Lecture 14- Transepithelial Bicarbonate Handling

Question 1

Proximal tubule - HCO3 reabsorption

- Plasma HCO3??

Answer 1

25mM

Question 2

How much fluid is filtered in the kidney per day ??

Answer 2

180 L

Question 3

How much HCO3 is filtered per day in the kidney ??

Answer 3

4.5 moles per day

Question 4

Of HCO3 filtered, what percentage is reabsorbed by the proximal tubule ??

Answer 4

= 300g NaHCo3

Question 5

What stimulates HCO3 absorption

Answer 5

Angiotensin II, Endothelin I, Noradrenaline and adenosine

-Linked to increases in Ca and protein kinase C.

Question 6

What downregulates HCO3 reabsorption ?

Answer 6

ANP, parathormone and dopamine – linked to cAMP/PKA pathway.

Question 7

HCO-3 Reabsorption in the PT

Answer 7

Apical step due to CO2 transport
Stoichiometry of 1-3 to get bicarbonate reabsorption
- see diagram
- Basolateral > CAII
Na and 3HCO-3 into the blood
- Apical > Na in H+ out
H+ out ATP in this H > HCO3- + H+ > H2O + CO2
Inside CO2 + H2O&raquo_space; 3HCO-3 and H+ recycled

Question 8

What is the key regulator of bicarbonate transport?

Answer 8

Control by a CO2 receptor- regulator of the pathway
PT detecting CO2 levels
Balance of HCO3/CO2

Question 9

Experiment see lecture- page 1 notes

Answer 9

Experiment above – out of equilibrium
Achieved only for short time
Passing through the mesh gives turbulent flow and causes mixing
????

Question 10

SEE EXPERIMENT
Taken this technique and applied to the PT-
Removing HCO3

Removing CO2

Calcium Changes

Answer 10

Removing HCO3 increased HCO3 reabsorption – maybe reduce backflux through tight junctions and increased exit gradient

Removing CO2 reduced HCO3 reabsorption – hypothesis CO2 receptor ‘sensing’ blood CO2 levels.

Calcium changes – Adding HCO3/CO2 to the lumen has no effect – addition to the bath increases Ca2+.

Question 11

Apical step of bicarbonate reabsorption

Answer 11

CO2 transport

Question 12

Identity of the CO2 receptor -

Answer 12

Possible target that could be the sensor for CO2 - protein tyrosine phosphatase gamma

Question 13

Identity of the CO2 receptor - EXPERIMENTS

Answer 13

Inhibitor of receptor protein kinase you abolish the link between CO2 levels and Bicarbonate transport

Question 14

Localisation experiments

Answer 14

Phosphatase present in the PT at basolateral membrane

Question 15

KO mouse Experiment-

Answer 15

condition 1 - Fixed ph and bicarbonate levels and changed CO2 - KO phosphatase inhibits bicarbonate transport
KO- loss of response to CO2, no longer sensitive to changes in basolateral bicarbonate but Wt is

Question 16

High CO2 in the blood

Answer 16

Acidosis

body compensates by increasing bicarbonate reabsorption

Question 17

General rule of absorption

Answer 17

Low co2 low bicarbonate reabsorption

Increase in CO2 increase in bicarbonate reabsorption

Question 18

structure of basolateral binding site of phosphatase

Answer 18

Homolgous to carbonic anhydrase

• potentially site for CO2.HCO3 to bind to
• lacks then enz activity of carbonic anhydrase

Question 19

Hypothesis

Answer 19

When the CO2 binds to the phosphatase it causes an inhibition of phosphatase activity so receptor protein kinase activity is up regulated

Question 20

Why is bicarbonate transport important

Answer 20

Regulation of pH

helps drive water reabsorption

Question 21

Microfusion techniques - Experiment

Using split-drop micropuncture techniques.

Answer 21

Exposed the PT of Rabbit kindey
injected an oil block into the PT to stop perfusion in the middle of oil drop they’ve put a drop of fluid which they can change conc within it
- measured volume of fluid drop
- PT absorbs fluid so droplet gets smaller vol is proportional to the water permeability

Question 22

Microfusion techniques - Experiment

Using split-drop micropuncture techniques. RESULTS

Answer 22

Droplet gets smaller as PT carrys out reabsorption – proportional to water flux aross the tubule
Remove bicarbonate and the rate of volume change goes down
No bicarbonate – water reabsorption in the PT significantly reduced

Question 23

NHE3 Exchanger

- KO mouse

Answer 23

Apical membrane
- secretes protons across the membrane
- NHE3 KO mice have a decreased arterial blood pressure
Decrease in Plasma PH and HCO3 – consistent with mild proximal tubular acidosis

Question 24

NHE3 KO mouse - microperfusion techniques

Answer 24

Using in vivo mircroperfusion techniques on the surface proximal tubules
Bicarbonate transport goes down
Waterflux goes down
Conclusion: NHE3 drives ~60% of HCO3 reabsorption and ~70% of H2O reabsorption in mouse proximal tubule.
70% driven by the proton exchanger – highlights it importance

Question 25

Pancreatic duct - Bicarbonate secretion

Answer 25

Apical- Cl/ HCo3 exchanger Cl channel Baslateral - Na/H exchanger CO2 in combines with H20 >> H2 CO3 (carbonic acid) which provides the H+ (recycled) for exchange and HCO3 to the apical membrane ** Model doesnt work in humans*** - Reach a point where the chloride bicarbonate exchanger shuts down as it reaches a critical point (electrochemical gradient)

Question 26

Modified Model-

Answer 26

Electrogenic sodium bicarbonate transporter - 1 Na to 2 bicarbonate - Bicarbonate exit pathway through a channel to help bicarbonate secretion - question is the cl channel on the apical membrane CFTR ??

