Physiology of the Kidneys Body Fluids & Acid Base Balance MDC John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida College of Medicine

PHYSIOLOGY OF THE KIDNEYS BODY FLUID & ACID BASE BALANCE 2010 Suggested reading assignment: Berne and Levy, Chapter 2 (fluid compartments); Chapters (Renal Physiology) and Chapter 36 (Acid Base Regulation) Dr. Dietz: Office - MDC 3538 Ph: (Office – 3rd floor of the new research wing behind conference room MDC 3510)

DATETIMETOPIC Fri., April 248:00-9:50BODY FLUID COMPARTMENTS BODY FLUID IMBALANCES Mon., April 278:00-9:50FILTRATION & CLEARANCE RENAL BLOOD FLOW Tues., April 281:00-2:50PROXIMAL TUBULE CONCENTRATION & DILUTION GLUCOSE, PAH, UREA Wed., April 298:00-9:50SODIUM EXCRETION WATER EXCRETION K+ EXCRETION Ca2+ & Mg2+ EXCRETION Schedule 2010 Fri., May. 18:00-9:50 RENAL HORMONES BODY FLUID REGULATION Mon., May. 48:00-9:50ACID BASE CHEMISTRY H+ EXCRETION Tues., May 51:00-1:50 ACID-BASE BALANCE Wed., May 68:00-9:50Clinical Correlation Dr. Bryan Bognar, Internal Medicine 11:00-11:50Clinical Correlation, Kidney/Acid Base Dr. Valerie Panzarino, Pediatric Nephrology Thurs. May 710:00-11:30Renal/Acid-Base Small Group Clinical Cases

Kidney function in disease states, with injury or transplantation, aging, ureteral obstruction, hypertension or hypotension. Clinical measurements of kidney function. Renal Tubular Acidosis (RTA): Types 1, 2 and 4. Effects of renal failure on blood volume, pressure, calcium balance, creatinine and urea, red cell production, acid – base status. Adrenal insufficiency. Changes in body fluid balances with changes in salt intake, I.V. solutions, over-hydration, dehydration, diarrhea and/or vomiting, heart failure, hemorrhage. Hyper- and hypokalemia. Edema formation. Diabetes Insipidus. Acute Renal Failure Glucose handling by the kidneys in diabetes mellitus. Syndrome of Inappropriate ADH secretion. Mechanisms of action for diuretics. Renovascular Hypertension. Acid – Base Disorders: metabolic acidosis, metabolic alkalosis, respiratory acidosis, respiratory alkalosis, mixed acid-base disturbances. Some relevant clinical concepts discussed in this section of the course

RENAL WWW RESOURCES Genetic abnormalities in renal transporters: The Merk Manual Chapter on Water, Electrolytes and Acid-Base: Fluid Physiology: Renal Pathology: Urinalysis: Online Practice Tests: Renal Physiology Objectives: Several Reviews of Renal Physiology: Acid-Base Calculator: Acid-Base cases online: Fluid balance in space:

Medical Physiology Course Outline Cell Transport Excitable Membranes Respiration Acid Base Kidney Heart & Circulation Endocrinology

Where are we going ? Fluid compartments iv solutions disturbances Filtration/ blood flow normal Pathophys Reabsorption Salt/water regulation Reabsorption Na/K/Ca urea General Fluid Regulation How the kidneys fit with everything else Acid Base Regulation How the kidneys and lungs work together

Body Fluid Compartments Normal Compartment Volumes Changes with I.V. Solutions Pathophysiological changes Molarity vs Osmolarity Osmotic Pressure Membrane Permeability (cell vs capillary)

BODY FLUIDS At the end of this section you should know: Normal volumes for the basic fluid compartments as Liters or as a % of body weight. Solute concentrations of the basic compartments. How compartments are measured. How various i.v. solutions affect the different fluid compartments. How changes in fluid intake or output affect the different fluid compartments. How to calculate precise changes in fluid volumes and use the DY diagrams.

THREE EMERGENCY ROOM PATIENTS 80 year old male with AD - over medicated because of irritability and has had nothing to drink for 3 days. 3 week old infant with vomiting and diarrhea for 2 days. Gunshot wound - loss of 2 liters of blood.

BODY FLUIDS DISTRIBUTION: Total body water (TBW) varies from 50-70% of body weight (ave. = 60%). In an average person (70Kg) this is: 0.6 X 70Kg = 42Kg or 42L

PERCENTAGE OF WATER IN TISSUES

Intracellular (ICF): 40% of Bd. Wt. 28L Extracellular (ECF): 20% of Bd. Wt. 14L Plasma: 4-5% of Bd. Wt. 20% of ECF

MEASURING THE SIZE OF COMPARTMENTS Standard Equation Compartment Volume = Amount Injected Final Concentration

Molar and Equivalent Concentrations HCO 3 -

Osmolality: millimoles of solute/Kg of H 2 O; Osmolarity: millimoles of solute/L of H 2 O Activity of H 2 O decreases as Osmolality increases. Colligative Properties of Solutions: Depend on number of molecules and not on their nature. Osmolality A B  Boiling Point  Freezing Point  Vapor Pressure  Osmotic Pressure

APPROXIMATE IONIC COMPOSITION OF THE BODY WATER COMPARTMENTS

CapillaryInterstitiumIntracellular Na mMNa mMNa + 10 mM K mM K mM Proteins 1.5 mM Proteins 0.1 mM Proteins 3 mM Osm. = mOsm/L 285 mOsm/L 285 mOsm/L Pc = 25 mmHg P = 0 mmHg   = 28 mmHg   = 3 mmHg Permeable to most solutes but imperm- eable to large proteins Impermeable to most solutes Comparison of the Three Major Compartments Colloid Osmotic Pressure, Protein Osmotic Pressure, Oncotic Pressure

CHANGES IN BODY FLUID COMPARTMENT VOLUMES

“The total body content of sodium is the principal determinant of extracellular and intravascular fluid volume” Na + & anions – Extracellular K + & anions – Intracellular D 5 W = Water – adds no effective osmoles At equilibrium, osmolality is the same in all compartments Calculate TBW first, then ECF and ICF

Problem We infuse 1 liter of D5W (5% Dextrose in water). How many total osmoles have we infused? What is the new osmolality after infusion?

