Ion channels and the kidney (L1-4)

Question 1

In what ways are ion channels classified?

Answer 1

Classified via selectivity (what ions do they let through?), Gating (what opens and shuts them), and regulation (what regulates the channel?)
Molecular families are based on amino acid sequence and structure.

Question 2

What is meant by the Nernst potential of an ion? How is it calculated?

Answer 2

When ion channels are open, they drive the membrane potential towards the Nernst (reversal) potential for the channel. Nernst = the conditions when an ion is in equilibrium across a membrane. This is when the voltage difference equals the equilibrium potential (Eion)
Eion = potential net flow of ions
Eion = (RT/vf) x Ln ([ion out]/[ion in])

Question 3

Explain the Nernst potential of ions at body temp

Answer 3

At body temp - RT/F = 61.5. SO potassium at body temp, Ek = -89mV and Ena = +66mV. When sodium is higher than potassium, it’s about 5x more selective. When potassium is higher than sodium, it’s about 50x more selective. Ecl of chloride is very similar to Ek (-87mV). The fact that K is more selected for, the resting membrane potential ends up being closer to Ek, then when the sodium channels open, the membrane potential rises towards the Ena because those channels are more open.

Question 4

How do you find the total current carried by a population of channels?

Answer 4

I=N x Po x g x (Vm=Eion)
You can alter N by membrane shuttling or endocytosis of channels. Po can be altered by closing the channels e.g. via phosphorylation, calcium or G proteins. You can change the membrane potential through activation or inhibition of other channels

Question 5

How can you identify ion channel currents?

Answer 5

You can identify ion channel currents using whole cells patch clamp techniques- clamp to a specific potential and then measure the total current flow across the membrane. You can add a blocker of an ion channel o see if the current decreases. If the current becomes 0 the blocker is blocking the channel completely. NaV channels are closed at negative potentials (therefore their current is 0. They are activated quickly with depolarization, giving an increased current. They then close again with depolarisation

Question 6

What are the symptoms and probable causes of FHEIG?

Answer 6

Symptoms include bi-temporal narrowing, hypertrichosis (extra hair), this upper lip, bushy/long eyebrows. Delayed development of intellectual ability and motor skills, seizures and EEG anomalies. Its thought to be caused by mutations in K+ channels - Esp. KCNK4. Mutants have larger currents (gain of function) - the mechanism is still unclear. KCNK4 is expressed in the CNS and PNS. In the wt KCNK4, there are low levels of K+ in the interstitial space, and other K+ channels are open. In the mutants, K+ is lost into the interstitial space so conc is high- this increases K+ and causes a change in Ek (makes it more positive). This causes neighbouring cells to have a more depolarised resting potential (means APs are fired more easily)

Question 7

How does the glomerulus filter blood?

Answer 7

The glomerulus filters blood plasma that passes through the kidney. Water and small molecules have free passage. The passage of blood cells and proteins is restricted because they’re too big. Filters about 180L per day. Total plasma passes the filtration barrier about 65 times a day. The afferent arteriole brings blood into the glomerulus and the efferent takes it out. The glomerulus is surrounded by the Bowman’s capsule and collects any filtrate. The filtrate then slowly flows into the proximal convoluted tubules to begin its journey through the nephron.

Question 8

Describe the structure of the filtration barrier

Answer 8

Consists of epithelial cells (podocytes), a basement membrane and endothelial cells (within the capillary)
- flat
-large nuclei
-Circular fenestrations (holes between the cells)
The cells are in contact with each other
- Filters blood cells and platelets (stops them getting out of the capillary)

Question 9

What are the properties of the basement membrane of the Bowman’s capsule?

Answer 9

Continuous (surround glomerular capillaries)
Acts as the main filtration barrier
Has many glycoproteins
Made of things like collagen, laminin and fibronectin
Negatively charged. It filters based on molecular shape, size (mainly) and charge.
Large molecules are not transported and smalled more negatively charged molecules aren’t because they’re repelled by the membrane. The shape is important too as bulky molecules aren’t filtered

Question 10

What are the properties of podocytes?

Answer 10

Trabecula (big processes coming out of the cell body)
Pedicles (smaller processes coming off the trabeculae) - they act as feet on the capillaries
The Pedicles interdigitate (like intertwining fingers) - so there are still quite big gaps (slit pores) - They don’t really serve a filtering purpose because the slit pores are too big, but their main role is maintenance and phagocytosis of any molecules that aren’t meant to be there (antigens etc)

Question 11

What determines filtration?

