Distal nephron, diuretics and transport disorders

1. Distal nephron, diuretics and transport disorders Linda. Peterson@ubc.ca PHY 423 Session 3

2. Three disorders causing low plasma [K] Group 1 Bartter’s Syndrome/Disease Group 2 Gitelman’s Syndrome/Disease Group 3 Liddle’s Syndrome/Disease What specific nephron segment, cell type and transport molecule- how is function changed (loss or gain of function) What is the effect on transport in the specific segment where the affected molecule is located

3. What are the changes in potassium excretion and BP in these three disorders?

4. Inherited transport disorders causing hypokalemia (low plasma [K]) and metabolic alkalosis i.e. elevated pH due to increased [HCO3]. Are they the same or different disorders?

5. ENaC is the Epithelial Na channel. Although we will see that the channel is composed of alpha and beta subunits and there can be defects in either leading to abnormal properties, it will be sufficient to just refer to the Na channel as ENaC. ENaC is under the control of aldosterone, it is one of the aldosterone induced proteins. ENaC is the Epithelial Na channel. Although we will see that the channel is composed of alpha and beta subunits and there can be defects in either leading to abnormal properties, it will be sufficient to just refer to the Na channel as ENaC. ENaC is under the control of aldosterone, it is one of the aldosterone induced proteins.

6. This slide is to remind you of the key features of these distal nephron segments. The TAL reabsorbs about 15-20% of the filtered amount of NaCl but none of the filtered water. This is the major site of free water production in the kidney. So in this segment, NaCl concentration decreases while the electrolytes are reabsorbed without water. The dark thickened edges represent that water tight nature of this region. Note that the distal convoluted tubule DCT is the next region adjacent to the TAL. It begins at the macula densa and continues for about 1/3d of the distance to the junction of two nephrons forming the CD. The DCT shares the water tight property of the TAL, and it contributes to the dilution of urine by reabsorbing NaCl without water. Both segments transport other ions which we will look at in more detail. The Cortical Collecting duct includes several different regions extending from the DCT, the fusion of cortical collecting tubules (CCT) of two nephrons forming a Cortical collecting duct and then ending at the junction of the cortex and the medulla. We call the entire section the CCD. The CCD is unusual in that it is a mosiac of two different cell types which have different transport systems. This slide is to remind you of the key features of these distal nephron segments. The TAL reabsorbs about 15-20% of the filtered amount of NaCl but none of the filtered water. This is the major site of free water production in the kidney. So in this segment, NaCl concentration decreases while the electrolytes are reabsorbed without water. The dark thickened edges represent that water tight nature of this region. Note that the distal convoluted tubule DCT is the next region adjacent to the TAL. It begins at the macula densa and continues for about 1/3d of the distance to the junction of two nephrons forming the CD. The DCT shares the water tight property of the TAL, and it contributes to the dilution of urine by reabsorbing NaCl without water. Both segments transport other ions which we will look at in more detail. The Cortical Collecting duct includes several different regions extending from the DCT, the fusion of cortical collecting tubules (CCT) of two nephrons forming a Cortical collecting duct and then ending at the junction of the cortex and the medulla. We call the entire section the CCD. The CCD is unusual in that it is a mosiac of two different cell types which have different transport systems.

7. Transport in the TALNo Water is Reabsorbed Here Primary active step Secondary active NaK2Cl entry- it is electroneurtal- all the Cl exits through the BL Cl channel- the Na that entered- exits through the NaKATPAse But the K reenters the lumen due to the presence of a K channel- called ROMK what pathway? This creates a lumen positive voltage This drives 50% of the Na is reabsorbed passively i.e. free All of the Ca++, Mg++ and K+ is reabsorbed passively what is this pathway? i.e. no direct expenditure of ATPPrimary active step Secondary active NaK2Cl entry- it is electroneurtal- all the Cl exits through the BL Cl channel- the Na that entered- exits through the NaKATPAse But the K reenters the lumen due to the presence of a K channel- called ROMK what pathway? This creates a lumen positive voltage This drives 50% of the Na is reabsorbed passively i.e. free All of the Ca++, Mg++ and K+ is reabsorbed passively what is this pathway? i.e. no direct expenditure of ATP