Add 1 Liter of D5W = Water TBWECFICF

Add 1 Liter of D5W = Water TBWECFICF Glucose is metabolized so no osmoles are added.

Add 1 Liter of D5W = Water TBWECFICF

Add 1 Liter of D5W = Water TBWECFICF

Add 1 Liter of D5W = Water TBWECFICF

Add 1 Liter of D5W = Water TBWECFICF 1/3 ECF and 2/3 ICF

Addition of 1 L of H 2 O

CapillaryInterstitiumIntracellular Na mMNa mMNa + 10 mM K mM K mM Proteins 1.5 mM Proteins 0.1 mM Proteins 3 mM Osm. = mOsm/L 285 mOsm/L 285 mOsm/L Pc = 25 mmHg P = 0 mmHg   = 28 mmHg   = 3 mmHg Permeable to most solutes but imperm- eable to large proteins Impermeable to most solutes Comparison of the Three Major Compartments Colloid Osmotic Pressure, Protein Osmotic Pressure, Oncotic Pressure

Problem We infuse 1 liter of normal saline (0.9%) of 0.150M How many total osmoles have we infused? What is the new osmolality after infusion?

Add 1 Liter of Normal Saline 0.150M (0.9%) TBWECFICF

Add 1 Liter of Normal Saline 0.150M (0.9%) TBWECFICF 0.150M NaCl = 150 mM Na + & 150 mM Cl - in 1 L so there are 300 mOsm total solute added as an isotonic solution

Add 1 Liter of Normal Saline 0.150M (0.9%) TBWECFICF

Add 1 Liter of Normal Saline 0.150M (0.9%) TBWECFICF

Add 1 Liter of Normal Saline 0.150M (0.9%) TBWECFICF

Add 1 Liter of Normal Saline 0.150M (0.9%) TBWECFICF

Add 1 L of normal saline (0.9%) or 0.150M or 150 mM or 300 mOsm/L

CapillaryInterstitiumIntracellular Na mMNa mMNa + 10 mM K mM K mM Proteins 1.5 mM Proteins 0.1 mM Proteins 3 mM Osm. = mOsm/L 285 mOsm/L 285 mOsm/L Pc = 25 mmHg P = 0 mmHg   = 28 mmHg   = 3 mmHg Permeable to most solutes but imperm- eable to large proteins Impermeable to most solutes Comparison of the Three Major Compartments Colloid Osmotic Pressure, Protein Osmotic Pressure, Oncotic Pressure

Problem We infuse 2 Liters of a 0.45% solution of NaCl. How many total osmoles have we infused? What is the new osmolality after infusion?

Problem We infuse 2 Liters of a 0.45% solution of NaCl. How many total osmoles have we infused? What is the new osmolality after infusion?

Add 2 Liter of Half-Normal Saline (0.45%) TBWECFICF

Add 2 Liter of Half-Normal Saline (0.45%) TBWECFICF 0.075M NaCl = 75 mM Na + & 75 mM Cl - but 2 L so there are 300 mOsm total added as a hypotonic solution

Add 2 Liter of Half-Normal Saline (0.45%) TBWECFICF

Add 2 Liter of Half-Normal Saline (0.45%) TBWECFICF

Add 2 Liter of Half-Normal Saline (0.45%) TBWECFICF

Add 2 Liter of Half-Normal Saline (0.45%) TBWECFICF

Problem What would you expect to happen to fluid compartments if we infuse KCl?

Fluids Commonly Lost

LOSS OF GI FLUIDS

Volume Contraction IsotonicHypertonicHypotonic ECF ICF ECF ICF Osmolality Volume Expansion IsotonicHypertonicHypotonic ECF ICF ECF ICF ECF ICF Osmolality From H. Valtin ICF PATHOPHYSIOLOGICAL CHANGES IN EXTRACELLULAR FLUID VOLUME

80 year old male with AD - over medicated because of irritability and has had nothing to drink for 3 days. 3 week old infant with vomiting and diarrhea for 2 days. Gunshot wound - loss of 2 liters of blood. THREE EMERGENCY ROOM PATIENTS

CapillaryInterstitiumIntracellular Na mMNa mMNa + 10 mM K mM K mM Proteins 1.5 mM Proteins 0.1 mM Proteins 3 mM Osm. = mOsm/L 285 mOsm/L 285 mOsm/L Pc = 25 mmHg P = 0 mmHg   = 28 mmHg   = 3 mmHg Permeable to most solutes but imperm- eable to large proteins Impermeable to most solutes Comparison of the Three Major Compartments Colloid Osmotic Pressure, Protein Osmotic Pressure, Oncotic Pressure

Glomerular Filtration Glomerular Capillary Starling Forces (review from CV) Filtration as bulk flow (both ions and water) Clearance (volume of plasma/time) John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida, College of Medicine

POP QUIZ Which of the following are true concerning fluid compartments (Answer True or False)? A. ECF is 2/3 of total body water (TBW). B. The major ECF cation is Na+. C. A normal saline infusion increases ECF volume but decreases ICF volume. D. Hypertonic NaCl infusion increases ECF volume but decreases ICF volume. E. Plasma volume = the distribution of water minus the distribution of Na+. F ? ? ? ? ? F F T T

Glomerular Filtration At the end of this section you should know: the arrangement of the renal arterioles, capillary beds, tubular segments renal innervation. how glomerular capillaries and filtration forces compare to other capillary beds how GFR is affected by physiological and pathophysiological conditions. how GFR is measured clinically using creatinine and what is meant by renal "clearance". how GFR can change in disease or aging and by compensatory hypertrophy.

Renal Failure Patient Patient Data (Plasma)Change from Normal K +  Phosphate  Calcium  Sulfate  Urea  Hematocrit  Blood Pressure  Bicarbonate  pH 

FUNCTIONS OF THE KIDNEYS Remove wastes. Reg. Vol. and Composition of ECF. Acid-Base Balance. Blood Pressure Regulation. Removal of Foreign Substances. RBC Production. Vitamin D Activity.

