Diuretics (my version)

Question 1

Draw and label the structure of a renal tubule.

Answer 1

Question 2

Draw and label a PCT cross-section.

Answer 2

NOTES:

• Basal interdigitations
  
  • These are infoldings of the basal plasma membrane
  • Increase surface area
    
    • For movement of substances out of the cell → interstitium → blood
    • Provides more space for transporters (e.g. Na⁺/K⁺ pumps)
• ​Interstitium
  
  • Composed of:
    
    • Various cell types
    • ECM
    • Interstitial fluid

Question 3

Describe the transport of substances at the proximal tubule.

Answer 3

Sodium reabsorption

• 65-70% reabsorbed
• On the basolateral membrane, there are Na⁺/K⁺ pumps which pump Na⁺ out of the cell → blood (via interstitum)
• This maintains a concentration gradient for Na⁺ to diffuse into the cell from the lumen
  
  • Once inside the cell, these Na⁺ can then by pumped out by the Na⁺/K⁺ pumps

Water reabsorption

• Reabsorption of Na⁺ generates an osmotic gradient for movemet of water from the lumen → blood
• There is also an oncotic pressure in the capillaries which draws water in
  
  • Oncotic pressure = osmotic pressure generated by plasma proteins
  • Draws water in from the interstitial fluid but this in turn draws water in from the proximal tubule cells and lumen
• ​Reabsoption can be:
  
  • Transcellular - requires aquaporins
    
    • APQ1 on apical and basolateral memranes
  • Paracellular - between the proximal tubule cells
    
    • Tight junctions permeable to water (i.e. water can get through them)

NOTE: You can also get paracellular movement (reabsoption) of ions (e.g. Na⁺, Cl^-, HCO₃^-)

Glucose and amino acid reabsoption

• There are transport proteins in the apical membrane which cotransport Na⁺ and glucose/AAs (depending on the transporter) into the PCT cells
  
  • These transport proteins are called cotransporters
• This is a form of secondary active transport
  
  • Energy released from the movement of Na⁺ down its electrochemical gradient is used to transport of glucose/AAs against its concentration gradient

Bicarbonate reabsorption

• HCO₃^- + H⁺ enters into renal filtrate (via ultrafiltraton)
• Carbonic anhydrase bound to the apical membrane:
  
  • HCO₃^- + H⁺ → CO₂ + H₂O
• The carbon dioxide and water enter the proximal tubule cells
• Carbonic anhydrase inside the proximal tubule cells:
  
  • CO₂ + H₂O → HCO₃^- + H⁺
• The H⁺ is transported back into the lumen via the Na⁺/H⁺ antiporter
  
  • This is secondary active transport
    
    • H⁺ being transported against its concentration gradient
• ​The HCO₃^- is transported out of the proximal tubule cells for reabsorption into the bloodstream via the Na⁺/HCO₃^- cotransporter​
  
  • This is secondary active transport
    
    • HCO₃^- being transported against its concentration gradient

Question 4

Describe the transport of substances at the thin descending limb of the loop of Henle.

Answer 4

Freely permeable to water

• ​You have both paracellular and transcellular transport (like in the PCT)
• This allows water reabsorption (tubule → interstitium → blood)

Question 5

Describe the transport of substances at the thick ascending limb of the loop of Henle.

Answer 5

Impermeable to water

• On the apical side of the ascending tubule cells, there are Na⁺/Cl^-/K⁺ cotransporters (triple transporters)
  
  • ​This is secondary active transport
    
    • ​Na⁺ and K⁺ travelling down their concentration gradient
    • Cl^- transported against his concentration gradient
• On the basolateral membrane there are:
  
  • Na⁺/K⁺ pump to maintain Na⁺ gradient
    
    • Na⁺ out, K⁺ in
  • So there is a high concentration of K⁺ inside the cell which moves down its concentration gradient out into the interstitium via the K⁺/Cl^- cotransporter
    
    • ​K⁺ movement also allows Cl^- transport into the interstitium
• This allows reabsorption of Na⁺ and Cl^- (i.e. reabsorption into blood)

Question 6

Describe the countercurrent effect by the loop of Henle.

