PSC2002/L05 NaCl Absorption

Question 1

Give 4 pathways in which NaCl and water pas through the epithelium.

Answer 1

Passive facilitative entry of Na+ across apical membrane
Active exit of Na+ across basolateral (serosal) membrane
Paracellular diffusion of Cl- through tight junctions
Osmotically driven water absorption

Question 2

Describe the pump-leak model of NaCl and fluid absorption in epithelial cells. (4)

Answer 2

Na influx via EnaC (G(Na))
Na+ actively pumped across basolateral membrane via NA+/K+ ATPase
Causes paracellular transport of Cl- via tight junctions (TJ) to maintain electroneutrality
Increases NaCl concentration on basolateral side & drives osmotic movement through AQPs or TJs

Question 3

What is the role of ENaC in:
a) the kidney
b) the lungs
c) colon
d) sweat glands?

Answer 3

a) Na+ retention, control of whole-body Na+ and water balance, BP
b) Na+ and water reabsorption, control of airway surface liquid (ASL) and alveolar lining fluid (ALF)
c) Na+ and water reabsorption from diet
d) Na+ retention, reabsorption of Na+ by sweat ducts, not followed by water, produces a hypotonic sweat secretion

Question 4

What is the functional channel of ENaC?

Answer 4

Hetrotrimer of 3 (aBy) subunits coded by 3 genes

Question 5

How many transmembrane domains does ENaC have?

Answer 5

2, forming the pore of the channel

Question 6

Give a potent specific inhibitor of ENaC.

Answer 6

Amiloride

Question 7

What are the long extracellular loops the site for on ENaC?

Answer 7

CAPs and SPLUNC1 regulation

Question 8

What is the PY-motif in C-term site for in ENaC?

Answer 8

Ubiquitination

Question 9

Describe the role of ENaC in the kidney.

Answer 9

Determines final urinary salt composition through natriuretic hormones e.g., aldosterone

Question 10

How does aldosterone affect ENaC function?

Answer 10

Changes ENaC function in aldosterone-sensitive distal nephron (ASDN)
Stimulates Na+ reabsorption through ENaC by principle cells in ASDN
Increased NaCl and H2O absorption, increased blood volume and BP

Question 11

Where is the ASDN (aldosterone-sensitive distal nephron)?

Answer 11

Last third of DCT, CNT and cortical collecting duct (CCT)

Question 12

Describe the action of aldosterone on principal cells on the distal nephron. (3)

Answer 12

Binds to cytosolic mineralocorticoid receptor (MR)
Binds to genes with HREs in nucleus
SGK levels increase with 1 hr

Question 13

What does aldosterone increase? (4)

Answer 13

Surface ENaC levels 2-5 fold
Na+/K+ ATPase activity
ATP supply to support increased Na+/K+ ATPase activity
K+ excretion across apical membrane via ROMK

Question 14

What is the number of channels in the apical membrane controlled by?

Answer 14

Rate of insertion versus rate of retrieval/degradation

Question 15

How does aldosterone increase number of ENaC channels?

Answer 15

By decreasing rate of retrieval

Question 16

What is the retrieval and degradation of ENaC regulated by? (3)

Answer 16

Ubiquitin ligase NEDD4-2
Binds to PY motif of a, B or yENaC and adds ubiquitin group to lysine residue in N-term of ENaC subunits
Signals internalisation of ENaC followed by degradation

Question 17

How does aldosterone prevent ubiquitination of ENaC? (3)

Answer 17

Stimulates production of serum and glucocorticoid regulated kinase 1 (SGK1)
SGK1 phosphorylates NEDD4-2 and prevents binding to ENaC
Inhibits ENaC ubiquitination

Question 18

How is hypertension caused by dysregulated ENaC?

Answer 18

1. Excess aldosterone or mineralocorticoid action of other steroids
   Aldosteronism
   Glucocorticoid-remediable aldosteronism (GRA)
   Apparent mineralocorticoid excess (AME)
2. Liddle’s syndrome - gain of function in ENaC
   Mutations in cytoplasmic C-terminal region of B- or yENaC

Question 19

Describe Liddle’s Syndrome.

Answer 19

Gain of function mutations in ENaC
Rare, autosomal dominant genetic disease caused by mutations in genes encoding 3 subunits SCNN1A/B/G
Leads to cytoplasmic region changes in ENaC subunits
Prevents NEDD4-2 binding and increased number of ENaC causing hypertension
Leads to low blood K+ (hypokalaemia)

Question 20

Describe how Liddle’s Syndrome causes enhanced K+ excretion.

Answer 20

Enhanced ENaC function depolarises apical m.p.
Electrical gradient for K+ excretion through ROMK increased
Excessive loss of K+ in urine leading to low blood K+

Question 21

Give a drug which helps restore normal blood K+ and explain how this works.

Answer 21

Amiloride
Inhibitor of ENaC
Hyperpolarises apical m.p.
Reduces K+ loss and lowers BP
(K+ sparing diuretic)

Question 22

Where is ENaC expressed in the lungs? (2)

Answer 22

Apical membrane of surface epithelial and alveolar type II cells

Question 23

Describe the role of ENaC in the lungs.

Answer 23

Regulates ASL volume in conducting airways crucial for innate defence of lungs via mucociliary clearance
Regulates ALF volume essential for efficient gas transfer

Question 24

What is the effect of channel-activating proteases (CAPs) on ENaC in conducting airways?

Answer 24

Increase ENaC activity via cleavage of external loops of a and y ENaC

Question 25

What is the effect of anti-proteases on ENaC in conducting airways? Give an example.

Answer 25

Reduce ENaC activity by inhibiting proteases E.g., aprotinin

Question 26

What is the effect of SPLUNC1 on ENaC in conducting airways?

Answer 26

Reduces ENaC function Secreted into ASL and binds to ENaC Protects from internalisation and CAP activation

Question 27

What is the effect of ATP on ENaC activity in the lungs?

Answer 27

Reduces ENaC activity

Question 28

Describe the experimental evidence that the protease Cathepsin B (CTSB) regulates ENaC activity in primary cultures of human airway epithelial cells. (3)

Answer 28

Basal cell isolation and expansion from lungs Differentiation at an air-liquid interface Pseudostratified single epithelial layer Inhibiting cathepsin B reduces fluid absorption across monolayers of primary human airway cells