Proximal Tubule Distal Tubule Thin descending Thin ascending Loop of Henle Thick ascending (diluting segment) Early Late Collecting Duct Cortical Medullary

Blood Supply to the Kidneys Glomerular capillaries Peritubular capillaries Vasa recta Note the 3 capillary beds: Afferent Arteriole Efferent Arteriole

Blood Supply to the Kidneys

Innervation of the Kidneys Sympathetic Afferent & Efferent Arterioles Juxtaglomerular cells Tubule

Basic Theory of Urine Formation Filtration Reabsorption Secretion Excretion

Organ to Cellular to Molecular

Glomerular Filtration Production of a protein - free filtrate of plasma

THE GLOMERULAR MEMBRANE Three Sieves in Series Capillary Endothelium Basement Membrane Bowman’s Capsule Epithelium (podocytes)

Permeability Based on Molecular Size

Starling Forces for Skeletal Muscle Capillary P C 32 P T 1 mmHg  C 25  T 2 mmHg 15 mmHg 25 mmHg Blood Flow

Starling Forces Across the Glomerular Capillary GFR = 125 ml/min = 180 L/day NFP = mmHg outward P GC 60 P BC 15 mmHg  GC 25  BC 0 mmHg 58 mmHg 35 mmHg Blood Flow

Filtration Coefficient (Kf) Permeability Surface Area

P GC 60 P BC 15 mmHg  GC 25  BC 0 mmHg 58 mmHg 35 mmHg Blood Flow Regulation of GFR

Changes in Kf (Permeability or Surface area): Mesangial Cell Contraction or Relaxation + ANP, NO - AII, Endothelin, Norepi, Epi, ADH

A volume of plasma from which a substance is completely removed by the kidneys per unit time. Clearance Where UF = urine flow; Ux = urine concentration of X; Px = plasma concentration of X; Cx = clearance of X C x = UF  U x = Volume/Time eg. ml/min or L/day P x

Freely FilteredNot Metabolized Not ReabsorbedDoes Not Change GFR Not Secreted Not Produced Measurement of GFR (Inulin M.W. = 5,000) Amount Filtered = Amount Excreted GFR · P IN = UF ·U IN GFR = UF ·U IN = C IN P IN (Filtered Inulin = Excreted Inulin) Excreted Inulin Plasma Inulin (volume/time)

1 ?1 Inulin in the Kidney Renal Plasma Flow (RPF) = 600 RPF = 475 GFR = 125 I = 1 I = RPF = 599 Peritubular Capillaries Salts & Water Reabsorbed but no Inulin Reabsorbed Vasa Recta Capillaries Salts & Water Reabsorbed but no Inulin Reabsorbed Renal Artery Renal Vein Urine Flow = I = (All flows are in ml/min) (I = Inulin concentration in mg/dl) RPF = 415 RPF = 60 Glomerulus ? 0.8?

Changes in Plasma Creatinine in Renal Failure

Compensatory Hypertrophy 1) Changes in GFR a. Disease b. Transplantation 2) Changes in GFR with age “The Four Stages of Life”

Changes in GFR in Chronic Renal Disease

Daily Creatinine Excretion Production varies based on weight & gender Excretion equals creatinine production Excretion can be normal even in renal disease GFR = UF ·U creatinine (Creatinine Excretion) P Creatinine

Problem: 24 hour urine collection for GFR 1. Tell patient to go home and collect all urine in container provided. 2. Patient goes home. Patient pees in container. 3. Patient goes to Walmart to buy dental floss, uses store restroom. 4. Patient goes home. Patient pees in container. Etc. 5. Patient gets up at 3:00am and uses toilet. 6. Next day: Patient returns container to office. Draw a blood sample. Volume collected – 1,800 ml (normal range) Collection time – 24 hours Urine creatinine = 70 mg/dl (normal range) Plasma creatinine = 1.4 mg/dl (high end of normal) Creatinine Clearance (GFR) = Should you order a renal biopsy? calculated as 90 L/day or 62 ml/min.

Daily Creatinine Excretion Normal PersonKidney disease Creatinine (Cr) Production (1mg/minute) Plasma Cr = 1 mg/dl Plasma Cr = 4 mg/dl Amount of Filtered Cr = 1 mg/minute Amount of Filtered Cr = 1 mg/minute Volume Filtered 25 ml/min [Cr] = 4 mg/dl Volume Filtered 100 ml/min [Cr] = 1 mg/dl Amount of Excreted Cr = 1 mg/minute Amount of Excreted Cr = 1 mg/minute Recall that all of the filtered Creatinine is excreted. Blame Tania Condarco

RENAL BLOOD FLOW (RBF) NORMAL = ml/min. (both kidneys) = 20-25% of C. O. RENAL PLASMA FLOW (RPF) = RBF (1-hematocrit) = ml/min. (both kidneys) FILTRATION FRACTION (FF) =GFR/RPF = 125ml/min/650 ml/min = 20 % Regulation of Blood Flow (review of CV) Clearance (again?) John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida College of Medicine

At the end of this section you should know: the distribution of renal blood flow, extra-renal RBF regulation and autoregulation. how renal plasma flow (RPF) is measured using clearance. how to calculate RBF, RPF and Filtration Fraction (FF). how renal oxygen consumption is effected by changes in RBF or GFR. RENAL BLOOD FLOW (RBF)

Distribution of Blood Flow Cortex ml/min (75%) Outer Medulla ml/min (20%) Inner Medulla - 60 ml/min (5%)

Measurement of Renal Plasma Flow (RPF) (Fick Method) X Artery / min = (X Vein / min) + (X Ureter / min) RPF (ml/min) · [ X] Artery = (RPF (ml/min) · [X] Vein ) + (UF (ml/min) · [X] Ureter ) RPF = UF · U X (X Artery - X Vein ) RPF = UF · U X P X RPF = ERPF (C PAH ) 0.9 If X Vein = 0 then C PAH = UF · U PAH = ERPF P PAH Infuse a substance in a patient to achieve a steady plasma concentration

PAH in the Kidney RPF = 600 RPF = 475 GFR = 125 PAH = 1 RPF = 599 Peritubular Capillaries Salts & Water Reabsorbed /no PAH Reabsorbed/ PAH secreted into tubule Vasa Recta Capillaries Salts & Water Reabsorbed but no PAH Reabsorbed or Secreted Renal Artery Renal Vein Urine Flow = PAH = (All flows are in ml/min) (PAH = para-aminohippurate concentration in mg/dl) RPF = 415 RPF = 60 Glomerulus PAH = 1 PAH = 0 PAH = 0.1

