Glomerular Disease Overview

by Carlo Raj, MD

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    00:01 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.

    00:38 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.

    01:48 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.

    02:12 Identify proteins and such.

    02:14 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.

    02:26 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.

    03:36 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.

    04:45 So the granular would then be a "lumpy-bumpy." All this means is immune complex deposition.

    04:54 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.

    05:58 Let us now continue.

    06:01 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.

    06:16 What color would this be? Black and white. Fusion of your podocytes. Close your eyes.

    06:22 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.

    06:46 What about in immunofluorescence? What does immunofluorescence fluoresced or recognized? Immunoglobulins.

    06:56 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.

    07:12 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.

    07:39 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.

    08:03 You go as far as that from now, for your boards and wards , you are in fantastic shape.

    08:08 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.

    09:39 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.

    10:10 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.

    10:29 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.

    12:08 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.

    12:37 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.

    12:56 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.

    13:11 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.

    14:19 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.

    15:02 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.

    15:13 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.

    15:26 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.

    16:47 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.

    17:06 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.

    18:32 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.

    About the Lecture

    The lecture Glomerular Disease Overview by Carlo Raj, MD is from the course Glomerulonephritis. It contains the following chapters:

    • Glomerular Disorders
    • Electron Microscopy
    • Pathogenesis of Glomerular Disease
    • Mechanisms Producing Glomerular Disease

    Included Quiz Questions

    1. It involves the deposition of immune complexes.
    2. It is a type II hypersensitivity disorder.
    3. It is not visible with light microscopy until severe kidney damage has ensued.
    4. It shows a linear pattern on immunofluorescence.
    5. It involves anti-glomerular basement membrane antibodies.
    1. Immunofluorescence
    2. Electron microscopy
    3. All answers are correct
    4. Two answers are correct
    5. Light microscopy
    1. Identifies patterns and type of immunodeposition.
    2. Classifies type of glomerular disease.
    3. Allows better visualization of structures compared to light microscopy.
    4. To show the structure of the glomerulus.
    5. Allows for visualization of filtration barriers.
    1. Between the glomerular basement membrane and the cells lining the lumen containing blood.
    2. Between the glomerular basement membrane and the cells lining the lumen containing urine.
    3. Within the glomerular basement membrane.
    4. Subendothelial deposits are the result of type II hypersensitivity reactions, so there would be no immune complexes.
    5. Between the cells lining the lumen containing urine and the cells lining the lumen containing blood.
    1. They both can detect immune complex deposition.
    2. They both can detect the site of complex deposition.
    3. They both show submicroscopic defects in the glomerulus.
    4. They both may aid in the diagnosis of minimal change disease.
    5. They both rely on the recognition of immune complexes by chemical tests.
    1. Epithelial
    2. Subendothelial
    3. Mesangial
    4. Intramembranous
    5. Subepithelial
    1. Immune complex deposits may be mesangial, subepithelial or subendothelial.
    2. It shows a linear pattern on immunofluorescence.
    3. It involves antibodies directed against the glomerular basement membrane.
    4. It is a type II hypersensitivity reaction.
    5. Activated T-cells are primarily responsible for glomerular damage.
    1. It is visible with immunofluorescence of biopsy specimens.
    2. It involves fusion of the podocytes of visceral epithelial cells.
    3. It is associated with T-cell activation.
    4. It is a type of nephrotic syndrome.
    5. Cytokines are responsible for the lose of the negative charge of the glomerular basement membrane.

    Author of lecture Glomerular Disease Overview

     Carlo Raj, MD

    Carlo Raj, MD

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