Systemic lupus erythematosus (SLE) is an autoimmune disease in which approximately 50% of patients have kidney involvement (lupus nephritis). Membranous glomerulopathy makes up a subset of lupus nephritis. On renal biopsy, immunofluorescence shows granular capillary loop deposits. Electron microscopy shows subepithelial immune complex deposits. EXT1/2 and NCAM1 are two antigens which have been shown to be present in membranous lupus nephritis. This study looked at EXT1 and NCAM1 glomerular staining over time in patients with multiple renal biopsies. A total of 31 patients with multiple biopsies were studied. EXT1 positive patients had lower levels of chronicity compared to EXT1 negative patients at diagnosis, but both groups showed a similar rate of decline in kidney function over time. EXT1 staining can be dynamic, as a proportion of patients lost or gained expression of EXT1 in subsequent biopsies.
Dr. Rebecca May- Hi, I’m Dr. Rebecca May, and this is Arkana Advances. Follow along as we explore all renal research happenings at Arkana Laboratories.
Hello, welcome to Arkana Advances, where we discuss exciting new research in renal pathology performed by our own physicians. I’m Dr. Rebecca May, and today we have Dr. Paul Miller, who is one of our newest pathologists here at Arkana. He will be discussing his recent article in nephrology dialysis transplantation titled EXT1 and NCAM1 associated membranous lupus nephritis in a cohort of patients undergoing repeat kidney biopsies.
Thanks so much for joining us, Dr. Miller, and welcome to Arkana.
Dr. Paul Miller- Thanks Rebecca. Happy to be here.
Dr. Rebecca May- So, first, can you give us a brief description of lupus and lupus nephritis?
Dr. Paul Miller- I can. Uh, lupus is an autoimmune disease that affects all organs in the body and it can be quite debilitating for people that have it.
About half of people with lupus develop lupus nephritis and a good proportion of those patients go on to develop end stage kidney disease.
Dr. Rebecca May- So, membranous glomerulopathy makes up a subset of lupus nephritis cases. Um, can you describe what membranous glomerulopathy is?
Dr. Paul Miller- Membranous glomerulopathy is a specific type of lupus nephritis where the immune complex deposits that cause lupus nephritis actually deposit on the sub epithelial side of the glomerular basement membrane.
And what that means is that they’re depositing between the podocytes and the basement membrane that makes up the capillary loops of your functional unit of the glomerulus. And people with membranous… Uh, nephropathy have profound nephrotic syndrome, which means that they’re spilling protein into their urine.
It can cause edema, and it can be quite debilitating for patients.
Dr. Rebecca May- And this is the highest cause of nephrotic syndrome in lupus, right, lupus, uh, membranous lupus nephritis?
Dr. Paul Miller- That’s right. Yes.
Dr. Rebecca May- And, um, can you talk about some of the autoantigens? There’s been recent changes in this field, but, uh, the autoantigens involved in membranous and membranous lupus nephritis.
Dr. Paul Miller- In recent years, uh, several different autoantigens have been discovered that cause lupus nephritis. Or cause membranous nephropathy, I should say. Uh, you know, the prototypical one is PLA2R. And, uh, membranous nephropathy related to PLA2R is actually an autoimmune disease, where your body is creating antibodies to the PLA2R protein, which is in the podocytes, and then you get immune complex deposits on the subepithelial side of your basement membrane.
And lupus, uh, for a very long time, we didn’t know What auto antigen or if there was an auto antigen involved in membranous lupus nephritis. But now there’s actually, you know, two antigens that have been discovered, uh, that are related to membranous lupus nephritis. One is exostosin one and two, and the other is NCA one.
Dr. Rebecca May- And ext1 and 2 form a pair, right? So if you have one, you definitely have the other.
Dr. Paul Miller- That’s right. Yeah, ext1 and 2 are more or less interchangeable. If you have one, you have the other. There’s been no cases where there’s a lupus membranous nephritis case that’s positive for ext1 or negative, and negative for ext2 or vice versa.
Dr. Rebecca May- So it makes it a little easier to test for them. How many cases do we still not know the antigen in membranous lupus nephritis. What percentage of patients is that?
Dr. Paul Miller- So it’s about two thirds. About one third of patients will have, uh, EXT one and two positive membranous lupus nephritis, and the other two thirds will be negative for EXT one and two.
Uh, NCAM1 is less than 10% of all lupus nephritis, but there’s still, you know, a. A large proportion of patients with membranous lupus nephritis where either there isn’t an auto antigen or we don’t know what it is.
Dr. Rebecca May- Yeah, there’s still a lot to learn in that area. So what do we know about how the antigen might affect the prognosis of the patient?
Is it different with the different antigens?
Dr. Paul Miller- It is, yes. So when EXT one and two were first discovered, it was discovered as an auto antigen in. You know, autoimmune causes of membranous nephropathy. And then a recent study out of the Mayo Clinic looked at patients with membranous lupus nephritis specifically, and they, they looked at them longitudinally.
