Moving to the glomerular region. Up until
this point, what we have done over and over
again is giving you an overview as far as
laying down the foundation and then we build
upon well. With glomerular diseases,
first and foremost,
think about where you are, you are in the
glomerulus and there are numerous issues or
factors that may then result in glomerular
disease. Let us begin. First and most importantly
at this point, is the fact that your operative
word here is biopsy. So the fact that you
are taking a biopsy specimen based on the
symptoms of your patient. Remember once again.
Do you remember when we did our session with urinalysis?
And with urinalysis, you used information
that you had derived based on the history
that you got from the patient, either through
a clinical vignette or from the attending or
what not, and you put all this together so
that you can arrive at the proper diagnosis.
Well, with the biopsy, there are a couple of
important imaging studies that you will have
to conduct. You can do an H &E stain and with
this, we mean hematoxylin and eosin. And with
this, this then allows us to classify the
type of glomerular disease. This, for the
most part, will be light microscopy. What
does light microscopy mean to you? Well, as
we go to the various diseases, when it is relevant
and pertinent, we will then take a look at
diseases in which it is best to classify them
through light microscopy. Well, let us say
that you don't find exactly as to which you
need to or perhaps there is further investigation
that is warranted and then you are going to
other types of stains and this includes something
like immunofluorescence. This is stain number 2,
upon what? Biopsy. What was the first one?
Light microscopy and we are using H&E stain.
With immunofluorescence, it is the
fact that as the name implies, you are literally
immune, well you are looking for the immuno
particles and you are fluorescing them and
with this particular stain, you probably have
seen the color, that bright green type of
appearance whenever there is immunoglobulins
that are being deposited in your glomerulus.
Identify proteins and such.
The linear pattern is something that it is
part of your immunofluorescence and let us takes
for example you have Goodpasture. From immunology,
you have heard Goodpasture being type II hypersensitivity.
What does that mean to you? It means that
it is an antibody dependent issue, isn't it?
Well when you are antibody dependent, what
does that mean? It means that you are literally
attacking your target. So for example, another
name for Goodpasture would be anti-glomerular
basement membrane antibodies. There are no
complexes here that you are forming. Is that
clear? No complexes. So what are they doing?
These immunoglobulins are then literally attacking
the glomerular basement membrane and so therefore
if you have these immunoglobulins attacking
the basement membrane, therefore, what
kind of pattern would you expect to see on
immunofluorescence? You are going to light
up the glomerular basement membrane in a linear
pattern and as we walk through this and I
give you specificlly a Goodpasture or antiglomerular
basement membrane disease, you will see this
pattern under immunofluorescence. Now, if
that is attacking the glomerular basement
membrane, what if you actually had immune
complexes? For example, we have type III hypersensitivity
that may occur with SLE. We have type III
type hypersensitivity in which what does that
mean even? Remember immunology once again.
It means antigen-antibody complexes. You see
the difference. In Goodpasture discussion above,
what was it? A type II hypersensitivity. What
does that mean? You are attacking the glomerular
basement membrane and type III you have immunofluorescence
here that is then recognizing the immune complexes
of antigen-antibody that is depositing where?
Either under the side of the epithelium. When
do you say epithelium you tell me what side
of the glomerular basement membrane are you
on, if I say epithelium? Good, urine side. Whereas
if I say subendothelial between the basement
membrane and the endothelium, that is the
side of the blood. Is that clear? Now, when
you have such complexes that can be deposited
either underneath the endothelial cell or
maybe perhaps under the epithelial cell what
have you formed? Immune complexes. What kind
of hypersensitivity? Type III. Cannot be detected
by electron microscopy and if it cannot so,
therefore, what do you do? You do an immunoflorescence
in which you find a pattern known as granular.
So the granular would then be a "lumpy-bumpy."
All this means is immune complex deposition.
If by chance you cannot find certain things
under electron microscopy such as your linear
pattern, then you'll have to use your immunofluorescence
to find that linear pattern. But let us say
that you have electron microscopy for the
granular pattern, it recognizes immune
complexes perhaps and you want further confirmation,
then upon immunofluorescence, it is then called
your "lumpy-bumpy". Three major patterns of
imaging that are important based on the type
of pathology that you will get and as we go
through both nephritic and nephrotic, I will
be asking you this question such as does this
weren't require LM, light micrioscopy on H&E?
Number 2, biopsy. Immunofluorescence, based on protein
deposition and is it a linear pattern or granular
pattern? And then the third and final one would
be electron microscopy and we had a previous
discussion where we went into great detail
about electron microscopy where we then identify
the glomerular basement membrane, the epithelial
side and the endothelial side.
Let us now continue.
Now, electron microscopy is then going to
help you identify few things. Light microscopy
may have shown you or increased your suspicion
of glomerular disease. You do electron microscopy.
What color would this be? Black and white.
