L12 - Molecular composition and functions of the basement membrane Flashcards
Last two slides missing, proteinuria figures not added
Permselectivity definition
Restriction of permeation of macromolecules across a glomerular capillary wall on the basis of molecular size, charge, and physical configuration.
Factors that govern permselectivity of glomerular barrier
result of supramolecular organization of 3 major components:
1) Podocyte foot processes
2) Glomerular basement membrane
3) Endothelial surface
Basement membrane (definition, location, components, source, function)
Basement membranes are specialized extracellular matrices that underlie all epithelial cell sheets and tubes. They also surround individual muscle cells, fat cells and Schwann cells, separating these cells and cell sheets from the underlying or surrounding connective tissue. All basement membranes contain type IV collagen together with proteoglycans (primarily heparan sulphates) and the glycoproteins laminin and entactin Basement membranes are largely synthesized by the cells that rest on them, and they provide a strong connection between the epithelia and the underlying connective tissue. Basement membranes also act as filtration barriers for substances moving between parenchymal cells and the connective tissue space, and provide a scaffold for the migration of cells during embryogenesis and regeneration
Property of glomerular endothelial cells
Fenestrated
ESL full name, alternative name
Endothelial cell surface layer; also known as glycocalyx
glomerular filtration rate (GFR)
125 ml/min in humans
plasma flow rate (Qp)
close to 700 ml/min
filtration fraction (calculation and estimated value)
Filtration fraction = GFR / Plasma flow rate
Estimated values = 20%
Serum albumin concentration vs primary urine albumin concentration
The concentrations of albumin in serum (40 g/l) and estimated concentration in primary urine 4 mg/l (i.e., 0.1% of that in plasma). The sieving coefficient of albumin across the glomerular barrier in humans is estimated to be 10% of that in rodents.
normal solute concentrations in plasma vs bowmen’s capsule
Change of concentration of small solutes after ultrafiltration
No significant changes in small solute concentration, because they are small enough to be freely diffusible through glomerular barrier
Location of cells and proteins in bowmen’s capsule
retained on the capillary side with the blood
Sd = Slit Diaphargm
Fp = Foot process
GBM = Glomerular basement membrane
En = Fenestrated Endothelial cells
Slit diaphargm location
Between podocyte foot processes
Slit diaphargm basic structure
Slit diaphargm is made of interdigitating nephrin molecules from two opposite foot processes
Nephrin Structure (domains from intracellar direction to slit diaphargm, Terminal location)
1) Intracellular domain/cytoplasmic domain
2) Transmembrane domain
3) Fibronectin III domain (FN-III)
4) Immunoglobin loop 8 and 7
5) Spacer domain
6) Immunoglobin loop 6 to 1
C-terminal intracellular; N-terminal extracellular
cytoplasmic domain of nephrin (location, function)
Location: In cytoplasm of foot processes of podocytes
Function: Interact with molecules (e.g. CD2AP or cytoskeletal molecules such as actin) to anchor nephrin in place
Nephrin Immunoglobulin loop structure
In each of the immunoglobin loops 1-8, two cysteine residues are available to form disulphide bonds, thus stabalizing the loop formation
Nephrin spacer domain (formation, function)
Formation: As there is only one cysteine residue located at the spacer domain, no disulphide linkages can be formed to achieve the loop configuration
Function: Forms part of the pores of slit diaphragm, allowing passage of molecules
GBM Type IV collagen intracellular biosynthesis
1) Translation of pre-procollagen by ribosomes on rough endoplasmic reticulum; containing the repeating triplet sequence Gly-X-Y, where X and Y represent amino acids other than glycine. Proline is commonly found in the X position and 4-hydroxyproline in the Y position of the Gly-X-Y triplets.