8. Mutations in any one of the circled transport proteins will cause Bartter’s syndrome. It is clear that Calcium and Magnesium reabsorption will not occur if the NaCl transport system is blocked by mutations in Bartter’s patients or use of loop diuretics. Calcium excretion is clearly above normal but, Mg excretion is not elevated in either case. What accounts for this? The DCT is a very important site for Mg reabsorption after the TAL. It is mediated by a Mg selective channel in the apical membrane. When the TAL is dysfunctional or blocked by a loop diuretic, the increased load of NaCl increases DCT reabsorption and this causes and increase in Mg is reabsorbed by the DCT which is not affected. The DCT can not reabsorb the amount of Ca that is delivered so it mitigates the total amount of Calcium lost but none the less there will be increased loss of Calcium. Magnesium excretion will be normal in these two clinical situations. Mutations in any one of the circled transport proteins will cause Bartter’s syndrome. It is clear that Calcium and Magnesium reabsorption will not occur if the NaCl transport system is blocked by mutations in Bartter’s patients or use of loop diuretics. Calcium excretion is clearly above normal but, Mg excretion is not elevated in either case. What accounts for this? The DCT is a very important site for Mg reabsorption after the TAL. It is mediated by a Mg selective channel in the apical membrane. When the TAL is dysfunctional or blocked by a loop diuretic, the increased load of NaCl increases DCT reabsorption and this causes and increase in Mg is reabsorbed by the DCT which is not affected. The DCT can not reabsorb the amount of Ca that is delivered so it mitigates the total amount of Calcium lost but none the less there will be increased loss of Calcium. Magnesium excretion will be normal in these two clinical situations.

9. Transport in the Distal Convoluted Tubule (DCT) No water is reabsorbed Here First primary active step NaCl entry is electroneutral, all Na exits via atpase and Cl exits through the cl channels Notice there is a Ca channel and a Mg channel. If the NaCl cotransporter is blocked or impaired- then Na entry via the Na Ca exchanger increases. This creates a driving force for Ca entry- First primary active step NaCl entry is electroneutral, all Na exits via atpase and Cl exits through the cl channels Notice there is a Ca channel and a Mg channel. If the NaCl cotransporter is blocked or impaired- then Na entry via the Na Ca exchanger increases. This creates a driving force for Ca entry-

10. the most common mutation that has been identified in patients with Gitelman’s is in the NaCl cotransporter. Why does Ca excretion decrease in patients with Gitelman’s syndrome? When Na entry through the NaCl cotransporter is not possible due to the mutation, then Na enters the cell via the Na-Ca exchanger which is in the basolateral membrane. This decreases the intracellular Ca concentration and thus Ca enters through the apical Ca channel. This is one mechanism that explains why Ca excretion is not elevated in patients with Gitelman’s, rather Ca excretion tends to be lower than in the normal population. The DCT is a very important site for Mg reabsorption after the TAL. It is mediated by a Mg selective channel in the apical membrane. The increased excretion of Mg has been attributed to downregulation of this apical Mg channel in the DCT cell which is associated with both blockade (thiazide diuretic) or mutation of the NaCl cotransporter. the most common mutation that has been identified in patients with Gitelman’s is in the NaCl cotransporter. Why does Ca excretion decrease in patients with Gitelman’s syndrome? When Na entry through the NaCl cotransporter is not possible due to the mutation, then Na enters the cell via the Na-Ca exchanger which is in the basolateral membrane. This decreases the intracellular Ca concentration and thus Ca enters through the apical Ca channel. This is one mechanism that explains why Ca excretion is not elevated in patients with Gitelman’s, rather Ca excretion tends to be lower than in the normal population. The DCT is a very important site for Mg reabsorption after the TAL. It is mediated by a Mg selective channel in the apical membrane. The increased excretion of Mg has been attributed to downregulation of this apical Mg channel in the DCT cell which is associated with both blockade (thiazide diuretic) or mutation of the NaCl cotransporter.

