1. Biochemistry of the kidney mirka.rovenska@lfmotol.cuni.cz

2. The main roles of the kidney  Participation in amino acid / nitrogen metabolism  Excretion of water, salts, and waste products (urea, uric acid…)  Regulation of electrolyte and acid-base balance (homeostasis)  Participation in biosyntheses (creatine, carnitine, glucose, AA)  Participation in hormonal regulation

3. AA/nitrogen metabolism during fasting  During fasting, AA are released from skeletal muscle: Ala, Gln  Sk. muscle oxidizes Val, Ile, Leu to produce energy and Gln  The amino groups of Val, Ile, Leu, Asp, and Glu are transferred out of skeletal muscle in Ala and Gln Ala supplies gluconeo- genesis in the liver, Gln is taken up by the kidney and gut

4. Branched-chain AA  BCAA are the major AA that can be oxidized in tissues other than liver  α- keto acids of the BCAA are either released into the blood and taken up by liver, or oxidized to CO 2, or con- verted to Gln

5. Gln and Ala as nitrogen carriers  Gln is generated mainly in the skeletal muscle from Leu, Ile, Val, and also by the lungs and brain from the removal of NH 4 + : Glu + ATP + NH 4 +  Gln + H 2 O + ADP + P  The kidney, the gut, and cells with rapid turnover rate (e.g. cells of the immune system) are the major sites of Gln uptake  Here, Gln serves as a fuel and a nitrogen donor for syntheses; in the kidney, the glutaminase reaction is particularly important  Much of the unused nitrogen from Gln is built into Ala which carries the nitrogen to the liver where it is converted to urea

6. Ammonia release in the kidney  1) By glutaminase  2) By glutamate dehydrogenase  α -ketoglutarate is used as a fuel by the kidney: oxidized to CO 2 or converted to glucose

7. Glutamine as a fuel for the kidney  The carbon skeleton of Gln forms α- ketoglutarate, which is oxidized to CO 2, converted to Glc, or released as S or A  Glucose is used principally by the cells of the renal medulla  Lactate is oxidized in renal cortical cells which have a higher mitochondrial capacity and a greater blood supply

8. Ammonium is excreted into the urine

9. Kidney in the regulation of the acid-base balance

10. H + ion secretion  In the proximal tubules, H + ions are secreted by secondary active transport with Na + :  In the distal tubules and collecting duct, H + ions are secreted by H + -ATPase.

11. Removal of H + ions from tubular fluid  Reaction with ammonia  Reaction with HCO 3 -  Reaction with HPO 4 2- to form H 2 PO 4 -

12. Proton removal by ammonia  NH 3 diffuses through the membrane of the renal tubule cells into the urine  Ammonia increases proton excretion by combining with a proton to form ammonium ion  Shunting Gln from liver to kidneys also conserves bicarbonate (needed for urea formation)

13. Glutaminase reaction in the regulation of acid-base balance  In the kidney, NH 3 carried by Gln is excreted into the urine  This process removes protons formed during fuel oxidation and helps to maintain the body‘s pH  During metabolic acidosis: the kidney becomes the predominant site of Gln uptake the activities of renal glutaminase and Glu-dh increase the urinary excretion of NH 4 + increases severalfold the liver synthesizes less urea, making more Gln available for the kidney

14. Reaction of H + with HCO 3 -  Secreted H + ions promote HCO 3 - resorption: H + react with HCO 3 - in the primary urine to form H 2 O and CO 2, which diffuses back to the tubule cells and then into the blood

15. Excretion by the kidney

16. Urine formation   180 l of primary urine is filtered out of the 1500 l of blood flowing through the kidneys every day  Primary urine is concentra- ted and 0,5-2 l of final urine is excreted per day  Concentrating urine and transporting it through membranes require large amount of energy (ATP)

17. Urine  95% water  Organic components: urea uric acid creatinine AA and their derivatives (e.g. hippurate) metabolites of hormones xenobiotics pigments  Inorganic ions (Na + and Cl - represent 2/3 of electrolytes)

18. Creatinine excretion  Creatine phosphate can readily regenerate ATP:  Creatine phosphate is unstable and spontaneously cyclizes, forming creatinine, which is excreted in the urine

19. Creatinine as an indicator  The amount of creatinine excreted each day is constant and depends on body muscle mass  It can be used as a gauge for urinary excretion of other compounds and as an indicator of renal excretory function Creatinine clearance (U Cr x V) / P Cr or estimates based on serum Cr are used to assess the glomerular filtration rate  The amount of Cr can also be used to determine whether the sample truly represents a whole day‘s urinary output (140 – age). mass [kg] E.g.: C Cr = 48,9. serum Cr [µmol/l]

20. Regulation of the electrolyte balance

21. Electrolyte and water resorption CompoundResorption [%]Site of resorption Na + 99,4PT, HL, DT, CD K+K+ 93,3PT, HL, DT, CD Cl - 99,2PT, HL, DT, CD HCO 3 - 100PT, DT Urea53PT, HL, DT, CD Uric acid98PT Glucose100PT Water99,4PT, HL, DT, CD

