Good evening everybody and welcome to tonight's members webinar. My name is Bruce Stevenson and I have the honour and privilege of chairing tonight's webinar. Our speaker tonight is no stranger to us and I've heard him speak many times and you are really in for a treat.

I don't think that we have got any new members on with us tonight. So for those of you that, want to pose a question, just pop it in the Q&A box, and we will keep it over to the end, as we always do. And if we run out of time, then we will arrange to get those answered for you afterwards.

So our speaker tonight, Doctor Benou is a graduate of the National Veterinary School of Toulouse in France. After a one-year clinical pathology specialty internship in Toulouse, he moved to Kennedy. Initially as a rotating intern at the University of Montreal, then as a rotating intern at the Ontario Veterinary College.

He subsequently completed a small animal internal medicine residency and a PhD at the Ontario Veterinary College. Benno is a diplomat of the American College of Veterinary Internal Medicine. Prior to his appointment at the UCD School of Veterinary Medicine, Beno worked as a clinician in small animal internal medicine department at the Ontario Veterinary College, and also at the University of Montreal, as well as in a private practise in both Ontario and in Quebec.

His area of interest includes haematology and immune-mediated disorders, bone marrow disease, as well as urology, nephrology in companion animals. Beno now finds himself in Ireland, and Benno, it's welcome back to the webinar vet and over to you. Hello, good evening, everybody.

Thank you for, for joining us and me tonight to discuss again some nephrology topics together. So today we are going to discuss proteinuria and overall glomerular diseases in in dogs. And one of the first steps before we, we dive a bit further is to remind ourselves of what are the glomeruli and what are the normal structure.

So the glomerulus is the, the initial part of the functional unit of the kidney. And so the glomerulus is the initiation of the nephron and the, the, the journey that will make the The plasma into into urine. Each glomerulus is a modified capillary bed, and that means that there will be a lot of blood circulation allowing for filtration of the plasma across the the glomerulus in order to form that ultrafiltrate that will then one day become urine.

The filtration barrier at the level of the glomerulus is composed of three layers. One is the endothelium of the blood cell of the blood vessels, is penetrated, allowing for molecules to go through directly. There is also a glomerular basement membrane, which is the most important part of that philtre and will be the major determinant for the passage into filtrate based on molecule size and ionic charges for each molecules.

And finally, there is a visceral epithelial . Surface that is composed of the protocyte from the these very specific cells, the protocytes themselves that will form slits drags, that are at the level of the junction between each of their extensions. So you can see kind of like a A see through model of the, of that philtre here with the three layers of the vascular level, the basement membrane here, and then this protocytes that are attached, and you can imagine that the filtrate can go in between each of the protocytes.

We know that very small molecule can pass freely, and by small we mean molecules that are around 5 kdaltons. The upper limit for that free travel across the glomeruli is probably around 60 to 70 kilodalton, and there is no a passive filtration above this. So albumin, which we know is 69 kiloton, is is excluded from the filtrate and we know that in our patients, we should not find any proteins in their urine because it is retained within the bloodstream.

These, the layers of each some specificity and the specificity of the of the protocyte and their food processes is the fact that they are very heavily riched in negatively charged proteins, and so they are gonna repulse negatively charged proteins and maintain them within the bloodstream, and facilitate the passage instead of positively charged proteins or positively charged molecules. So through these three layers, you have a triage and a selection of molecules that are allowed to pass and be excreted in the urine. What we call a nephropathy is a disease of the kidney with an involvement of the glomeruli as an initial injury.

And we know that looking at postmortem study, this is going to be present in half to all of the kidneys of dogs that are examined in postmortem. So it's something that is probably naturally occurring as a degenerative process in all patients. However, we also recognise a pathological pathway for nephropathy and glomerulopathies, which encompasses three main categories.

One is an immune-mediated glomerunephritis, so an immune-mediated trigger that will lead to damages at the level of the glomeruli. Another one is amyloidosis, which is a deposit of amyloid within the glomeruli that will damage these filtration system that we just presented and discussed. Finally, glomerulosclerosis, which is that degenerative, disease and the fact that ageing glomerulus, we lose slowly function and be be more permissive to some molecules.

There is a big retrospective study that was performed in 2013 looking at the prevalence of immune mediated disease among glomerular diseases in dogs. A big case, a series of 500 cases has been reviewed, and they showed that about half of them were immune mediated, and the other half were either glommeloclerosis or amyloidosis. And that is very important because it means that you can't just assume.

When you suspect a glomerulopathy, what the underlying cause is, and you can't just blindly treat your patient without knowing what's going on because obviously treatments between an immune media disease or amyloidosis are completely different and probably your patient with amyloidosis does not need to receive immunosuppressant and eventually taking the risk of complication due to the administration of the drug. The population of patients is usually a middle-aged adult dog, with average in study around 8 years, and it's been shown, experimentally that an early marker of lameal disease would be the presence of what we call microalgumen within the urine, which would be kind of a very efficient way to detect proteins that is not fully available commercially yet. I think some labs offer it, but it's not been completely validated.