Typical Clearance Values PAH (p-amino-hyppurate) 650 ml/min Creatinine130 ml/min Inulin125 ml/min Urea55 ml/min Na+2 ml/min H 2 O1 ml/min glucose0 ml/min Albumin0 ml/min

Blood Pressure Intrinsic: autoregulation 1. Myogenic 2. Tubuloglomerular feedback prostaglandins Extrinsic: nerves hormones Control of Renal Blood Flow

Renal Oxygen Consumption:

POP QUIZ Which of the following are true concerning renal filtration and blood flow (Answer True or False)? A. Glomerular capillary hydrostatic pressure is lower than that of muscle capillaries. B. GFR can be measured by the clearance of creatinine. C. Afferent arteriole dilation will decrease GFR. D. Sympathetic stimulation will decrease RBF and GFR. E. A decrease in blood pressure from 100 to 80 mmHg will result in a proportional decrease in RBF. F ? ? ? ? ? F F T T

Filtration Reabsorption - NFP + NFP - NFP Starling Forces Ion channels & Transporters Cell Membrane Permeability Concentration gradients Osmolality REABSORPTION

At the end of this section you should know: the mechanisms for an isotonic reabsorption of fluid in the PT. (i.e. permeability, active and passive transporters). the role of peritubular capillaries in PT reabsorption. how active Na+ transport provides the driving force for reabsorption. how inulin can be used as a marker for water reabsorption in the tubule. how glucose and a.a. are reabsorbed by secondary active transport. PROXIMAL TUBULE

General Reabsorption in the Tubule

1,500 g 6 g Renal Handling of Salt Filtration Excretion

PROXIMAL TUBULE

Proximal Tubule Reabsorption Early Late

Proximal Tubule Reabsorption

PROXIMAL TUBULE SUMMARY 2/3 of salts and water reabsorbed All glucose and a.a. reabsorbed Reabsorption is isotonic: PT Osmolality is isotonic at the beginning & the end

CONCENTRATION & DILUTION Loop of Henle & Collecting Duct Permeability and Solute Transport in the John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida College of Medicine

CONCENTRATION & DILUTION At the end of this section you should know: permeability aspects of the Loop of Henle, DT & CD. the importance of the high medullary interstitial osmolality. the reabsorption of Na+, Cl-, urea and water in the Loop, DT and CD. changes in osmolality along the tubule and actions of ADH on the CD.

Low permeability to solutes High permeability to water H2OH2O H2OH2O 1200 Thin descending limb of the loop of Henle

permeable to solutes H2OH2O H2OH2O Thin ascending limb of the loop of Henle low permeability to H 2 O NaCl Urea

Na + K+K+ 2 Cl - Na + K+K+ 2 Cl Thick Ascending limb of the loop of Henle (TAL) Diluting segment impermeable to H 2 O Special carriers co-transport ions from tubule to interstitium Na + K+K+ 2 Cl - Na + K+K+ 2 Cl -

Na + K+K+ 2 Cl - Na + K+K+ 2 Cl Distal Tubule Na + K+K+ 2 Cl - Na + K+K+ 2 Cl - Impermeable to H 2 O Special carriers co-transport ions from tubule to interstitium 60 NaClNaCl

Na + K+K+ 2 Cl - Na + K+K+ 2 Cl Na + K+K+ 2 Cl - Na + K+K+ 2 Cl - 60 NaClNaCl Cortical Collecting Duct 1200 H2OH2O H2OH2O H2OH2O H2OH2O Medullary Collecting Duct Variable permeability to H 2 O Regulated by Antidiuretic Hormone (ADH)

Antidiuretic Hormone (ADH) Arginine Vasopressin (AVP) Plasma Osmolality (osmorecptors)   Blood Pressure (baroreceptors) Kidney V 2 receptor  H 2 O reabsorption Collecting Duct Arterioles V 1 receptor Vasoconstriction

Collecting Duct ADH cAMP Lumen (hypotonic) Interstitium (hypertonic) V 2 H 2 O

Summary of ADH Actions on the Kidneys Increases permeability of entire Collecting Duct to Water. Increases permeability of Medullary CD to Urea. Decreases Vasa Recta blood flow. Increases expression of the Na/K/2Cl transporter in the TAL. ?Question? How can each of these effects increase the concentration of the urine?

Na + K+K+ 2 Cl - Na + K+K+ 2 Cl Na + K+K+ 2 Cl - Na + K+K+ 2 Cl - 60 NaClNaCl 1200 H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O

High medullary interstitial osmolality Reabsorption of Na + and Cl - in the thick ascending limb (diluting segment). Low flow through vasa recta. Counter current multiplication. Counter current exchange. Importance of urea.

300 - The Counter Current Multiplier H2OH2O H2OH2O

Counter Current Exchange (a function of the vasa recta capillaries) Na+, Cl- & Urea out Na+, Cl- & Urea in

A Simple Counter Current Exchanger Bath B  Bath A  Which bath will stay warm longer ? The tube of cold water carries heat away from the bath.

Loop Diuretics

Numbers in ( ) refer to the volume of filtrate in ml/min remaining to be reabsorbed

Renal Regulation of Special Substances Urea, Glucose, Phosphate, Sulfate, Water, Sodium, Potassium & Calcium John R. Dietz Physiology & Biophysics University of South Florida College of Medicine

Renal Regulation of Special Substances (Urea, Glucose, Phosphate & Sulfate) At the end of this section you should know: How urea is reabsorbed. Principles of secondary active transport and how it applies to carrier mediated secretion and reabsorption. Transport maximum and how it is calculated. Renal handling of glucose in diabetes.