So over, you know, a protracted period of time or the disease course, and what they found was that patients that were positive for EXT one and two actually did better than patients that were negative for EXT one and two. They develop end stage kidney disease, uh, less frequently and they had less chronicity on their biopsies, uh, which are.
You know, when you’re looking at biopsies, chronicity is really the most important finding because it means that that portion of your kidney has you know, essentially stopped working and there’s nothing you can do to treat it. So, you know, EXT positivity may actually be a positive prognostic indicator and may, if you’re positive, you may do better.
Dr. Rebecca May- So it’d be really important information for the patients to know, right, if they have one of these antigens, that it could affect their prognosis. So you decided to study EXT and NCAM in membranous lupus nephritis patients. And what questions were you hoping to answer?
Dr. Paul Miller- So first and foremost, we wanted to know.
Whether we could, whether in our different patient population, the differences in prognosis between exostosin positive and exostosin negative patients was the same as what had been shown in the Mayo population. We were also interested in whether exostosin positivity stays the same over the course of disease, or whether Uh, you know, at one point you could be positive, and at another point you could be exostosin negative, and what that meant for the patient.
So those were the two main questions we wanted to answer.
Dr. Rebecca May- So how did you design your study to answer these questions?
Dr. Paul Miller- So we, uh, looked through all of the biopsies that we had at Stanford. And we look for patients that had, had more than one biopsy with membranous lupus nephritis in both of those biopsies.
So that way we could test for their exostosin status at two different time points in their disease course. Uh, we found a total of 31 patients. that met these criteria, and we stained each of the biopsies for exostosin 1 and 2 as well as NKM1.
Dr. Rebecca May- And this was about 18 years of renal biopsies at, at Stanford, correct?
And that’s where you did your renal fellowship.
Dr. Paul Miller- That’s right, yeah. Yeah, before this year I was, uh, training at Stanford, but I’m happy to be here now.
Dr. Rebecca May- And so your definition of discordant was if the first and last or the intervening biopsies did not match, um, basically if they were, um, negative for EXT and became positive or positive and then became negative.
So what was your breakdown of EXT and NCAM positive patients among this cohort of 31 patients?
Dr. Paul Miller- Alright, so out of the 31 patients, about a third of them ended up being exostosin positive. Uh, about 10 percent were NCAM positive, and then the remaining were negative for exostosin and NCAM, which really matched very nicely with what had been shown previously.
Uh, we have three patients that were discordant, two of which were positive for exostosin their first biopsy, negative on their second biopsy, and one patient was positive. negative on their first biopsy and then positive on their second biopsy.
Dr. Rebecca May- So, were there any differences between the EXT positive group and the EXT negative group in terms of their demographics?
Dr. Paul Miller- So, the EXT positive group, you know, interestingly, uh, was all, uh, EXT negative group had a large portion of male patients as well. You know, this is something that we found that hadn’t been shown. Previously, uh, you know, it’s a small number, so it’s, you know, it’s definitely possible that this is just a result of the fact that we only had 31 patients.
Um, but it is interesting, uh, that, uh, you know, more females were in the EXT positive group, especially because it’s well known that autoimmune disease is more prevalent in, uh, you know, female patients.
Dr. Rebecca May- And what about, um, the biopsies between the EXT positive biopsies and the EXT negative biopsies? What were, what was different about them in your study?
Dr. Paul Miller- So the EXT positive biopsies, interestingly, had much less chronicity in them. So the way that we measure chronicity, one is by how many of the glomeruli are sclerosed down. So global glomerulosclerosis is what we call it. And there was more global glomerulosclerosis in the EXT negative cohort compared to the EXT positive cohort.
There was also more scarring of the kidney, so interstitial fibrosis and tubular atrophy, in the EXT negative cohort. Um, so these findings matched what had been shown previously, and really reinforced this idea that EXT positivity is a prognostic indicator, and that if you are EXT positive and you have membranous lupus nephritis, uh, you’ll have less chronicity in your biopsy, uh, and do better over the course of time.
Dr. Rebecca May- So, what happened over the course of time between EXT positive and negative?
Dr. Paul Miller- So both the EXT positive and EXT negative patients, they, they lost kidney function over time. And they lost kidney function at a similar rate over time.
Dr. Rebecca May- But the EXT positive were sort of starting at a better spot.
Dr. Paul Miller- They were. Right. So, because, again, because of the biopsies that showed less chronicity, they actually had much better kidney function, you know, at that first time point. Uh, so they were starting at a. you know, a better spot. And, you know, even though they were progressing, they still had better kidney function over time.
We had one ext positive patient that progressed to end stage kidney disease and several of the ext negative patients progressed to end stage kidney disease. Our numbers are a little bit small to show that the progression to end stage kidney disease was statistically significant between the two groups.