Dog with nephrotic syndrome and we will go through the definition of nephrotic syndrome a little bit later, but, you, you already are familiar with the term and these dogs are usually younger than the other ones with median age of 6 years. There was no sexual predisposition in any other retrospective study looking at glomeropathies, and again, some immune-mediated glomeropathies were secondary to infection disease, and that would be more present in younger dogs. The presence of neoplasia leading to glomerulopathies was more prevalent in older dogs.

There is no need to panic. You don't need to remember the table by heart. I don't, but definitely these are breeds for which familial glomerular diseases have been identified and characterised fairly well.

So if you have a pure bred dog that you diagnosed with glomerulopathy, obviously probably looking for a genetic trait and . You know, warning the breeder, asking about the the the relatives is probably important if they belong to any of these, of these breeds. Labrador and golden retriever are often overrepresented, but there is a massive bias given the, the, the difference in the number of dogs in the population.

They are so popular that the number that they represent in studies is sometimes a little skewed towards high number, but there are no predisposition for them that have been identified to this day. The patients that you will diagnose with loopathies are very, very varied, and they, there is a lot of variability in the history and the clinical signs that they will display. The reason for consulting are very valuable.

Some proteinuria might be incidental findings, some proteinuria might be because you're looking for them. And so. And I would say in consultation, you will see anything from a pre-anesthetic, you know, checkup for a dental to a dog that presents to you with ascites and peripheral edoema.

A lot of these patients are asymptomatic at the time of diagnosis or have very mild signs of chronic kidney disease. Very unspecific signs that seem to be quite common though are weight loss and lethargy. It's important to note that as you can see in that slide, there is no real acute kidney injury type presentation for glomeropathies in dogs.

I would say the little caveat is in advanced nephrotic syndrome. It is possible sometimes that there is an AKI complicating the overall already damaged kidney situation. Again, physical examination will be very variable, often unremarkable if it's an incidental finding to patients with more specific conditions, more specific signs of advanced chromeopathies, and the ventral nephrotic syndrome, as I've mentioned, ascites, spiritual edoema, eventually changes in the kidney's size or shape.

And What we are going to talk for a long part of the talk today is proteinuria. So proteinuria is the presence of protein in the urine, and as I said, slide number 2, it should not be that. So that means that there is something along the urinary tract that has been damaged, that is not working properly and that leads to a leak of protein in the urine.

It's been shown to be a negative prognostic indicator for both dogs and cats with renal disease, and it's been shown that dog with an elevated UPCR and renal disease would live less than dogs with a low UPCR. In cats with no azotemia that was detected, proteinuria was a precursor of azotemia, and these cats developed azotemia within 12 months after the diagnosis of the azotemia. So it's important to understand how we diagnose, how we monitor proteinuria, and then how we manage it, and what are the steps associated with that.

Proteinuria has been shown to be associated with intestitial fibrosis, so which is just degeneration of the renal tissue. Tubular degeneration, atrophy, secondary to direct toxicity and tubular cells. It's possible that there are formation of protein issues cast if the urine are very, very rich in proteins that you can see on your urinalysis, and that may cause an obstruction of the tibus as well, and eventually a worsening of the situation.

And finally, it's been also associated with mechanism for decreased profusion of the tuber interstition and sular necrosis throughout the kidney, which shows to you that any kind of renal disease has the pro the propulsion to self-promote and self entertain itself, leading to a maintenance or a progression of the damages that were initially present. When you assess proteinuria, there are 3 key elements that have to be present. First is the persistence.

If you identify proteinuria once, you probably want to repeat another sample, another time to make sure that it's repeatable and still present. Then it's important to try to differentiate between pre-renal proteinuria, renal proteinuria, and post renal proteinuria. Preurinnal proteinuria, meaning that you have such a high concentration of protein in the urine that in the, in the blood that it has to be excreted somewhere and it goes into the urine.

This is very rare and mostly associated with neoplastic condition, if you think of the Ben Jones protein for the multiple myeloma patient, for example. Renal proteinuria is the main topic tonight and it's really a hallmark of glomerunephritis, glomerulopathies in in dogs. So it's a dysfunction at the level of the glomerulus, and impairment in the filtration membrane that leads to a loss of protein through the glomeruli.

And finally, post renal, and that's probably one of the most important thing to know is that any inflammation along the urinary tract because you have a urinary tract tumour, because you have an infection, because you have a stone, because you're bleeding, any of that can lead to a false increase in proteins in the urine, and that needs to be ruled out before you conclude that the patient is having a glomeru arthritis. Finally, the magnitude of the protein area is also important because obviously for some numbers, we won't be very, very worried versus for others, we would need immediate attention and immediate treatment. Quantification of the protein is important, and for that the urine protein creatinine ratio, or UPCR which you will see probably in every single slide from now on, is necessary to, to give you a definitive answer for proteinuria.

The dipstick is good and we will get to that in like just the next slide, but you need a quantification to, to be able to assess the magnitude of your proteinuria. You need to rule out pre-renal and post-renal causes before you put the money into the UPCR because obviously the, the magnitude doesn't really matter if we know that there is something else that we have to address to address the protein area. So if you have a urinary tract infection, it doesn't really matter how high your proteins are, you want to get rid of the infection and recheck, and if it's persistent, then address it eventually.

