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Kidney Treks 2013 Rotation D1 Marquita Kilgore, Robyn Levine, Adam Milam, Maya Ratnam, & Deepak Ravindranathan Acid-Base Module: Ur-ine for a great time! Slide 2 Outline I. Background Acid-Base Physiology Collecting Duct Physiology II. Introduction to Experiments Research Objectives Hypotheses III. Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis IV. Conclusion Results Summary Future Direction Slide 3 Outline I. Background Acid-Base Physiology Collecting Duct Physiology II. Introduction to Experiments Research Objectives Hypotheses III. Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis IV. Conclusion Results Summary Future Direction Slide 4 pH pH is tightly regulated We can ingest large loads of alkali, acids, and yet our blood pH remains constant! How is that possible? Slide 5 H + + HCO 3 - H 2 CO 3 H 2 O + CO 2 The Bicarbonate Buffer System Slide 6 pH= pK + log 10 Which for the bicarbonate system becomes: pH= 6.1 + log 10 7.4 = 6.1 + log 10 [Base] [Acid] [HCO 3 - ] [H 2 CO 3 ] The Henderson Hasselbach Equation (20) Slide 7 Background pH is maintained at a constant range by modifying reabsorption of HCO 3 - and/or acid excretion The majority of HCO 3 - reabsorption occurs in the proximal convoluted tubule, but approximately 10% of reabsorption occurs in the collecting duct Acid-Base regulation mainly occurs in the collecting duct Slide 8 Background Metabolic acidosis- occurs when the body produces too much acid or the kidney is not able to excrete acid Metabolic alkalosis- occurs when rise in pH of tissue is due primarily to bicarbonate Slide 9 Background After an acid load, the kidney will excrete H+ ions and resorb HCO 3 - in order to return to homeostasis H + ions may be excreted in the form of ammonium or other acids After a basic load, the kidney will excrete HCO 3 - to return to homeostasis Slide 10 Peritubular Capillary\ CO 2 + H 2 O Carbonic anhydrase K+K+ ATP H+H+ HCO 3 - Cl - ATP Na + K+K+ ATP H+H+ Na + K+K+ HCO 3 - Cl - CO 2 + H 2 O Carbonic anhydrase H+H+ H+H+ H+H+ H+H+ H+H+ Intercalated Cells of Collecting Duct Slide 11 Principal Cells of Collecting Duct Na + ATP K+K+ K+K+ ROMK/BK Na + ATP K+K+ Na + ENaC K+K+ ROMK/BK Slide 12 Outline I. Background Acid-Base Physiology Collecting Duct Physiology II. Introduction to Experiments Research Objectives Hypotheses III. Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis IV. Conclusion Results Summary Future Direction Slide 13 Experiment 1: Human Acid-Base Regulation Objective Examine the effect of acid- and base-loading on HCO3 - reabsorption/secretion and net acid excretion in human in vivo studies. Hypothesis Acid-loading will decrease urine pH, whereas base-loading will increase urine pH. Slide 14 Experiment 2: H + Transport by Turtle Urinary Bladder Objectives Examine the electrical nature of H + secretion. Examine the dynamics of Na+ and H+ ions flowing across the epithelial membrane. Examine how changes in CO 2 availability affect pump rate. Hypotheses H + secretion is electrogenic. Blocking ENaC channels will inhibit Na + flow and measured current will result from H + flow. Increasing CO 2 availability will increase H + pump rate. Slide 15 Outline I. Background Acid-Base Physiology Collecting Duct Physiology II. Introduction to Experiments Research Objectives Hypotheses III. Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis IV. Conclusion Results Summary Future Direction Slide 16 Experiment 1 Conditions Condition 1: Ammonium Chloride (NH4Cl) Condition 2: Sodium Citrate (NaC6H5O7) Control: Saline (NaCl) Our goal was to alter serum bicarbonate by + / - 3mEq/L by adding either acid or base, while controlling with saline Slide 17 Experiment 1 Design 1 Urine Sample #1: Morning Baseline Void Treatment w/ acid, base, or saline 2 Urine Sample #2-4 at 2 hour intervals post-treatment (i.e., 2 hr, 4 hr, 6hr) 3 pH Measurement Group Analysis Slide 18 Key Factors in Treatment Calculations Total Body Water (TBW) Female: 50% Total Body Weight Male: 60% Total Body Weight Assume bicarbonate ions flow between all compartments Bicarbonate: 24 mEq/L Slide 19 Sample Treatment Calculation 110 lb Female in acidic group ~50 kg & 50% body water (50 kg)(1 L/kg)(0.50) = 25 L body water (24 mEq/L HCO3-)(25 L) = 600 mEq HCO3- (21 mEq/L HCO3-)(25 L) = 525 mEq HCO3- 600 525 = 75 mEq HCO3- (75 mEq HCO3-)(53 mg/ml NH4Cl) = 3869 mg NH4Cl 3869 mg/500 mg = 8 tablets of NH4Cl Slide 20 Acid load decreases urine pH and basic load increases urine pH. Acidic Group n = 3 Basic Group n = 3 Control Group n = 2 Slide 21 Experiment 2 Design 1. Monolayer (Bladder Tissue) 2. Epithelial sodium channel 3. Electron transport ApicalBasal Na I e-e- V Generated I due to V= Rate of Na + transport through ENaC Image created by IPE 2013 participant, Elizabeth Chen Slide 22 Experiment 2 Design 1. Amiloride: blockage of ENaC ApicalBasal Na I e-e- V Image created by IPE 2013 participant, Elizabeth Chen Slide 23 Experiment 2 Design ApicalBasal HCO3 - I e-e- V Generated I due to V= Rate of H + transport through H- ATPase Image created by IPE 2013 participant, Elizabeth Chen Slide 24 Experiment 2 Results Time (minutes) Amiloride added J Na + J H+ = I sc Slide 25 Experiment 2 Results Time (minutes) Addition of Bicarbonate Slide 26 Outline I. Background Acid-Base Physiology Collecting Duct Physiology II. Introduction to Experiments Research Objectives Hypotheses III. Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis IV. Conclusion Results Summary Future Direction Slide 27 Results Summary Experiment 1 The kidney adapts to acid- and base-loading by modifying the reabsorption of filtered HCO 3 - and/or the quantity of net acid excreted. Experiment 2 Acid-secreting intercalated cells of the collecting duct generate an electrical gradient. When ENaC is blocked, current flow continues in the opposite direction Increased CO 2 availability leads to increased H + pump activity Slide 28 Looking ahead Measure NH4+ concentration in urine Assess H+ secretion by a specific primary active transporter (pH, bafilomycin) Assess H+ transport at steep pH gradients Examine how certain antifungal antibiotics induce renal tubular acidosis Slide 29 Acknowledgements Drs. John Schwartz & Mark Zeidel Drs. Martina McGrath, Bryce MacIver, & Finnian McCausland American Society of Nephrology Slide 30 Questions? Slide 31 Experiment 2 Turtle Experiment Sample 1Sample 2 Time (mins) uAmVuAmV 0-1912 -8133 3-2014 -7735 6-1914 -6740 9-1714 -5340 12-1512 -4038 15-1311 -3234 Amiloride Added 18-108 0-2 21-87 2-6 24-54 3-8 27-32 4-9 3000 4-10 Bicarbonate Added 332-2 11-27 363-3 17-39 3920-44 7-6 4221-48 10-10 4523-50 13