Answer 11

Size, shape and charge. F/P = filtrate to plasma ration - a freely filtered molecule will have the same conc on the filtrate and plasma, so will have a ration of 1. A non-filtered molecule will have no conc in the filtrate so will have a ratio of 0. Therefore F/P ration gives an indication of how likely it is that something is filtered. You can see on a graph that F/p decreases as size increased. And a natural (charged) dextran (chains of glucose) doesn’t filter as much as uncharged dextran
The charge doesn’t really affect very small molecules because they don’t interact with the basement membrane as much (they just move through).

Question 12

Explain the forces governing glomerular filtration

Answer 12

The filtration coefficient Kf is a constant that gives an idea of the measure of the permeability of a membrane
Starlings forces govern Glomerular Filtration Rate (GFR)
GFR is proportional to the forces favouring filtration - the forces opposing filtration
Forces favouring: Pcap (the hydrostatic pressure in the capillary - pushes plasma out of the capillary, and the oncotic pressure of the Bowman’s capsule (osmotic pressure induced by proteins which are making plasma move into the BC)
Forces opposing filtration are the Pbc (the hydrostatic pressure of the BC, which is opposing movement of plasma out of the capillary) and the oncotic pressure of the capillary, which is trying to draw plasma into it.
Therefore, GFR is proportional to (Pcap+nbc)-(Pbc+ncap)
Along the glomerular capillary - Pbc stays small and constant because fluid enters from glomerulus then flows away. Volume is slightly lower at the end of the capillary. Oncotic pressure increases in capillary because there is a higher conc of proteins as you move along (all the other stuff gets filtered out). n is negligible because its generated from proteins and obvs proteins aren’t in the filtrate (except for a very small amount). So filtration occurs over the length of the whole capillary - unlike in other places where the pressure reverses at the second half. Experimentally, it’s hard to know Kf in a live patient because you have to dissect it out. So you use a clearance technique.

Question 13

What is the normal GFR, how is it regulated?

Answer 13

Normal GFR is about 125 ml/min, and for a single nephron it’s about 50nl/min.
GFR is maintained at a constant level by autoregulation. BP drops which cause renal blood flow to drop, therefore filtration drops because the hydrostatic pressure decreases, so the nephron adjusts this to increase GFR. A drop if GFR is characteristic of renal failure. GFR is controlled by the afferent arteriole. When renal blood flow increases, autoregulation increases the resistance in the afferent arteriole which decreases the renal blood flow and pressure in the glomerulus. This, therefore, causes a decrease in GFR (think of a car going through a 6 lane toll then driving off into 4 lanes). When arterial BP decreases, causing a decrease in renal blood flow and GFR, autoregulation causes a decrease in resistance, allowing more blood to flow through the glomerulus and increase GFR

Question 14

What are the 2 theories to how GFR is autoregulated?

Answer 14

1. Myogenic theory
   - Autoregulation is the property of the afferent arteriole smooth muscle - there are stretch receptors in the smooth muscle itself
2. Tubuloglomerular feedback theory
   - autoregulation controlled by the juxtamedullary apparatus
   - Macula densa cells sense the change in the rate of flow via cilia projection. The release vasoactive chemicals which affect the afferent arteriole because they’re close to it. E.g. Increase in GFR and flow, macula densa release vasoconstrictors to decrease blood flow to the glomerulus

Question 15

What is osmolality?

Answer 15

A measure of how concentrated a solution is
Osmolality = [X] x n in mOSmol/KgH2O
n = number of particles the molecule dissociates into in solution. e.g. 100mM glucose in solution has osmolality of 100 because it doesn’t dissociate into anything, but 100mM of NaCl would be 200 osmolality because it dissociates into 2 molecules (Na and Cl) (theoretically, in reality, things don’t fully dissociate

Question 16

Explain how counter-current multiplication works

Answer 16

Changes in osmolality give us the ability to control the concentration of urine. This happens in the inner medulla part of nephrons (loop of Henle and collecting duct). Juxtamedullary nephrons are particularly important in concentrating urine because they go down deeper into the kidney. Only birds and mammals have a loop of Henle so can concentration our urine. These segments handle water, sodium and chloride differently. The tubular fluid goes down the descending limb, up ascending limb, into the distal tubule then down the collecting duct. The descending limb and collecting duct are water permeable, but the ascending limb is not. However, water movement out of the collecting duct only occurs in the presence of arginine vasopressin. Sodium and chloride leave the ascending limb. This movement is critical for setting up countercurrent multiplication. Osmolality rises as you go deeper into the medulla because water has left but ions haven’t - this increases the osmotic driving force for water to leave.