So your analysis is really an easy. An easy task to do and, and like I, I always feel sad because I feel like it's always very It's put kind of last after the blood when I think we get a lot of very valuable information from our urine, and I think it should, in an ideal world be performed for every single patient that has a biochemistry. Urine dipstick is extremely easily available and it's quite good for proteinuria in the sense that it's sensitivity is all right, but the specific ECG is very good, so we know that when it's positive, it's positive most likely.

Similarly, the opposite in cats, where the sensitivity is very good, but the specificity is much more questionable for proteins in the urine. Some old tests were performed before, such as the silo biometric assay where working with the different pH of the urine, you would see precipitation of proteins if they were in excess, but that was clearly not a quantitative test at all, and for some safety, safety reasons within the, within the workplace, it's been, discontinued. And then finally, the quantification test that we've been already mentioning before, the urine protein rein ratio, I think it's something that is indicated if you have a repeatable proteinuria on your dipstick.

If you have looked at your sediment and you have not an active sediment, there is no bioa, no hematuria, no bacteria, and ideally you want a negative urine culture before you perform your UPCR. This is a ratio expressed based on the concentration of the creatinine, which helps you to get rid of the dilution factor and allows you to compare your UPCR from one sample to the other, regardless of the urine specific gravity at each time. This table is coming from the Iris kidney website, and I think this is a wonderful resources for all veterinarians, general practitioners, specialists.

You have all the information you need about renal disease in dogs and cats, including the staging, and the search and quantification of proteinuria is part of the normal staging for chronic kidney disease in dogs and cats. We try to get a representative sample for a 24 hour urine protein loss. My recommendations would be to ask your client to bring 3 samples from 3 consecutive mornings, and you mix them, you mix 1 mL of each together and then you send out for the UPCR on that pool sample.

The normal UPCR in dogs should be below 2.2. And finally, this is another paper from 2016.

It's a it's a review on proteinuria and I think it's really well done. This is a table that's kind of showing you all of the different location where your proteins might come from. And all of the potential underlying causes.

I think what is very important is to see the pre-renal and post-renal, and to see that within the renal category, there are several options, and several locations where damages might have happened to lead to proteinuria. However, we know that if you have an impaired tubular function, you will have other signs than just the proteinuria, such as glucosuria, electrolyte imbalance in your, in your urine. So you should be able to suspect that.

Similarly with interstitial, you have usually an advanced chronic kidney disease and you might be able to pick that up on a biopsy. And then you have all of the glomerular diseases that we will discuss after this. It's been shown in dogs that for any UPCR that is above 2, there is a certainty that there is some degree of glomerular impairment.

If it's below 2, it can either be glomerular or tubular. And if there is a hypoalinemia that's associated with your elevated UPCR, you have even further evidence of glomerular lesions because we know that this is where the argument is gonna leak through and pass into the urine. A urine protein electrophoresis can be performed to help you to distinguish between glomerular and tubular proteinuria.

However, this is rarely performed in clinical practise. Other laboratory findings are again very unspecific. Isotenuria can be present, but will be very variable in dogs with glomerular disease.

Azotemia might be present, however, if you have an azotemia with an intact concentrating ability, that means that you have a glomerular lesion and that your tubules are still able to concentrate the urine. It's been shown that about 2/3 of dogs with glomerul nephritis have almost a normal urine specific gravity, and that isoin urea was present in just, just above 25% of the patient. We've said it before, but Croa can be present in dogs with glomerular disease.

You can have a hyaline or a granular cast with the quantity of proteins that are accumulated in the tubules, but also waxy and fatty casts have been reported. Tambo salt protein can be . Can be promoted and their precipitation is what will lead to these hyaline cast formation within tubules in these dogs.

Renal hematuria is very common in humans with proteinuria, but seems to be rare in dogs, and in humans, they even can look at the morphology of the erythrocyte, which seems to change and there may be misshapen as well, leading to suspicion of glomeru damage and leakage of red blood cells through the glomeruli. Hypoproteinemia can be identified as well on on on a serology biochemistry. It's usually secondary to hyperpoalinemia, the abdomen being lost in the urine.

60% of dogs with glomer nephritis and 70% of dogs with amyloidosis have been reported to be hypoproteinemic. Azotemia and hyperphosphatemia, any form of metabolic disease that will be called chronic kidney disease have been reported as well to be associated with proteinuria. Usually, these dogs have a severe disease and they have lost tubular and glommelar function.

However, it's been shown that about half of the dog with glomerul nephritis and 25% of the dog with amyloidosis were known as aemic. Probably they were picked up at an early stage. On haematology, you might find a non-regenerative anaemia, eventually thrombocytosis, and if you do measure fibrinogen on your coagulation profile, you might find a hyperfibrinogenemia, which is associated with these microbleeds, stimulation of the production of, of platelets.

