Acid-Base Module: “Ur-ine” for a great time! Kidney Treks 2013 Rotation D1 Marquita Kilgore, Robyn Levine, Adam Milam, Maya Ratnam, & Deepak Ravindranathan

Outline Background Introduction to Experiments Methods & Results Acid-Base Physiology Collecting Duct Physiology Introduction to Experiments Research Objectives Hypotheses Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis Conclusion Results Summary Future Direction

Outline Background Introduction to Experiments Methods & Results Acid-Base Physiology Collecting Duct Physiology Introduction to Experiments Research Objectives Hypotheses Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis Conclusion Results Summary Future Direction

pH pH is tightly regulated We can ingest large loads of alkali, acids, and yet our blood pH remains constant! How is that possible?

The Bicarbonate Buffer System H+ + HCO3- H2CO3 H2O + CO2

The Henderson Hasselbach Equation pH = pK + log10 Which for the bicarbonate system becomes: pH = 6.1 + log10 7.4 = 6.1 + log10 [Base] [Acid] [HCO3-] [H2CO3] We can use the bicarbonate and carbon dioxide concentrations to calculate the pH of plasma. This is done using the Henderson Haselbalch equation. As shown here, the normal bicarbonate concentration of 24 millimole per liter and carbon dioxide concentration of 1.2 millimoles per liter provide the normal pH value of 7.4. We should note that a 1.2 mmol solution of carbon dioxide has a partial pressure of 40 mmHg. (20)

Background pH is maintained at a constant range by modifying reabsorption of HCO3- and/or acid excretion The majority of HCO3- 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 Despite our

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 The kidneys role is to maintain homeostasis and avoid acidosis or alkalosis

Background After an acid load, the kidney will excrete H+ ions and resorb HCO3- 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 HCO3- to return to homeostasis The kidney will also vary the apical membrane expression and activity of proximal tubule Na+/H+ exchanger, renal ammoniagenesis and the expression of a proton pump (H+-ATPase) in the apical membrane of the intercalated cells of the collecting duct

Intercalated Cells of Collecting Duct K+ ATP H+ ATP Na+ K+ CO2 + H2O Carbonic anhydrase H+ HCO3- Cl- H+ H+ H+ Peritubular Capillary\ H+ ATP H+ ATP Na+ K+ CO2 + H2O Carbonic anhydrase HCO3- Cl-

Principal Cells of Collecting Duct Na+ ATP K+ ROMK/BK ENaC Principal Cells of Collecting Duct

Outline Background Introduction to Experiments Methods & Results Acid-Base Physiology Collecting Duct Physiology Introduction to Experiments Research Objectives Hypotheses Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis Conclusion Results Summary Future Direction

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.

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 CO2 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 CO2 availability will increase H+ pump rate.

Outline Background Introduction to Experiments Methods & Results Acid-Base Physiology Collecting Duct Physiology Introduction to Experiments Research Objectives Hypotheses Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis Conclusion Results Summary Future Direction

Experiment 1 Conditions Our goal was to alter serum bicarbonate by +/- 3mEq/L by adding either acid or base, while controlling with saline Condition 1: Ammonium Chloride (NH4Cl) Condition 2: Sodium Citrate (NaC6H5O7) Control: Saline (NaCl) Understand: saline neutral effect

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

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 In addition to the background previously given, there are 3 things you should understand for experiment 1’s methods. Bicarbonate: 24 mEq/L

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

Acid load decreases urine pH and basic load increases urine pH. Acidic Group n = 3 Basic Group n = 3 Control Group n = 2 Change in acidic group is less dramatic than basic group, Acidic group had pills most likely did not absorb as well as basic group liquid Varying lunch and dinner during urine analysis

Experiment 2 Design Monolayer (Bladder Tissue) Apical Basal Monolayer (Bladder Tissue) Epithelial sodium channel Electron transport Na Generated I due to ΔV= Rate of Na+ transport through ENaC I e- An Ussing chamber is used to measure the short-circuit current as an indicator of net ion transport taking place across an epithelium The Ussing chamber was created using the bladder of the turtle which is analogous to collecting duct in mammalians (monolayer)/ A voltage clamp (silver-silver chloride) was attached to the Ussing chamber which is displayed as the red line in the diagram. The voltage clamps allows for measurement of the short circuit (Sodium channel, ENaC and H-ATPase). The voltage clamp was turned off in order to measure the open circuit transepithelial potential. Current and voltage were taken in 5-minute time intervals. V Image created by IPE 2013 participant, Elizabeth Chen

Experiment 2 Design 1. Amiloride: blockage of ENaC e- Apical Basal Na To determine the current generated from the ENaC versus HATPase we attempted to inhibit Na entry. We added diuretic amiloride to the basolateral side and so no change in voltage or current. We then added diuretic amiloride to the apical membrane…the change is noted on the next slide. V Image created by IPE 2013 participant, Elizabeth Chen

Experiment 2 Design Apical Basal Generated I due to ΔV= Rate of H+ transport through H-ATPase HCO3- I e- We also added bicarbonate to apical side to increase PCO2. We examined the effect of the increase of PCO2 on voltage and current. V Image created by IPE 2013 participant, Elizabeth Chen

Experiment 2 Results JNa + JH+ = Isc Time (minutes) Amiloride added To determine the current generated from the ENaC versus HATPase we inhibited Na entry across the apical membrane via the addition of the diuretic amiloride. When we block the ENaC channel we inhibit Na from crossing from apical border to basolateral border. We see an increase in current because of the unopposed H-ATPase current. Increasing PCO2 (by addition of bicarbonate) will increase current of H+. Time (minutes)

Experiment 2 Results Addition of Bicarbonate Time (minutes)

Outline Background Introduction to Experiments Methods & Results Acid-Base Physiology Collecting Duct Physiology Introduction to Experiments Research Objectives Hypotheses Methods & Results Experiment 1: Human Analysis Experiment 2: Turtle Analysis Conclusion Results Summary Future Direction

Results Summary Experiment 1 The kidney adapts to acid- and base-loading by modifying the reabsorption of filtered HCO3- 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 CO2 availability leads to increased H+ pump activity

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

Acknowledgements Drs. John Schwartz & Mark Zeidel Drs. Martina McGrath, Bryce MacIver, & Finnian McCausland American Society of Nephrology

Questions?

Experiment 2 – Turtle Experiment Sample 1 Sample 2 Time (mins) uA mV -19 12 -81 33 3 -20 14 -77 35 6 -67 40 9 -17 -53 -15 -40 38 15 -13 11 -32 34 Amiloride Added 18 -10 8 -2 21 -8 7 2 -6 24 -5 4 27 -3 -9 30 Bicarbonate Added -27 36 17 -39 39 20 -44 42 -48 10 45 23 -50 13 To determine the current generated from the ENaC versus HATPase we inhibited Na entry across the apical membrane via the addition of the diuretic amiloride.