Answer 6

Countercurrent - because fluid is flowing in the acending and descending limb in opposite directions

REMEMBER: Higher osmolarity = more concentrated

​Step 1 (pictures A and B)

• Loop fluid and interstiium are initially isotonic
• Na+ leaves the ascending limb and enters medullary interstitium
• Fluid in ascending limb decreases in osmolarity
  
  • i.e. becomes less concentrated

Step 2 (picture C)

• More concentrated medullary interstitium draws water from the permeable descending limb
  
  • Fluid can’t be drawn out of ascending limb when Na⁺ is pumped out as the ascending limb is impermeable to water
• Fluid in descending limb increases in osmolarity
  
  • i.e. becomes more concentrated

Step 3 (picture D)

• More fluid enters and forces fluid from descending to ascending limb
  
  • So now the fluid in the ascending limb has an increased osmolarity because it came from the descending limb
  • And at the descending limb, water moved out into the medulla, increasing the osmolarity of the tubular fluid

Step 4 etc (picture E and F)

• You get more Na⁺ leaving the ascending limb and entering into the medularry interstitium
• This causes more water to leave the descending limb
• Then fluid from the descending limb gets pushed forward into the ascending limb
• Process repeats - results in a very concentrated interstitium

NOTE:

• Na⁺ moves from interstitium to blood
  
  • Down its concentration gradient as interstitium is very concentrated
• This provides the osmotic gradient for water to move into blood
• Therefore, the countercurrent effect allows water reabsorption

REMEMBER:

• Movement of Na⁺ out of the ascending limb concentrates the medullary interstitium surrounding both the descending limbs AND collecting duct
• Therefore, this counter current effect is also very importanct for water reabsorption from the collecting duct

Question 7

Describe the transport of substances at the distal tubule.

Answer 7

EARLY DISTAL TUBULE

You get Na⁺ and Cl^- reabsorption

• This is done by the Na⁺/Cl^- cotransporter on the apical membrane
  
  • Secondary active transport (lumen → distal tubule cell)
  • Na⁺ movement down electrochemical gradient
  • Cl^- transported against concentration gradient
• DCT cell → interstitium via Na⁺/K⁺ pump and K⁺/Cl^- cotransporter
  
  • ​Once in interstitium it enters into the blood

You do not get water reabsorption because the early distal tubule is not freely permeable to water

LATE DISTAL TUBULE

​Water reabsoption

• ADH stimulates aquaporin 2 insertion into the apical membrane
  
  • Allows water movement: lumen → distal tubule cell
  • REMEMBER: ADH release is dependent on plasma osmolarity detected by central osmoreceptors
• Aquaporin 3/4 always present in the basal membrane
  
  • Allows movement: distal tubule cell → interstitium (so that it can move into the blood)

Sodium reabsorption

• Late distal tubule cells sensitive to aldosterone
  
  • ​Steroid hormone - works as a transcription factor
• Aldosterone increases production of:
  
  • Na⁺/K⁺ pump (to maintain Na⁺ gradient) - basal membrane
  • Na⁺ channel (passive movement) - apical membrane
• Sodium reabsorption provides the osmotic gradient to drive water reabsorption

Question 8

Describe the transport of substances at the collecting duct.

Answer 8

Same as late distal tubule

​Water reabsoption

• ADH stimulates aquaporin 2 insertion into the apical membrane
  
  • Allows water movement: lumen → collecting duct cell
  • REMEMBER: ADH release is dependent on plasma osmolarity detected by central osmoreceptors
• Aquaporin 3/4 always present in the basal membrane
  
  • Allows movement: collecting duct cell → interstitium (so that it can move into the blood)

Sodium reabsorption

• Collecting duct cells sensitive to aldosterone
  
  • ​​​Steroid hormone - works as a transcription factor
• Aldosterone increases production of:
  
  • Na+/K+ pump (to maintain Na+ gradient) - basal membrane
  • Na+ channel (passive movement) - apical membrane
• Sodium reabsorption provides the osmotic gradient to drive water reabsorption

Essentially you end up with a concentrated urine because most of the water is reabsorbed

Question 9

What are the two ways in which diuretics can work?

