5_Nephropathology.docx

**THEME 5: NEPHROPHATHOLOGY** **Introduction** One of the best known functions of our kidneys is the removal of waste products from the blood and their excretion via the urine. The filtering units of this genius disposal system are the nephrons. They consist of the glomeruli, which contain a size...

**THEME 5: NEPHROPHATHOLOGY** **Introduction** One of the best known functions of our kidneys is the removal of waste products from the blood and their excretion via the urine. The filtering units of this genius disposal system are the nephrons. They consist of the glomeruli, which contain a size and charge-selective filtration barrier and the downstream tubules, which possess a set of transporters for reabsorption and secretion of ions, carbohydrates and amino acids. Nephrons can be injured by a variety of different factors: these include hemodynamic (hypertension), metabolic (diabetes), inflammatory (infection, autoimmunity) or genetic (polycystic disease) alterations. Injury to the nephrons may lead to loss of proteins in the urine, formation of edema, accumulation of waste products in the blood and eventually to loss of renal function. Independent of the underlying etiology of the disease, glomerular injury will sooner or later progress to glomerulosclerosis and degeneration of the tubular system. Sclerosis in the kidney is characterized by excessive accumulation of extra cellular matrix components, accompanied by loss of cells via apoptosis and progressive scarring of renal tissue. Nephrosclerosis can be regarded as a progressive tissue reaction after kidney injury, in which the natural capacity of the renal tissue to repair and to remodel its original architecture has been lost. In this theme, a pathologist, a nephrologist, a scientist, and a patient will tell you about the different aspects of renal disease. You will learn how renal injury and nephrosclerosis develop, how kidney disease is detected and treated in the clinic, how kidney disease impacts patients' life, and how scientists search for new targets that can be used to develop novel treatments for kidney disease. **Theme-related objectives** *By the end of this theme you will be able to:* - Recognize and describe different types of lesions in the glomeruli and the tubules (1). - Understand the etiology of the presented kidney diseases (2). - Explain the relationship between the various sites of immune injury and the subsequent pathological reaction in the kidney (3) - Deduce the most likely pathogenesis of renal injury based on results of clinical tests for renal injury (3). - Describe the clinical symptoms of patients with nephritic/nephrotic syndrome (3). - Explain nephrosclerosis as a final common pathway of injury that is independent from the underlying etiology (3). - Indicate treatment options of clinical symptoms associated with nephron damage (4). - Select and motivate the appropriate experimental models to study human renal disease (4). **Reading list and study guidelines** Afbeelding met tekst, schermopname, Lettertype, lijn Automatisch gegenereerde beschrijving **Microscopy and pathology of nephrosclerosis.** End stage renal disease is characterized morphologically by what is called glomerulosclerosis **Glomerulosclerosis** = fibrotic scarring of glomeruli  The main cause of glomerulosclerosis is aging, because of less blood flow and thus decreased oxygen supply **Glomerulosclerosis nomenclature** **Focal** = \50% of all glomeruli is involved **Segmental** = majority of involved glomeruli show sclerosis in \50% of cut surface Typical for old age = focal global glomerulosclerosis Most other causes: focal and segmental glomerulosclerosis (FSGS) **Incidence of focal global glomerulosclerosis with age** **Example of focal segmental sclerosis**  **Glomerulosclerosis causes** Aging -\> focal global glomerulosclerosis Most other causes -\> focal segmental sclerosis - **Ischemia**: aging, arteriosclerosis (thickening of blood vessel wall) - **Inflammation**: glomerulonephritis, (chronic) rejection - **Endocrine**: diabetic nephropathy - **Epithelial** **damage**: genetic (congenital nephrotic syndrome, PKD), minimal change disease/FSGS) - **Infection**: HIV, CMV. Hepatitis C - **Hyperperfusion**: nephron loss, cell damage - **Unknown**: primary FSGS (primary focal segmental glomerulosclerosis) **Inflammation as a cause of glomerulosclerosis** Relation between glomerular localization of immune complexes and clinical picture \> The inflammation is usually of the activation of the humoral immune system, so antibodies \> The involvement of the cellular system is mostly secondary The [immune complex] is usually the cause of further downstream inflammation Antibodies have been fixed in the glomeruli capillary walls The location of the immune complex causes a different disease phenotype **Anatomy of the kidney** Nephrons consists of the glomeruli and the Henle mechanisms   Baumans space? Baumans capsule? Mesangium surrounds it all? Afbeelding met kunst, tekening Automatisch gegenereerde beschrijving  **Different locations at which you can find the immune complexes** 1. Subendothelial -\> under the endothelial and the basal membrane 2. Basal membrane 3. Subepithelial 4. Mesangial Epithelial cell Mesangium Endothelial cell Blood area Primary urinary area 1. **Subendothelial** These immune complexes come from the blood flow Which antigens can be involved in these immune complexes? Two groups of antigens: - **Alloantigens** (Coming from breakdown products of microorganisms -\> infections) glomerulonephritis - **Autoantigens** (autoimmune diseases, for example lupus erythematosus) glomerulonephritis *The result of subendothelial deposits of antigens?