Lecture 14- Transepithelial Bicarbonate Handling Flashcards
Proximal tubule - HCO3 reabsorption
- Plasma HCO3??
25mM
How much fluid is filtered in the kidney per day ??
180 L
How much HCO3 is filtered per day in the kidney ??
4.5 moles per day
Of HCO3 filtered, what percentage is reabsorbed by the proximal tubule ??
= 300g NaHCo3
What stimulates HCO3 absorption
Angiotensin II, Endothelin I, Noradrenaline and adenosine
-Linked to increases in Ca and protein kinase C.
What downregulates HCO3 reabsorption ?
ANP, parathormone and dopamine – linked to cAMP/PKA pathway.
HCO-3 Reabsorption in the PT
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»_space; 3HCO-3 and H+ recycled
What is the key regulator of bicarbonate transport?
Control by a CO2 receptor- regulator of the pathway
PT detecting CO2 levels
Balance of HCO3/CO2
Experiment see lecture- page 1 notes
Experiment above – out of equilibrium
Achieved only for short time
Passing through the mesh gives turbulent flow and causes mixing
????
SEE EXPERIMENT
Taken this technique and applied to the PT-
Removing HCO3
Removing CO2
Calcium Changes
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+.
Apical step of bicarbonate reabsorption
CO2 transport
Identity of the CO2 receptor -
Possible target that could be the sensor for CO2 - protein tyrosine phosphatase gamma
Identity of the CO2 receptor - EXPERIMENTS
Inhibitor of receptor protein kinase you abolish the link between CO2 levels and Bicarbonate transport
Localisation experiments
Phosphatase present in the PT at basolateral membrane
KO mouse Experiment-
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
High CO2 in the blood
Acidosis
body compensates by increasing bicarbonate reabsorption
General rule of absorption
Low co2 low bicarbonate reabsorption
Increase in CO2 increase in bicarbonate reabsorption
structure of basolateral binding site of phosphatase
Homolgous to carbonic anhydrase
- potentially site for CO2.HCO3 to bind to
- lacks then enz activity of carbonic anhydrase
Hypothesis
When the CO2 binds to the phosphatase it causes an inhibition of phosphatase activity so receptor protein kinase activity is up regulated
Why is bicarbonate transport important
Regulation of pH
helps drive water reabsorption
Microfusion techniques - Experiment
Using split-drop micropuncture techniques.
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
Microfusion techniques - Experiment
Using split-drop micropuncture techniques. RESULTS
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
NHE3 Exchanger
- KO mouse
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
NHE3 KO mouse - microperfusion techniques
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
Pancreatic duct - Bicarbonate secretion
Apical-
Cl/ HCo3 exchanger
Cl channel
Baslateral -
Na/H exchanger
CO2 in combines with H20»_space; 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)
Modified Model-
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 ??
Experiment – is Bicarbonate transferred by CFTR in the pancreatic duct
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
Pancreatic duct system –
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
Interaction between SLC26A6 and CFTR
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
CFTR – mutations that affect HCO3 transport.
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.
CFTR Bicarbonate mutations
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
CF and bicarbonate
In CF cases where there are problems associated with the pancreas there is a defect leading to a reduction in bicarbonate transport
NBC- Sodium- Bicarbonate Cotransporter
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
NBC- Sodium- Bicarbonate Cotransporter findings
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?
Does the difference in the N terminals of the kidney and pancreatic isoforms account for the difference in stoichiometry ?
Study set up
- 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
Does the difference in the N terminals of the kidney and pancreatic isoforms account for the difference in stoichiometry ?
Results
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
Role of Protein kinase phosphorylation in NBC Stoichiometry
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
Effect of Calcium on NBC activity
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
