Kidney and acid base physiology : part 3

Distal tubule (DT) and collecting duct (CD)

Distal tubule and the collecting duct are the chief sites of K⁺ secretion. Along with that about 7% of filtered NaCl and about 10-15 % of water (in principal cells only in the presence of ADH hormone) is also absorbed here. As for the details, let us just go through the following points and then compare with the previous two sections:

1.      The key player is Na⁺-Cl^- symporter in the early Distal tubule which moves NaCl inside the cell.

2.      This segment is totally impermeable to water hence its name ‘cortical diluting segment.

3.      Thiazide diuretics also act here and inhibit NaCl transport inside the cell.

4.      Principal cells are involved in the potassium secretion where it reaches a high concentration via Na⁺/K⁺ ATPase.

5.      Na⁺ also gets absorbed here through epithelial sodium channels (ENaC) channel present on the apical surface.

6.      The inward movement of Na results in a relatively low voltage in the lumen as compared to the voltage across the basolateral surface. This lumen negative transepithelial voltage acts as a driving force for K⁺ from blood to the lumen via voltage gated K⁺ channels on the apical surface (which are inhibited by high cytosolic Ca⁺⁺ or H^+.

7.      Sensitivity of the K channels in the principal cells can explain stimulation of K⁺ excretion during metabolic alkalosis or increased bicarbonate excretion.

8.      Intercalated cells have very less Na/K ATPase activity and absolutely no conductance channel on the apical surface for ions; all they possess is gastric parietal type H⁺/K⁺ ATPases which are active constitutively and Colonic type H⁺/K⁺ ATPases which get activated only during dietary NaCl depletion, potassium depletion and acidotic condition.

9.      In the intercalated cells of the CD and late DT, H⁺-ATPase and H⁺/K⁺ ATPase are present on the apical membrane to facilitate the H⁺ ion exit from the cell into the lumen.

10.  The HCO₃^- leave the cell via AE1 (as described previously). Aldosterone increase the H⁺ secretion by stimulating H⁺-ATPase.

11.  Resorption of K⁺ ions occurs only when there is a need for K⁺ conservation. On dietary loading , area of basolateral surface and activity of Na⁺/K⁺ ATPase increases.

12.  Stimulation of K transport by mineralocorticoids

a.       Activate Na/K pump

b.      Increased rate of electrogenic cationic exchange

c.       Hyperpolarisation of cell negative basolateral membrane voltage

13.  There are two types of K⁺ channels on the apical membrane: the secretory type ( with a small conductance and a high open probability; inhibited by increased cytosolic H⁺ and Ca⁺⁺ concentration) and maxi-K⁺ type (large conductance and low open probability; high cytosolic Ca⁺⁺ concentration activate this channel).

14.  Amiloride inhibits K⁺ secretion by reducing the lumen - negative transepithelial voltage by blocking the Na⁺ channels from the luminal side.

15.  Aldosterone penetrates the cell from the interstitial side and combines with the mineralocorticoid receptor MR. This complex enters the nucleus and promotes mRNA synthesis which then directs the synthesis of Aldosterone-induced-proteins (AIP).

16.  The AIPs include Na+ channels and Na/K-ATPase and increase ATP production by mitochondria. All these act to promote sodium absorption and increase K+ and H+ secretion.

17.  Spironolactone binds to MR and prevents aldosterone effects.

Kidney and acid base physiology:part 2

Transport of ions at the loop of Henle

Water is absorbed passively at the thin descending limb of the loop of Henle and is (?) accompanied by diffusion of sodium ions in the loop of Henle.

Limited absorption of NaCl can occur through the thin ascending limb passively.

Key players at The Thick Ascending limb

As in the previous post, the accompanying diagram is self explanatory and the steps involved in the H⁺/ HCO₃^- transport and Na⁺/K⁺ movement are more or less same barring a few exceptions which can be summarized as follows:

 i.                    About 15% of the filtered HCO₃^- can be reabsorbed here at the basolateral  (BL) surface.

ii.                  The Na⁺-HCO₃- cotransporter at the BL surface is NBCn1.

iii.            The HCO₃- can exit the cell in exchange for Cl^- (Anion Exchanger2, AE2) or by a K⁺-HCO₃ symporter.

iv.                There is no Carbonic anhydrase in the brush border (luminal side).