Question 27

Experiment – is Bicarbonate transferred by CFTR in the pancreatic duct

Answer 27

Wt mouse and Ko mouse Isolated Pancreatic ducts Measured PH in the cell - link PH change to bicarbonate movement If bicarbonate moves into the cell it will bind to a proton and cause alkalisation Conditions – Cl- free, Hco3 – free and Dids inhibits NBS – cAMp to stimulate Cftr – BSAOLATERAL SIDE Change K – increase you get depolarisation Hyper Polarisation conditions bicarbonate moves out of the cell Depolarisation > acidification because bicarbonate moves out In KO change in membrane potential has no effect on pH inside the cell CONCLUSION – Bicarbonate movement facilitated by CFTR

Question 28

Pancreatic duct system –

Answer 28

Produce Nacl rich secretion Across the apical membrane Cl reabsorbed and effectively replaced by bicarbonate secretion Further down the duct the CL bicarbonate is inhibited and taken over by CFTR

Question 29

Interaction between SLC26A6 and CFTR

Answer 29

Rest – fairly inactive – regulatory domains bound PKA stimulation – Phosphorylation inihibition of CFTr release therefore acts a a CL/HCO3 channel At same time Interaction with the regulatory domain of SLC26A promotes Chloride bicarbonate exchange

Question 30

CFTR – mutations that affect HCO3 transport.

Answer 30

Depending on the CF mutation patients can be pancreatic sufficient or insufficient. Study of the transport properties of various CF mutations – some associated with pancreatic insufficiency.

Question 31

CFTR Bicarbonate mutations

Answer 31

Focus upon CFTR linked HCO3 transport. There are also links between anion exchange and CFTR. I148T – shows normal Cl transport – but HCO3 transport disrupted – linked to insufficiency. R117H – defect Cl transport but CFTR linked HCO3 transport still present – pancreatic sufficient. Stimulate with Forskolin

Question 32

CF and bicarbonate

Answer 32

In CF cases where there are problems associated with the pancreas there is a defect leading to a reduction in bicarbonate transport

Question 33

NBC- Sodium- Bicarbonate Cotransporter

Answer 33

characterised in salamander proximal tubule by Boron and Boulpaep First cloned by expression cloning methods from salamander proximal tubule. Isoforms isolated from human kidney and human pancreas. These clones are splice variants from the same gene

Question 34

NBC- Sodium- Bicarbonate Cotransporter findings

Answer 34

Difference between kNBC and pNBC is in the N terminal. The initial 41aa of kNBC are replaced by 85aa in pNBC. kNBC shows a stoichiometry of 1Na:3HCO3 whereas pNBC shows a stoichiometry of 1Na:2HCO3. Does the difference at the N terminal account for this property?

Question 35

Does the difference in the N terminals of the kidney and pancreatic isoforms account for the difference in stoichiometry ? Study set up

Answer 35

- used a PT cell line and CCD cell line - both lines were deficient in NBC activity - Cells were transfected with pNBC or kNBC - Cells mounted in Ussing Chambers and voltage clamped - Transepithelial Na gradient was varied and the reversal potential measured

Question 36

Does the difference in the N terminals of the kidney and pancreatic isoforms account for the difference in stoichiometry ? Results

Answer 36

Take pancreatic isoform pNBC and put in PT cell line Change conditions and measured reversal potential pNBC- has 1Na:3HCO3 stoichiometry pNBC in collecting duct cells – 1Na:2HCO3 stoichiometry of the transporter linked to cell type its expressed in same results were obtained for kNBC Looks like nothing to do with N terminals

Question 37

Role of Protein kinase phosphorylation in NBC Stoichiometry

Answer 37

pNBC- WT> before stimulation shows 1:3 stochiometry and after stimulation shifts to 1:2 S1026A ( serine to an alanine so mimic unphosphorylated state) > PKA site inactive 1:3 stochiometry with no change after PKA stimulation S1026D> Mimic PKA phophorylation. stimulation of PKA causes no shift but transporter is natively in the 1:2 stochiometry configuration * * Same results for kNBC * ** Stochiometry of NBC can be altered by phosphorylation of a PKA site

Question 38

Effect of Calcium on NBC activity

Answer 38

Experiement - using xenopus oocyte expressing NBC and Macropatch techniques - low Ca Stochiometry 1NA : 2HCO3 - High Ca Stochiometry 1Na: 3HCO3 increase in Ca causes a shift from 1:2 to 1:3 PKA sends it the other way