Abatacept for Glomerular Diseases: A New Era of Intelligent immunosuppression?

At ASN Kidney Week there was some interest in abatacept as a targeted therapy for glomerular diseases. T-cell activation requires 2 signals; (i) binding of the T-cell receptor to the antigen-MHC complex on the antigen-presenting cell and (ii) a co-stimulatory signal involving CTLA-4 on the T-cell and B7-1 on the antigen presenting cell. Abatacept is a fusion protein composed of the Fc region of IgG1 fused to the extracellular domain of CTLA-4 which inhibits the T-cell co-stimulatory pathway via B7-1 binding. 

          FSGS

The headlines must go to the small case series of abatacept in FSGS published in NEJM. The rationale for its use was the observation that B7-1 expression is not apparent in normal human podocytes but is found in certain diseased podocytes including a subset of FSGS patients. The series included 4 patients with recurrent FSGS post-transplantation (rituximab-resistant) and one with glucocorticoid-resistant primary FSGS. All patients achieved either partial or complete remission.

In vitro studies demonstrated that α3-Integrin knockout mice constitutively expressed B7-1 in podocytes and abatacept blocked B7-1 mediated podocyte migration in these cells. The molecular mechanism of B7-1-induced podocyte dysfunction was shown to be disruption of activation of the glomerular protein β1-integrin. The authors conclude that B7-1 immunostaining of biopsies may identify a subgroup of patients who would benefit from treatment with abatacept.

2       Lupus Nephritis

The late-breaking session included a randomized controlled trial of Euro-lupus regime cyclophosphamide (i.e. low dose IV) with or without abatacept for proliferative lupus nephritis [Access Trial]. Azathioprine was introduced at 3 months and stopped at 6 months in the abatacept group if they had achieved a remission. Overall, there was no difference in remission rate between the groups. Despite the neutral outcome, 2 points should be taken from the study: (i) The Euro-lupus regime appeared to work in a US cohort of patients where almost 80% were either Hispanic or African American. (ii) Abatacept patients who achieved remission maintained this at 1 year despite coming off immunosuppression at 6 months. However, with the growing confidence in Mycophenolate-based therapy for lupus nephritis and the lack of improved remission with Abatacept in this study, its place in the treatment of proliferative lupus nephritis remains uncertain.

3       Diabetic Nephropathy

An oral presentation on abatacept in Diabetic Nephropathy [FR-OR010] reported increased B7-1 expression in both murine podocytes cultured in high-glucose and on human glomerular podocytes from biopsy specimens. The use of Abatacept in diabetic mice prevented an increase in albuminuria.

Bottom Line: The FSGS case series beautifully illustrates how targeted therapies may be applied to immune-mediated renal diseases. While this case series is very small, it demonstrates the potential for reclassifying disease based on pathogenesis (i.e. B7-1-mediated) rather than crude pathological patterns (focal segmental sclerosis). This is similar to the recent re-classification of MPGN into complement or immune complex-mediated forms. With new targeted therapies like abatacept (and eculizumab for complement mediated glomerulopathies), we may be entering an era of intelligent immunosuppression based on molecular pathogenic signals rather than crude histological patterns.

Genetic Defects of the Glomerular Basement Membrane

The glomerular basement membrane is a thin layer of extracellular membrane proteins that is an important part of the filtration barrier, particularly glomerular permselectivity, by preventing proteins from crossing into the filtrate. The major proteins in the GBM are laminin, type IV collagen, nidogen and the heparan sulphate proteoglycan agrin. Interestingly, in the past, I was taught that the highly negative charge on agrin played the major role in mediating charge selectivity. However, recent studies have shown that mutations in the gene encoding agrin, leading to a reduction in charge along the GBM, have no effect of glomerular function in mice. Similarly, deletion of agrin has little effect on permselectivity further suggesting that the role of the proteoglycans in selective filtration is minor at best.

There are two conditions associated with genetic defects in glomerular basement membrane proteins:

Syndrome	Gene(s) affected	Protein	Phenotype
Alports Syndrome	COL4A3 COL4A4 COL4A5	Type IV Collagen, α3, α4, α5 subunits	Initial normal formation of GBM but eventually hematuria, proteinuria and eventual ESRD with characteristic splitting of the GBM. COL4A5 mutations are commonest and are X-linked. Other forms are autosomal.
Pierson Syndrome	LAMB2	Laminin β2	Autosomal recessive disorder with variable phenotype depending on the particular mutation. However, ocular abnormalities (microcoria) are present at birth and the majority of affected individuals progress to ESRD within the first few weeks/months of life. Extrarenal manifestations including hypotonia and neurodevelopmental defects have been reported.

Please see this excellent review in Nature Reviews Nephrology for further information.