There is also probably an anaemia of chronic disease that is associated with with that chronic kidney disease, underlying. And finally, the nephrotic syndrome. So nephrotic syndrome is a dog that has to tick all of these boxes with alwaboinemia, proteinuria, hypercholesterolemia, and then peripheral edoema or ascites.

It's only present in about 15% of dogs with glomeru nephritis, and it carries a worse prognosis than dogs that don't have nephrotic syndrome. However, if you remove the last line, the peripheral edoema and the ascites, you will realise that a lot of your patients with proteinuria actually take the three first lines and those 3 1st boxes. So it's been shown that the 3 1st categories are actually present in about half of the dogs with proteinuria.

In terms of investigation, obviously, we recommend to do diagnostic imaging. Most of the time it is to rule out any concurrent affection, neoplastic, stones, anything else going on. DI is very unspecific.

There is no image of kidneys with . With proteinuria, like they don't have a different aspects than kidneys with chronic kidney disease, meaning that they can be completely normal to completely atrophied and remodelled. Further investigations should be carried on if you have a very elevated UPCR that is persistent and it should be probably taken.

On right away if you have a UPCR that is above 3.5%, 3.5, sorry, when you, when you diagnose your patient.

One of the things that is important to investigate would be an infectious disease, and depending on which area you're practising the infectious disease, pressure might be different, or depending where your patient has been travelling, the, the infectious disease pressure might be different. Tick bone disease, Lyme disease, leosis, babeciosis have been associated with proteinuria, and although they are not the most common diseases in England or in Ireland, a lot of these pets have travelled to mainland Europe and been exposed eventually to some of these pathogens, or some dogs through rescues or just because their owners have moved may have come from a country with less maniasas, for example, or ravioses. So it's very important to have a thorough history in these patients to make sure that we are not missing on a potential aetiology.

Inflammatory and neoplastic diseases can be investigated via cytology if there is anything abnormal that has been seen on ultrasound. I don't personally do a cytology of the kidneys in all of my patients with proteinuria, but I will definitely do it if I feel like the kidneys are enlarged or if I suspect that there is potentially an infiltrative disease within the kidney or a mass, obviously. If the protein in your area is present with other abnormalities digestive of lupus, such as polyarthritis, thrombocytopenia, severe anaemia.

You could quantify your anti-nuclear antibodies to try to see if there is an immune mediated origin to your proteinuria and therefore a very specific treatment for that. Depending on the papers, thoracic radiographs can be indicated. I think it's important to know if your patient has some degree of cardiac disease, because if you give them fluid, you might overload them.

So in some patients it might be more indicated than others, but it's something that has been discussed in terms of staging slash investigation in proteinuria patient. Finally, the renal biopsies, renal biopsies, I think are very important to get a definitive diagnosis and. As we said in in slide 3, half of them are immune mediated, but the other half isn't, and therefore, it's hard to pick, you know, if you have a dog that's proteinuric and you initiate the first type of treatments which we will review in a few minutes, and they don't respond, and then you are on the verge of giving an immunosuppressive medication.

Well, in half of the cases, your patient doesn't need it. So that's when the biopsy, I think, becomes very important. They can be performed at the very beginning of the investigations because we are curious and we want to know what we're dealing with, or they can be performed further along down the road, depending on the owners, the finances, and everything.

If you have a proteinuria that is refractory and does not respond to the initial treatment. Contraindication for biopsies are very few. One is end stage CKD, because probably there is not much more that we would be doing with, with that patient anyway, given the survival of dogs in stage 4 CKD.

Patients with coagulopathy, the kidneys has received 25% of the blood flow and therefore, it's going to bleed very easily if you perform the biopsies in a kidney that has in a patient that has a coagulopathy, and hypertension is suspected to promote bleeding, so it would appear to be another contraindication for kidney biopsies at the same time. Ideally you want to sample the cortex and you want to avoid to sample the medulla, to avoid to have urine leakage, and to avoid to have too many damages and risks of bleeding. So there are several techniques.

The ones that are, I think, recommended and the most popular are ultrasound guided true biopsies, as you can see on the left image, and you can see the white line of the needle entering the kidney. You can see the cortical mediaulla junction very well, so you know that your needle is well placed and within the cortex only, not damaging the medulla. On the right hand side, you can see a surgical biopsy using the same truecu forceps because it's safe and it limits the damages and the risk for bleeding compared to a wedge biopsy.

And the surgeon is inserting the needle parallel to the capsule in order to try to avoid the medulla and to only sample the cortex. Different picture, same story, you can see on the left image, the correct way to position your needle, in order to sample the cortex only and to leave the medulla intact. What you retrieve are these little wormy white thing on the right that are under a microscope there.

Usually we take about 3 biopsies, and we then dispatch them in different fixing solutions for the lab. What you want to see is this white material and these red dots that indicate the presence of nephrons, and you know that if you have that, the quality of your biopsy is good and they should be able to achieve a diagnosis. Anaesthesia has been associated with a better quality of samples and a lot of radiologists will refuse to do true-cut biopsies if the patients are not under general anaesthesia.

