GLOMERULAR ULTRAFILTRATION-ANATOMY AND PHYSIOLOGY.

1. GLOMERULAR ULTRAFILTRATION-ANATOMY AND PHYSIOLOGY. Dr.AbhijitKishoreKorane.Department of Nephrology,Apollo Hospitals , Chennai.

2. HISTORY

3. HISTORY 384-322 BC:- ARISTOLE- Stated surplus liquid becomes separated from blood in red meat. 1843:- CARL LUDWIG- labeled golmerulus as a simple filter. 1897:- LUDWIG FILTRATION HYPOTHESIS, ( Hydraulic pressure across the glomerular epithelium must be slightly above the ureteral pressure.) 1924:- WEARNS AND RICHARDS. First time performed the renal micropuncture studies.

4. HISTORY • 1941:- Experiments on gunieapig established that glomerularultrafiltrate is essentially protein free. 6) VAN LEIW AND COWORKERS, Exogenous molecules <14 A are freely filtered, neutral molecules of <20A are freely filtered and molecules larger than 50A are excluded from the filtration(size selectivity). 7) 1968:- On SANGERS theory for movement of substances across the membrane. In a two component system containing solvent and a non-electrolyte solute, a trans capillary flux of water and solute, with each of the component driven by the force equivalent to its difference in free energy across the membrane.

5. FACTS ABOUT GLOMERULAR ULTRAFILTRATION • Human kidney:- 0.5% of the body weight. • Human kidney receives 20% of the total cardiac output. • Filtration fraction of capillaries in other organs:- 0.25%. • Filtration fraction in kidney capillaries:- 20%. • Surface area of filtration:- Total:- 1.2 meter square. Per glomerulus:- 0.6 mm square. • Hydraulic permeability of glomerular capillaries:- 2.5 to 4.0 micro liter/min/mm of hg.cm square. • Plasma transmitting in human kidneys in one contains 50,000gms of proteins. • Albumin excreted per day is less than 150mg. • Width of the filtration unit:- 360nm. • Thickness of GBM:- 200nm. • Width of filtration slit:- 39nm.

6. ANATOMY OF GLOMERULAR ULTRAFILTRATION BARRIER

7. ANATOMY OF GLOMERULAR ULTRAFILTRATION. The average diameter of the glomerulusin the human kidney is approximately 200micro mm. The filtration barrier between the blood and the urinary space is composed of:- 1) Fenestrated endothelium. 2) Glomerular basement membrane. 3) Slit pores between foot processes of the visceral epithelial cells.

8. GLOMERULAR ENDOTHELIAL CELLS Glomerular capillary from interior. • Glomerular capillaries are lined by a thin fenestrated layer of endothelial cells. • Endothelium is perforated by pores which in human kidney ranges from 70 to 100nm. • Fenestrated region of endothelium represents 54% of total surface area. • Thin diaphragms extends across the fenestrae.

9. GLOMERULAR ENDOTHELIAL CELLS(SURFACE CHARGE) Endothelium is negatively charged due to presence of, -Polyanionic surface glycoprotein's, -Podocalyxin, -Sialoprotein of glomerular epithelial cells.

10. GLOMERULAR ENDOTHELIAL CELLS SYNTHESISES Nitric oxide(EDRF) Endothelin-1 (vasodilator) (vasoconstrictor) VEGF:- - Receptors for VEGF are found on the glomerular endothelial cells. - VEGF affects microvascular permeability of the glomerular endothelial cells. - Increases endothelial cell permeability and induces formation of endothelial fenestrae. - Important in endothelial cell survival and repair of endothelial cells.