Answer 9

• Inhibiting the reabsorption of Na⁺ and Cl^-
  
  • ^​i.e. Increasing excetion
  • These drugs indirectly increase the osmolarity of the tubular fluid
• Increasing the osmolarity of the tubular fluid
  
  • i.e. Decreasing the osmotic gradient across the epithelia (i.e. tubule cells)
  • These drugs directly increase the osmolarity of the tubular fluid

Question 10

What are the 5 main classes of diuretics? Give examples for each class.

Answer 10

The classes are named based on their mechanism of action

• Osmotic diuretics
  
  • ​e.g. mannitol
• Carbonic anhydrase inhibitors
  
  • ​e.g. acetazolamide
• Loop diuretics
  
  • ​e.g. frusemide (furosemide)
• Thiazides
  
  • ​e.g. bendrofluazide (bendroflumethiazide)
• Potassium sparing diuretics
  
  • ​e.g. amiloride, spironolactone

Question 11

Where does each class of diuretics act?

Answer 11

• Osmotic diuretics
  
  • Proximal tubule
  • Loop of Henle - descending limb
  • Collecting duct
    
    • Directly increases osmolarity of tubular fluid so reduces water reabsorption wherever water is reabsorbed
• Carbonic anhydrase inhibitors
  
  • ​Proximal tubule
• Loop diuretics
  
  • Loop of Henle - ascending limb
• Thiazides
  
  • ​Early distal tubule
• Potassium sparing diuretics
  
  • ​Late distal tubule

Question 12

How do loop diuretics work?

Answer 12

• They inhibit Na⁺/Cl^-/K⁺ cotransporters on the apical membrane of the ascending limb cells
• This inhibits Na⁺ and Cl^- reabsorption
  
  • i.e. Less ion movement: ascending limb cell → interstitium
  • Makes sense as there needs to be Na⁺ and Cl^- in the ascending limb cells in the first place for them to be transported out
• This results in:
  
  • Increased tubular fluid osmolarity
  • ​Reduced osmolarity of medullary interstitium
• Therefore, you get reduced water reabsorption due to reduced water being drawn out of the:
  
  • Descending limb
  • Collecting duct (main effect)
• ​Very effective - promotes 15-30% water loss

Question 13

Apart from its effect on water reabsorption, what are some other effects of loop diuretics?

Answer 13

Increased K⁺ loss (similar to thiazide diuretics)

• This is because you are delivering more Na⁺ to the distal tubule to be reabsorbed there as you have inhbited Na⁺ reabsorption at the ascending limb
• At the distal tubule, Na⁺ reabsorption is driven by the Na⁺/K⁺ pump
  
  • This pump is required to maintain the Na⁺ gradient to drive Na⁺ movement: lumen → distal tubule cell
  • Once Na+ is in the cell, it can be pumped out ​
    
    • The more Na+ in the cell, the more that can be pumped out (i.e. more activity of the Na+/K+ pump)
• ​The Na⁺/K⁺ pump causes K⁺ movement: interstitium → cell
• This results in K⁺ to move into the lumen (tubular fluid) and be excreted

Reduced Ca²⁺ and Mg²⁺ reabsorption

• Due to loss of potassium recycling

Question 14

What is potassium recycling? How do loop diuretics cause a loss of potassium recycling?

