MEGALIN AND CUBILIN IN RENAL PROXIMAL TUBULE

A SEMINAR PRESENTED

BY

FAJUKO, OLUWADAMILOLA PEACE

AT
THE SCHOOL OF MEDICAL LABORATORY SCIENCES,

UNIVERSITY COLLEGE HOSPITAL, IBADAN.

8TH
AUGUST, 2019 1
 OUTLINES
• DEFINITION OF TERMS
• INTRODUCTION
• FUNCTION OF NEPHRON IN THE KIDNEY
• MEGALIN RECEPTOR
• CUBILIN RECEPTOR
• ROLE OF MEGALIN AND CUBILIN IN THE PROXIMAL RENAL
TUBULE
• DETECTION OF CUBILIN, MEGALIN AND ALBUMIN BY
IMMUNOHISTOCHEMICAL (IHC) STAINING

2
 INTRODUCTION
• Under normal physiological conditions of kidney, Blood is filtered through
the glomerular barrier and the ultrafiltrate contains proteins, charged
molecules (ions), hormones, salts. Reabsorption occurs at the renal tubule
and excretion of electrolytes and proteins takes place by different
mechanisms. Finally, the urine is collected in the renal pelvis (Moestrup
and Verroust 2001).
• The reabsorption of proteins including albumin takes place in the renal
proximal tubular lumen.

3
INTRODUCTION CONTD

• Albumin is preferentially reabsorbed by receptor-mediated

endocytosis in the proximal tubule because of its size and
charge.
• This was initiated by binding to the multiligand receptors
Megalin and Cubilin Christensen and Birn (2002).
• In proximal tubular epithelial cells (PTEC), proteins were
further processed to lysosomes for degradation and later
receptors recycle onto the apical membrane of PTEC
(Christensen and Birn (2001).

4
FUNCTION OF NEPHRON IN THE KIDNEY

5
 MEGALIN RECEPTOR
• Identify by Kerjaschki and Farquhar (1982).
• The endocytic transmembrane protein megalin (600 kDa) belongs to the
low-density lipoprotein (LDL) type receptor family (Raychowdhury et al
1989).
• Megalin was identified identified as a target antigen in rat model of
Heymann nephritis.
• Location; colocalized with cubilin in Renal PTEC, visceral yolk sac, small
intestine and cytotrophoblast of the placenta (Christensen et al 1998).
• In addition, megalin was observed in ependymal cells, thyroid cells,
oviduct, type-2 pneumocytes, parathyroid secreting cells, epididymis,
choroid plexus, the endometrium, ciliary epithelium of the eye (Zheng et al
1994), embryonic tissues of trophoectodermal cells and neuroectoderm
(Gueth-Hallonet et al 1994). 6
MEGALIN RECEPTOR CONTD

• Ligands of Megalin are vitamin carrier proteins, albumin,

myoglobin, lactoferrin, lipoproteins, hormones, enzymes and
drugs. The expression of megalin depends upon receptor-
associated protein (RAP) (Birn et al 2000).
• RAP function to escort protein and can function as receptor
antagonist (Bu and Rennke 1996) and protects the newly
synthesized receptor during the transport to the cell surface
(Willnow et al 1996).
• Binds to other LDL- receptor family members and have a role in
receptor folding (Bu et al 1995).
7
 CUBILIN RECEPTOR
• Cubilin is a peripheral membrane protein (460 kDa), which is expressed on
the apical surface of renal PTECs, visceral yolk sac and Co-localized with
Megalin.
• Previously known as intrinsic factor-vitamin B12 receptor as well.
• Hereditary megablastic anemia 1 or Imerslund-Grasbeck syndrome are
caused by gene defects of cubilin.
• Vitamin B12 malabsorption and proteinuria in evidenced (Aminoff et al
1999).
• Found in small intestine and thymus as well (Hammad et al 2000) and also
in lysosomes (Seetharam et al 1997).

