Lecture 12 Bicarbonate Handling

Question 1

What is the average physiological plasma bicarbonate concentration and pH

Answer 1

25mM with a pH of 7.4

Question 2

What volume of plasma is filtered by the kidney per day

Answer 2

180L

Question 3

If the amount of HCO3- filtered per day is around 4.5moles and 80% of this is reabsorbed how many grams of NaHCO3 is reabsorbed

Answer 3

Mr = 84 n = 4.5 m = 84*4.5 m = 378 378*0.8 = reabsorbed reabsorbed = 302.4g

Question 4

The apical step of HCO3- reabsorption involves what transport

Answer 4

CO2 transport

Question 5

Which region of the nephron is responsible for reabsorbing the majority of HCO3-

Answer 5

Proximal tubule accounts for 90%

Question 6

Describe the cell model for HCO3- handling in the PCT

Answer 6

The sodium hydrogen exchange protein (NHE3) on the apical membrane of PCT cell uses Na+ influx as a driving force to pump H+ out into the tubular fluid against concentration gradient. This H+ removed combines with HCO3- to form carbonic acid H2CO3 in a reaction catalysed by carbonic anhydrase IV (CAIV). The H2CO3 formed then dissociates into H2O and CO2 and these then move into the cell via AQP1 in the case of H2O and diffusion for CO2. Once inside the cell they recombine to reform H2CO3 a reaction catalysed by intracellular carbonic anhydrase II (CAII). This H2CO3 then dissociates again back into H+ and HCO3-. The H+ is recycled across the apical membrane through the action of NHE3. Meanwhile HCO3- is reabsorbed across basolateral membrane by a sodium bicarbonate cotransporter (SLC4A4/NBC). This bicarbonate efflux from the cell at the basolateral membrane also drives Na+ out

Question 7

What isoform of carbonic anhydrase catalyses the formation of H2CO3 in the lumen of the PCT

Answer 7

Carbonic anhydrase IV

Question 8

Which enzyme catalyses the formation of H2CO3 inside the cells of the PCT

Answer 8

Carbonic anhydrase II

Question 9

Complete the cell model below with the species being transported

Answer 9

See completed diagram

Question 10

Describe the differences in the stoichiometry of SLCA4/NBC and the effect of this on HCO3- handling

Answer 10

Under basal conditions in some tissues 3 HCO3- molecules are reabsorbed by NBC for each 1 Na+ reabsorbed hence NBC has a stoichiometry of 1:3 (Na+:HCO3-). However NBC has another configuration with a stoichiometry of 1 Na+ to 2 HCO3-. This configuration of the transporter actually moves NaHCO3 into the cell as opposed to out

Question 11

Angiotensin II Endothelin I Noradrenaline and adenosine all stimulate HCO3- reabsorption. What mechanism is thought to mediate this

Answer 11

Linked to increases in Ca2+ and PKC activity

Question 12

Which factors act to decrease HCO3- reabsorption by increasing cAMP and activating PKA

Answer 12

ANP parathormone and dopamine

Question 13

What two out of equilibrium solutions were used to determine if there was a CO2/HCO3- receptor responsible for regulating HCO3- secretion

Answer 13

The first out of equilibrium solution had a physiological pH physiological HCO3- but no CO2. The other solution had a physiological pH physiological CO2 but no HCO3-

Question 14

What the results below show are the effects of perfusing the basolateral membrane of dissected PCT cells with either of the two out of equilibrium solutions

Answer 14

Perfusing the basolateral membrane with a non-physiological solution where HCO3- had been removed increased the HCO3- reabsorption from the apical or luminal solution to 100pmol/min/mm. In contrast removing CO2 from the basolateral membrane solution reduced HCO3- reabsorption to 600pmol/min/mm.

Question 15

What has the hypothesis that aimed to explain why changing the concentration of HCO3- in the basolateral perfusate changed the HCO3- reabsorption by the PCT

Answer 15

Hypothesis was that a CO2 receptor sensing blood CO2 levels and changing HCO3- reabsorption depending on the level of CO2

Question 16

What was the evidence for the role of Ca2+ in a potential response of the basolateral membrane to changes in CO2 levels

Answer 16

Addition of CO2 and HCO3- to the lumen had no effect on Ca2+ levels indicated by a FURA-2 dye. However addition of CO2 and HCO3- to the basolateral membrane results in a Ca2+ signal. Specifically the addition of only CO2 at a physiological pH to the basolateral membrane was also sufficient to cause a Ca2+ flux

Question 17

What does the fact that there is a Ca2+ signal upon changing the concentration of CO2 in the basolateral membrane perfusate of PCT cell tell us about a potential receptor

Answer 17

There is likely to be a basolateral CO2 receptor are to intracellular calcium

Question 18

Protein tyrosine phosphatase γ was the receptor thought to be sensing changes in basolateral CO2 levels in the PCT. What sequence alignment data backed up this hypothesis

Answer 18

RPTPγ had a 30-35% homology to the binding site of carbonic anhydrase which is known to bind CO2