Lonely Glomeruli

One of the difficulties in doing molecular research on the kidney is the diversity highly specialized cells that exist in the glomerulus. As a result, it is important to be able to isolate glomerular tissue from the surrounding kidney. A recent paper in KI detailing a method for isolated podocytes reminded me of a relatively simple technique that I was taught a few years ago for glomerular isolation in mice.

The technique was first described in this paper from 2002 but in brief, it involves injecting the mouse heart with deactivated magnetic beads (after euthanizing them of course). Some of these beads (which are just 5µm in diameter) get trapped in the glomerular capillaries. The kidneys are then removed, minced, digested and passed through a 100µm strainer to remove any larger particulate matter. Finally, the remaining tissue is suspended and exposed to a magnet to pull the glomeruli (with the beads inside) out of the mixture. The glomeruli are then left stuck to the wall of the tube next to the magnet and they can be easily removed.

The picture below is a low-power view of the glomeruli following isolation. You can see that there is very little non-glomerular tissue present, which is remarkable given that the glomeruli make up such a small proportion of renal tissue.

Below is a higher power view of 3 more glomeruli following isolation. You can clearly see the microbeads trapped in the glomerular capillaries. Cool science. (Click on any image to enlarge)

Treatment of IgA Nephropathy - Update

Following on from yesterday's update on membranous nephropathy, I thought that this would be a good time to give an update on the treatment of another, very common glomerular disease: IgA nephropathy. IgA has been covered extensively on the RFN before including the history, pathogenesis, diagnosis, features and treatment: collected in this group of posts. This month an excellent review of the treatment of IgA nephropathy was published in Nature Reviews Nephrology which highlights some of the difficulty and controversies in the treatment of IgA.

As the authors point out, one of the major difficulties in determining the best treatment (and assessing the comparative effectiveness of therapies) is the heterogeneity in the presentation and prognosis of patients with IgA. It is extremely common, with up to 1.6% of zero-hour allograft biopsies having evidence of asymptomatic IgA. As a result, many patients are diagnosed incidentally and never have any clinically significant disease. One important issue relates to determining who is going to progress. The recently introduced MEST classification came about in an effort to define histological markers in IgA that predict progression. However, follow-up studies have indicated that the only consistent marker of progression was the degree of interstitial fibrosis and atrophy. It should be noted that this classification does not include crescents which are associated with a poor prognosis. Traditional clinical markers of progression include hypertension, increased BMI, smoking and, most importantly, persistent proteinuria of >1g/24 hours.

The treatment of IgA depends on the clinical presentation. Patients with normal blood pressure, normal renal function and no proteinuria can generally be watched in the clinic with no specific treatment and, in fact, it is debatable if these patients should be biopsied in the first place. The figure below (originally from JASN and reproduced in NRN) provides a suggested algorithm for the management of patients with IgA depending on the stage at presentation.

It is important to state that the only treatment that has been consistently been shown to be beneficial in patients with IgA is RAAS blockade.The role of corticosteroids is controversial and the current KDIGO guidelines suggest the use of steroids only in patients who have been on optimal therapy for 3-6 months, have persistent proteinuria >1g and have an eGFR of >50 ml/min/1.73m2. Low dose steroids are of no benefit and the use of higher dose steroid regimens is associated with significant morbidity so this recommendation comes with significant caveats. Other immunosuppressants including cyclophosphamide, MMF and azathioprine have been trialed in IgA and despite some encouraging results in open-label and retrospective studies, prospective studies have failed to show a consistent benefit. However, there are ongoing RCTs that will be reported in the next 12-24 months that may finally give us the definitive answer with regard to these agents.

Tonsillectomy, more commonly performed in Japan, is not recommended, either by these authors or in the KDIGO guidelines. Fish Oil may be of benefit in patients with persistent proteinuria >1g but is probably of limited use in other groups. One of the limitations of the studies of fish oil in IgA is that they were generally performed prior to the widespread use of ACEi and it is uncertain if they add any additional benefit to these agents. Again, the dose needs to be sufficient (supranormal doses).

Finally, crescentic IgA is a different entity that generally is associated with rapid progression. It is only diagnosed when more than 50% of glomeruli have crescents; the presence of a single crescent in one glomerulus should not prompt the institution of aggressive immunosuppressive therapy. Again, there are no RCTs that demonstrate a benefit of cyclophosphamide but there are some retrospective studies that have shown some effectiveness. That said, in the presence of rapidly progressive disease and active glomerular inflammation, the recommendation is that a combination of steroids and cyclophosphamide should be used.

Update on Membranous Nephropathy

U Penn established an annual one-day seminar in nephrology symposium. This year was titled “New Horizons in Nephrology: Updates in Glomerular Disease”.