11. Podocytes are the largest cells of the Glomerulus.

12. VISCERALEPITHELIALCELLS-PODOCYTES.

13. PODOCYTES FOOT PROCESSES SLIT PORE ZO-1 protein is specific to the tight junctions

14. PODOCYTES

15. PROTEINS OF THE FILTRATION BARRIER • Proteins in filtration barrier. 1) Nephrin(NPHS1 gene on Chr.19) 2) CD2 associated protein(CD2AP). 3) Podocin. MEMBRANE COMPONENT IDENTIFIED ON THE SURFACE OF THE PODOCYTES • Negatively charged surface coat rich in sialic acid on the foot processes. • Podocalyxin. • C3b receptor, Heymann nephritis antigen, Gp330 or megalin. • Podoplanin- maintains shape of podocytes. VISCERAL epithelial cells are responsible for synthesis and maintenance of glomerular basement membrane

16. GLOMERULAR BASEMENT MEMBRANE Lamina rara externa Central dense layer, lamina densa. Lamina rara interna.

17. GLOMERULAR BASEMENT MEMBRANE Width of GBM, - Osawa and coworkers:- 315nm. - Jorgensen and Bentzon:-329nm. - Steffes and coworkers:- 373nm in males and 326 nm in females.

18. BIOCHEMICAL COMPOSITION OF GLOMERULAR BASEMENT MEMBRANE Composed of typeIV collagen, laminin, fibronectin, entactin, nidogen,heparinsulphateproteoglycans. Type IV collagen is the major constituent of GBM. Six chains alpha 1 to alpha 6 forms the GBM.Alpha 1 and 2 are most abundant and mutation of Alpha 3,4,5 are known to cause Alports syndrome. CHARGE ON THE GBM:- GBM is negatively charged. Ananionic site is demonstrated to be due to glycosaminoglycans rich in heparansulphate. GBM is both charge and size selective.

19. HYDRALLIC RESISTANCE FOR ULTRAFILTRATION 48% by the filtration slits between the glomerular epithelial foot processes 50% by the Basement membrane. 2% by capillary endothelium

20. THE ULTRAFILTERATION BARRIER Podocytes Filtration slit Lamina rara externa Lamina densa Lamina rara interna Type IV collagen Endothelium

21. HYDRALLIC PRESSURES IN GLOMERULAR CAPILLARIES AND THE BOWMANS SPACEThe Starling forces affecting the glomerular ultrafiltrationhe Starlin forces affecting the glomerular filtration

22. The Starling forces affecting the glomerular ultrafiltration

23. METHODS OF DETERMINATION OF GLOMERULAR PRESSURES INDIRECT TECNIQUE:- “STOP-FLOW TECHNIQUE using MICRO-PUNCTURES” (Pgc=60 to 65 mm of Hg)

24. METHODS OF DETERMINATION OF GLOMERULAR PRESSURES DIRECT ESTIMATE:- Trans capillary hydraulic pressure(∆P)of =Stop flow pressure+ Systemic colloid osmotic pressure Estimates ∆P of 70 to 90mm of hg. Direct estimation of hydraulic pressure in the single capillaries and the tubules- SERVO-NULL technique.(BRENNER and coworkers,Pgc-46mm Hg.) Hydraulic pressure in the proximal tubule is determined by the LANDIS technique first demonstrated in 1950. Thus estimation of hydrallic pressures and estimation of efferent arteriolar protein concentration can give hydraulic and oncotic pressures in the glomerularultrafiltration.

25. GLOMERULAR CAPILLARY COLLOID OSMOTIC PRESSURE (∏c)– The Filtration pressure equilibrium.

26. DETERMINATION OF THE GLOMERULAR ULTRAFILTRATION Rate of fluid movement across the capillary walls is determined by the STARLINGS EQUATION. Jv= K(∆P- ∆ ∏), Where, Jv= rate of fluid movement across the capillary wall K= hydraulic permeability of the filtration barrier. ∆P= trans capillary hydraulic pressure gradient. ∆ ∏= transcapillary colloid osmotic pressure gradient. ∆P= Pgc-Pt = Hydraulic pressure in – Hydraulic pressure in glomerular capillaries bowmans space.

27. ∆ ∏= ∏gc - ∏t = colloid pressure in glomerular – colloid pressure in capillary bowmans space Where ∏t is 0. Single nephronglomerular filtration rate(SNGFR). SNGFR= Ks(∆P- ∆ ∏), s= is product of surface area for filtration. SNGFR=KfPuf Kf= glomerularultrafiltration coefficient(product of surface area of filtration and hydraulic permeability of filtration barrier) Puf= difference of mean trans capillary and colloid osmotic pressure gradient.