Ideally, you should see 5 of these pinky glomeruli in each sample of the cortex to ensure that your assessment by the lab will be good. So I would recommend to use the bigger needle you are comfortable using. I usually try to use 16 to 14 to 16 gauge needles to obtain my biopsies.

In general, you place one of them in formalin, but also one of them in neutraldeide because you want to do some advanced testing on these biopsies, and I would recommend that you send these biopsies to a specialist in renal biopsies. Two of them are available. One is called the European Veterinary Renal Pathology Services.

It is in Milan. You can contact them via their website and you can order the reagent in the submission form. It takes 48 hours to come, and then you can plan your biopsies.

Similarly, the Ohio State University is, the host of the International Veterinary Renal Pathology Services. This is led by Dr. Rachel Sentolo.

She's one of the leaders in renal pathology at the moment in veterinary medicine. But honestly, what they perform is very similar. They will, do a routine, hematoxin and using staining, like any, histology sample, you submit to the lab.

But they will also do all of these other, stainings. Such as the PAS, the methamine silver, the trichrome strength stain, the Congo red, to identify the presence of any pathogen, any immune deposition, or any lipid deposition for the congore, red. And they will also perform immunoistochemistry for specific immunoglobulin, immunoglobulin M, G, and A, and also complement 3 to try to really locate any sort of abnormal immune-mediated reaction towards the glomeruli.

And finally, with the glutaralion sample, they will perform transmission electron microscopy to characterise even better the deposition of all of these immune complexes that we just mentioned. So now, if we go through that 500 dog publication from Doctor Chenolo, she has identified in a quarter of her biopsies roughly, membrane no proliferative glominal nephritis. This is a disease that is well characterised in humans.

And that is associated in humans with a 50% chance to develop a nephrotic syndrome. . And in one study about nephrotic syndrome, it was shown that The dogs with nephrotic syndrome that had membrano proliferative glom nephritis at the highest UPCR and it's true that's a disease in which we see UPCR being very, very high, it's been reported as a famil disease in Bernese Mountain dog, and it's often associated with some infectious agent such as Lyme.

Lyme disease, so Lyme nephritis can be associated with, with that membrane or peripheative gloom nephritis. It's important to find an underlying cause and if and treat it, but then if you have treated your underlying cause, you still could consider to immunosuppress that patient because there is an aberrant and exaggerated immune reaction within the glomeruli for these patients. Membranous nephropathy is another category of disease that was also present in about 25% of the biopsies in that study.

There is no breed predisposition, however, dopamine were overrepresented, and the protein era here again might be massive at the time of presentation. It's been also present in about 40% of, sorry, and about 40% of dogs with that disease have developed a nephrotic syndrome due to the protein loss. It's most of the time thought to be idiopathic and doesn't seem to be associated with any specific trigger.

There is no inflammation that is associated with the deposition of the immunoglobulins. So it's thought to be a very primary immune-mediated reaction and . Immunosuppressive therapy may or may not be efficient for membranes nephropathy.

Proliferative glomul nephritis is another category and that is more rare. You can see that depending on the publications, we are ranging between 2 and 16% of renal biopsies. It's associated with proteinuria and azotemia in most patients.

And it's an immune complex glomonephritis. If there is a source that is identified for this immune complex, it should be treated, but most of these dogs will require immunosuppression. IgA nephropathy is associated with immunoglobulin A.

Immunoglobulin A as the sole antibody is very rare but has been described in some young patients, often associated with the azotemia and hematuria, and even hypertension in one of the cases. Underlying GI liver disease should be treated and see if that's enough to control the proteinuria. However, most of these cases will require some sort of immunosuppression.

It's one of these, proteinuria that will respond very well to omega 3 fatty acids. Amyloidosis reported about 15% of cases in dogs. It's very common in older dogs and has been reported to be very prevalent in Beagle and English foxhound.

Nephrotic syndrome is strongly associated with amyloidosis with over half of the cases of dogs with nephrotic syndrome that had amyloidosis. And it's important to understand that it is similar but yet different to the amyloidosis in the Sharpie fever, this kind of like familial fever complex. They will develop amyloidosis or through the kidneys, although the amyloidosis in Sharpie will be more present in the medulla when for most of the other dogs it's mostly present in the cortex.

So the mechanism there are probably quite different. Amyloidosis is secondary to the deposition of amyloid A, which is due to the polymerization of the SAA. SAA is a protein, it's an acute phase protein.

It's a protein that is produced by the liver to demonstrate the an ongoing and active inflammation. It's used in horses and cats as a marker of inflammation, like we use C-reactive proteins in dogs. There is no treatment really for amyloidosis except supportive care, trying to treat the proteinuria with the the drugs that we know are efficient for, for proteinuria.

In some patients, colchicine has also been used to try to reduce the progression of the fibrosis, and it's been recommended in Sharpi fever. The prognosis is relatively poor, and these dogs will live around between 2 months and 20 months, usually we say between 2 months and a year. And finally, glomerulosterosis, which we had mentioned earlier, and these are Prevalent lesions depending on the study, and they are clearly just a degenerative lesion that should appear with age.