Dr. Beck from Boston University gave the update on membranous nephropathy (MN). Previously, we discussed his article in the NEJM in detail, and today we will go over some key points and updates:

• 75% of MN is considered primary (idiopathic).
• IgG4 subclass is predominant in idiopathic MN.
• Anti-PLA2R is highly specific for primary MN
• Co-localization of PLA2R and IgG4 within immune deposits on biopsy.
• The majority of patients with primary MN have circulating autoantibodies against PLA2R.

There is a clear association of anti-PLA2R with disease activity.

- Positive in nephrotic state.

- Declines prior to the decrease in proteinuria.

- Absent in remission.

- Returns with (or prior to) relapse.

- Associated with recurrent MN

Methods of testing:

- Western blot

- Indirect immunofluorescence test

- ELISA : Will likely be the test of choice. Available in Europe, not yet in the US.

Treatment:

- Do NOT use corticosteroid monotherapy or MMF monotherapy for initial therapy of MN.

- No single definitive immunosuppression could be easily identified.

Current Initial therapy :

• Ponticelli regimen: methylprednisolone 1g/d iv x 3 doses followed by oral prednisone 0.5mg/kg/d in months 1,3,5; oral cyclophosphamide (2mg/kg/d) in months 2,4,6.
• Alternative initial therapy for those who refuse or have contraindications to cyclophosphamide is Calcineurin inhibitor (CNI) for at least 6 months.
• Stop if no remission in 6 months.
• If remission is attained, reduce dose every 1-2 mo by 50% and continue for at least 12 months 

Posted by Tarek Alhamad

suPARBaby?

The American Journal of Kidney Diseases published a fascinating letter this month from Jochen Reiser's group concerning the transmission of the suPAR from a mother to her newborn infant. The patient in question had a history of primary FSGS and after her baby was born, the infant had proteinuria which eventually resolved. At the time, this was published as a letter in the NEJM and was used as evidence for the existence of a soluble FSGS "factor" which had yet to be identified.

Now, more than 10 years later, they got their hands on blood from the mother and the infant. The level of suPAR in the mother was 4635pg/ml and in the child was 5225 pg/ml. This compares with a mean of 2884 pg/ml in controls. Unfortunately, the family moved away from the area so there were no follow-up samples. However, the patient's pediatrician reported that the baby's urine was negative for albumin at one year of age.

Although this does not definitively prove that suPAR is the causative agent in primary FSGS, and there is some debate about the true pathogenicity, this, along with recently published data in JASN, adds to the weight of evidence suggesting that this may be the elusive factor we were looking for. Interesting times indeed.

International Update on Glomerular Disease

On April 27th 2013, a course on glomerular disease will take place at the North Shore University Hospital in Manhasset, New York. This course is being organized by Dr Kenar Jhaveri who is well known to the blogging community. There is an impressive list of speakers and this one day course is designed to provide an update on the management of glomerular diseases.

Best of all, registration is free for medical students, residents and fellows in training. Here is a link to the course description. and application form.

A Gel Coating Your Hairy Endothelium

To follow up on one of Nate’s posts from 2010, hair grows not only on your skin. He describes a glomerular capillary to be hairy. Glycocalyx, a hairy structure attached to the glomerular endothelium, is a mixture of glycosaminoglycans and proteoglycans. The picture of glycocalyx accompanying that post is quite impressive.

Now we know these hairs are coated with another gel matrix called endothelial surface layer (ESL). ESL, together with glycocalyx, is believed to function as a barrier to prevent protein passage from blood to urine. A recent article addressing this topic was published in JASN.

By using an animal model, the authors showed that loss of ESL increases the sieving co-efficient for albumin and that the degree of albuminuria correlates with the degree of ESL loss (by the way their confocal microscopy images of ESL are pretty cool).

The implication of the study is that it’s not just podocyte or GBM that are responsible for the development of proteinuria; ESL, glycocalyx and endothelium appear to play an important role as well. For example, loss of ESL has been reported in patients with diabetes. Maybe it’s not just on the skin where hair loss occurs.

For those who are interested in this topic, there is a nice review article for further reading.  

Posted by Tomoki Tsukahara

Conference Review: Renal Biopsy in Medical Diseases of the Kidneys

I'm not a huge conference goer.   I've been to ASN a few times and have made a brief appearance at the Advanced Nephrology for the Consultant down in San Diego but otherwise haven't seen much of the conference circuit.  But when I got the Columbia flyer for the Renal Biopsy in Medical Diseases of the Kidneys a few years ago I knew it was going to be something I'd have to eventually check out.  It features the core of the GN clinical and pathology brain trust lecturing on what they know best: what GNs look like and how to manage them.  Falk and Jennete on ANCA, Appel and D'Agati on SLE, Rennke on, well, anything.  So this summer I finally got myself organized and funded and took the trip out to Manhattan to see the show.