* - The antigens are stuck, circulating molecules will bind and you will get activation of the complement system - The type of complement system can tell what type of antigens - Then you get recruitment of inflammatory cells - The first cells that arrive are the [granulocytes] - Once the granulocyte is activated, it will degranulate -\> proteases - The proteases destruct the immune complexes but they also destruct other cells. - The cells also get degraded, now you get holes in the capillary walls - You get leakage of blood cells into the urine, **hematuria** (also some proteinuria, but this is not characteristic) - Granulocytes also release growth factors upon degranulation, to restore the tissue - The growth factors are also not specific, they drive proliferation - It will become harder to push fluid out of the glomeruli, so you will get an increased pressure of the blood Blood pressure is measured, measures different in blood flow in efferent and afferent. If you have decreased blood flow towards the glomeruli, then the [renin-angiotensin system (RAS)] will be activated and you get **hypertension**. At the same time you will get a **renal dysfunction**. **Nephritic syndrome** - Hematuria - Renal dysfunction - Hypertension Treatment with inflammation reducing drugs, steroids. And cytostatics against the proliferation. Subendothelial complexes  Afbeelding met schermopname Automatisch gegenereerde beschrijving  Erytrocyte cast = upper urinary tract increased prolliferation due to GF Afbeelding met verven, tekening, schets, kunst Automatisch gegenereerde beschrijving  Afbeelding met kunst Beschrijving automatisch gegenereerd met lage betrouwbaarheid 2. **Glomeruli basal membrane** (=anti-GBM nephritis) In the glomeruli basal membrane - **Autoantibodies** (because of autoimmune disease against epitopes in the basal membrane) - **Alloimmune disease after transplantation** The basal membrane is a dynamic structure, it consists of ECM molecules such as collagen, laminin, fibronectin. These antigens can be a target in an [autoimmune disease]. So you have **autoantibodies** against epitopes in the basal membrane. Some patients have autoantibodies against epitopes present in different basal membranes. They can have both glomeruli nephritis and inflammation in the longs, this is called **good pasture syndrome**. Most patients will only produce antibodies against glomeruli basal membrane, this is called **anti-glomeruli basal membrane disease**. If you transplant a healthy kidney into a patients who lost their kidney due to a disease which was caused by a genetic alteration in a molecule that is an important components of the glomeruli basal membrane, for instance collagen, than this patients immune system will recognize the epitope of the non-self-antigen. Those patients will also get anti-glomeruli basal membrane disease. This often happens in patients with **Alport disease**, this is a disease with mutation in collagen 4 -\> alloimmune disease after transplantation. - There are no antibody complexes, there are circulating free antibodies that bind in situ to their target - They will activate complement - They will attract granulocytes via chemotaxis, granulocytes will be activated and degranulate - This will produce proteases -\> this causes holes -\> **hematuria** - There will also be GF -\> proliferation -\> hampering of blood flow -\> activation of renin-angiotensin system -\> **hypertension** -\> this will lead to renal **dysfunction**. **Nephritic syndrome** - Hematuria - Renal dysfunction - Hypertension Only with IHC and electron microscopy you will see difference between subendothelial and basal membrane: Subendothelial or subepithelial Basal membrane Granular binding pattern Linear binding pattern  [Treatment ] - Anti-inflammatory agents, steroid - Cytostatics - But here also: **plasmapheresis** (because you have circulating free antibodies) 3. **Subepithelial deposits** You would think that circulating immune complexes cannot pass the basal membrane. But if you look at the equilibrium of free antigens and free antibodies against immune complexes then this is possible. Ag + Ab \ AgAb If due to chemical characteristics, your circulating 'immune complexes' have an equilibrium that consists more of free antigens and free antibodies (Ag + Ab) instead of complexes, then the free antigens and free antibodies can pass the basal membrane and later on aggregate in the subepithelial space. This can involve: - Alloantigens - Autoantigens (So this can also be lupus) Other reason for subepithelial deposits are autoantibodies. Antibodies that are directed to epitopes that are normally present in the subepithelial space. This are molecules that are involved in binding of glomeruli epithelial cell to the basal membrane, such as integrin - You will get complement activation and granulocytes are recruited - But the granulocytes are to large to pass the basal membrane -\> First traded