Question 19

What evidence from animal models suggests that RPTPγ is the receptor involved in changing HCO3- reabsorption in response to differences in basolateral CO2/HCO3-

Answer 19

In response to [CO2] increases on the basolateral side HCO3- reabsorption is stimulated in wild type mice. However in RPTPγ knockout mice as [CO2] increases on the basolateral side there is no change in HCO3- reabsorption. Similarly in wild type mice as [HCO3-] increases on the basolateral side HCO3- reabsorption is reduced. Whereas in the RPTPγ knockouts as [HCO3-] increases on the basolateral side there is no change in HCO3- reabsorption. This indicates that RPTPγ is required to change HCO3- reabsorption in response to change in the levels of CO2 and HCO3- at the basolateral membrane

Question 20

Why is HCO3- reabsorption important

Answer 20

HCO3- reabsorption is vital in maintaining blood plasma pH but it also seems to be driving a lot of H2O reabsorption

Question 21

What evidence is there for a role of HCO3- in H2O reabsorption

Answer 21

In the absence of bicarbonate water reabsorption by the PCT is greatly reduced

Question 22

What is the contribution of NHE3 in reabsorption in the PCT

Answer 22

NHE3 drives 60% of HCO3- reabsorption and 70 of H2O reabsorption

Question 23

What is the evidence from animal models for the role of NHE3 in driving H2O and HCO3- reabsorption

Answer 23

NHE3 knockout mice have a decreased arterial blood pressure. In addition they also have a decrease in plasma pH and HCO3 consistent with mild proximal tubular acidosis. This is due to a huge reduction in HCO3- reabsorption and an even greater reduction of PCT fluid reabsorption

Question 24

Below is a diagram of the pancreatic duct cell model. Outline what is going on apart from the region in the red box

Answer 24

CO2 is taken up across basolateral membrane where once inside it combines with H2O to form H2CO3. This H2CO3 then dissociates back into H+ and HCO3-. The H+ is recycled across the basolateral membrane through the action of NHE3. Meanwhile HCO3- is transported across the apical membrane by an anion exchanger most likely SLC26A6. Here HCO3- moves out for Cl- coming in. Cl- then recycles through an apical Cl- channel to keep the process going

Question 25

What is the significance of NBC in the basolateral membrane of the pancreatic duct cells

Answer 25

in humans pancreatic luminal fluid can have [HCO3-] as high as 140mM. This means that SLC26A6 is unable to drive more HCO3- secretion alone and hence requires additional transporters to provide this extra HCO3- efflux. The basolateral NBC is in the 1:2 configuration and is hence electrogenic. It acts to bring 2HCO3- and 1Na+ into the cell which acts to raise intracellular [HCO3-] so as to drive additional efflux.

Question 26

The electrogenic NBC in the pancreatic duct cells can drive HCO3- secretion up to a luminal HCO3- concentration of 100mM. What protein mediates the remaining HCO3- efflux and how

Answer 26

The additional driving force for HCO3- is through CFTR. CFTR is permeable to HCO3- in a 1:4 ratio of HCO3- to Cl- so that when Cl- levels drop to low concentrations CFTR can secrete HCO3-. This additional driving force by CFTR can drive HCO3- secretion upto 140mM

Question 27

What is the effect of raising extracellular K+ concentrations on the membrane potential of pancreatic duct cells

Answer 27

Causes depolarisation

Question 28

What is the effect of decreasing extracellular K+ concentrations on the membrane potential of pancreatic duct cells

Answer 28

Causes hyperpolarisation

Question 29

How can you investigate HCO3- transport in the pancreas cells through CFTR experimentally

Answer 29

Perfuse the apical and basolateral membranes of pancreas cells expressing CFTR with a solution devoid of Cl- but that contains HCO3-. Then stimulate CFTR with cAMP as there is no Cl- in either perfusates CFTR will only be able to transport HCO3-

Question 30

What does the data below show about the effect of changing basolateral K+ concentrations on intracellular pH how is this thought to be mediated by HCO3- efflux through CFTR

Answer 30

Dropping extracellular K+ concentrations causes an acidification of the cells. This is consistent with HCO3- moving out of the cell through CFTR as a result of the hyperpolarisation that would be caused by decreasing extracellular K+. Conversely raising extracellular K+ concentrations causes an alkalinisation of the cells. This is consistent with HCO3- moving into the cells through CFTR as a result of the depolarisation that would be caused by increasing extracellular K+. These changes must be due to HCO3- movement through CFTR as when a non-functional mutant form of the CFTR protein is expressed instead there are no changes in pH following raising or lowering extracellular K+

Question 31

How does the driving force for HCO3- secretion in the pancreatic duct change along its length

Answer 31

As you move along the pancreatic duct HCO3- secretion goes from being mediated by SLC26A6 to being mediated by CFTR to produce this HCO3- rich fluid

Question 32

Describe the changes seen in HCO3- secretion by the pancreas before and after eating a meal