11. Inherited transport disorders causing hypokalemia (low plasma [K]), and metabolic alkalosis i.e. elevated plasma pH due to an increase in [HCO3].

12. What is the underlying defect in Liddle’s Disorder?

14. Please notice this is the first cell along the nephron where Na enters via a channel! Na enters via a carrier in all the other upstream segments. Please notice this is the first cell along the nephron where Na enters via a channel! Na enters via a carrier in all the other upstream segments.

15. Inherited transport disorders causing hypokalemia (low plasma [K]) and metabolic alkalosis i.e. an increase in plasma pH due to an increase in [HCO3]. Hypokalemia due to increased K loss is a direct effect of the change in ENaC in the Principal cell. No other explanation is required.Hypokalemia due to increased K loss is a direct effect of the change in ENaC in the Principal cell. No other explanation is required.

16. Potassium loss in patients with Liddle’s Disorder occurs in the CCD-due to increased K secretion by Principal cells as a direct consequence of the mutation in ENaC. Why is potassium lost in patients with Bartter’s and Gitelman’s syndromes?

17. Potassium is freely filtered and then extensively reabsorbed in the proximal tubule. Only 25% of the filtered K enters the loop of Henle. The last part of the proximal tubule has the capacity to secrete K and in end stage renal failure it makes an important contribution to renal excretion. The Medulary and cortical TAL have the capacity to reabsorb K. Although the Na,K 2Cl co-transporter moves K into these cells, K re-enters the lumen via K conductance channels in the luminal membrane. The purpose of this movement is to create a lumen positive potential since the co-transporter effectively moves 2 negative chloride ions and only one positively charged sodium into the cell. The Na must exit the cell via the Na-K-ATPase pump on the basolateral surface which costs metabolic energy. The lumen positive potential effectively drives 50% of the Na reabsorption, and a large fraction of Mg and Ca reabsorption along with some K, through the paracellular pathway i.e. between the cells. This movement is passive and requires no additional expenditure of energy. No water is reabsorbed in the TAL regardless of the presence or absence of ADH. The last part of the distal convoluted tubule named DCT2 has the capacity to secrete K along with the connecting tubule CNT, and the initial collecting duct, ICT and the cortical collecting duct CCD. For the purposes of this course, we will refer to this region as the CCD, knowing that it refers to all these slightly different regions containing cells that can secrete K. Although there are differences between them, we will find they share more similarities than differences. K transport in the ICT, CNT and CCD is regulated and responds to your needs to conserve or excrete potassium, whereas all upstream regions are not involved in the regulation of K balance.Potassium is freely filtered and then extensively reabsorbed in the proximal tubule. Only 25% of the filtered K enters the loop of Henle. The last part of the proximal tubule has the capacity to secrete K and in end stage renal failure it makes an important contribution to renal excretion. The Medulary and cortical TAL have the capacity to reabsorb K. Although the Na,K 2Cl co-transporter moves K into these cells, K re-enters the lumen via K conductance channels in the luminal membrane. The purpose of this movement is to create a lumen positive potential since the co-transporter effectively moves 2 negative chloride ions and only one positively charged sodium into the cell. The Na must exit the cell via the Na-K-ATPase pump on the basolateral surface which costs metabolic energy. The lumen positive potential effectively drives 50% of the Na reabsorption, and a large fraction of Mg and Ca reabsorption along with some K, through the paracellular pathway i.e. between the cells. This movement is passive and requires no additional expenditure of energy. No water is reabsorbed in the TAL regardless of the presence or absence of ADH. The last part of the distal convoluted tubule named DCT2 has the capacity to secrete K along with the connecting tubule CNT, and the initial collecting duct, ICT and the cortical collecting duct CCD. For the purposes of this course, we will refer to this region as the CCD, knowing that it refers to all these slightly different regions containing cells that can secrete K. Although there are differences between them, we will find they share more similarities than differences. K transport in the ICT, CNT and CCD is regulated and responds to your needs to conserve or excrete potassium, whereas all upstream regions are not involved in the regulation of K balance.