v.        The most remarkable difference is the presence of Na⁺-K⁺-2Cl^- cotransporter here which is responsible for the downhill transport of Na⁺ and Cl^- and uphill transport of K⁺ inside the cell.

vi.                K⁺ and Cl^- ions exit at the basolateral surface through separate channels whereas Na⁺ leaves the cell primarily through the Na⁺/K⁺ ATPase.

vii.              Some K+ is able to leak through the apical surface into the lumen and results in a slightly positive (+6 mV) inside the lumen. This acts as a driving force for cations like Na⁺, Mg⁺⁺, Ca⁺⁺ and even K⁺ to pass through a paracellular route to capillaries.

viii.            This segment is totally impermeable to water so known as the ‘diluting segment’.

ix.           Thus 20% Na⁺ and K⁺ are reabsorbed in this segment where half of that Na⁺ absorption is passive (paracellular) and the rest is active.

x.                  The site of action of Furosemide is the Na⁺-K⁺-2Cl⁺ cotransporter to which it reaches through the luminal side after being secreted in the PT by organic anion transport (shown in the diagram in the previous post).

Kidney and acid- base physiology:part 1

As we move up the ladder, I feel that it is the right time to discuss the role of kidney in maintaining the acid base balance. Just as the gut is important for HCO₃^-  secretion , kidney plays a role in hydrogen ion removal from the body. Each day the bicarbonate loss in the faeces imparts an acid load (Net endogenous acid production, NEAP) to the body which is in a way made up for by the kidney’s acid secretion (Renal net acid excretion, RNAE).

Thus a balance is reached in normal physiological conditions when NEAP equals the RNAE.

As I was about to start with this topic, I found that sodium-potassium transport is invariably linked to and coexists with the H⁺/HCO₃^- movement in/out of the tubular cells. I felt that this is the right time when I highlight the site of action of various diuretics too.

The normal HCO₃^- in plasma is about 24 meq/l and GFR is 180 l/day, so the filtered load of the bicarbonate is about (180*24=4300 meq/day). Approximately 80 % of the HCO₃^- is reabsorbed in the proximal tubule (PT) with the thick ascending limb (ThAL) and distal convoluted tubule (DT) contributing to additional 16 % reabsorption. The remaining bicarbonate is reabsorbed by the collecting duct (CD).

The following diagram depicting the reabsorption of the various ions at basolateral surface and secretion at the apical luminal surface is self -explanatory.

The salient features of the events at (proximal tubule) PT are as follows:

1. Main transporter for H⁺ ion secretion at the PT are H⁺ - ATPase and Na⁺/H⁺ antiporter (exchanger; NHE3).

2. The NHE3  is responsible for driving approximately 2/3 of HCO₃^-  reabsorption at the corresponding basolateral surface; rest 1/3 is contributed by the H⁺ - ATPase.

3.      The H+ are generated inside the cell by the activity of Carbonic anhydrase (II).

4.    Carbonic anhydrase (IV) is present in the brushborder at the apical surface also where it catalyses the production of H₂O and CO₂ from the luminal carbonic acid.

5.      Thus the bicarbonate in the tubular fluid are neutralized by the H⁺ secreted at the apical surface inside the lumen and there is actually no change in the pH of the tubular fluid.

6.      The HCO₃^- which is reabsorbed at the basolateral surface is actually a new molecule; for one H⁺ secreted from the cell, one molecule of HCO₃^- is reabsorbed in the blood.

7.      The HCO₃^- transporters at the basolateral surface are 3HCO₃^--Na⁺ symporter (NBCe1) mainly and HCO₃^-/Cl^- exchanger (to some extent).

8.      The Na⁺ entering the cell by NHE3 at the apical surface is extruded out by Na⁺-K⁺ ATPase athe basolateral surface.

9.      The Na⁺ movement inside the cell at the apical surface is also coupled with glucose cotransport (SGLT-1, SGLT-2 at late and early PT respectively). At the basolateral surface, the carrier for the facilitated diffusion of glucose are  GLUT-2,GLUT-1 at early and late PT respectively).

10.  The glucose, lactate, phosphates and amino acids are completely reabsorbed in the PT.

11.     The K⁺ and Cl^- entering the cell are extruded or reabsorbed at the basolateral surface by separate K⁺-Cl^- cotransporter.

12.     Na⁺, K⁺, Cl^- are also reabsorbed through paracellular pathways and the lateral surface of the PT cells have selective K⁺ channels for the K⁺ ion movement into the cells.