Answer 14

Potassium recycling

• K+ which is transported from the into the ascending limb cell via the Na+/Cl-/K+ cotransporters
• This K+ diffuses back out of the cells into the lumen (via K+ channels)
• This creates a postive lumen potential
  
  • The inside of the lumen is more positive than the outside of the lumen (i.e. inside the ascending limb cells)
• This potential drives the paracellular transport of ions (Na+, Mg2+, Ca2+) from the lumen into the interstium
  
  • Essentially these ions flow down an electrical gradient (positive → negative)
  • The flow is due to repulsion from the large amount of positive charges in the lumen

How loop diuretics cause a loss of potassium recycling

• By inhibiting the triple transporters, you no longer have this potassium recycling
  
  • i.e. K+ movement: lumen → cell → lumen
• ​Therefore you no longer have the positive lumen potential
  
  • By inhibiting the triple transporter, you are preventing K+loss from the lumen in the first place
  • BUT you also have Cl- in the lumen which balances out the positive charges
  • When the transporter is active, you get Cl- movement into the ascending limb cell
  • Then, when you get K+ being recycled back into the lumen, then you get that positive lumen potential
  • Therefore, inhibiting the triple transporter means you have loss of the positive lumen potential

Question 15

How do thiazide diuretics work?

Answer 15

• Act on the early distal tubule
• They inhibit the Na⁺/Cl^- cotransporter
• This inhibits Na⁺ and Cl^- reabsorption
  
  • i.e. Less ion movement: distal tubule cell → interstitium
  • Makes sense as there needs to be Na+ and Cl- in the distal tubule cells in the first place for them to be transported out
• ​This results in:
  
  • Increased tubular fluid osmolarity
  • Therefore, decreased water reabsorption in the collecting duct
    
    • ​You don’t get water reabsorption in the early distal tubule because it is not freely permeable to water
    • Therefore, water reabsorption can only occur in the late distal tubule and collecting duct (main)
• ​​Less effective than loop diuretics - promotes 5-10% water loss
  
  • Less effective because they are not interfering with the countercurrent effect which is more significant

Question 16

Apart from its effect on water reabsorption, what are some other effects of thiazide diuretics?

Answer 16

Increased K+ loss (similar to loop diuretics)

• This is because you are delivering more Na+ to the late distal tubule to be reabsorbed there as you have inhbited Na+ reabsorption at early distal tubule
• At the late distal tubule, Na+ reabsorption is driven by the Na+/K+ pump
  
  • This pump is required to maintain the Na+ gradient to drive Na+ movement: lumen → late distal tubule cell
  • Once Na⁺ is in the cell, it can be pumped out
    
    • ​T**he more Na⁺ in the cell, the more that can be pumped out (i.e. more activity of the Na+/K+ pump)
• ​The Na+/K+ pump causes K+ movement: interstitium → cell
• This results in K+ to move into the lumen (tubular fluid) and be excreted

Increased Mg²⁺ loss and Ca²⁺ reabsorption

• The cause of this is unknown

Question 17

What effect do thiazide and loop diuretics have on renin secretion? Which of these two diuretics has the most powerful effect?

Answer 17

Normal physiology

• Macula densa cells line part of the distal tubule
  
  • i.e. They make up part of the wall (not the whole way around)
• They sense Na⁺ concentration/load in the distal tubule
• If the macula densa cells sense decreased Na⁺ load, they stimulate renin production by the juxtaglomerular cells
  
  • ​Renin promotes Na⁺ reabsorption
    
    • Renin required for angiotensin II production
    • Angiotensin II stimulates aldosterone production by the adrenal glands
    • Aldosterone stimulates Na⁺ reabsorption

Thiazide and loop diuretics

• Long term, they promote water and Na⁺ loss from the plasma (i.e. reduced blood volume and plasma Na⁺ concentration over time)
  
  • So over time, the renal perfusion pressure (pressure in the afferent arteriole) and Na⁺ load in the distal tubule decreases
  • Both of these things stimulate renin release

NOTE:

• Acutely, they increase Na⁺ load in the distal tubule → therefore, would suppress renin secretion
  
  • Increase tubular Na⁺ load by preventing Na⁺ reabsorption
  • Only thiazide diuretics would this effect, as loop diuretics inhibit the triple transporter

More powerful effect - LOOP DIURETICS

• Macula densa cells have Na+/Cl-/K+ cotransporters on their apical membrane
  
  • These are inihibited by loop diuretics
• Therefore, if Na⁺ can’t enter the macula densa cells from the lumen via the triple transporters, the macula densa cells will sense that as low tubular Na⁺ concentration
• Therefore, they will have a more powerful effect on renin secretion
  
  • With thiazide diuretics there will be a reduced Na⁺ load which will be sensed
  • But with loop diuretics, barely any Na⁺ will be able to be sensed due to the triple transporter being blocked, which will stimulate even more renin secretion

Question 18

Why are thiazide and loop diuretics a problem long-term?

