diuretics

Question 1

NCC

Answer 1

thiazides

Question 2

KCC

Answer 2

stilbenes
- block potassium chloride transport in the nephron

Question 3

NKCC

Answer 3

loop diuretics but also block KCC
- NKCC was found to be a new transporter, about 40 years ago that’s in the loop of henle.

When there have been advances in molecular techniques, we now know that through cloning experiments that there isn’t just one type of NKCC.
- There’s actually two so we’ve got two distinct isoforms that fall from different genes.

Question 4

how did they try distinguish between NKCC and then the 2 separate transporters of NCC and KCC

Answer 4

drugs were then used to try and distinguish between NKCC and then the two separate Transporters of
NCC and KCC.
- And what was found when they looked at the amino acid sequences is there was really similar amino acid sequences between the three different Transporters.
- So NCC had about 45 to 50 percent homology with NKCC and about 25% with KCC.
- So there were similarities in the amino acid sequences

Question 5

NKCC1

Answer 5

• belongs to SLC12 family of Cl- transporters
  larger - BSC2 1200 amino acid residues
• found on basolateral membrane except choroid plexus
• widespread distribution in epithelia and non epithelial cells
• bumetanide inhibition greater than for NKCC2 - 4x more potent

Question 6

NKCC2

Answer 6

smaller - BSC1 1100 amino acid residues
- found on apical membrane
- only found in kidney
- promotes transepithelial transport; reabsorption of NaCl in nephron and establishing the countercurrent multiplier

Question 7

NKCC1 role in volume regulation. - cell shrinks

Answer 7

when the cells are exposed to hypertonic solution the cell shrinks

Question 8

NKCC1 role - what happens once the environment is restored

Answer 8

Once the environment is then restored as the body’s trying to maintain homeostasis that cell needs to go back to its original size and the way it does that is through this process of regulatory volume increase
- So by activating the NKCC 1 on the basolateral membrane, sodium, potassium and two chloride ions enter the cell and water will follow and the cell will then return to its normal size.
- So this plays a crucial role in Regulatory volume increase in the recovery of cells following any increase in the osmolality of your ECF

Question 9

what does ROM K provide

Answer 9

• We have this ROM K (renal outer medulla potassium channel) which we also need to have present and this provides the potassium which can then be recycled.

Question 10

where else has NKCC2 ben identified

Answer 10

was found in the thick ascending limb of the loop of henle, but NKCC2 has also been identified in the juxtaglomerular apparatus cells of the macula densa
- NKCC 2 in this area here is involved in tubuloglomerular feedback.
- So they must be having a sensing role that can actually help with that activation through renin Angiotensin system.

Question 11

different types of NKCC2

Answer 11

3 different isoforms found in different regions of the nephron and have slightly different roles

Question 12

isoform F of NKCC2

Answer 12

lowest affinity for transported ions
- expressed in outer medulla where salt conc. is high
main Istform for CC multiplier

Question 13

isoform B of nkcc2

Answer 13

has the highest affinity for transported ions
- expressed where there is a low concentration of sodium and chloride
- highest affinity for bumetanide (loop diuretic)

Question 14

isoform A of NKCC2

Answer 14

medium affinity for transported ions. yet, greatest transport capacity

Question 15

how did each of these isoforms of NKCC2 utilise Canopus

Answer 15

each of these different isoforms utilized xenopus oocyte - the large cells that are really easy to inject them with messenger RNA and get them to express different proteins with different Transporters. And
then you can monitor movement of ions and also functional ion movement

Question 16

KNOCK OUT STUDIES FOR NKCC2

Answer 16

a mouse was produced. It was a knockout.
- So we removed the ability to produce NKCC 2 in these animals
- and what happened.First of all is the mice were born
○ So the fact that they were born normal tells us that NKCC2 doesn’t have any effect or any impact in utero
- However day one they became severely dehydrated. - impact of NKCC2 on the counter current multiplier.
-By day seven most of them have gone into renal failure
-They also have metabolic acidosis, which is what seen in humans.
- They couldn’t excrete protons - This could have been due to the renal failure not directly due to the NKCC2
- They had high plasma renin
- by day 14 they were dead

Question 17

knock out studies NKCC1

Answer 17

day 0: normal - no effect in utero
day 2-3: development of interesting phenotypes:
- deafness
- shaker Waltzer defect - shaking head from side to side - suggest role in vestibular function
- reduction in saliva production
- fall in systolic blood pressure - no change in blood bolume though
- male infertility - role in spermatogenesis

Question 18

what are both NKCC1 AND 2 inhibited by

Answer 18

both of these are inhibited by Loop Diuretics,
- bumetanide is much more potent in NKCC1
- and bumetanide generally is about 40 times more effective at blocking the NKCC Transporters than furosemide.
- Both furosemide and bumetanide work by blocking the chloride transport.
○ So they compete for chloride on the transporter
○ if chloride can’t bind to the transporter, then you don’t get activity. so normally they given orally and once in the bloodstream in the GI tract, they get bound to plasma proteins

Question 19

why, if loop diuretics block both NKCC 1 and 2 (and NKCc1 is so widespread) do we not see more side effects with their use?

Answer 19

As loop diuretics are bound to plasma proteins, the concentration in the plasma is always low; therefore they have very little activity on NKCC1

Because they are bound to plasma proteins they are inactive until they get to the renal tubule where they are secreted into the tubule and then it starts to have its actions

Question 20

if loop diuretics inhibit NKCC2 then what happens

Answer 20

reduces salt reabsorption, so increased conc. in distal convoluted tubule.
- this will be detected by the macula densa.
- If we’ve got an increase in sodium that will then stimulate renin release
- that’s going to increase sodium reabsorption through the aldosterone system, which is then going to cause us to retain more water, which is completely counteracting the actions of the NKCC2 Inhibitors

Question 21

if loop diuretics counteract the actions of the NKCC2 inhibitors how do they work

Answer 21

the point of taking a loop diuretic is to reduce blood volume but if we’ve got Loop Diuretics that Inhibit NKCC2 here, then it’s completely useless.
- But we know now that Loop Diuretics also inhibit NKCC 2 in the macula densa, which HAS a sensing role and therefore the Loop Diuretics also inhibit that so it’s not going to sense that increasing sodium therefore we don’t get Activation of the renin-angiotensin system.

Question 22

where are OATs found

Answer 22

basolateral and apical membrane

furosemide and bumetanide are examples of drugs that actually utilize these organic anions transporters

Question 23

where are majority of OATs found

Answer 23

in kidney

Question 24

how do the loop diuretics utilise the OATs

Answer 24

msot diuretics act on the luminal surface of nephron
- arrive at site of action by filtration or by secretion by OATs in PCT
- some drugs compete for OATs so if taken with diuretics this may affect the diuretic activity

Question 25

which drugs utilise OATs

Answer 25

bumetanide/ furosemide/ thiazides utilise hOATs

Question 26

loop diuretic resistance

Answer 26

• patients with compromised renal function
• proteinuria
• presence of high levels of pprotein in tubular fluid bumetanide rebinds to this - so becomes effective
• unable to have actions on NKCC2

Question 27

excretion of diuretics

Answer 27

water-soluble diuretics are excreted in the urine via tubular secretion
- lipid soluble metabolised by the liver