13.     More water is reabsorbed in the early PT as compared to Cl^- so Cl^- concentration goes on increasing and is reltively higher in the late PT. This creates a concentration gradient for the Cl^- ions which diffuse to the interior through paracellular pathway leaving the lumen slightly positive. This positivity then drags the Na⁺ to diffuse in the blood.

14.     Thus about 67% of filtered Na⁺, K⁺, Cl^- are reabsorbed at the PT along with water.

Acid base and GIT: part 2

The pancreatic secretion of electrolytes

The aqueous component of pancreatic cells is contributed by acinar cells ( isotonic, rich in Na, K, Cl and bicarbonates, stimulated by CCK and Ach; concentration similar to that in plasma), intralobular ductal cells (bicarbonates double and chlorides less of that in plasma, spontaneous secretion) and extralobular ductal secretion ( rich in Bicarbonates and poor in chlorides, stimulus is SECRETIN)

The basolateral transporters are called house keepers as they maintain the normal pH and maintain the cell  membrane volatage.

ref: Kim D, Steward MC. The role of CFTR in bicarbonate secretion by pancreatic duct and airway epithelia. J Med Invest. 2009;56 Suppl:336-42. Review

Secretin secretion from the duodenal S cells is stimulated by gastric distension and acid secretion of stomach  that signals the ductal cells to secrete bicarbonate-rich, clear, watery fluid ( hydraulic secretion).

The ''exocrine'' pancreas is an abdominal “salivary gland” and releases 1.5 l of pancreatic juice daily, with the pH increasing with increased secretion rate. The maximal secretion rate about 20-50 µl/min. The pancreatic juice is a clear fluid, isosmolar with plasma. Bicarbonates in the pancreatic secretion can approximate to the [H⁺] in gastric juice (150 mM). Pancreatic juice (pH 8) thus buffers the extremely acid gastric juice and protects the duodenal mucosa.

At high secretory rate bicarbonates increase and chloride decreases and vice-versa. Thus both share a reciprocal relationship.

This is because at low flow rates, the pancreatic juice which is rich in Na and Cl comes from the acinar and ductal cells whereas at high flow rates, the proportion of ductal cell secretion rises which has got a high HCO₃ concentration.

Thus Bicarbonate secretion can vary from 80 to 120 mEq/l depending on the secretory rate. Na and K secretion (unlike saliva) are similar to plasma and remain unaffected by the secretory rate.

A schema of transporters including the SKOU pump in pancreatic ductal cell

The secretion of HCO₃ being the primary process, can be understood as:

A: Entry of Bicarbonate in to the duct cell    

i.                    Through Na/ HCO₃ cotranspoter (NBC) located on the basolateral membrane.

ii.                  Through the H₂ CO₃  reaction inside the cell through Carbonic anhydrase catalysed reaction where CO₂ + H₂O  combine to form H₂CO₃ and then breaking into H⁺ and HCO₃^-. The H+ are extruded through either an active H+-ATPase pump or by Na/ H exchanger.

B: Release of HCO₃ into the lumen

1.         Depends on Cl- transport across the apical membrane (as seen to be defective in cystic fibrosis).
2.          Secretin opens the Chloride channels which is proved by 20- fold decrease in the resistance of the apical membrane to chloride ions. The chloride channels can be CFTR Cl^- channels or Ca ⁺² sensitive Cl^- channels.
3.    The chloride recycles through the Cl/ HCO₃ exchanger which extrudes the HCO₃ ions into the lumen.
4.     A rise in [Ca²⁺] opens up luminal chloride channels and a basolateral K channel through which these ions can leave the cell.
5.       The fall in intracellular Cl and K activates a basolateral Na/K/Cl cotransporter through which NaCl enters the cell.
6.      It is followed by paracellular transport of Na and accompanied by H₂O (the acinar lumen has  about -6mV potential)
7.     If the luminal [Cl-] concentration falls or there is a reduction in the activity of Cl/HCO₃ exchanger, then HCO₃ can be extruded from the CFTR channels directly into the lumen.

        i.           