28. ULTRAFILTRATION COEFFICIENT (Kf):- From number of studies in Munich-Wistar rats estimates of Kf averages 3.5+/-0.2 nl/min. When, SNGFR= Ultrafilteration coeifficent * net ultrafiltration (Kf) pressure(Puf) Thus, Ultrafiltration coefficient(Kf)= SNGFR/Puf. Deen and collegues were the first to provide measurment of Kf in rats.(4.8 nl/min mm of hg)

29. FILTRATION BARRIER AND FILTRATION OF MACROMOLECULES THE SIEVING COEFFICIENT:- The filtration of the macromolecules is quantified in terms of the SIEVING COEFFICENT. It is the ratio of various proteins in the Bowmans space to plasma proteins. SIEVING COEFFICIENT OF ALBUMIN= 0.062%, SIEVING COEFFICIENT of low mol.wt. proteins= 99%. Indicates size selectivity of the GLOMERULAR FILTRATION barrier.

30. Glomerular “Sieve”

31. GBM selectivity based on molecular size DEXTRAN SIEVING COEFFICIENT Permeability of the filtration barrier depends on the size selcecivity. Restriction to filtration of neutral dextran do not occur until the effective radius is 20A and approaches zero as dextran radii approaches 40A.

32. Size selectivity of neutral dextran:- Molecular radius of discrete dextran fraction is calibrated by the Stokes-Einstein radius. A value of 1.0 indicates a dextran clearance equal to that of Inulin. Size selectivity of GBM can be explained by:- • Isoporus model, • Heteroporus model. ISOPORUS MODEL:- Solutes are regarded as solid spheres whose movement across the equal sized pores take place by convection and diffusion and is interfered by, size, shape, charge and hydrodynamic effect related to the presence of nearby pore wall.

33. Thus the movement of the uncharged solute across the glomerular capillary depends on, 1) Concentration of solute in glomerular capillary plasma. 2) Diffusivity of solute in bulk solution. 3) Local glomerular Tran capillary volume flux. 4) Fraction of capillary surface area occupied by the pores. 5) Length of the pores. 6) Glomerular capillary ultra filtration coefficient. 7) The glomerular pore radius.

34. Hetereporus pore model:- The normal glomerular capillary wall behaves as a isoporus filter with a pore radius of about 50 to 55 A. But in a diseased glomerulus's there is appearance of second population of pores with larger radius(Deen and colleagues). The Shunt Pathways:- Increased in diseased glomerulus's, do not have size selectivity.

35. PORE DENSITY:- It is the apparent number of pores per unit area of Glomerular capillary wall. = Fraction of the capillary surface area occupied by the pore/Length of the pore. The PORE Theory:- The physical basis of molcularseiving is explained by using the pore theory. It is based on the KEDEM-KATCHALSKY flux equation.

36. KEDEM-KATCHALSKY flux equation Jv=Lp(∆P- σs∆ ∏), Js=Ps ∆Cs+Jv(1- σs)Čs. Jv:- Efflux per unit area of volume(water), Js:- Efflux per unit area of solute. ∆P:- gradient for hydrostatic pressure. ∆ ∏:- gradient for osmotic pressure. ∆C:- concentration difference across the membrane. σs –reflection coefficient of the membrane for s. Lp:- hydraulic permeability per unit area of the membrane. Ps:- permeability of the membrane to the solute. Čs:- mean concentration of solute(s). Filtration barrier allowing transfer of solutes depends on, membrane geometry, stokes Einsten radius of solutes, temperature, viscosity.

37. Charge selectivity of glomerular capillary wall • In normal kidney fractional dextran clearance is less than neutral dextran, conversely positively charged molecules are freely filtered at the same radius. • Graph shows fractional clearance of neutral dextran against molecular size.