Answer 32

Under baseline conditions you want to minimise HCO3- and Cl- secretion as you don’t need a HCO3- rich fluid to buffer the acidic conditions during digestion. Hence HCO3- secretion is only stimulated after a meal to aid the digestive process

Question 33

Outline the interaction between CFTR and SLC26A6

Answer 33

The R domain of CFTR interacts with the NBD domains to limit Cl- movement under rest conditions. Following activation of PKA there is a phosphorylation of the R domain of CFTR which alleviates its inhibition of the NBD domain. This results in an alleviation of Cl- transport inhibition by CFTR. The R domain of CFTR then interacts with SLC26A6 to stimulate HCO3- secretion

Question 34

How can CFTR mutations result in pancreatic insufficiency

Answer 34

If a mutation in CFTR prevents the interaction of the R domain of the protein with SLC26A6 then it may be unable to trigger HCO3- secretion. It is this loss of HCO3- secretion by the pancreas that leads to pancreatic insufficiency

Question 35

What are the two classes of mutations in CFTR based on their effects on the pancreas

Answer 35

Pancreatic insufficiency mutations in CFTR can show normal Cl- transport although this is often defective but have disrupted HCO3- transport in the pancreas. Conversely pancreatic sufficient mutations will have defective Cl- transport but CFTR linked HCO3- transport is still seen.

Question 36

What causes pancreatic insufficiency in CF

Answer 36

Loss of HCO3- secretion by CFTR in the pancreatic duct

Question 37

Give an example of a pancreatic insufficient CFTR mutation where Cl- transport is normal

Answer 37

I148T

Question 38

Give an example of a pancreatic sufficient CFTR mutation where Cl- transport is abnormal

Answer 38

R117H

Question 39

What is significant about the kidney and pancreatic Na+/HCO3- cotransporters

Answer 39

They are splice variant isoforms of the same gene

Question 40

What is the main difference between the kidney and pancreatic isoforms of NBC

Answer 40

The difference between kNBC and pNBC is in the N terminus. The initial 41 amino acids of kNBC are replaced by 85 amino acids in pNBC

Question 41

What is different about the stoichiometry of pNBC and kNBC

Answer 41

kNBC shows a stoichiometry of 1Na:3HCO3- and tends to be involved in HCO3- reabsorption whereas pNBC shows a stoichiometry of 1Na:2HCO3- and is involved in HCO3- secretion

Question 42

What evidence shows that the differences in the N terminus of kNBC and pNBC do not account for the differences in stoichiometry

Answer 42

Expressing pNBC in a proximal cell line causes a switch in stoichiometry to 1Na+:3HCO3-. But expressing pNBC in a collecting duct cell line results in a stoichiometry of 1Na+:2HCO3- this is the same stoichiometry it shows in its endogenous cells of the pancreas. These stoichiometry’s are also obtained from expressing kNBC in those cell lines. Hence it is not the difference in isoform itself but more likely due to the cells it is expressed in and the environment/conditions in that cell

Question 43

What evidence is there to show the role of PKC in switching NBCs stoichiometry

Answer 43

Before stimulation of PKA by adding db-cAMP the stoichiometry of NBC is 1:3. However after addition of db-cAMP the stoichiometry of NBC switches to 1:2

Question 44

How does PKA phosphorylation of NBC effect the function of NBC

Answer 44

Phosphorylation of NBC by PKA switches its stoichiometry to 1:2

Question 45

How do ANP parathormone and dopamine downregulate HCO3- secretion

Answer 45

These factors act to increase cAMP levels inside the cell. Increases in cAMP lead to an activation of PKA which in turn phosphorylates NBC. This changes the stoichiometry of NBC to 1:2 switching the cotransporter to uptake HCO3- and Na+ rather than secrete them

Question 46

Which residue in pancreatic NBC is phosphorylated by PKA to cause the stoichiometric switch

Answer 46

S1026 is phosphorylated by PKA to cause it to secrete HCO3-

Question 47

What is the result of the S1026A mutation in pNBC

Answer 47

S1026A switches a serine in the PKA phosphorylation site to an alanine. This renders the PKA site inactive and hence pNBC remains in a 1:3 stoichiometry even after PKA stimulation

Question 48

What is the result of the S1026D mutation in pNBC

Answer 48

S1026D switches a serine in the PKA phosphorylate site to aspartate. Aspartate (D) at this position mimics phosphorylation as it is negatively charged. This means that the pNBC transporter is natively in the 1:2 configuration and as such stimulation of PKA causes no change in stoichiometry

Question 49

What is the effect of PKA phosphorylation of kNBC at S982

Answer 49

S982 in kNBC is equivalent to S1026 in pNBC. Hence phosphorylation of this residues by PKA switches the stoichiometry of the transporter to 1Na+:2HCO3-

Question 50

What is the effect of Ca2+ levels on HCO3- reabsorption how is this mediated

Answer 50

Increases in Ca2+ stimulates HCO3- reabsorption and reverses the effects of PKA. The increase in Ca2+ shifts the stoichiometry from 1:2 to 1:3