Reabsorption of Urea 50% of Filtered Urea Reabsorbed Tubular Concentrations of Urea are in mmoles/L Maximal ADH H2OH2O H2OH2O H2OH2O H2OH2O

Reabsorption of Glucose in the Proximal Tubule LumenBasal Membrane Na + K+K+ Glucose Na + SGLT 1 GLUT 2

Glu Sodium Powered Secondary Active Transport Na + Lumen Cell Membrane Stanley J. Nazian, Ph..D., This slide was stolen without remorse from Na + Glu

Reabsorption of Glucose in the Proximal Tubule Lumen Capillary Na + K+K+ Glucose Na + SGLT 1 GLUT 2

Facilitated Diffusion Interstitium Cell Membrane Glu

Secondary Active Transport

Reabsorption of Glucose Excreted Filtered Reabsorbed Excreted = Filtered - Reabsorbed 2 3 1

Secretion of Weak Acids and Bases Excreted = Filtered + Secreted

Fanconi’s Syndrome hyperaminoaciduria glucoseuria hyperphosphaturia hypersulfaturia bicarbonaturia natriuresis severe water loss

POP QUIZ Which of the following are true concerning renal tubular absorption (Answer True or False)? A. Most of the filtrate is absorbed in the thick ascending limb of the loop of Henle. B. Fluid leaving the Proximal Tubule is iso-osmotic to plasma. C. Renal medulary hyperosmolality is dependent on mainly passive mechanisms. D. Most reabsorption in the renal tubules is directly or indirectly dependent on Na/K APTase. E. ADH increases water absorption in most parts of the renal tubule. F ? ? ? ? ? F F T T

ANGIOTENSIN II SYMPATHETICS ALDOSTERONE HEMODYNAMICS ANP PROTEIN OSMOTIC PRESSURE Regulation of Na+ Excretion John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida College of Medicine

At the end of this section you should know how each of the following affect Sodium reabsorption: Blood pressure GFR Aldosterone Angiotensin II atrial natriuretic peptide (ANP) and BNP Protein osmotic pressure Renal nerves (epinephrine or norepinephrine. Renal Regulation of Special Substances (Sodium and Chloride)

Sodium (Na) Natriuretic? Egyptian valley: Wadi El-Natron Natrium (Lt.) from Natrum (Gr.) from Egyptian - Natron increases sodium excretion

Regulation of Na+ Excretion Decreases in GFR decrease Na+ Excretion Changes in Filtration Pressure with decreased blood pressure. Changes in Permeability: (Mesangial Cell Contraction or Relaxation) + ANP, NO - AII, Endothelin, Norepi, Epi, ADH

Atrial Natriuretic Peptide (Factor) ANP (ANF) Decreases Na + Reabsorption Regulation of Na+ Excretion Medullary Collecting Duct Na + ANP (or BNP) cGMP Lumen Capillary Na + K + X

Electron Micrograph of Rat Atrium J. D. Jamieson; G. E. Palade The Journal of Cell Biology, Vol. 23, No. 1. (Oct., 1964), pp

Rat atrial extracts increase sodium excretion when injected into anesthetized rats Atrial Natriuretic Factor (ANF) Atrial Natriuretic Peptide (ANP) DeBold et al., Life Sci.1981

Volume Expansion Atrial Stretch Vasodilatation  Blood Pressure Increased Na + & H 2 O Excretion ANP atrial natriuretic peptide Actions mediated by cGMP

Similar Structures of the Natriuretic Peptides Family Atria Other tissues Brain Atria Ventricles CNS Vasculature

Dendroaspis angusticeps Green Mamba Dendroaspis natriuretic peptide (DNP)

SODIUM EXCRETION ATRIAL STRETCH ANP BLOOD VOLUME EXPANSION ALDOSTERONE BLOOD PRESSURE Actions of Atrial Natriuretic Peptide GFR Actions mediated by cGMP

Active ANP Neutral Endopeptidase Inhibitors Increase ANP Neutral Endopeptidase Neutral Endopepidase Inhibitor Inactive ANP

Sympathetic Increase Na+ Reabsorption Arterioles JG Cells Kf (Surface Area) Tubule PP1 Na + K + Lumen Capillary Na + P P Active APTase Inactive APTase Norepinephrine and Angiotensin II Ca 2+ /Calmodulin - Tubule Sympathetics (Norepi or Epi) increase the active Na/K APTase and thus increase Na reabsorption. AII has a similar effect but different receptor.

Angiotensin II Increases Na+ Reabsorption Angiotensin II constricts both arterioles but has more effect on efferent.  FF = GFR  RPF  Result: Protein Osmotic Pressure Increased in peritubular Capillaries  Aldosterone  Kf (surface area)  Proximal Reabsorption  Filtration Fraction (FF)

Regulation of Na+ Excretion Aldosterone Increases Na + Reabsorption Distal Tubule and Cortical Collecting Duct Lumen Capillary -70 mV Na + K + + K + K +

Decreased Protein Osmotic Pressure Increases Na+ Excretion (glomerulus)

Decreased Protein Osmotic Pressure Increases Na+ Excretion (Proximal Tubule)

Increased Na + Intake Results in Increased Blood Flow to Cortex Juxtamedullary Nephron Salt Sparing Cortical Nephron Salt Wasting

ANGIOTENSIN II SYMPATHETICS ALDOSTERONE HEMODYNAMICS ANP PROTEIN OSMOTIC PRESSURE Regulation of Na+ Excretion

Regulation of Water & K + Excretion John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida College of Medicine

Renal Regulation of Special Substances (Water and Potassium) At the end of this section you should know: How water excretion is controlled by ADH How water excretion is markedly affected by solute excretion (osmotic diuresis). Regulation of ADH secretion (review) that potassium excretion is controlled by secretion in the DT and cortical CD. how changes in plasma K+ or aldosterone affect K+ secretion. how potassium excretion is altered by diuretics.

Collecting Duct ADH cAMP Lumen (hypotonic) Interstitium (hypertonic) V 2 H 2 O

Primary control of H 2 O excretion by ADH. Osmotic diuresis The reabsorption of solutes is not dependent on the reabsorption of water. The reabsorption of water is dependent on the reabsorption of solutes. Increased solute excretion nearly always results in increased urine flow Regulation of Water Excretion

Osmotic Diuresis

Regulation of Water Excretion ADH effects urine flow (UF) by altering free water excretion. If Uosm > Posm = net H 2 O reabsorption (or negative free water excretion). If Uosm < Posm = net H 2 O excretion (or positive free water excretion). If the urine is isoosmotic to plasma then free water excretion = 0. Most factors (other than ADH) alter UF by changing solute excretion.