Fusion of your podocytes. Close your eyes.
Think about your podocytes. Where are you?
You are on the side of the urine. You are
on the other side of the glomerular basement
membrane and that podocyte has food processes
in which they then become fused. Prototype,
minimal changed disease. On electron microscopy,
you actually find the fusion of the food processes.
Such a finding would never take place at microscopy.
What about in immunofluorescence? What does immunofluorescence
fluoresced or recognized? Immunoglobulins.
The fusion of food processes has nothing to
do with immunoglobulins, does it? No. So,
therefore, why would you ever want to use
immunofluorescence when you have fusion of
podocytes? You don't. Use common sense. Know
as to what information that you are getting.
Why it has been given? That it would then
lead you into your diagnosis. Damage to what?
Your visceral epithelial cell a.k.a. podocyte.
What else? Well with electron microscopy, apart
from the fusion of your podocytes, it detects
IC, stands for immune complex. So, immune
complex deposition, just takes us back to our
previous discussion with immunofluorescence.
So, for example, let us say that electron
microscopy did show you deposits, where? Maybe,
on the side of the epithelial. Maybe, on the
side of the endothelial. The electron microscopy
shows you such dark deposits. Then on immunofluorescence
what kind of pattern is this known as? Granular.
You go as far as that from now, for your boards and wards ,
you are in fantastic shape.
Now as you go in further specialization, let us
say in pathology and understand there's certain
techniques that you can use along with immunofluorescence
that will then tell you if you are on the
side of your endo or epithelial. The sites
are designated as follows. Now pay attention
here. Subendothelial. You are on the side
of the blood. If you are underneath subendothelial,
you will be between as the name says here
or as the statements say here between the
glomerular basement membrane and the endothelial
cell. Is that clear? If it is isn't, I would
recommen that you go back and take a look
at the picture in which we walked through normal
electron microscopy, identify glomerular basement
membrane and the endothelial cell and what
would you find in electron microscopy? A dark
deposit. Now, what if you it did with the immunofluorescence?
Is that even relevant? Of course, it is because
it is immune complex. And here how would you
then describe the pattern? Granular. Another
one for electron mircroscopy. However, this
will be subepithelial, so what are you under?
Your podocyte and your glomerular basement
membrane. This is once again an immune complex.
On immunofluorescence, what kind of pattern
is this called again? Very good, granular. Or you can
have issues within the basement membrane.
For example, you have heard of membranous
glomerulonephritis, haven’t you? MGN, membranous
glomerulonephritis. What does that mean to
you? Intramembranous issues, electron microscopy
or maybe mesangial. Mesangial would be much
better seen in terms of its changes if you
did a light microscopy with H&E stain. The
basis of what you need to know in terms of
moving forward, laboratory investigations
and biopsy is here in these discussions.
It is only once you have mastered the foundation
of your investigation, you will then move
into the particular pathologies. To summarize,
three major biopsies stainings, we have our
light microscopy H&E, we have immunoflurorescence,
and number 3 electron microscopy. Let's move on.
Here in a cartoon form, we are showing you pathogenesis
of glomerular disease and I will show you
two major issues. The first one that you are seeing here,
let me just set up this picture. This picture
that you are seeing is the glomerulus.
Next, what you are seeing there in blue, purple?
That is going to be your podocyte. How can
you confirm that? A podocyte should have food
processes. You see that box around that podocyte
in that blue cell. We are going to take that section
and we are going to blow it up over anterior
right. So now we have amplified it or blow it
up, and that dark blue structure or the violet
structure that we are seeing is a huge podocyte
with a nucleus. And then we are seeing food
processes. Identify those first. Next, you
see that orange line. It looks like paint
or if you take a brush looks like you paint
it with an orange brush. Well, that's your glomerular
basement membrane, isn't it? So now what we
do is along with that orange line, which is
your glomerular basement membrane and the
epithelial cell, are you either going to deposit
some of these immunoglobulins either underneath
the epithelial site or you are going to deposit
it where? Underneath endothelial site. And what
you are clearly seeing here, ladies and gentleman
is immune complexes that are being deposited
underneath the endothelial cell. Between the
endothelial cell and the basement membrane
on that first picture where it's circulating, you
noticed that that is a subendothelial deposit.
First and foremost, would light microscopy
show you this? No. Would electron microscopy
show you this? Absolutely. And number 3, immunofluorescence,
but then recognizes beign what kind of pattern?
Granular. Let us now move
into the membrane a little bit. The one in
the middle that you see there is you have
immunoglobulins and what do these immunoglobulins
do? These immunoglobulins are literally only
attacking the glomerular basement membrane.
That is it. We will call this in situ. What
does in situ mean? Membrane. Doesn't it?
So literally here all you have is immunoglobulins
that are attacking some antigen of my glomerular
basement membrane. This is a type II hypersensitivity.