22.  Calcium and phosphate ions: almost completely resorbed by active transport; resorption is regulated by:  parathormone – produced by the parathyroid gland,  Ca 2+ resorption,  phosphate resorption  calcitonin – produced by the thyroid gland,  resorption of both Ca 2+ and phosphate  calcitriol – formed in the kidney;  resorption of both Ca 2+ and phosphate

23.  Sodium ions: More than 97% resorbed – by passive transport as well as secondary active transport Resorption is regulated by:  aldosterone –  Na + reuptake  atrial natriuretic peptide (ANP) –  Na + reuptake  angiotensin II – increases resorption of Na + and HCO 3 - in the proximal tubule (see below)  Water: passively resorbed (water follows the osmotically active particles, particularly Na + ) regulated by ADH in the collecting duct: ADH stimulates the transfer of aquaporins into the plasma membrane

24. ADH (vasopressin)  Nonapeptide released by the posterior pituitary in response to stimuli from baroreceptors and osmoreceptors, which sense a fall in blood pressure or a rise in extracellular Na +  Stimulates aquaporin insertion in the luminal PM:

25. Diabetes insipidus  In the absence of ADH, the cells of the collecting duct are relatively impermeable for water and the diuresis increases  Causes of diabetes insipidus: failure to produce ADH (e.g. due to damage of the pituitary gland or hypothalamus) inability of the kidney to response to ADH:  mutation in the gene encoding for the receptor  mutation in the gene encoding for aquaporin 2

26. Biosyntheses in the kidney

27. Gluconeogenesis in the kidney  Under the more extreme condi- tions of starvation (2 days and more), renal gluconeogenesis also becomes significant  Glc produced by the kidney cortex is used mainly by the kidney medulla, but some may enter the bloodstream to supply brain and RBCs  The main substrate: AA (Gln)  Regulated by cortisol

28. Serine biosynthesis  Serine biosynthesis: the major sites are the liver and kidney

29. Conversion of Gly to oxalate  Gly is degraded to glyoxylate which can be oxidized to oxalate  Oxalate is sparingly soluble and tends to precipitate in kidney tubules, leading to formation of kidney stones

30. Arg synthesis by the gut and the kidney  The gut releases citrulline which is converted to Arg in the kidney  Arg can be converted to creatine in the kidney or released into the blood  The liver uses Arg to generate ornithine for the urea synthesis

31. Creatine synthesis  Creatine synthesis begins in the kidney and is completed in the liver  Creatine travels through the bloodstream to other tissues, particularly skeletal muscle, heart, brain

32. Carnitine synthesis  Carnitine is derived from Lys residues which are N-methylated to form trimethyllysyl residues  Trimethyllysine is released upon protein degradation  Kidney and to a lesser extent liver carry out the complete pathway and supply other tissues, espe- cially muscle and heart

33. Secretory function of the kidney Participation in the hormonal regulation

34.  The kidney secretes: renin 1,25-dihydroxycholecalciferol (vitamin D, calcitriol) erythropoietin

35. Renin-angiotensin system  Generates angiotensin II  Renin – aspartic protease formed in the kidney; acts on angiotensinogen in blood  ACE – found on the surface of endothelial cells, mainly pulmonary and renal

36. The effects of AngII lead to increased blood pressure and sodium and water retention

37. Aldosterone  Producing cells: adrenal zona glomerulosa  Functions: causes Na + reabsorption in the kidney, mainly in the distal tubule causes water retention raises blood pressure by increasing fluid volume

38. Pharmacological intervention  The renin-angiotensin system can be influenced by: angiotensin I analogs that inhibit ACE antagonists that block the binding of AngII to its receptors inhibitors of renin  These drugs decrease blood pressure Captopril: ACE inhibitor

39. Calcitriol  7-dehydrocholesterol is activated in the skin by sunlight to generate vitamin D 3 (cholecalciferol), which is hydroxylated: in the liver in position 25 then in the kidney in position 1 C-C bond cleavage opens the B-ring

40.  Hormone-receptor complex binds to response elements in DNA  Result: increased rate of transcription of genes that encode a number of Ca 2+ -binding proteins (calbindins, Ca 2+ -ATPase…) stimulates the absorption of Ca 2+ and phosphate from the intestinal lumen also plays an important role in bone mineralization in the kidney, weakly stimulates Ca 2+ reabsorption

41. Erythropoietin (EPO)  Decreased supply of oxygen to the tissues signals the kidney to release erythropoietin  In bone marrow, erythropoietin (glycoprotein) stimulates the multiplication and maturation of erythroid progenitors Reticulocyte: the nucleus is extruded, but ribosomes+mRNA are retained and enable the synthesis of Hb

42. Renal eicosanoids  PGE 2, PGI 2 dilate renal blood vessels and increase blood flow. PGE 2, PGD 2 increase water and salt excretion.  20-HETE regulates Na + excretion and diuresis  12- and 15-HETE influence the renin-angiotensin system (they probably mediate the feed-back inhibition of renin) PGE 2