Question 17

Explain the transverse and vertical gradient hypothesis

Answer 17

An artificial loop of Henle was filled with tubular fluid at 290mOsm/kgH2O - It was thought that water moved out of the ascending limb and moved into the descending limb, so osmolality of ascending limb goes down and the ascending goes up. And because fluid is continuously moving, the changed osmolalities move around and it happens again. Eventually, you get the highest osmolality at the apex, which decreases upwards. Means you end up with transverse and vertical gradients (vertical up the limb, and transverse across the limb. However, this model isn’t correct because we now know that water moves from the ascending limb into the interstitial fluid, which then causes the gradient and driving force for the solute to move out the descending limb. Sodium and chloride loss from the ascending limb starts the whole process for countercurrent multiplication. High interstitial fluid osmolality across the medulla which produces driving force for water movement out. The more Na and Cl moving out, the more concentrated our urine Vasopressin regulates aquaporin 2 in collecting duct and sodium/chloride handling in thick ascending limb.

Question 18

What are the features of the thin descending limb?

Answer 18

Extremely water permeable
A very small leak of NaCl into limb but is negligible really.
Aquaporin 1 water channel are constitutively open, water moves out via gradient
KO humans and mouse leads to problems with urine concentration, probably because the movement of water out helps concentrate sodium and chloride which provides driving force for NaCl absorption in ascending limb, which then leads to driving force of reabsorption in collecting duct.

Question 19

What are the features of the thin ascending limb?

Answer 19

H2O impermeable. NaCl permeable - passive process

Transport systems not well understood because it’s not easily accessed.

Question 20

What are the features of the thick ascending limb?

Answer 20

On the basolateral (next to capillary) membrane:
- Na/K ATPase - moves 3 Na out and 2K in - creates a driving force for Na.
-K channels for recycling of K
- CLCK (regulated by Barttin) - moves chloride out.
On the apical membrane (facing into the tube)
- NKCC2 - transports 1 Na, 2 Cl and 1 K into the cell from limb.
ROMK recycles potassium across the apical membrane (this is important for NKCC2 function)

Question 21

Explain what causes Bartter’s syndrome

Answer 21

Genetic inheritance
Mutated ROMK, NKCC2 or Barttin - Leads to decreases absorption of Na or CL
Causes salt wasting, polyuria due to water the following salt into wee. - Less of a driving force for collecting duct so less water reabsorption. Hypokalemia (low potassium absorption). Metabolic alkalosis, hypercalciuria and nephrocalcinosis.

Question 22

What is the function of principle cells?

Answer 22

Cells of the cortical and outer medullary collecting duct
Apical - Sodium channel (ENaC - sodium in), ROMK (potassium out) and AQ2 (water in)
Basolateral - Na/K/ATPase, Aquaporins 3 and 4 (water out), Kir2.3 (K out). Aquaporins 3 and 4 are constitutively active - Activation of the vasopressin receptor leads to insertion of vesicle containing AQP into the apical membrane (shuttling hypothesis). Problems with aquaporin 2 APV system leads to diabetes insipidus

Question 23

Explain how urea aids the concentration of urine

Answer 23

Interstitial osmolality is made up of 50% NaCl and 50% urea. You need to get rid of urea but it also helps concentrate urine - so it is important. The early part of the collecting duct has a v low urea permeability but is permeable to water in the presence of AVP. Therefore, conc of urea in tubular fluid starts to go up bc it’s not being reabsorbed but water is. In the later collecting duct (medullary) in presence of APV there is now a urea permeability. Bc conc of urea is so high now, there is a driving force for it to move into the interstitial fluid. This helps concentrate the interstitial fluid and therefore drives water reabsorption. A little bit of urea leaks back into the thin ascending and descending limb, but its so small, it’s just recycled back. In the inner medullary collecting duct, there are urea transport proteins. Apical (tubular fluid) UT-A1. Basolateral (interstitial fluid) - UT-A3. Urea just follows gradient through the cell.
Massive conc in later part of the collecting duct. KOs of UT-A1/3 shows that in wt mice that are water-deprived, the osmolality of their urine goes up. Whereas, in KO mice deprived of water, showing no transport of urea means the mice can’t regulate their urine concentration as easily (they have the same urine osmolality as KOs with free access to water). KOs osmolality is half because they’ve lost half of the driving force.