? You have a patient with hypertension who you have been treating with a loop diuretic for 1 month. His initial plasma [K+] was 4.5 mEq/L and has decreased to 3.0 mEq/L ? Why would a diuretic cause hypokalemia ? ? How much potassium has the patient lost ? ? How can your knowledge of physiological processes help you treat the hypoklemia ? Potassium

Volume (L) ECFICF 140 mmoles/L 4.5 mmoles/L  4000 mmoles  60 mmoles Potassium Intake = 100 mmoles/day Potassium Excretion = 100 mmoles/day 90% urine 10% feces

Regulation of Potassium Excretion 100% of the filtered K + is reabsorbed by the beginning of the distal tubule K + Reabsorption K + Secretion

Regulation of Potassium Excretion Aldosterone Increases K + Secretion & K + Stimulates K + Secretion Directly Distal Tubule and Cortical Collecting Duct Lumen Capillary -70 mV Na + K + K + + K +

? You have a patient with hypertension who you have been treating with a loop diuretic for 1 month. His initial plasma [K+] was 4.5 mEq/L and has decreased to 3.0 mEq/L ? Why would a diuretic cause hypokalemia ? ? How much potassium has the patient lost ? How can your knowledge of physiological processes help you treat the hypoklemia ? Potassium

Diuretics and Potassium ? Why would a diuretic cause hypokalemia ?

Renal Contribution to Calcium Homeostasis

Renal Regulation of Special Substances (Calcium, Phosphate, Magnesium) At the end of this section you should know: mechanisms controlling Ca2+ absorption. mechanisms controlling phosphate excretion. Mg2+ is absorbed in the TAL and excretion affected by diuretics. changes in calcium balance in renal failure.

Renal Contribution to Calcium Homeostasis 1. Calcium reabsorption. Parathyroid hormone (PTH) -increases reab. DT and CD. Vitamin D also increases reab. in DT. 2. Phosphate excretion. PTH-decreases reab. PT. 3. Active form of Vitamin D is made in the kidneys

Regulation of Calcium Reabsorption Early Distal Tubule Na + Cl - Na + K + Lumen Capillary AT P -ase Na + Ca mV PTH Ca ++ PTH PTH stimulates calcium entry from lumen to cell and calcium movement from cell to plasma exchanged for Na+. Thiazide Diuretics X

Regulation of Calcium Ca ++ Blood Parathyroid Gland PTH Ca ++ Vit. D 3  Ca ++ absorption Ca ++ PO 4 - Ca ++ PO 4 - PTH  Ca ++ and PO 4 - resorption PTH  Vit. D 3 Ca ++ reabsorption & PO 4 - excretion Ca ++ PO 4 -

Calcium handling in renal disease Vitamin D 3 Plasma Ca 2+ PTH Bone Plasma HPO 4 2- ? ? ? ? ?

Ca ++ Blood Calcium handling in renal disease Parathyroid Gland PTH Ca ++ PO 4 - Ca ++ Vit. D 3  Ca ++ absorption Ca ++ PO 4 - PTH  Ca ++ and PO 4 - resorption PTH  Vit. D 3 Ca ++ reabsorption & PO 4 - excretion Ca ++ PO 4 -

1. Reabsorbed primarily in the Thick Ascending Limb. 2. Reabsorption stimulated by: PTH Calcitonin ADH Hypomagnesemia 3. Excretion markedly increased by diuretics. Renal Contribution to Magnesium Homeostasis ?

Thick Ascending Limb of Loop of Henle Na + K + Lumen (+) Capillary Na + K + 2 Cl - K + Ca 2+ Mg 2+

Renal Hormones & Fluid Balance Review of everything John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida, College of Medicine

Renal Hormones & Fluid Balance At the end of this section you should know: Regulation of erythropoietin secretion. Regulation of renin secretion. Prostaglandins as renal vasodilators help regulate RBF. Mechanisms for monitoring body fluid volume and osmolality. Regulation of both salt and water intake and output. Physiologic changes to salt deficit or excess. Fluid balance changes to hypertension or hypotension.

RBC Production Oxygen Delivery { Hypoxia Ischemia Erythropoietin Kidney - Heme receptor (Peritubular Capillaries) Bone Marrow Erythroid Precursor Cells to Normoblasts

Renin-Angiotensin system Regulation of Blood Pressure and Sodium Output The American Heritage® Dictionary of the English Language

Stimulation of Renin Secretion  Blood pressure activates renal vascular receptor (baroreceptor) and  renin.  Blood pressure also  GFR and delivery of Cl - to Macula Densa in the distal tubule which  renin.  Blood pressure causes a reflex activation of renal sympathetic nerves which  renin. Juxtaglomerular Apparatus

Renin-Angiotensin-Aldosterone System

Emerging! ACE (angiotensin converting enzyme) Converts AI (10 aa) to AII (8 aa) Actions of AII vasoconstrictor releases aldosterone anti-natriuretic causes heart remodeling causes cardiac fibrosis ACE 2 Converts AII to A 1-7 (7 aa) Actions of A 1-7 vasodilator decreases aldosterone natriuretic prevents heart remodeling prevents cardiac fibrosis IMHO this will lead to new treatments for hypertension and CHF.

 Blood Pressure  Renin (Kidney)  Angiotensin I  Angiotensin II Angiotensinogen (Liver) Angiotensin Converting Enzyme (ACE)  Aldosterone  Na + Reabsorption  FF  ADH & Thirst (water reabsorption) Vasoconstriction 

Hypertension Ureteral Constriction Renal Artery Constriction

 Blood Pressure  Renal Prostaglandins  RBF Vasoconstriction (eg. AII and Norepi) Dilation RBF Infuse Angiotensin II into the renal artery 1 min.

Acute Renal Failure Pre-Renal Renal Post-Renal

CONTROL OF BODY SALT AND WATER What factors is the body trying to control and why? Where are the receptors? What are the effectors? What are the responses? ECF VOLUME ECF OSMOLALITY

Intake Output WATER

CONTROL OF WATER INTAKE Primarily by THIRST Stimuli: 1) Increased Osmolality 2) Decreased Arterial Pressure 3) Decreased Blood Volume 4) Angiotensin II WARNING: This is NOT an example of good bed-side manner

CONTROL OF WATER OUTPUT Primarily by ADH Stimuli: 1) Increased Osmolality 2) Decreased Arterial Pressure 3) Decreased Blood Volume 4) Angiotensin II 5) Trauma 6) Surgery 7) Drugs eg. opiates and anesthetics.

