renal WEEK 16 Nephron • Structural and functional units that form urine • ~1 million per kidney • Each nephron consists • Renal corpuscle: an initial filtering component • Renal tubule: Extends from the renal corpuscle Slide 13 of 41 PHA112 Renal WEEK Nephron 16 Slide 14 of 41 PHA112 Renal WEEK Nephron 16 Slide 15 of 41 PHA112 Renal WEEK Nephron 16 • Cortical nephrons make up about 80–85% of the 1 million microscopic nephrons • Their renal corpuscles are located in the outer portion of the cortex, with short loops of Henle that penetrate only a small way into the medulla. • The ascending limbs of their loops of Henle consist of only a thick segment, lacking any thin portions. • Nephrons with short loops receive their blood supply from peritubular capillaries that arise from efferent arterioles. Slide 16 of 41 PHA112 Renal WEEK Nephron 16 • The other 15–20% of the nephrons are juxtamedullary nephrons . • Their renal corpuscles lie deep in the cortex, close to the medulla, and they have long loops of Henle that extend into the deepest region of the medulla. • The ascending limbs of their loops of Henle consist of both thin and thick segments. • Nephrons with long loops receive their blood supply from the vasa recta that arise from peritubular capillaries before becoming peritubular venules Slide 17 of 41 MPharm PHA112 Renal WEEK Nephrons 16 Slide 18 of 41 PHA112 Renal WEEK Nephrons 16 • The distal convoluted tubules of several nephrons empty into a single collecting duct. • Collecting ducts unite and converge into several hundred large papillary ducts which drain into the minor calyces, major calyces, renal pelvis, and ureters. Slide 19 of 41 PHA112 Renal WEEK 16 Functions of Nephron Slide 20 of 41 PHA112 Renal WEEK 16 Slide 21 of 41 Renal Corpuscle Glomerular (Bowman’s) capsule and glomerulus PHA112 Renal WEEK 16 Slide 22 of 41 Renal Corpuscle Glomerular (Bowman’s) capsule and glomerulus PHA112 Renal WEEK 16 Slide 23 of 41 Glomerular Filtration PHA112 Renal WEEK Glomerular Filtration 16 Total Amount in Plasma Amount in 180 L of filtrate (/day) Amount returned to blood/d (Reabsorbed) Amount in Urine (/day) Water (passive) 3L 180 L 178-179 L 1-2 L Protein (active) 200 g 2g 1.9 g 0.1 g 3g 162 g 162 g 0g 1g 40 g 24 g 30 g (about 1/2) (about 1/2) Glucose (active) Urea (passive) 0.03 g Creatinine Slide 24 of 41 PHA112 1.6 g Renal 0g (all filtered) 1.6 g (none reabsorbed) WEEK 16 Glomerular Filtration Net filtration pressure = 55-30-15=10mmHg Slide 25 of 41 PHA112 Renal WEEK 16 Glomerular Filtration Regulation Slide 26 of 41 PHA112 Renal WEEK 16 Glomerular Filtration Regulation • Two hormones contribute to regulation of GFR • Angiotensin II is a potent vasoconstrictor of both afferent and efferent arterioles (reduces GFR). • A sudden large increase in BP stretches the cardiac atria and releases atrial natriuretic peptide (ANP). • ANP causes the • glomerulus to relax, • increasing the surface • area for filtration Slide 27 of 41 PHA112 Renal WEEK 16 Glomerular Filtration Regulation Slide 28 of 41 PHA112 Renal WEEK Reabsorption Routes 16 Tubule cell Fluid in tubule lumen Na+ Peritubular capillary Na+ Na+ Paracellular reabsorption ATP ADP Na+ Na+ Na+ Na+ Key: Transcellular reabsorption Diffusion Basolateral membrane Apical membrane Slide 29 of 41 Sodium–potassium pump (Na+/K+ ATPase) Tight junction Interstitial fluid PHA112 Active transport Renal Reabsorption in PCT WEEK 16 Fluid in tubule lumen Proximal convoluted tubule cell ATP 2 Na+ Glucose Na+ Peritubular capillary Na+ Na+ ADP Key: Glucose Tight junction Glucose Interstitial fluid PHA112 Na+–glucose symporter Glucose facilitated diffusion transporter Diffusion Sodium–potassium pump Brush border (microvilli) Slide 30 of 41 Other Na+ symporters in the PCT follow similar way • HPO42- (phosphate), SO42- (sulfate) ions • all amino acids and lactic acid Renal WEEK 16 Reabsorption in PCT Na+ reabsorption and H+ secretion via secondary active transport through apical membrane CO2=Carbon dioxide H2CO3=carbonic acid CA=carbonic anhydrase Slide 31 of 41 PHA112 Renal WEEK 16 Reabsorption in PCT HCO3 reabsorption CO2=Carbon dioxide H2CO3=carbonic acid CA=carbonic anhydrase Slide 32 of 41 PHA112 Renal WEEK 16 Reabsorption in PCT Passive reabsorption in second half of the PCT Slide 33 of 41 PHA112 Renal WEEK 16 Reabsorption in Thick ascending limb of Loop of Henle Reabsorbs 15% of filtered water;20-30% filtered Na+ and K+; 35% of filtered Cl-;10-20% of filtered HCO3- and variable amount of filtered Ca2+ and Mg2+ Slide 34 of 41 PHA112 Renal WEEK 16 Reabsorption in Distal convoluted tubule and in collecting duct Reabsorption of Na+ and secretion of K+; Slide 35 of 41 PHA112 Renal Hormones and Homeostasis WEEK 16 •Five hormones affect the extent of Na+, Cl–, Ca2+, and water reabsorption as well as K+ secretion by the renal tubules. These hormones, all of which are key to maintaining homeostasis of not only renal blood flow and B.P., but systemic blood flow and B.P., are: • angiotensin II • antidiuretic hormone (ADH) • aldosterone • atrial natriuretic peptide (ANP) • parathyroid hormone (PTH) Slide 36 of 41 PHA112 Renal WEEK 16 Slide 37 of 41 Hormones and Homeostasis PHA112 Renal Hormones and Homeostasis WEEK 16 • Antidiuretic hormone (ADH) is released by the posterior pituitary in response to low blood flow in this part of the brain. • ADH affects facultative water reabsorption by increasing the water permeability of principal cells in the last part of the distal convoluted tubule and throughout the collecting duct. • In the absence of ADH, the apical membranes of principal cells are almost impermeable to water Slide 38 of 41 PHA112 Renal Hormones and Homeostasis WEEK 16 • Parathyroid hormone (PTH) is released by the parathyroid gland. • Stimulates cells in early distal convoluted to reabsorb Ca2+ into the blood • Inhibits HPO42- (phosphate) reabsorption in proximal convoluted tubule thereby promoting phosphate excretion Slide 39 of 41 PHA112 Renal WEEK 16 Summary of filtration, reabsorption and secretion in nephron and collecting duct Slide 40 of 41 PHA112 Renal WEEK 16 Further Reading refer to the following textbooks • Ross and Wilson Anatomy and Physiology in Health and illness 13th Edition • Gerard J. Tortora and Byran H. Derrickson Principles of Anatomy and Physiology 13th Edition • Frederic H. Martini Fundamentals of Anatomy & Physiology 7th Edition • Lauralee Sherwood Human Physiology From Cells to Systems 7th Edition • Robert G. Carroll Elsevier’s Integrated Physiology Slide 41 of 41 PHA112 Renal