Question 24

Explain how countercurrent flow occurs in the kidney

Answer 24

Takes place in the Vasa Recta, the specialist blood supply to the kidney.• Efferent arterioles lead on to vasa recta. They have loops which act like the loop of Henle (dips down into medulla)
Acts to stop wash-out of interstitial fluid. If we had a conventional blood supply (blood vessels go down into medulla and out) Blood plasma has an osmolality of 290, meaning lots of NaCl and urea will enter blood vessel and will be gone. (all the hard work of the kidney getting the interstitial fluid concentrated is gone )
So instead, blood vessels go down and loop back. Means their osmolality follows that of the loop of Henle. Therefore, is driving force for water to go out and conc gradient for solutes to go in at beginning of vasa recta, but plasma is moving around, so now the solutes are at the end, and therefore move back out again and water moves back in.
Therefore, wash out is prevented by vasa recta going back up into cortex. Very important for water reabsorption. 9there is still a tiny bit of washout but is negligible)
UT-B is a urea transporter found on red blood cells. When solutes (incl. urea) enter the vessel, The urea is transported by RBCs. When plasma gets to ascending limb, urea moves out of RBC and back into the interstitial fluid
Non-functioning UT-B means patients can’t concentrate urine because urea in descending limb urea enters RBCs but then gets trapped. Therefore, much lower osmolality in interstitial fluid so less water reabsorption

Question 25

What is the prevalence and symptoms of Liddle’s syndrome?

Answer 25

Described in 1963 by Dr Liddle. It is autosomal dominant (only need 1 faulty copy). Patients have renal sodium retention - so absorb too much sodium across the collecting duct - therefore they also reabsorb too much fluid. This leads to hypertension (main problem). Patients also get hypokalaemia (secondary effect ) and metabolic alkalosis (pH is too high). Low renin and aldosterone levels - hypertension causes the body to decrease these levels because they are involved in increasing BP. The main problem is in ENac on the apical principle cell. ENac is made of 3 subunits - a mutation in any have been shown to cause Liddles. The problem in Liddles is that too much sodium is being reabsorbed, so the mutations in ENac are GAIN OF FUNCTION

Question 26

Explain the mutations in ENac and how they cause Liddle’s

Answer 26

ENac = Epithelia Na Channel
It has alpha, beta and gamma subunits in a ratio of 1:1:1 - all 3 are needed for normal function.
Liddle’s mutations are usually in the COOH tail of the beta or gamma subunits. Deletion of proline-rich motifs which is important in endocytosis of ENac. The proline-rich motif allows ENaC to interact with a ubiquitin ligase called NED2 which allows ENac to be tagged for removal from the membrane. Therefore, in Liddles, ENaC removal can’t occur at the rate it normally would (much slower). In the presence of low renin and aldosterone you would expect the number of ENaC channels to be low, however, in Liddle’s the number stays high. You can see an increase in the number of channels is we overexpress ENaC in an over-expression system (E.g. in a Xenopus oocyte) - take the eggs out and inject them with RNA of protein of interest - they make the protein. rRNaC (rat) - deflections downward show that the channels open much more frequently- and there are more. A bar graph shows a mutation in beta subunit shows an increased ENaC current

Question 27

How does ENaC mutation cause hypertension and other symptoms?

Answer 27

ENaC mutations lead to excessive sodium and therefore water reuptake. This is what leads to hypertension - water is reabsorbed through AQ2 on the apical side and AQ3 and 4 on the basolateral. Increased sodium reabsorption means that the NaK ATPase on the basolateral side of the cell has to work harder to remove sodium from the cell, which means it up takes more potassium from the blood (secretion). This extra potassium in the cell goes out ROMK and is lost in the urine. This causes hypokalaemia. Metabolic alkalosis also occurs because in the alpha-intercalated cells (also in the collecting) there is a negative membrane potential due to increased sodium in the tubular fluid. This drives hydrogen ion secretion into the urine - so you get alkalosis. Both hyperkalaemia and alkalosis are secondary to the fact too much sodium is being absorbed.

Question 28

How does aldosterone control BP in a normal person?

Answer 28

In a normal individual, if you have a rise in blood pressure, you get a decrease in renin and angiotensin release, which leads to low aldosterone. This leads to a loss of ENac from the apical membrane of the principal cells (and also a decrease in the production of ENaC) and therefore a reduction in sodium reabsorption and therefore water reabsorption. This leads to a lower circulatory volume, which decreases BP.