This is antiglomerular basement membrane diseases.
There are no complexes that are being formed
and no deposits. So, therefore, on let us
say electron microscopy, maybe you find changes within
the membrane. Great, but then here, immunofluorescence.
Immunofluorescence will show you, you see that
orange basement membrane are completely containing
immunoglobulins. So, therefore, the entire
basement membrane is going to be "laid up."
So this gives us our linear pattern. Then,
moving over to the far right, the two things
that are occuring here. Take a look at the
difference between the one on the middle and
the one on the right. The one on the right,
not only are you attacking the membrane, but
where else are you depositing? It is depositing
underneath what is that blue cell that we
called earlier? That is a visceral epithelial
cell. That is a podocyte and so, therefore,
the fact that you have involvement of the
membrane and you have deposits underneath
the epithelial cell. This is no doubt membranous
glomerulonephritis, involvement of the membrane
in what kind of hump or deposit? A subepithelial
deposit. So therefore, electron microscopy
would be relevant here and immunofluorescence
will show you what? A granular pattern.
Through these pictures on immunofluorescence
will then give you granular pattern, which
two? The one on the left with subendothelial,
the one on the right with subepithelial and
the one in the middle immunofluorescence would
be what pattern? Linear. Just
to make sure we have complete our discussion.
If you take a look at the big picture on your
left of the entire glomerulus or a section
of the glomerulus, the middle there that you
are seeing in green is a mesangium. And then
just to make sure we're clear that orange cell
that we're seeing with subendothelial is the
endothelial cell and so therefore that would
be your capillary lumen. Everything is in
properly annotated here. Spend a little time.
Make sure that you take a look at the text
from the previous discussions, arrive here,
take a look at the picture and let's now
continue forward with more pathology.
So real quick, let me just have your highlight subepithelial
deposit. Take a look at it. Underneath the
epithelial cell maybe immune complexes, what
would you then call this? Immunofluorescence.
This would be granular. On the bottom, subendothelial.
Underneath the endothelial cell, immune complexes
once again granular. Everything else has been
properly annotated here for you. These are
circulating in immune complexes. Let me give
you an example now. Let us build down a little
bit of foundation. Subepithelial, I will give
you PSGN, which one? Post-streptococcal glomerularonephritis,
subepithelial. Subendothelial would
be something like a SLE prototype known as
DPGN, diffuse proliferative glomerularonephritis.
How did I know that? Years and years and years
of learning and practicing. You will get this
done as well in which at that point you might
have to memorize a little bit. Here strictly
what are you doing? The immunoglobulins are
attacking the basement membrane only. May
I ask you something? Do you find any immune
complexes here? No. So therefore, on immunofluorescence,
what kind of pattern would you call this?
Very good. Linear. Welcome to Goodpasture,
type II hypersensitivity. And then finally
a picture here is showing you not only involvement
of the membrane. Take a look, but then also
what kind of deposit would you call that underneath
that epithelial cell? Obviously, it is subepithelial.
What would be my diagnosis here? Take a look. This
is membranous glomerulonephritis. Further
exapanding upon the overview picture that
we began with and then further dissecting
into each individual pathologies.
Type III hypersensitivity, immune complexes.
Examples for this with SLE. Now let us go
into more detail. The SLE, if it is involving
your kidney, how many kidneys you have? And
I've talked to you about this earlier, it
is just whatever if it helps you remember. You have
two kidneys thus you call this double-stranded
DNA, anti-DNA complex. But this is an anti-double-stranded
DNA issue. This to you means that is SLE induced
kidney damage. Immune complexes, what do they
do? They activate the complement system. What
does that mean? Well, remember antigen-antibody
complex. What if you have such a complex taking
place with IgM, then you tell me as to what
kind of complement pathway you are then going
to stimulate? Is it alternative? Is it you
are going to be classical or is it would be
.... ? Antigen-antibody complex will, in fact,
be your class IV. So
C5a is produced, which is chemotactic, neutrophils
damage the glomeruli, occurs in nephritic
type of glomerulonephritis. Nephritic, primarily,
looking for cytochyme. We will talk more about
this as we go through nephritic and nephrotic.
That is important that you understand, with
those immune complexes, you are then going
to stimulate this complement system. We will
then focus upon that when the time is right.
Another type of damage here. We talked
about Goodpasture. I showed you linear pattern
or should I say I showed you how a linear
pattern will be developed in Goodpasture, antiglomerular
basement membrane antibodies.
T-cell production of cytokines. The cytokines
cause glomerular basement membrane to lose
its negative charge. The cytokines damage
the podocyte. We put all this together, what would you
end up getting? Minimal change disease in the
nephrotic syndrome. Please do not forget the
conductor of damage in minimal change disease
representing damage to the glomerular basement
membrane, which losing its negative charge
and also fusion of food processes is primarily
involved with T-cell production of cytockines.