C: Holding of HCO₃ in the lumen

Because of electrical gradient, HCO₃ has a tendency to go back in the cell at 145 mM concentration.The IC concentration of HCO₃ is about 10-15 mM and the cell membrane potential is about  -60mV (even at max stimulation??).Another important event which takes place during this instance is the inactivation/ closure of basolateral Cl/ HCO₃ exchanger ( I have not drawn in this figure). This decreases Cl concentration in the lumen and favors HCO₃ extrusion through the CFTR channel which otherwise shows only 25 % conductance for HCO₃ as compared to Cl conductance.

suggested reading:

1. secretin receptors

2. VIP and Secretin

3. Potassium ion in pancreatic secretion

Acid Base and GIT: part 1

GIT and Acid base homeostasis

Acid- base changes in the GIT are actually in sync and infact only to bring about efficient absorption of the dietary component and hence do not play a role in acid-base homeostasis (unlike kidney). The small amount of alkali lost as a by-product of acid-base transport events in the guts is easily regenerated by renal net acid excretion. Abnormal gut function brings about a upheaval in maintenance of acid-base homeostasis.

Normal Physiology of Gut Fluid and Electrolyte Transport

During the course of each day, secretion as well as absorption of fluid and electrolytes occurs along the gastrointestinal tract.

Normally 7 to 8 L of fluid is secreted each day, far exceeding dietary consumption, and almost all of these secretions, as well as any ingested fluid, are absorbed by the end of the colon.

Saliva

Via parotid, submandibular, and sublingual glands; in addition, there are many very small buccal glands. The parotid glands secrete almost entirely the serous type of secretion, while the submandibular and sublingual glands secrete both serous secretion and mucus. The buccal glands secrete only mucus. The submandibular glands contribute about two thirds of resting salivary secretion, the parotid glands about one fourth, and the sublingual glands the remainder. Saliva has a pH between 6.0 and 7.0, a favorable range for the digestive action of ptyalin.

At rest salivary secretion is low, amounting to about 30 mL/hr. Stimulation increases the rate of salivary secretion, most notably in the parotid glands, up to 400 mL/hr. The most potent stimuli for salivary secretion are acidic-tasting substances ( citric acid),  smell of food and chewing. Anxiety, fear, dehydration, and medications (e.g., antihistamines) inhibit secretion.

Primary secretion (from the acini)  is isotonic as plasma as if formed as a result of ultrafiltration. But actually as the final secretion (ductular) occurs, Cl^- and Na⁺  are reabsorbed and and K⁺ and HCO₃^-  are secreted (via CFTR channel??). The changes in the ductular cells result in a hypotonic secretion. When salivary flow is rapid, there is less time for ionic composition to change in the ducts.  Aldosterone increases the K⁺ concentration and reduces the Na⁺ concentration of saliva in an action analogous to its action on the kidneys.

The hypotonicity facilitates taste sensitivity and results in various organic compounds form a protective coating on the oral mucosa. Salivary bicarbonate , calcium and phosphate act as buffer and  neutralize acids that would otherwise destroy tooth minerals 

For details readers are advised to read an excellent paper by Melvin JE (1999)

Gastric secretion of acid

Acid is secreted by parietal cells. Various transport proteins are present in the parietal cells.

1.       H⁺/ K⁺ ATPase

2.       K⁺ selective channel

3.       Cl^- channel

4.       Cl^-/ HCO₃^- exchanger  :   At the serosal surface

5.       Na⁺/ K⁺ ATPase   :     At the baso- lateral surface  

The pH and volume of the gastric secretion is regulated primarily by gastrin so maximum acid secretion occurs only after a meal [ pH= 1, volume= 7 ml/ min].

A fall in pH also increases the amount of HCO₃ absorbed [ alkaline tide]. Eventually HCO₃^- secreted back into GI tract by pancreas.

It is believed that K⁺ is merely recycled at the canalicular membrane on the luminal side otherwise intracellular K⁺ levels may rise to dangerous levels.

         

Rest of  the details  will be discussed in the next post.

Poorvi

Ammonia buffer

Ammonia buffer

Most ammonia (60%) is synthesized in proximal tubule cells by deamidation and deamination of the amino acid glutamine [rest comes from glycine and alanine].

Ammonia is secreted into the urine by two mechanisms.

1.   As NH₃ (because of its lipid soluble nature), it diffuses into the tubular urine; as NH₄ ⁺, it substitutes for H⁺ on the Na⁺/H⁺ exchanger. In the lumen, NH₃ combines with secreted H⁺ to form NH₄⁺, which is excreted [because of its polar nature and therefore can not diffuse back into the tubular epithelium].

For each mEq of H⁺ excreted as NH₄⁺, one mEq of new HCO₃^_ is added to the blood. The hydration of CO₂ in the tubule cell produces H⁺ and HCO₃ ^_.