Question 29

What are the treatments of Liddle’s?

Answer 29

Treat Liddles with amiloride (or a derivative). This blocks ENaC. Treatment in an infant shows a decrease in BP.
Spironolactone is an aldosterone receptor antagonist - shows a reduction in BP which shows aldosterone is already low because you get no change even if you block it. Basically, a control which shows ENaC is causing hypertension. Amiloride also causes an increase in potassium, and a decrease in blood pH, and an increase in renin and aldosterone (shows no more hypertension)
However, overall conc of sodium stays the same, but total content (no of moles) goes down and that causes water reabsorption to go down. Amiloride treatment is reversible (stop taking it and hypertension comes back))

Question 30

How does APV control water reabsorption?

Answer 30

Principle cells in collecting duct have vasopressin 2 receptors. APV stimulates receptors leading to activation protein kinase A and phosphorylation of AQP2 channels in vesicles. This inserts into the apical membrane and makes new AQP2 channels. There’s an osmotic driving force which causes water to move into the cell and out the other side into capillary (through unregulated AQP3/4) - regulation of water is through AQP2 because 3 and 4 are constitutively active

Question 31

What are the symptoms of diabetes insipidus? What causes it?

Answer 31

Diabetes insipidus causes polyuria (too much wee) with compensatory polydipsia (thirst)
Dehydration can cause a serious issue
In inherited its between 1:25k/30k
Hereditary is due to problems with AVP-AQ2 system.

Question 32

What are the different types of diabetes insipidus?

Answer 32

1. Primary polydipsia - suppressed AVP production caused by excessive water intake - temporary. Increased urine flow rate
2. Gestational - in some pregnant women. Decreased AVP levels because of metabolism by placental enzymes. Stops after birth
3. Central - problems with AVP production - can be acquired or congenital
4. nephrogenic - Impaired effect of AVP - so AVP is made, but kidneys can’t react to it - can also be acquired or congenital.

Question 33

Compare aquired and congenital diabetes insipidus

Answer 33

Congenital (also known as neurohypophysial) forms less than 10% of the total - more than 67 mutations in the gene for vasopressin. Makes a version of vasopressin that can’t be trafficked/isn’t released/ is inactive. Acquired can be caused by infection e.g in the pituitary or kidney, trauma e.g. injury or surgery.
Acquired nephrogenic is more common than central. Caused by lithium, impacts on the ability to reabsorb water. Antibiotics, antifungals and chemotherapy agents can also cause nephrogenic. Hypokalemia can also lead to a reduction in AQP2 levels. Hypocalciuria also impacts on AQP2 levels. Can also be caused by some types of chronic renal failure.
Mutations in vasopressin are X-linked - only in males.
Mutations in the AQP2 gene causes an impact in trafficking (dominant) or function os protein (recessive)
Symptoms appear in infants - hypernatremic, dehydration, poor feeding, skin dryness, depressed anterior fontanel (skins with dehydration)

Question 34

What are the treatments of diabetes insipidus?

Answer 34

Treat central with desmopressin (easy) - just replaces vasopressin (in a nasal spray)
Nephrogenic is very difficult to treat. Can’t respond to vasopressin so need a different response. E.g. when there is a mutation in vasopressin 2 receptor (misfolded) - you use cell permeable antagonists which target the misfolded VR2 to the membrane and correct the folding. . When in the membrane, the antagonist then comes off and is replaced with APV. Or, you can use cell permeable receptor agonists - to stimulate signalling pathway. They cross the membrane, go into the cell and stimulate misfolded vasopressin receptor instead of in membrane. Allows for downstream signalling to happen. Or, cell permeable agonists that aid correct folding and trafficking and induce V2R signalling (sort of a mix of the other two).

Question 35

Explain how you can use compounds that bypass VR2 signalling to treat diabetes insipidus?

Answer 35

They still cause activation of AQP2. They target prostaglandin receptors which leads to stimulation of cAMP, which leads to AQP2 insertion. They target a different number of things:
1. Prostaglandin receptor agonist induces AQP2 expression and membrane abundance.
2. Stimulating cGMP breakdown levels (PDE5 inhibitors prevent the breakdown of cGMP)
3. stimulating cAMP levels (PDE4 inhibitors prevent the breakdown of cAMP). Statins (usually help control cholesterol) prevent internalisation of AQP2.
5. Heat shock protein 90 inhibitors allow misfolded AQP2 to reach the membrane.
The one you use really depends on the mutation and its also very trial and error.