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

2. 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

3. 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

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?

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

6. The Henderson Hasselbach Equation
pH = pK + log10
Which for the bicarbonate system becomes:
pH = log10
= 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 We should note that a 1.2 mmol solution of carbon dioxide has a partial pressure of 40 mmHg.
(20)

7. 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

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

9. 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

10. Intercalated Cells of Collecting DuctK+
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-

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

12. 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

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.

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 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.

15. 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

16. 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

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

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

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

20. 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

21. 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

22. 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

23. 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

24. 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)

25. Experiment 2 Results
Addition of Bicarbonate
Time (minutes)

26. 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

27. 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

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

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

30. Questions?

31. 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.