2.   Two H⁺s are consumed when the anion α₂-ketoglutarate^2_ is converted into CO₂ and water or into glucose in the cell. The new HCO₃^_ returns to the blood along with Na⁺.

If excess acid is added to the body, urinary ammonia excretion is increased for two reasons.

·        More acidic urine traps more ammonia (as NH₄⁺) in the urine.

·        Renal ammonia synthesis from glutamine increases over several days which is a lifesaving adaptation because it allows the kidneys to remove large H⁺ excesses and add more new HCO₃^_ to the blood. With severe metabolic acidosis, ammonia excretion may increase almost 10-fold.

Renal ammonia synthesis is increased through an increase in the glutaminase activity.

Phosphate buffer and protein buffers

Phosphate buffer

The pKa for phosphate, H₂PO₄ , is 6.8, close to the desired blood pH of 7.4, so phosphate is a good buffer.

 In the ECF, phosphate is present as inorganic phosphate in a very low concentration  (about 1 – 2 mmol/L), so it plays a minor role in extracellular buffering.

Phosphate is an important intracellular buffer  for the reasons:

1.    Cells contain large amounts of phosphate in such organic compounds as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and creatine phosphate. Although these compounds primarily function in energy metabolism, they also act as pH buffers.

2.    Intracellular pH is generally lower than the pH of ECF and is closer to the pKa of phosphate which is 6.8 (e.g.The cytosol of skeletal muscle has a pH of 6.9.).

Phosphoric acid is triprotic weak acid and has a pKa value for each of the three dissociations:

	pKa1 = 2		pKa2 = 6.8		pKa3 = 12
H3PO4	<= = = >	H+ + H2PO4-	<= = =>	H+ + HPO4-2	< = = = >	PO4-3 + H+

The three pKa values are sufficiently different so that at any one pH only the members of a single conjugate pair are present in significant concentrations.

The pKa2 is 6.8 and this makes the closed phosphate buffer system a good buffer intracellularly and in urine. The pH of glomerular ultrafiltrate is 7.4 and therefore phosphate is predominantly in the monohydrogen form so as to  combine with more H⁺ in the renal tubules efficiently. This makes the phosphate buffer more effective in buffering against a drop in pH than a rise in pH.

Protein buffer

Proteins act as efficient buffers because of their amino acid structure which  have a central carbon with four groups off of it.

The carboxyl and amino groups are what enable proteins to act as buffers.

Protein buffers in blood include haemoglobin (150g/l) and plasma proteins (70g/l). Buffering is by the imidazole group of the histidine residues which has a pKa of about 6.8. This is suitable for effective buffering at physiological pH (7.4).

Haemoglobin is quantitatively about 6 times more important than the plasma proteins as it is present in about twice the concentration and contains about three times the number of histidine residues per molecule. For example if blood pH changed from 7.5 to 6.5, haemoglobin would buffer 27.5 mmol/l of H⁺ and total plasma protein buffering would account for only 4.2 mmol/l of H⁺.

Deoxyhaemoglobin is a more effective buffer than oxyhaemoglobin and this change in buffer capacity contributes about 30% of the Haldane effect. The major factor accounting for the Haldane effect in CO₂ transport is the much greater ability of deoxyhaemoglobin to form carbamino compounds.

Haemoglobin has a special place in the pHstabilizing mechanisms of blood because

(a)  Haemoglobin and oxyhaemoglobin have different iso-electric points and different ionization constants so that at the pH of blood, absorption of about 0.7 g. of hydrogen ion [from carbonic acid mainly] (and the release of an equivalent amount of potassium ion in exchange) takes place during liberation of oxygen from 1 g molecule of oxyhemoglobin.

(b)  (b) Haemoglobin, more easily than other proteins, reacts reversibly with carbonic acid to form a still weaker carbaminoacid; and an increasing accumulation of carbamino-haemoglobin as the blood passes through the capillaries and a sharp reversal when, in the lungs, the equilibrium is disturbed, carbon dioxide being excreted, carbonic acid converted to carbon dioxide under the influence of carbonic anhydrase, and, consequently, carbaminohaemoglobin  is decomposed.

(Frazer &Stewart, J. clin. Path. (1959), 12, 195-206)

For more details readers are advised to go through the above mentioned paper and then directed to the link http://www.madsci.org/posts