First, a few general comments.  Staying in Manhattan is expensive.  Like $400 a night expensive.  The conference had a block of rooms reserved at hotel called the Lucerne for $200 a night.  I procrastinated and called the hotel after the deadline listed in the flyer and was politely told that all the rooms were gone and I could now book one for $440 a night if I was interested.  I declined and luckily had a family member take mercy on me and allowed me to stay with them on short notice.  If that hadn't come through I was ready to book a room through Airbnb which, if you haven't used it, is a cool site where you can find locals renting out space in their homes for cheap.

Second, it is hot and humid in Manhattan in the summer. Crazy hot. Bring wardrobe accordingly. I scuttled between air conditioning on the subway, lecture hall and my hosts place the whole week (I know, soft Californian behavior).

Third, take advantage and see Manhattan. I channeled my inner intern and skimped on sleep to hit the conference then experience the city.  Well worth it.

As for the conference it is certainly one of the best I've ever been to.  It's truly education focused, the talks are given in series rather than parallel like some of the big conferences so you're never at risk for missing anything.  The speakers are, for the most part, amazingly clear and avoid the common sin of nerding out on their own particular pet projects but rather focus on big picture relevance.

It's fun watching the older and younger members of the team interact as they clearly like each other and have a great back and forth.  You also get a sense of how some of the younger faculty like Glenn Markowitz, Andrew Bomback and Leal Herlitz are coming into their own and making their marks on the GN world.

The group aren't afraid to give their opinions when it gets to the grey areas.  Who doesn't want to hear how Gerry Appel manages not so clear cut cases of Lupus Nephritis?

Lots of path slides, which for those like myself about to take boards, is great review.  The faculty clearly take pride in their lectures and are up-to-date current in their lectures with recent literature incorporated.  Lots of interested cases discussed.  Very nice syllabus with all slides included.  If they want to kick up their game they could go for online access to high res versions of the path slides - though many of them you can find in the recent NephSap renal path edition.

My inner intern unfortunately caught up with me on the last day and I missed Saturday morning and had to catch a flight out before the path and case workshops in the afternoon.

My only gripe would be the daily supplied lunch - terrible.  I bailed and braved the heat to find something good to eat, which was actually a lot of fun.  All in all a wonderful conference that I highly recommend.  I kept up a pretty good twitter feed of pearls from the first three days of lectures which you can check out here.

Isolated C3 deposits on an inflammed glomeruli

Six months after a kidney transplant, a patient developed a rising creatinine, significant proteinuria (~2g/day), hematuria and low C3 levels. A kidney graft biopsy showed severe glomerulonephritis with mostly occluded capillary loops with immunofluorescence staining negative for immunoglobulins but strongly positive for C3 (representative picture on left). On EM, subendothelial and mesangial electron dense deposits were visualized. The features are characteristic of the so-called C3 glomerulonephritis (GN).

C3 GN is part of the same family as dense deposit disease (DDD) and fall into the new proposed classification of C3 glomerulopathy. Compared to C3 GN, DDD is characterized by extremely electro-dense intra-membranous deposits on EM. Both entities can present with features of MPGN, once significant chronic endothelium glomeruli damage occurs. These glomerular pathologies shared in common its pathogenesis, which involves complement dysregulation.

Classically, immunoglobulins that deposit in the glomeruli are the major triggers of glomeruli inflammation. In rare settings such as C3 GN, complement proteins are present in the glomerular lesions in the absence of immunoglobulins. Abnormal activation of the alternative pathway is implicated on this finding. Either an acquired or inherited defect in the control of alternative complement pathway should be investigated.

The following tests are generally recommended:

** Levels of C3 and complement factors B/H/I

** Presence of C3 nephritic factor (C3 NeF)

** Genotyping for certain complement mutations (common in kids)

Our patient underwent complement testing and was found to have circulating C3 nephritic factor. This factor is an IgG autoantibody that directly stabilizes the C3-convertase activating complex of the alternative pathway and thereby prevents the normal inhibitory action of complement factor H, leading to continuous activation of the complement cascade and consequent deposition of complement by-products in the glomeruli.

Although atypical hemolytic uremic syndrome (aHUS) is also strongly associated with a dysregulation of the alternative pathway, glomerular lesions in aHUS do not exhibit C3 deposits or electro-dense deposits on electron microscopy, differentiating from C3 glomerulopathies. How changes in complement regulation leads to very diverse presentations might be related to the underlying defect. Nonetheless, atypical HUS could be placed in one side of this spectrum of renal diseases associated with alternative complement dysregulation (figure below; adapted from Servais et al. 2007).

While the pathogenesis is complicated, even harder is the treatment challenge of C3 GN in our patient above. There are a couple of new promising approaches and markers that might help guide therapy. I will try to cover them on my next blog.