NaCl Intake Blood Volume Plasma Proteins Atrial PressureArterial Pressure GFR Regulation of Na + Excretion Na + Excretion ANP SecretionSympathetics Renin Angiotensin II Aldosterone ? ? ? ? ? ? ? ? ? ? ?

Puffy Face and Bird legs

CapillaryInterstitiumIntracellular Na mMNa mMNa + 10 mM K mM K mM Proteins 1.5 mM Proteins 0.1 mM Proteins 3 mM Osm. = mOsm/L 285 mOsm/L 285 mOsm/L Pc = 25 mmHg P = 0 mmHg  = 28 mmHg  = 3 mmHg Permeable to most solutes but imperm- eable to large proteins Impermeable to most solutes Comparison of the Three Major Compartments Colloid Osmotic Pressure, Protein Osmotic Pressure, Oncotic Pressure

Control of Water Intake & Output

Blood Volume Atrial Pressure Arterial Pressure GFR Hemorrhage Na + Excretion ANP Sympathetics Renin Angiotensin II Aldosterone ADH H 2 O Excretion ? ? ? ? ? ? ? ? ? ? ?

Congestive Heart Failure Clinical Correlation Dr. Bryan Bognar Internal Medicine

ACID-BASE BALANCE Regulation of Body pH 1)Chemical Buffering. 2) Respiratory Compensation. 3) Renal Compensation Why is pH regulated? How does a buffer work? CO 2 – Carbonic acid John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida College of Medicine

Patient: Found unconscious with no pulse & no respiration. Blood drawn for electrolytes and gases (While attempting CPR) pH = 7.02( )* O 2 = 30 mmHg(85-95)* CO 2 = 80 mmHg(35-45)* HCO 3 - = 19 mM(22-28)* Na + = 141 mEq/L( ) Cl - = 103 mEq/L( ) K + = 4.0 mEq/L( )

pH AcidBase

Acid-base Chemistry At the end of this section you should know: Basic buffer chemistry. How to use the Hen.-Hass. Equation. Buffers in plasma, RBCs and other cells. Three lines of defense against changes in pH. pH: norm = 7.40, range normal [H+] = 40 nM

Why do we regulate our pH? pH Normal = 7.40 ( ) Viable range = pH = 7.40 then [H+] = 40 nM = M pH = 7.20 then [H+] = 60 nM pH = 7.10 then [H+] = 80 nM

Henderson-Hasselbalch Equation: pH = pK + log HCO 3 - CO 2

Production of Acids by the Human Body: Volatile Acids: CO 2 + H 2 O  H 2 CO 3  HCO H + Non-Volatile Acids (fixed acids): H + + SO 4 2-

First Line of Defense: Fast Physicochemical Buffering. Strong acid + Buffer salt  Neutral salt + Weak acid H + + Cl - + Na + + HCO 3 -  Na + + Cl - + H 2 CO 3 H 2 CO 3  CO 2 + H 2 O HCl  H + + Cl - 100% HSO 4 -  H + + SO % H 2 CO 3  H + + HCO % Conjugate Base Dissociation of Acids

K = [H + ] [A - ] [HA] [H + ] = K [HA] [A - ] -log [H + ] = -log K - log [HA] [A - ] pH = pK + log __[A - ] __ [HA]

Titration Curves for the Bicarbonate and Phosphate Buffer Systems

pH = pK + log HCO 3 - = pK + log HPO 4 2- = pK + log Prot - pCO 2 H 2 PO 4 - H n Prot The Isohydric Principle: Special Characteristics of the Bicarbonate Buffer System: High [HCO 3 - ] [HCO 3 - ]/CO 2 = 24 : 1.2 or 20 : 1 Open System: Controlled pCO 2.

Henderson-Hasselbalch Equation: pH = pK + log [HCO 3 - ] [H 2 CO 3 ]  CO 2 + H 2 O pH = pK + log [HCO 3 - ] Dissolved CO 2 + H 2 CO : 1 pH = log 24 mMoles/L 0.03 x 40 mmHg*** pH = log 24 mMoles/L = log mMoles/L = 7.40

ADDITION OF A STRONG ACID (eg. HCl): First Line of Defense: The Henderson-Hasselbalch Equation Fast Chemical Buffering 12 H Cl Na HCO 3 -  24 Na Cl HCO H 2 CO 3  12 CO H 2 O pH = log 12 mmoles/L mmoles/L pH = log 12 mmoles/L 13 mmoles/L pH = 6.06

Alveolar ventilation increased further because of acidosis. pH = log 12 mmoles/L 0.75 mmoles/L (pCO 2 = 25mmHg) pH = 7.30 Second Line of Defense: Fast Respiratory Component. Alveolar ventilation increased to maintain pCO 2 at 40 mmHg (1.2 mmoles/L). pH = log 12 mmoles/L 1.2 mmoles/L pH = 7.10

Third Line of Defense: Slow Renal Compensation (H + secretion and HCO 3 - reabsorption) Over a period of days plasma HCO 3 - returns to normal. pH = log 24 mmoles/L 1.2 mmoles/L pH = 7.40

Acid Excretion John R. Dietz, Physiology & Biophysics University of South Florida, College of Medicine

Acid Excretion At the end of this section you should know: how H+ and HCO3- are formed in the renal tubular cells. the permeability of the tubule to H+ and HCO3- and how H+ is exchanged for Na+. the three substances that H+ combines with in the renal tubule. how physiological perturbations (e.g. CO2) affect H+ secretion, HCO3- reabsorption and NH4+ production. how H+ secretion is affected by aldosterone, K+ or in disease states.