Answer 18

• Long-term, thiazide and loop diuretics stimulate renin secretion
• Stimulation of the RAAS system leads to aldosterone production (stimulated by angiotensin II)
  
  • Aldosterone stimulates salt and water reabsorption (rentention)​
• You give diuretics as an antihypertensive agent to reduce blood volume and hence BP by reducing salt and water reabsorption
  
  • Thererfore, aldosterone production counteracts the effect of these diuretics
• This is why ACE inhibitors are often given alongside diuretics
  
  • ACE is required for angiotensin II production, which then stimulates aldosterone production

Question 19

What are the two classes of potassium sparing diuretics? Give an example for each class.

Answer 19

• Aldosterone receptor antagonists
  
  • e.g. spironolactone
• Inhibitors of aldosterone-sensitive Na⁺ channels
  
  • e.g. amiloride

Question 20

How do potassium sparing diuretics work?

Answer 20

Aldosterone receptor antagonists

• EXAMPLE: Spironolactone
• These bind to the mineralocorticoid receptor, preventing aldosterone from binding
• Therefore, they decrease the production of:
  
  • Na+/K+ pump (to maintain Na+ gradient) - basal membrane
  • Na+ channel (passive movement) - apical membrane

Inhibitors of aldosterone-sensitive Na+ channels

• EXAMPLE: Amiloride
• These only inhibit the Na⁺ channel on the apical membrane
  
  • If there is no Na⁺ entering the cell from the lumen, then there is no Na⁺ for the Na⁺/K⁺ pump to transport out of the cell → interstitium
  • Therefore you also get reduced activity of the Na⁺/K⁺ pump

Question 21

What effect do the potassium-sparing diuretics have on water reabsorption and potassium?

Answer 21

• They inhibit Na⁺ reabsorption
  
  • → Increased tubular fluid osmolarity
  • → Reduced water reabsorption in the collecting duct (and late distal tubule)
• Effectiveness - 5% water loss

Effect on potassium

• These drugs are called potassium-sparing, which means they prevent renal K⁺ loss
• They either:
  
  • Directly reduce Na⁺/K⁺ pump production (spironolactone)
  • Or lead to reduced Na⁺/K⁺ pump activity (amiloride)
• This means you have less K+ movement: interstitium → cell
  
  • Therefore less K+ to moving into the lumen (tubular fluid) and being excreted

Question 22

Apart from its effect on water reabsorption and potassium, what are some other effects of potassium-sparing diuretics?

Answer 22

• Amiloride also inhibits the Na⁺/H⁺ exchanger (proximal tubule)
  
  • ​Na⁺ movement: lumen → cell
  • H⁺ movement: cell → lumen (excretion)
• So reduced Na⁺/H⁺ exchange → reduced H⁺ excretion → increased H⁺ retention

Question 23

What are the common side effects of diuretics?

Answer 23

LOOP DIURETICS

• Hypovolaemia
  
  • 30% water loss
• Hyponatraemia
  
  • 30% Na⁺ loss
• Hypokalaemia
  
  • Increased Na⁺/K⁺ exchange due to increase Na⁺ delivery to collecting duct
• Metabolic alkalosis
  
  • Cl^- loss
    
    • ​Long-term use results in reduced Cl^- decrease in the plasma and hence tubular fluid
    • There are HCO₃^-/Cl^- exchangers on the apical membrane of collecting duct cells
    • Reduced Cl^- movement: lumen → cells
    • Therefore reduced HCO₃^- excretion (cells → lumen)
    • So you get increased HCO₃^- retention → alkalosis
• Hyperuricaemia