However, some dogs seem to develop it faster. In humans, it's also been associated with diabetes, and this is the poster child for your diabetes nephropathy in humans. There are no evidence of any kind of immune reaction associated with it.

It's really just a sclerosis, a degeneration of the glomeruli. Therefore, immunosuppression is not recommended at all for these patients. This is the table from that big paper from Chaolo and again, just to point out that the immune complex glomeronephritis that you can see on the first line represents 48%, so half, roughly half of the patients in that cohort.

Now, the question is how do we treat gloopathies, starting by how do we address the protein area. So the therapeutic intervention to treat proteinuria are based on the inhibition of the RAS system. You treat the underlying disease, you reduce your proteinuria by inhibiting yourra system, and eventually you manage your chronic kidney disease if chronic kidney disease is present.

The inhibition of the RAS system is the first step to control proteinuria because it's going to halter the hemodynamic forces that will influence the passage of protein through the glomeruli. So if you alter these renal hemodynamics and you decrease the hydrostatic pressure within the glomeruli, you reduce the proteins that are pushed out of the capillaries into the ultrafiltrate. The rash targeting agents that we use are anti-hypertensive drugs.

Such as ACE inhibitor or angiotensin receptor blockers. It's important to realise that they are not the strongest anti-hypertensive drugs, with usually a reduction of blood pressure by 10 to 15%. So it's fairly safe to use them in dogs that have already compromised renal function.

The effect on the reduction in proteinuria is usually greater than the effect that we are trying to achieve for blood pressure control. So the inhibition of the RAT system should be considered your, your standard of care in pets with renal proteinuria. And usually, if your dog has a renal disease, we start treating them when they are above 0.2 0.4, depending who you are talking to.

In order to do that, we use either ACE inhibitor or ARB as I just said. The most, Common ones are Benazapril, probably and enalapril. In ARB category, Chinesearan is nowadays, I think the leader there because of the veterinary products that are available with that molecule.

So ACE inhibitors have been associated with positive outcomes in both dogs and cats with proteinuria, and they have been shown to significantly reduce proteinuria, and delay the onset of azotemia in in dogs that were administered ACE inhibitors. They are associated with the high GFR and the low UPC in dogs that had chronic kidney disease versus dogs that we were treated with a placebo. And finally in CAS it's been associated with a significant decrease in the UPCR as well.

So the ACE inhibitor will decrease the afferent arterial resistance, which means that the blood within the glomeruli can flow faster and therefore the hydrostatic pressure overall is decreased within the glomeruli itself. Art artificially, it's also kind of decreasing the GFR because there is less time for filtration that is allowed for, for the blood. Usually the administration is SID initially and then increased to twice a day if you don't have the effect that we are looking for.

It's very important to make sure that your patients are stable and you volemic before initiation, because again, you kind of change the hemodynamic within the kidneys and you will alter the GFR of these patients. Angiotensin receptor blockers or ARB, they will block the angiotensin 2 receptor one, which has a vasopressor effect but also controls the aldosterone secretion. In humans, it's been shown that losartan will reduce proteinuria by 35% within 6 months of initiating therapy.

In dogs and cats, town of Sartan is the one ARB that is widely available through the label Sementtra, and it's been shown to be very efficient and very good at reducing proteinuria in both dogs and cats. Although it's also been shown that it's a good anti-hypertensive drug and that it will reduce blood pressure more than benazepril, at least in CAT, so you have to be careful when you are using it, and in terms of treating the proteinuria, it's been shown that it was equivalent or superior to Benazepril to reduce your PCR over time in in cats. And it's been shown in dogs as well recently in the Jain paper, and I think slowly it's coming up as people wanting termmisartan to be the first line of choice for both the hypertension treatment, but also proteinuric cats and proteinuric dogs, even though costs might be an issue for a large breed dog.

What about combining both therapies if we don't get enough reduction in the proteinuria using only one of the two drugs? Well, there definitely would be some added benefits to combine both. However, there are also some risks associated with it.

I think we have to be realistic that a complete inhibition of the RAS cascade is impossible. And there will be mechanism to escape either ACE inhibitors, or ARP. It's also shown in humans that Both drugs can act synergistically, however, the, the changes in GFR are real and the combination is associated with more acute kidney injury, moreover in elderly patients.

So it's, it's important that you switch from one drug to the other, rather than combining them right away and that you are overly careful with patients that were already aemic before. There is no proper study on the combination yet in veterinary medicine, but we can only expect from the little data we have, we can only expect the same type of complications. So, when we inhibit the RAS system to control proteinuria, the risk is an aldosterone breakthrough, which is a resistance mechanism when we try to inhibit the production or the action of aldosterone.

There is a possibility that angiotensin too, even though it's inhibited by ACE inhibitors, can be synthesised via other enzymes in other locations. It's also possible that there is an elevation in re activity that will then overstimulate the production of enzymes and eventually lead to some degree of secretion of angiotensin too. And even when you use both molecules together, it's been shown in humans that the blockade of your a system is again, never 100%, but probably more close to 80%.