But there was There was more progression in the EXT negative group, and if you extrapolate that out to what had been previously shown, uh, I think, you know, it’s suggestive that that’s the case.
Dr. Rebecca May- And what about the EXT discordant patients? Can you talk about what you found in those cases? And I was really surprised to see that data.
It’s, it’s, um, very interesting because I wasn’t expecting it to, to be different. This really suggests we should be staining repeat biopsies.
Dr. Paul Miller- So, yeah, it was, it was surprising and, you know, it had not been shown before that patients could change their EC status, um, at different time points. So, we again had three patients who this was true for.
Two of those three patients did progress to end stage kidney disease. And the reason, you know, for being EXT positive and then EXT negative or vice versa is, is unknown. Uh, but. It’s either the stain may not be entirely sensitive and we’re just not picking it up at low levels later in the disease course, or the way that the immune system is interacting with the kidney and the production of autoantibodies is dynamic and variable over time.
So, just because… your ext positive at one point doesn’t necessarily mean that that’s like a fundamental component of your disease process over the entire course of your disease.
Dr. Rebecca May- That’s a really interesting thought. What about the NCAM1 patients?
Dr. Paul Miller- So the NCAM1 patients, we had two that were NCAM1 positive at, you know, both their biopsies.
Those patients tended to do well, but again, the numbers were very small, so it’s hard to say, you know, too much about any prognostic differences in the NCAM1 subset of patients compared to, you know, the rest of the, um, the cohort.
Dr. Rebecca May- Can you discuss some of the limitations of the study? You’ve already touched on a little bit that it was a smaller cohort because you were looking at multiple biopsies. But any other limitations that you, that you see in, in the study?
Dr. Paul Miller- Yeah, so I think, you know, it’s a smaller cohort. Uh, we you know, are using, uh, an immunohistochemical stain to describe the EXT status. So, you know, we don’t actually have autoantibodies in the serum. They haven’t been discovered in exostosin.
And so, you know, we only have really one piece of data that’s, you know, describing someone as positive or negative. And, you know, and PLA2R, for example, you could both stain the biopsy and look for autoantibodies in the patient’s serum, which really confirms that diagnosis of PLA2R positive or PLA2R negative.
So, you know, our study did lack that just because it hasn’t been found yet. Our study looked at patients that had multiple biopsies, and in order to need a second biopsy with lupus nephritis, there needs to be something going on clinically to justify that biopsy. So, by looking at patients with multiple biopsies, we selected for a group of patients that actually had a more severe disease course than just the general population of patients with lupus nephritis that only required one renal biopsy during their disease course.
By doing that, you would expect that we would be you know, selecting for more EXT negative patients and patients that are doing worse. It is interesting that even after selecting for a more severe phenotype of Lupus nephritis that we still were able to show these prognostic differences between XT positive and XT negative.
It suggests that even among patients that are, you know, have a severe, uh, disease process and you know, a worse disease course, that these differences still exist in that population.
Dr. Rebecca May- So, to summarize, uh, some of your findings at. First biopsy, EXT positive patients compared to the negative patients had higher estimated GFRs, lower interstitial fibrosis, lower global glomerulosclerosis, and even you showed a lower NIH chronicity score, but the decline in EGFR was similar between the groups.
So EXT may be predictive of better kidney function, at least at the first biopsy, and therefore may not progress to end stage renal disease as quickly. You’re also the first to show that EXT staining may actually be dynamic, and this could represent a distinct subset of disease, which is a really interesting finding.
So what questions are you interested in answering in the future?
Dr. Paul Miller- Alright, so I, right now we know that EXT positivity exists in a subset of membranous lupus nephritis, but we don’t really know why or what it’s doing in the disease. So, really understanding the pathophysiology of how exostosin positive membranous lupus nephritis affects the patient and is causing this better prognosis, uh, is critical.
And if we were ever to develop, you know, a a treatment that was specific to treat exostosin positive membranous, we would need to understand, you know, how this protein is interacting in the disease. Uh, so I think. Just understanding the fundamentals of what it means to have exostosin positive membranous nephritis is an open question and something that I would be very interested to learn about.
Dr. Rebecca May- Well, thank you so much, Dr. Miller, for doing this exciting research and for coming on the podcast. Follow us on Twitter at ArkanaLabs for more exciting kidney news and research. And you can also follow me on Twitter at RebeccaMay_RP.
Dr. Miller, where can people reach out to you if they have more questions?
Dr. Paul Miller- So you can find me at arkanalabs.com. I reach out to our lab here in Little Rock, and I’m happy to, you know, answer any questions.
Dr. Rebecca May- Thanks so much, Dr. Miller.
Dr. Paul Miller- Thank you, Rebecca.
Dr. Rebecca May- Thank you for listening. This podcast and more can be found in the iTunes store. For more information and educational programming like this, follow us on Facebook and Twitter, or visit us on the web at arkanalabs.com.
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