2) Cleavage of signal peptide, leading to formation of procollagen polypeptide chains known as α-chains
3) Procollagen α-chains undergo Intracellular modifications (cleavage of signal peptides, hydroxylation, glycosylation, chain association, disulphide bonding, hydrogen bonding), which take place when the procollagen chains are translocated across the ER membrane into the lumen and are being synthesized
4) At the C-terminal, 3 carboxy terminals propeptides interact intertwine and associated, stabilized with disulphide bond formed between cysteine residues - forming the NC1 domain
5) The 3 propeptides interact through hydrogen bonding and disulphide bond formation, forming the procollagen triple helix (collagenous domain)
GBM Type IV collagen extracellular biosynthesis
Overview: Extracellular processing (cleavage of the N and C propeptides, self-assembly of the collagen molecules into fibrils by nucleation and propagation, and formation of covalent crosslinks) then converts the procollagens to collagens and incorporates the collagen molecules into stable, crosslinked fibrils or other supramolecular aggregates.
1) Procollagen is converted to type IV collagen protomer, which contains a globular carboxy-terminal domain (A)
2) Through C-terminal dimerization, tropocollagen dimers are formed (carboxy terminal heximer, B),known as the NC1 domain
3) At the N-terminals, aggregation of 4 dimers’ amino terminal domains forms a 7S domain. A type IV collagen tetramer is formed (C) through this N-terminal tetramer formation
4) Tetramers then form a suprastructural lattice through Lateral association of triple helical domains, which provides structural support to the basal lamina
Laminin structure and characteristics
Structure:
1) contain three chains - A chain, B1 chain and B2 chain
2) Joined to form a 4-armed structure
3) Longest arm ends with three C-terminals of A, B1, B2 chains (see image)
4) Such interaction leaves free ends to join respective partners
Characteristics:
1) Insoluble
2) glycoprotein in nature
HSPG Core proteins (name and properties)
1) Agrin - carries 2 heparan sulfate
2) Perlecan - 3 heparan sulfate attached at the N-terminal domain I; contains series of immunoglobins
Endothelial glycocalyx and ESL
Overview: Several, or all, of the components of the ESL are produced by the endothelium at certain rates and removed to blood or urine at similar rates during steady-state conditions.
Glycocalyx intracellular contents: Some molecules have parts embedded into the cytoplasm of the endothelial cells. They include i) GPI (Glycophosphatidylinositol) anchor (a glycoprotein that can bind with C-terminals of proteins); ii) Syndecan core protein; iii) Glypican core protein; iv) Hyaluronan binding receptors that bind with hyaluronan.
Glycocalyx: Membrane-bound proteoglycans (PG), such as syndecan and glypican, which carry chondroitin sulfate (CS) side chains (syndecan) and/or heparan sulfate (HS) side chains (glypican and syndecan) form the glycocalyx.
ESL: The ESL is formed by secreted proteoglycans such as perlecan (mainly HS) and versican (mainly CS) together with secreted glycosaminoglycans (GAG) (e.g., hyaluronan) and adsorbed plasma proteins (e.g., albumin and orosomucoid). Several other macromolecules are important components of the ESL and the glycocalyx.
Interaction of endothelial cells with matrix and podocytes
The podocytes produce substances such as ang1 (received by endothelial Tie2 receptors) and VEGF (received by endothelial VEGFR1 and VEGFR2) that affect the endothelial cells. Integrin receptor a5b1, a5b3 also located on endothelial cells that interact with GBM matrix content.
Graph of F/P ratio against molecular weight for plasma protein (-ve) and dextran (neutral)
___ : plasma proteins, negatively charged
—– : dextran, neutral
F/P : glomerular filtrate/plasma ratio for test substance
___ : plasma proteins, negatively charged
—– : dextran, neutral
F/P : glomerular filtrate/plasma ratio for test substance
With the increase in molecular size, F/P ratio decline as less molecules can pass through glomerular filtration barrier (size barrier). The negatively charged molecules generally has a lower filtration rate as they are repelled by the negatively charged GBM and ECL (charge barrier)
Explain the graph of differentially charged dextran
-ve charged dextran sulphate repelled by negatively charged glomerular barrier
+ve charged DEAE dextran attracted towards glomerular barrier and thus has a higher filtration rate
change on graph if nephritis present
Charge selectivity is lost as the glomerular barrier is no longer as negatively charged due to structural losses.