38. Charge selectivity of glomerular capillary wall Anionic Dextrin sulphate Cataionic Dextran sulphate

39. Selectivity of glomerular capillary wall based on molecular configuration To compare the Seving coefficient of the molecules with different conformations, the effect of molecular shape or configuration must be taken into account Bohers and coworkers, compared excretion of neutral dextran with Ficoll(copolymers of sucrose) and epichlohydrin, and found that at any given effective radius, the flexible coils of dextrans are filtered more rapidly than ficoll.

40. Which is more important:Size/Shape or Charge? • Estimates from Mathematical Models: – 30% ↓ in charge density → 25-fold ↑ in albumin filtration. – 100% ↑ in “pore” radius → 5-fold ↑ in albumin filtration. • Loss of fixed negative charges probably results in a physical rearrangement of proteins that contribute to the size barrier. • But does this really matter ? Not really as both size- and components of the filtration barrier are compromised → protein in the ultrafiltrate • Proteinuria (or albuminuria) is the hallmark of glomerular injury!

41. Primary determinants of Glomerular ultrafiltration. Primary determinants of glomerularultrafiltration are:- 1) Glomerular plasma flow rate(Qa), 2) Transcapillary hydraulic pressure differences (∆P), 3) Glomerular capillary ultrafiltration coefficient(Kf), 4) Colloid osmotic pressure(∆ ∏). • Glomerularplama flow rate(Qa):- Increase in Qa is associated with increase in SNGFR. In initial stages there is proportionate increase in SNGFR but further increase in Qa is associated with proportionately lower increase in SNGFR as filtration disequilibrium is reached.

42. Relation of Glomerular plasma flow rate and SNGFR

43. TRANSCAPILLARY HYDRALLIC PRESSURE DIFFRENCE(∆P) Increase in transcapillary hydraulic pressure Increase in SNGFR Rate of rise of SNGFR is not linear, because of filtration disequilibrium(associates increase in colloid osmotic pressure).

44. Relation of ∆P with SNGFR

45. Relation of Kf and SNGFR • Kf depends upon glomerular capillary hydraulic pressure(inverse relation between PGc and Kf), increase in hydraulic pressure shifts the filtration pressure disequilibrium to efferent arteriole and decreases in hydraulic pressure shifts the disequilibrium towards afferent arteriole.

46. GLOMERULAR CAPILLARY ULTRAFILTRATION COEFFICIENT

47. COLLOID OSMOTIC PRESSURE(∆ ∏) SNGFR vary reciprocally with colloid osmotic pressure. These experiments are done with keeping values of Kf, Qa, ∆ P constant. Although decrease in ∏a is not associated with increase in SNGFR due to decrease in Kf which is due to increase in Puf pressure secondary to decrease in colloid oncotic pressure and decrease in ultrafiltration coefficient.

48. SECONDARY DETERMINANT OF GLOMERULAR ULTRAFILTRATION Pre and post glomerular resistance Studies in Munich-Wistar rats has shown large pressure drops between renal artery and the glomerulus. Major drop occur between, Renal artery and glomerulus(25mm of hg). Glomerular capillaries and efferent arterioles. This models are used in studying effect of different vasoactive substances on afferent and efferent vessels.

49. CONTROL OF GLOMERULAR ULTRAFILTRATION BY HORMONES AND VASOACTIVE SUBSTANCES A variety of hormones and vasoactive influence glomerularultrafiitration by affecting, 1) Pre and post glomerular resistance, 2) Glomerular hydraulic pressure, 3) Glomerulartranscapillary hydraulic pressure gradient. A variety of growth factors can affect filtration by promoting mesangial cell proliferation and expansion of mesangial cell matrix, leading to obliteration of capillary loops and reduction of ultrafiltration coefficient. Vasoactive substances act on podocytes leading to decrease in number or reduction of the size of the filration pore, thereby reducing conductivity of the filtration barrier.

50. Renal Vasoconstrictors Renal vasculature and mesangium responds to variety of vasoconstrictors, reducing the glomerularultrafiltration coefficient. • ANGIOTENSIN II:- Plays an important role in regulating glomerular plasma flow rate and filtration rate. Renin release depends upon intracellular calcium levels, cGMP and cAMP levels.