8
CUBILIN RECEPTOR CONTD

• In rats, both megalin and cubilin expression was observed in

glomerular podocytes (Christensen and Verroust 2006).
• Ligands for Cubilin include; intrinsic factor-vitamin B12, vitamin
D binding protein, albumin, myoglobin, hemoglobin,
transferring, apolipoprotein A1 and high-density lipoprotein.
• Cubilin interacts with RAP at sub cellular level (Birn et al 1997) .
• The endocytic function is by interaction with megalin (Kozyraki
et al 2001).

9
 CUBILIN RECEPTOR CONTD
• It has little structural homology with other known endocytic receptors.
• The extracellular domain contains 27 CUB domains.
• The CUB domains most likely constitute the ligand binding domains,
• The binding site for intrinsic factor-cobalamin has been located within CUB
domains 5–8 whereas the binding site for RAP is located within CUB
domains 13–14 (Padiyar et al 2010).

10
MEGALIN AND CUBILIN STRUCTURES

11
ROLES OF MEGALIN AND CUBILIN PROXIMAL
RENAL TUBULE
• In renal PTECs, Megalin and Cubilin mediate the reabsorption of proteins
(albumin, carrier bound proteins, vitamins), which are present in the
glomerular ultrafilterate (Christensen and Nielsen 2007).
• Megalin binds directly to its ligand and internalizes it for lysosomal
digestion but the Cubilin-ligand complexes require Megalin for further
process (Verroust and christenten 2002).
• The ligand–receptor interaction is Ca2+ dependent (Verroust and christenten
2002).

12
ROLES OF MEGALIN AND CUBILIN
PROXIMAL RENAL TUBULE CONTD
• Megalin deficient mice show increased excretion of albumin in
the urine, as a result of defective tubular reabsorption (Willnow et
al 1996).
• Dogs with Cubilin deficiency show dysfunctional tubular
reabsorption also (Fyfe et al 1991).
• Patients with hereditary malabsorption of vitamin B12 often
suffer from proteinuria due to structurally abnormal expression of
Cubilin (Aminoff et al 1999).

13
ROLES OF MEGALIN AND CUBILIN PROXIMAL
RENAL TUBULE

14
 EXPRESSION AND SYNTHESIS OF MEGALIN AND
CUBILIN
• Megalin is expressed in the S-shaped body later giving rise to both the
glomeruli during development, megalin is only expressed in the proximal
tubules and, to a lesser extent, the glomerulus Sahali et al 1993).
• In the kidney proximal tubule megalin can be localized to the brush border,
coated pits endocytic vesicles (Bachinsky et al 1993), and the membrane
recycling compartment, dense apical tubules.
• The membrane expression is high in small and large endosomes in the
proximal tubule cells but is virtually absent in late endosomes/prelysosomes.
However, smaller amounts of intact and degraded megalin have been
identified in the matrix of the lysosomes. In rats the expression of megalin in
the proximal tubule brush border varies between different segments, with the
highest expression in segment two. Megalin has also been identified in the
glomerular podocytes of rat kidney. 15
EXPRESSION AND SYNTHESIS OF MEGALIN AND
CUBILIN
• Cubilin is also identified in lysosomes. Similar to megalin, cubilin is
expressed in the S-shaped body during renal development whereas
later expression is confined to the proximal tubule only. So far it has
not been identified in the glomerulus.
• The posttranslational processing of cubilin may involve furin-
mediated cleavage in the trans-Golgi network, as suggested by the
finding that affinity-purified human cubilin appears to be truncated at
a recognition cleavage site for furin in the NH2-terminal region.
• Posttranslational processing may be tissue specific, as it was recently
shown that ileal cubilin undergoes more extensive NH2-linked
glycosylation than renal cubilin.