chemotaxis - So there will be NO nephritic syndrome - In the end you will irritation immune complexes - The epithelial cells will produce proteins, the protein production of the basal membrane will start to show mistakes and the basal membrane will change, it will become a sieve (zeef) - You will get lesions of the basal membrane -\> **membranous glomerulopathy** - Proteins will start to leak out -\> proteinuria - Liver cannot keep up the normal protein concentration, this will lead to **hypoproteinemia** - You will get to much fluid in extravascular tissue -\> **edema** (mostly at lower areas, for example in the ankles) **Nephrotic syndrome** - Proteinuria - Hypoproteinemia - Edema (Pitting edema, if you push in the tissue than you will see a pit) Spike formation????? Thickening of the basal membrane Afbeelding met verven, kunst, tekening Automatisch gegenereerde beschrijving  It can start as subendothelial and then also can become subepithelial Treatment - Anti inflamamtory drugs are less effective! - Protein free diets 4. **Mesangial** The mesangial space is characterized by mesangial space and mesangial matrix Circulating antibodies will sometimes be able to find the mesangial space - **IgA nephropathy** (abnormality in the structure of antibodies, mainly IgA) - **Autoantibodies against mesangial antigens** \> Abnormal IgA can accumulate in the mesangium \> Or autoantibodies against mesangial antigens, such as laminin Complement activation will be slower, because the mesangial space is harder to reach. And Fc tails are not freely be available to bind to by Granulocytes. You will get hematuria and inflammation but slower. You will often get **microscopic hematuria**, and this is often found by accidence. At a late stage, in the long term, the smoldering inflammation can lead to scarring. You will also get accumulation of ECM, this will lead to an increase of mesangial area and this leads to a decrease of the filtration area -\> decrease of renal function -\> hypertension Afbeelding met schermopname Automatisch gegenereerde beschrijving **Overview**  5. **Lecture -- Clinical nephrology......................................................................................................................................................** **Kidney function** - Production of urine - Excretion of wastes - Blood-pressure regulation - Production of erythropoietin (hormone that signals to the bone to produce red blood cells) - Activation of vitamin D - Acid/base regulation **Kidney failure** - Volume overload - Accumulation of wastes - Hypertension - Anemia - Bone mineral disease - Metabolic acidosis **Acute renal insufficiency (renal)** - Tubulo-interstial - Allergic - Auto-immune - Hypoxic - Allograft rejection - Glomerular - Nephritic - IgAN, TBMN, Alport - Vasculitis (renal/systemic) - Allograft glomerulitis - Nephrotic - DM-N - FSGS (sec/prim) - Minimal change, MN, myeloma kidney - Allograft glomerulopathy - If the problem is in the kidney but not in the glomerulus then the urine will stay normal - If the glomerulus is involved, then you start seeing abnormalities in the urine \> Nephritic diseases will lead to red blood cells in the urine \> Nephrotic diseases will lead to leakage of protein in the urine (this will look foamy) **Glomerulus** Healthy Disease, nephritic  Afbeelding met schimmel Beschrijving automatisch gegenereerd met gemiddelde betrouwbaarheid Bowman's capsule Capillaries **Nephritic (crescent)** Bowman's capsule starts to proliferate  **IHC for diagnosis** People with lupus have an antibody complex Afbeelding met schermopname, groen Automatisch gegenereerde beschrijving **Nephrotic** - Podocytes line the blood vessels on the outside - Podocytes prevent leakage of proteins in the blood - People with nephrotic disease have a form of podocytopathy - People with diabetes have a high blood sugar, you will filter to much glycose. The podocytes become glycosylated and they don't function anymore to block protein. **Nephritic versus nephrotic** - If you have nephritic condition, the inside layer is inflamed (vasculitis, lupus) - The integrity of the endothelium is compromised - With nephrotic disease you will have antibodies against podocytes or glycosylated podocytes - There is nothing wrong with the endothelium **Clinical relevance** [Nephritic patient] [Nephrotic patient] - Hypertension - Normotensive - Rapid decline in function - Slow decline in function, if any [Complication nephritic] [Complication nephrotic] - Fluid overload - VTE (venous thrombosis, clotting factors and fibronectin - Hyperkalemia factors are usually balanced. But when loss of proteins, then \> Monitor for dialysis \> Prophylactic anti-coagulants **Membranous nephropathy -- patient demonstration** Depositions of IgG, dysfunction of podocytes -\> nephrotic 3 causes of membranous nephropathy: Autoimmune - Anti-PLA2r AB Parainfectious - HepB - Leus Paraneoplastic 80% of the patients have autoimmune response to A2 receptor antibody on podocytes (anti-PLA2r AB) Clinical demostration **Lecture - Experimental pathology...............................................................................................................................................