Henderson-Hasselbalch Equation: pH = pK + log HCO 3 - CO 2 pH =

H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+

CO 2 + H 2 O H 2 CO 3 HCO H + HCO 3 - Cl - HCO 3 - Na + H+H+ ATPase H+H+ C.A. BloodCellLumen ATPase H+H+ K+K+ Renal Transport of HCO 3 - & H +

H + exchanged for Na + H + ATPase H + - K + ATPase Various Mechanisms of H+ Secretion

CO 2 + H 2 O H 2 CO 3 HCO H + Bicarbonate Reabsorption C.A. Filtered H 2 CO 3 CO 2 + H 2 O C.A. Primarily in Proximal Tubule - 0 % of Acid Excretion BloodCellLumen HCO 3 - Na + HCO 3 - Na + H+H+ H+H+

CO 2 + H 2 O H 2 CO 3 HCO H + Bicarbonate Reabsorption C.A. Filtered H 2 CO 3 CO 2 + H 2 O C.A. Primarily in Proximal Tubule - 0 % of Acid Excretion BloodCellLumen HCO 3 - Na + HCO 3 - Na + H+H+ H+H+

CO 2 + H 2 O H 2 CO 3 HCO H + Titratable Acid Excretion C.A. Na + Filtered H + + HPO 4 2- Primarily in Distal Tubule & CD - 33 % of Acid Excretion Na + + H 2 PO 4 - or H SO HPO 4 2- or SO 4 2- H+H+ BloodCellLumen HCO 3 - Cl - ATPase

CO 2 + H 2 O H 2 CO 3 HCO H + Ammonium Excretion C.A. H + + NH 3 Primarily in Distal Tubule & CD - 66 % of Acid Excretion NH 4 + H+H+ NH 3 produced in the cortex from glutamine NH 3 [H+] is 1000 X greater in the lumen than the cell HCO 3 - Cl - ATPase

H+ Secretion in the Nephron

Factors that Stimulate H+ Secretion Acidosis Metabolic or Respiratory Hypokalemia Aldosterone

Regulation of H+ Excretion CO 2 + H 2 O H 2 CO 3 HCO H + HCO 3 - Na + H+H+ C.A. BloodCellLumen ATPase

Renal Responses to a Metabolic Acidosis Decreased filtered bicarbonate Increased H + secretion Increased NH 3 production

H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ Increased Renal Acid Secretion in Acidosis

Long-term Renal Compensation for Acidosis

Respiratory Alkalosis

CO 2 + H 2 O H 2 CO 3 HCO H + HCO 3 - Na + H+H+ C.A. BloodCellLumen HCO 3 - Respiratory Alkalosis ATPase

H+H+ H+H+ H+H+ H+H+ H+H+ HCO 3 - Decreased Acid Secretion with Respiratory Alkalosis (Hyperventilation)

Renal Tubule Acidosis (RTAs) 1.Type 1 - Distal RTA 2.Type 2 - Proximal RTA 3.Type 4 - Lack of Aldosterone

Clinical Aspects of Acid-Base Balance pH = pK + log HCO 3 - CO 2 John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida College of Medicine

Clinical Aspects of Acid-Base Balance At the end of this section you should know: The physiological changes in the 4 primary acid base disturbances. How to read a Davenport diagram Compensation for acid-base disturbances. Causes of Acid-Base disturbances. Acid-Base Balance Disturbances

Buffering by Hemoglobin CO 2 HCO 3 - /H 2 CO 3 Solution (HCO 3 - = 24 mM) HCO 3 - /H 2 CO 3 + BUF - /HBUF (Hemoglobin) Remember: CO 2 + H 2 O  H 2 CO 3  H + + HCO 3 -

TRANSPORT OF CO 2 AND BUFFERING BY HEMOGLOBIN Most CO 2 is transported in the blood as HCO 3 -

pH = pK + log HCO 3 - CO 2 Normal Acid-Base Conditions

A RESPIRATORY ACIDOSIS SOME POSSIBLE CAUSES: 1) COPD, CHF and other lung diseases. 2) Anesthetics and Drugs  pH = pK + log HCO 3 - CO 2 

RESPIRATORY ALKALOSIS SOME POSSIBLE CAUSES: 1) Pain. 2) Hysteria. 3) Hypoxia.  pH = pK + log HCO 3 - CO 2  B

METABOLIC ACIDOSIS SOME POSSIBLE CAUSES: 1) Loss of Base (eg. diarrhea). 2) Gain of Acid (diabetes, renal failure).  pH = pK + log HCO - 3  CO 2 C

 pH = pK + log HCO - 3  CO 2 METABOLIC ALKALOSIS SOME POSSIBLE CAUSES: 1) Over Ingestion of Base. 2) Loss of Acid (Vomiting, Gastric Suction Tube). D

Anion Gap Plasma [Na + ] - ( [Cl - ] + [HCO 3 - ] ) or approximately 8-16 mEq/L (5-11 mEq/L) Metabolic Acidosis Normal anion gap acidosis - HCO 3 - decreases and replaced by Cl - shift: Eg. Diarrhea or simple gain of H + + Cl - Increased anion gap acidosis - HCO 3 - decreases and replaced by other anions so no Cl - shift: Eg. lactic acidosis and diabetic keto-acidosis

Something to remember for Years 2-4

Renal and Respiratory Compensatory Responses to Disturbances in Acid-Base Balance

AcidosispHAlkalosis Metabolic (Bicarbonate) Respiratory (CO 2 ) Willie’s Acid-Base Box Patient pH = 7.27 HCO 3 - = 25 CO 2 = 60 From William Lipscomb, Ph.D.

AcidosispHAlkalosis Metabolic (Bicarbonate) Respiratory (CO 2 ) Willie’s Acid-Base Box Patient pH = 7.34* HCO 3 - = 30* CO 2 = 60* From William Lipscomb, Ph.D. *After compensation

Patient: Found unconscious with no pulse & no respiration. Blood drawn for electrolytes and gases (While attempting CPR) pH = 7.02( )* O 2 = 30 mmHg(85-95)* CO 2 = 80 mmHg(35-45)* HCO 3 - = 19 mM(22-28)* Na + = 141 mEq/L( ) Cl - = 103 mEq/L( ) K + = 4.0 mEq/L( )

AcidosispHAlkalosis Metabolic (Bicarbonate) Respiratory (CO 2 ) Willie’s Acid-Base Box Patient pH = 7.02 HCO 3 - = 19 CO 2 = 80

Renal Failure Patient Patient Data (Plasma)Change from Normal K +  Phosphate  Calcium  Sulfate  Urea  Hematocrit  Blood Pressure  Bicarbonate  pH 

Physiology of the Kidneys Body Fluids & Acid Base Balance MDC John R. Dietz, Ph.D. Molecular Pharmacology & Physiology University of South Florida College of Medicine