THIAZIDE DIURETICS

• Hypovolaemia
  
  • 10% water loss
• Hyponatraemia
  
  • 10% Na⁺ loss
• Hypokalaemia
  
  • Increased Na⁺/K⁺ exchange due to increase Na⁺ delivery to collecting duct
• Metabolic alkalosis
  
  • Cl^- loss
• Hyperuricaemia

POTASSIUM SPARING DIURETICS

• Hyperkalaemia
  
  • ​Reduced activity of the Na⁺/K⁺ pump → reduced K⁺ excretion

Question 24

How can diuretics cause hyperuricaemia?

Answer 24

On the basolateral membrane of the tubule cells (proximal and distal), there are organic ion transporters (OATs)

How OATs work (extra):

• Na⁺/K⁺ pump on basolateral membrane pumps out Na⁺
  
  • ^​Primary active transport
• This creates an inward Na⁺ concentration gradient
  
  • Movement of Na⁺ down its electrochemical gradient drives the transport of a dicarboxylate into the distal tubule cell - cotransport
    
    • Secondary active transport
• ​This now creates an outward dicarboxylate concentration gradient
  
  • Movement of dicarboxylate down its concentration gradient drives the transport of a dicarboxylate into the distal tubule cell - antiport
    
    • Tertiary active transport
• NOTE: This is just how some OATs work - there are different subtypes

IMPORTANT:

• OATs can transport both uric acid and diuretics from the into the distal tubule cell to be excreted
  
  • ​Thiazide and loop diuretics
• Essentially, diuretics compete with uric acid for the OAT
• Therefore, you get reduced excretion of uric acid → increased levels in the plasma → hyperuricaemia
• ALSO: Transporting the diuretic into the tubular fluid allows it to access its target on the apical membrane

NOTE: Uric acid, dicarboxylates and diuretics are organic anions as they can be can be anionic in certain pH conditions

Question 26

What is the first line treatment for hypertension in most countries?

Answer 26

Thiazide diuretics

• In UK - CCB or thiazide diuretics first line treatment if over 55 years or Afro-Caribbean
• These groups of people are salt sensitive so thiazides are especially useful
  
  • ​Salt sensitive hypertension = high blood pressure responsive to high salt (Na⁺) intake
    
    • Impaired renal capacity to excrete sodium Na⁺

Question 27

Why is thiazide preferred over other diuretics?

Answer 27

Problem with thiazide and loop diuretics

• Initially (first 4-6 weeks), they are effective as an antihypertensive agent
  
  • Reduce plasma volume → reduce BP
• However, due to the fact that long-term they reduce tubular Na⁺ and therefore stimulate renin secretion
• This counteracts the effect of the loop diuretics
  
  • Renin stimulates aldosterone production which results in increased Na⁺ reabsorption, leading to increased water reabsorption and increased plasma volume
• _​_Therefore, even though loop diuretics are more effective than thiazides in terms decreasing plasma volume, for both, the diuretic effect is very sensitive to tolerance

However…

• Unlike other diuretics, thiazide diuretics when taken chronically, also seem to be good vasodilators
• Mechanisms:
  
  • Activation of eNOS (endothelial nitric oxide synthase)
  • Ca²⁺ channel antagonism
  • Opening of K_Ca channels (Ca²⁺-activated K⁺ channels) on vascular smooth muscle cells
    
    • Can lead to hyperpolarisation

Question 28

What is heart failure? What are the consequences of heart failure?

Answer 28

Heart failure is when CO insufficient to meet metabolic demand (i.e. inadequate perfusion)

• Systolic HF - ejection issue

Therefore, BP increases to compensate for this and increase tissue perfusion by:

• Activation of the RAAS
• SNS activation

However, long-term this leads to cardiac remodelling which includes:

• Hypertrophy
• Change in shape of the heart
• COME BACK TO THIS

Caridac remodelling worsens heart failure

Question 29

What is the role of loop diuretics in heart failure?

Answer 29

Significant heart failure causes congestion

• Reduced rate of blood