Aldosterone will therefore increase despite everything you try, and that has been very well characterised in humans. It's important that if you have a resistance to the medication, you investigate eventually for hyperaldosterinism, just to make sure that there is not another underlying condition that would kind of counteract what you are trying to achieve with your drugs. And it's also important to realise that if you have anal breakthrough, your proteinuria, your reduction in GFR and your hypertension won't be as well controlled as you would hope with your medications.

So now that we know everything about the drugs, how do we prescribe them? This is a nice diagram from that same review paper from B Clinics of North America, and I think it's quite similar to what most people do. You initiate your treatment and then often after, so you start with ACE inhibitor or ARB.

It doesn't matter which one of the two you pick. Usually I see these patients about a week later to make sure that there is no progression of CKD. Just because of the changes in the GFR.

At that stage, there is no need to spend money in doing a UPCR because it takes much more time to, to be affected and to change. So I usually repeat a UPCR two weeks at the earliest after initiating ARB or ACE inhibitors, at which point, obviously this is combined with a physical examination, a complete urinalysis, a biochemistry profile with at least creatinine, phosphorus, potassium. Blood pressure, if there is a worsening of the azotemia by a 30% increase in creatinine, if there is any signs of hyperkalemia, if there are any signs of hypertension, I usually discontinue the drug and then we consider a different agent.

So if I start one, I will switch to the other. In order to avoid the day to day variation in the UPCR of dogs, I use pooled samples, obtained over 3 days, and the owners collect the urine 3 consecutive morning, the 3rd morning being the morning of the consultation. You use that morning consultation sample to send your UA, and then you mix 1 mL of each day to send the pool sample for the UPCR.

What we want to achieve is to reduce the UPCR by by at least 50% and ideally we would like to break it down, bring it down into normal or close to normal values around 0.5%. And obviously in an ideal world, we want to do that without damaging at all the renal function of this patient.

So for that, as I just mentioned, we want to be careful, we want to monitor the creatinine and make sure it doesn't increase by more than 30%. If you have a dog that is already quite azotemic with a stage 3 or stage 4 CKD, you probably can't really afford to allow for any progression in the azotemia. If you can't achieve your UPCR, the targets that you wanted.

And if your creatinine remains good, and if your potassium remains low, then you can continue to increment the dosage every 4 to every 6 weeks, and I would not go any faster than that. It. And once you've maxed out one drug, then you can consider adding a second agent or switch your agents over.

This is the same table that we've seen a bit earlier just to remind you of the maximum doses that are that are possible for our pets. Again, it's the same review paper from clinics and I think it's a really good paper to have on hand. Some of that information is probably available as well on the IIS website.

Then you have to manage the hyperkalemia and some of these patients, it's a common complication of Of anti-proteinuric drugs, and you want to avoid any toxicity, any cardiac toxicity of your, of your potassium. So if your potassium is creeping up, you want to have an electrocardiogram to make sure that you don't have any induced arrhythmia, and then you want to probably tweak your drugs if your hyperkalemia is persistent to try by reducing the doses to reduce the hyperkalemia. However, if you have a chronic kidney disease and the potassium is difficult to regulate, it's possible that you need to have a potassium-rich diet, and often it's going to be a homemade diet because most of the renal diet available in veterinary medicine are supplemented in potassium.

You want to manage the hypertension, which is another comorbidity of proteinuria, and we know that the kidney is being a target organ for hypertension, make the fact that there are damages associated with hypertension potential factor that will worsen your kidney disease and therefore worsen your proteinuria. Luckily, the drugs we use for the proteinuria are also useful for your hypertension, but you probably should consider adding amlodipine, at least in cats, if you don't have a good control of your blood pressure. In dogs, there are some controversies, and some people consider that amlodipine is not ideal to manage protein your own dogs.

And it's true that I would not reach for amlodipine as my first line. However, I will admit that it's often my second line when ARVs or ACE inhibitors are not sufficient. Just as a reminder, the therapeutic goals are a blood pressure within 120 millimetres of mercury, 260 millimetres of mercury in your practise.

You want to change the fatty acid ratio and the protein content of the food as well for proteinuric patient. Supplementation with omega 3 fatty acids or feeding a diet that has a reduced omega 6 to omega-3 ratio, should be indicated in order to work on the properties of these omega 3 fatty acids. The anti inflammatory properties within the kidneys and also by changing these ratio, this lipiddic ratio, that will also alter eventually the, the potential leak of protein at the level of the, of the glomeruli.

To that alone is not going to resolve or significantly improve the proteinuria long term, but probably confers some synergistic effects with the drug and some protection and some reduction in inflammation at the level of the kidneys. There is one breed in which probably the dietary management and the lipid metabolism management is important, and these are the schnauzers because it's been shown that schnauzers have a primary proteinuria, secondary to hypertroglyceridemia. The severe hypertroglyceridemia leads to glomerular damages, and that can be controlled with diet only.

So, as we've said, About 50 to 60% of dog with proteinuria might have some degree of immune complexes identified. 48% will have a primary immune mediated condition. The rest might have a condition that is triggered by an infectious agent that needs to be addressed first.