18. Increased Na delivery in Bartter’s and Gitelman’s patients stimulates K secretion Increased K excretion is an indirect effect of the two disorders K loss in Bartter’s is >>> Gitelman’s consistent with the difference in Na delivery Na entry through apical Na channels depolarizes the apical membrane Apical membrane depolarization creates a favourable electrical gradient for K exit via apical K channels Expressed only in the apical membrane-Epithelial Na Channels-ENaC DCT2, CNT and Principal CellsNa entry through apical Na channels depolarizes the apical membrane Apical membrane depolarization creates a favourable electrical gradient for K exit via apical K channels Expressed only in the apical membrane-Epithelial Na Channels-ENaC DCT2, CNT and Principal Cells

19. Na entry through apical Na channels depolarizes the apical membrane Apical membrane depolarization creates a favourable electrical gradient for K exit via apical K channels Na entry through apical Na channels depolarizes the apical membrane Apical membrane depolarization creates a favourable electrical gradient for K exit via apical K channels Expressed only in the apical membrane-Epithelial Na Channels-ENaC DCT2, CNT and Principal CellsNa entry through apical Na channels depolarizes the apical membrane Apical membrane depolarization creates a favourable electrical gradient for K exit via apical K channels Expressed only in the apical membrane-Epithelial Na Channels-ENaC DCT2, CNT and Principal Cells

20. Apical Na Channels Na entry through apical ENaC channels drives K secretion If ENaCs close? K secretion ceases If the number of Na channels increases, K secretion increases-In Liddle’s Disease, the number of Na channels is greatly increased and they are not subject to regulation i.e. by aldosterone. The amount of Na reabsorption through these channels is increased, and K secretion is greatly above normal causing hypokalemia and k depletion. The patient becomes hypertensive. Aldosterone is depressed and so is renin and Angiotensin II as you might suspect. Strictly speaking the term channel activity refers to the open state of the channel i.e. open or closed. Frequently when one says that channel activity is increased it is actually due to increased numbers of channels. Loss of function mutations in ENaC will cause the same clinical picture as hypoaldosteronism and is referred to as pseudohyperaldosteronism. In these rare genetic diseases, aldosterone is actually elevated as well as renin and aldosterone. If the number of Na channels increases, K secretion increases-In Liddle’s Disease, the number of Na channels is greatly increased and they are not subject to regulation i.e. by aldosterone. The amount of Na reabsorption through these channels is increased, and K secretion is greatly above normal causing hypokalemia and k depletion. The patient becomes hypertensive. Aldosterone is depressed and so is renin and Angiotensin II as you might suspect. Strictly speaking the term channel activity refers to the open state of the channel i.e. open or closed. Frequently when one says that channel activity is increased it is actually due to increased numbers of channels. Loss of function mutations in ENaC will cause the same clinical picture as hypoaldosteronism and is referred to as pseudohyperaldosteronism. In these rare genetic diseases, aldosterone is actually elevated as well as renin and aldosterone.

21. Factors affecting K secretion 1. Aldosterone * 2. Sodium delivery * Responsible for PHYL 423 Aldosterone is the most important regulator of K secretion- it must be present for normal rates of K secretion (excretion). However to answer our question, we will look at sodium delivery first.

22. How does Na delivery affect K secretion? The rate of apical Na entry depends on the Na gradient across the apical membrane

23. Na entry through apical Na channels depolarizes the apical membrane Apical membrane depolarization creates a favourable electrical gradient for K exit via apical K channels Na entry through apical Na channels depolarizes the apical membrane Apical membrane depolarization creates a favourable electrical gradient for K exit via apical K channels Expressed only in the apical membrane-Epithelial Na Channels-ENaC DCT2, CNT and Principal CellsNa entry through apical Na channels depolarizes the apical membrane Apical membrane depolarization creates a favourable electrical gradient for K exit via apical K channels Expressed only in the apical membrane-Epithelial Na Channels-ENaC DCT2, CNT and Principal Cells

24. How does Na delivery affect K secretion? The rate of apical Na entry depends on the Na gradient across the apical membrane At low flow rates, [Na] in the lumen decreases as Na is reabsorbed At high flow rates, [Na] in the lumen does not decrease as Na is reabsorbed despite maximal rates of Na transport out of the lumen. Na is replaced as quickly as it is reabsorbed.