16
 LABORATORY IDENTIFICATION OF MEGALIN AND
CUBILIN USING IMMUNOHISTOCHEMISTRY (IHC)
STAINING
• After optimization of antigen retrieval method and primary antibody
dilution, we performed the IHC staining on the renal biopsy sections.
Deparaffinisation and rehydration was followed by AR method.
• For megalin, citrate buffer with pressure cooker (citrate D) was used as AR
method and the primary antibody was diluted in 1:60. For cubilin, citrate at
80C as AR method was used.
• The biopsy sections were incubated with the primary goat anti-human
cubilin antibody diluted in 1:100. For albumin uptake citrate D as AR
method was used and the primary rabbit anti-human serum albumin
antibody was added in dilution of 1:400 with respective incubation time of 1
hour at room temperature.
17
 LABORATORY IDENTIFICATION OF MEGALIN AND
CUBILIN USING IMMUNOHISTOCHEMISTRY (IHC)
STAINING CONTD.
• The primary antibodies were administered into each renal biopsy in 100 µl
for 60 minutes at room temperature. After subsequent incubation with the
primary anti-megalin and anti-albumin antibodies, sections were incubated
for 1 hour at room temperature with the secondary anti-rabbit antibody
conjugated with peroxidase.
• For the detection of cubilin, rabbit anti-goat antibody as bridge antibody
was used in 1:500 dilution. All primary and secondary antibodies were
diluted in “antibody diluent” from DAKO. As secondary antibody, Dako
EnVisionTM+ System (HRP labeled) was added for 50 minutes and the
sections were kept in dark.
18
LABORATORY IDENTIFICATION OF MEGALIN
AND CUBILIN USING
IMMUNOHISTOCHEMISTRY (IHC) STAINING
CONTD.
• The staining was completed by a 3-10 minutes incubation with
3,3′ -diaminobenzidine (DAB) + substrate-chromogen, which
results in brown-colored precipitate at the antigen site. Later,
biopsy sections were concisely counterstained with haematoxylin
and mounted. Finally the staining was visualized by light
microscope

19
LABORATORY IDENTIFICATION OF MEGALIN AND
CUBILIN USING IMMUNOHISTOCHEMISTRY (IHC)

20
21
LABORATORY IDENTIFICATION OF MEGALIN AND
CUBILIN USING IMMUNOHISTOCHEMISTRY (IHC)

22
23
 CONCLUSIONS
• Megalin and cubilin constitute two large, endocytic receptors heavily expressed in
the endocytic apparatus of the kidney proximal tubule. Absence or dysfunction of
either receptor is associated with significant tubular proteinuria, showing that both
are important for normal absorption of filtered proteins, including albumin.
Although structurally very different, both receptors may be functionally linked.
Some ligands are common to both receptors, and megalin-cubilin interaction
seems to be important for the endocytosis and recycling of the “peripherally
attached” cubilin. Megalin is important for tubular uptake and metabolism of
several hormones and vitamin-carrier protein complexes, including the renal
activation by hydroxylation of vitamin D. In addition, megalin is involved in the
tubular uptake of potential nephrotoxic drugs, including aminoglyocosides. Thus
modification of receptor function may be a valuable prospect of future research
Finally, there is new evidence suggesting that megalin may be involved in signal
transduction. No doubt, future studies will help to unfold this potential new aspect
of megalin receptor function. 24
 REFERENCES
• 1.Moestrup SK, Verroust PJ (2001): Megalin- and cubilin-mediated endocytosis of
proteinbound vitamins, lipids, and hormones in polarized epithelia. Annu Rev Nutr 2001;
21:407-428.
• 2. Christensen EI, Birn H (2002): Megalin and cubilin: multifunctional endocytic
receptors.Nat Rev Mol Cell Biol 2002; 3:256-266.
• 3. Moestrup SK, Kozyraki R (2000): Cubilin, a high-density lipoprotein receptor. Curr Opin
Lipidol 2000; 11:133-140
• 23. Erik Ilsø Christensen and Henrik Birn (2001): Megalin and cubilin: synergistic endocytic
• receptors in renal proximal tubule. Am J Physiol Renal Physiol
• 280: F562–F573, 2001.
• 5. Kerjaschki D, Farquhar MG (1982): The pathogenic antigen of Heymann nephritis is a
• membrane glycoprotein of the renal proximal tubule brush border. Proc Natl Acad Sci U S A
• 79:5557–5561.
25
THANKS FOR LISTENING

26