This requires biopsies to be proven, and biopsy should therefore be considered in any patient that has a persistent glomerular proteinuria. Despite these initial steps of treatment with ACE inhibitors ARB, the use of these drugs is not going to impair the quality of your biopsies because they just change the hemodynamics and they won't change or alter the composition and architecture of the tissues. Again, if you realise kidney biopsies, I would stress that you send these biopsies to a specialised pathologist, such as Doctor Chenholu at Ohio State or Professor Arresu in the University of Milan.

Which immunosuppressant should you choose, and I think what we want depends a little bit on the patient. If your patient is unstable and you need a rapid onset of immunosuppression because you have a very severe proteinuria, you have a severe hypoalbinemia, you might have a nephrotic syndrome, you might have a severe aemia, you probably want to use mycophenoid morphil and a very short course of glucocorticoid. The corticoids are gonna help you control the ongoing inflammation and then the mycopheno mortal initiate your immunosuppression quite widely available and usually well tolerated.

If you have a slow progressing disease and a very stable patient, you have time and you could start microphenolate or you could consider cloro cell, both as a single agent, giving yourself these 4 to 6 weeks before rechecking anyway, so we know that this is something that we will do step by step at a very slow pace. Cylosporin, very interestingly is not really present in the discussions for this proteinuric talk, and there is only one study that has been ever performed and it showed no effect. But I have a hard time to believe that it doesn't work because a lot of the immune mity mechanisms that are described should be mechanisms that would respond to cyclosporin.

If you decide to immunosuppress a patient and they don't respond the way you want, I would recommend to discontinue immunosuppression after 8 to 12 weeks, either to switch to another immunosuppressant or to change the the way you are managing the patient because long-term immunosuppression, when it's not needed, can be very debilitating, but also. If it's needed, your patient should improve within that 8 to 12 week period. So just before we finish, I just wanted to discuss about the complication of proteinuria.

And as we've mentioned when we discussed the nephrotic syndrome, edoema would be one of them. The edoema is . A complication secondary to the low oncotic pressure associated with the low proteins within the blood.

That extravasation of fluid can happen about anywhere, but in patients with proteinuria, it's often in the, the, the, the limbs and the lower part of the limbs, and we say that the albumin has to be below 15 for these to open, to happen, . Like primarily The use of diuretics to treat the edoema is not recommended because your patient is probably hypovolemic and therefore the use of diuretic will be dangerous and quite ineffective. Another complication of proteinuria is thromboembolism, and it's quite a serious complication that has been shown in about 5% of dogs with glomeral arthritis, 14% of dogs with amyloidosis, and 13% overall of all forms of glomerular diseases.

These ambolis are often difficult to detect, and the prevalence might be higher. However, we know that there is definitely more risk for a nephrotic syndrome patient due to the severe hypoalbuminemia. So they can form a clot and throw a clot, and that clot loves to go like into the lungs.

So pulmonary thromboembolism is one of the most common locations for a thrombi to be lodged, but other arteries and other consequences such as brain, kidney, heart, or the aortic trifification have been reported for thrombombolic complications. Venus thrombus have also been described in the portal or the cable thing. And this is thought to be associated with the loss of proteins that are similar in size tomen, which are natural anticoagulants that are usually circulating within the blood to prevent an excessive clot formation.

So sorry, in order to, to do that, we use clopidogrel as a, as a prophylactic drug and we prescribe this to patients that are hypominimic until their argument is back up to normal values. So in conclusion, proteinuria is a hallmark of glomeal disease. It is not necessarily associated with azotemia and can be therefore considered a stage one chronic kidney disease.

However, we know that it's a negative prognostic indicator and that's likely the chronic kidney disease will progress once the proteinuria has been evidenced. It's important to know where the proteinur is coming from in order to be able to treat it at your best, including eventually biopsies if you don't have a definitive response to therapy, using the most common drugs we use. These drugs are the inhibitors of the RAS system with ACE inhibitors and RIB allowing you to control the hypertension, controlling the proteinuria.

Eventually coupling that with a diet change and finally, once you have your biopsies, eventually using immunosuppressant. If you have a severe hypoalinemia in your bloods, considering thromboprophylaxis, the clopidogrel is recommended and is routinely performed in most of our patients. The prognosis varies a lot depending on the underlying disease that you will find.

And again, that's something that the owners have to understand. Without the biopsies, you won't be able to be really precise in terms of prognosis and you can imagine that the prognosis between something that's slowly progressing like a sclerotic disease versus amyloidosis is important to know for the owners as well. Thank you very much for your attention, and I will be happy to take any questions.

Benno, thank you for your time tonight. That was an incredibly logical run through glomerular disease. It's yeah, it's, it's always so logical when, when we sit and listen to brilliant people like yourself and then you get into the clinic and you think, oh dear, what was it Beno said?

Fortunately, these are all recorded and we can go back to the website and watch them again. So thank you very much for your time tonight. My pleasure.

We don't have any questions for you. So, it's just left up to me to thank everybody for attending. Once again, thank you for your time and to my controller, Dawn in the background.

Thank you for all your help in making this run so smoothly. From myself, Bruce Stevenson, it's good night, everyone.