25. At high flow rate, [Na] in the lumen does not decrease despite high rates of Na reabsorption Increased Na delivery stimulates K secretion by maintaining a favourable Na gradient At high flow rate, [Na] in the lumen does not decrease despite high rates of Na reabsorption Increased Na delivery stimulates K secretion by maintaining a favourable Na gradient

26. Notice as Na delivery increases in the CCD, that potassium secretion increases. Then see the additional effect of aldosterone in regulating K secretion. Aldo is necessary for potassium secretion to occur and increases the amount of potassium secreted per cell for each increase in Na delivery.Notice as Na delivery increases in the CCD, that potassium secretion increases. Then see the additional effect of aldosterone in regulating K secretion. Aldo is necessary for potassium secretion to occur and increases the amount of potassium secreted per cell for each increase in Na delivery.

27. Aldosterone stimulates K secretion The primary purpose of aldosterone is to regulate K secretion ECF [K] regulates aldosterone release [Aldosterone] increases after meals in response to an increase in ECF [K] (<0.5 mM) Aldosterone increases K secretion for several hours to establish K balance Since Na delivery is normal, the total amount of K excreted is increased Primary Acute Effect Increases the number of ENaC in the apical membrane Stimulates the synthesis of the alpha subunit which combines with preformed beta and gamma subunits and translocates to the apical membrane Later and long-term effects Stimulates synthesis of Na-K-ATPase Stimulates synthesis of ROMK Basolateral membrane amplification and increase in cell volume The primary purpose of aldosterone is to regulate K secretion ECF [K] regulates aldosterone release [Aldosterone] increases after meals in response to an increase in ECF [K] (<0.5 mM) Aldosterone increases K secretion for several hours to establish K balance Since Na delivery is normal, the total amount of K excreted is increased Primary Acute Effect Increases the number of ENaC in the apical membrane Stimulates the synthesis of the alpha subunit which combines with preformed beta and gamma subunits and translocates to the apical membrane Later and long-term effects Stimulates synthesis of Na-K-ATPase Stimulates synthesis of ROMK Basolateral membrane amplification and increase in cell volume

29. Effects of Aldosterone Primary Acute Effect Increases the number of ENaC in the apical membrane Stimulates the synthesis of the alpha subunit which combines with preformed beta and gamma subunits and translocates to the apical membrane Stimulates the synthesis of the alpha subunit which combines with preformed beta and gamma subunits and translocates to the apical membrane

30. Longterm Effects of Aldosterone Stimulates synthesis of Na-K-ATPase Stimulates synthesis of ROMK- potassium channels Basolateral membrane amplification and cell volume increases due to increased synthesis of the Na-K-ATPase pump Basolateral membrane amplification and cell volume increases due to increased synthesis of the Na-K-ATPase pump

31. Normal or High Aldo + High Na Delivery is Pathophysiological

32. High Aldo + High Na DeliveryPathophysiological Increased Aldosterone + normal or increased Na delivery to the cortical collecting duct will dramatically stimulate K secretion K depletion (a reduction in total body K) will occur The effect on plasma [K] will depend on other factors that may affect K distribution

33. Changes in potassium excretion and [Aldo] in these three disorders

34. What are the changes in potassium excretion and [Aldo] in these three disorders?

35. Diuretics- mechanism of action

36. What are the changes in NaCl, water, K , Ca and Mg excretion, and BP caused by these diuretics relative to Normal and to each other?

37. Can you answer the questions in the course objectives handout? Can you draw a TAL cell, explain how it reabsorbs NaCl, K, Mg and Ca? Why doesn’t water follow? Can you identify the primary active step, carriers, and channels? Where is the defect in Gitelman’s syndrome?Can you describe the changes in ion transport in the segment and explain the underlying problem? If a patient is given furosemide can you describe the changes in electrolyte and water excretion and explain what causes them (direct and indirect effects)?