** **Diabetic nephropathy** \> Diabetic mellitus - Patients with diabetes produce high levels of glucose, but also oxygen radicals, this is toxic for the cells - The podocytes and endothelial cells become irritated and start producing more ECM - One of the first signs is thickening of the basement membrane, the filtration becomes more difficult - The filtration is increased by increasing the blood pressure - Due to this high pressure there is more stress on the cells, and they start to produce even more ECM - The kidney starts to leak proteins in the urine  **Vascular alterations in diabetic nephropathy** - Loss of fenestrae and glycocalyx (sugar layer on top of the endothelial cells, important for immune system and filtration) - Endothelial swelling - Abnormal angiogenesis - Arteriolar hyalinosis - Rarefaction (loss of capillaries) - Pro-inflammatory and pro-thrombotic changes - Oxidative stress - Abnormal growth factor expression Can protecting/restoring endothelial function be used to stop progression of diabetic nephropathy? You have to look at the roll of endothelial cell maintenance... **Vascular endothelial growth factor A** - Diabetic nephropathy characterized by dysfunction and injury of the microvasculature - Vascular endothelial growth factor-A (**VEGF-A**) is a fundamental regulator in maintaining the microvasculature Is VEGF an possible target for treatment? **VEGF: a major regulator of vascular function** \> Ther are several members in the VEGF family \> VEGF-A can bind to VEGF1 and VEFFR2, and also NP1/NP2 \> VEGFR2 is mainly expressed on endothelial cells, binding activates downstream effects: - Vasculogenesis - Angiogenesis - Cell migration, proliferation - Homeostasis - Permeability - Cell shape - Adhesion - Vasodilation - Cell survival  VEGF is produced by podocytes. It diffuses to the endothelial cells, were it binds to the VEGFR2 receptor. And it plays an important role in the maintenance of endothelial cells and glycocalyx. ***Microangiopathy in patients who were treated with Bevacizumab*** Bevacizumab is an antibody against VEGF This was given because people had a tumor It blocks the angiogenesis, and therefore inhibits tumor growth But some patients got proteinuria Afbeelding met kaart, schermopname Automatisch gegenereerde beschrijving ***Upregulation of VEGF from developing podocytes leads to glomerular defects by 3 weeks old*** They made mice with upregulated VEGF in the glomeruli They make more capillaries But they become sclerotic and get proteinuria as well So to much is also harmful ***Increase renal expression of VEGF and its receptor VEGFR-2 in experimental diabetes*** In diabetic mice they saw increase of VEGF ***Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes*** They treated the mice with antibodies against VEGF The proteinuria was decreased in these mice ***Reduction of VEGF-A and CTGF expression in diabetic nephropathy is associated with podocyte loss*** In the humans you see the opposite opposed to mice Instead of increase of VEGF in mice, they saw a decrease of VEGF in patients with diabetic nephropathy **Model system to study kidney disease**  **Animal models for kidney disease** [Advantages] [Disadvantages] - Fast development of disease - Scientific issues - Genetically identical - Ethical issues - Constant environmental conditions - Legal/regulatory issues - Monitoring over time - Genetic manipulation - Invasive and terminal experiments How to induce? \> Drug induced \> Spontaneously \> Environmental/food induced \> Genetic induces (KO insulin?) \> Or combinations **Drug induced** Induction of diabetes with streptozotocin Streptozotocin is toxic for beta cells in pancreas via GLUT 2 receptor ***VEGF protects the glomerular microvasculature in diabetes*** Model to alter VEGF expression Podocin is exclusively produced in podocytes It is activated with Dox, this activates the Cre recombinase Afbeelding met tekst, schermopname, Lettertype, diagram Automatisch gegenereerde beschrijving **Summery VEGF in diabetic nephropathy** Animal model for diabetic nephropathy - VEGF upregulated - VEGF inhibition has beneficial effect (reduces proteinuria) - Podocyte specific VEGF knockdown worsens diabetic nephropathy Human diabetic nephropathy: VEGF expression downregulated *If you knockdown VEGF at the genetic level then you have no VEGF at al. But if you use antibodies then the depletion is partial. That is a possible explanation why antibodies against VEGF have a beneficial effect, but the knockdown worsens the diabetic nephropathy.* **\ ** **How to inhibit the VEGF action?** - Small tyrosine kinase inhibitor (but not very specific) - Antibodies against VEGF receptor - Use mitogeen soluble VEGF receptors **Soluble Flt-1** VEGFr1 has a splice variant, which lacks the transmembrane domain, this is naturally present. Flt-1 is able to bind VEGF in the circulation and prevent binding to a receptor. This is a natural inhibitor. **SFlt-1 preeclampsia** Preeclampsia is known as pregnancy intoxication. These patients have high levels of the soluble Flt-1.  **Summary** Treatment with the natural VEGF inhibitor soluble Flt-1 reduce renal complications, endothelial activation and inflammation in type 1 diabetic mice. **Overall summary** VEGF plays a role in diabetic nephropathy and ani-VEGF treatment can improve renal dysfunction in animal models dor diabetic nephropathy. Effect depends on: - Pharmaceutical intervention - Timing - Dose