A Man's Best Friend

I'm sure many of you have a pet dog. Well, as Rover quietly licks himself in the corner, few would believe that he may hold the key to unlocking the genetic secrets of human disease, as suggested by this fascinating article in this weeks Nature Genetics.

Dogs evolved from the wolf about 15,000 years ago and, as a result of plenty of random mating over that time period, developed a 'short-range' haplotype structure due to genetic recombination. This resulted in lots of genetic diversity, with a wide variety of genetic mutations occurring at a low frequency across the species.

People only began breeding pedigree dogs in the past 200 years or so, whereby animals with desirable physical or behavioral traits were paired. This was effectively an extreme genetic experiment, which concentrated a fraction of the population genetic variation within a single pedigree, resulting in the 400 or so unique breeds we know today. Unbeknown to dog breeders, as well as collecting favorable polymorphisms for physical traits, these dogs were also silently accumulating disease-causing genetic mutations of strong effect due to genetic 'hitch-hiking'. Pedigree dogs demonstrate exceptionally high prevalence rates of complex disease within breeds (sometimes ~ 15%!), including many kidney diseases such as polycystic kidneys, MPGN and nephrolithiasis.

Within each of these breeds, there are very high rates of linkage disequilibrium, as there hasn't been enough time for long stretches of DNA to be broken down by recombination. This makes genome-wide association (GWA) much easier than in human studies, such that for a simple Mendelian trait one may need as few as 40 dogs and a gene-chip with 15,000 SNPs (as opposed to thousands of patients and around a million SNPs in humans).

In the aforementioned study, the investigators examined the Nova Scotia Duck Tolling Retriever, a hunting dog with a very high prevalence of autoimmunity, including ANA positive lupus. Using a simple 2-step GWAS approach they identified no less than 5 novel candidate genes associated with the development of lupus, effectively blowing the field wide-open. Most of the genes identified code for proteins involved in the NFAT pathway of T-cell activation, including a calcineurin subunit. The existence of a disease-causing single nucleotide polymorphism in calcineurin suggests a possible genetic explanation for differential treatment responses to cyclosporin / tacro in Lupus. Furthermore, as the NFAT pathway is known to be involved in peripheral tolerance, these results may result in a ground-shift in our understanding of the mechanism of this disease.

Continuation of Renal Fellow Network

Thank you to everyone who has made contact with the Renal Fellow Network over the past week or so. Your outpouring of love and support has been a great comfort to Nate's family and friends at this difficult time.

Many have asked about the future of the RFN project. After discussion with Nate's family and mutual colleagues, it is my intention to continue the blog. There are a few logistical issues to resolve, but I hope to resume blogging soon. I am pleased to announce that I have received several generous offers of collaboration from Nephrology trainees and Nephrologists of different backgrounds and research interests. I am confident that we can maintain the high standard of content that Nate and the RFN are known for.

Nathan Hellman Rest In Peace

It is with great sadness and sorrow that I am writing to inform you of the passing of Nathan Hellman, MD, PhD, and founder of the Renal Fellow Network Blog. I worked with Nathan in the Division of Nephrology of the Department of Medicine, Massachusetts General Hospital (MGH), Boston, where we were both fellows. He passed away on February 13, 2010 at the Massachusetts General Hospital after a short illness.

Born in Houston, Texas, on December 8, 1973, Nathan grew up in Duluth, Minnesota. He attended Yale University, graduating magna cum laude. After Yale, he went to Washington University, where he obtained his MD/PhD. He did his residency training in Internal Medicine at University of Pennsylvania and became a member of the Division of Nephrology in 2007 as a clinical fellow in Nephrology. He was completing his fellowship as a research fellow and member of Iain Drummond’s research group. He was to be appointed a faculty member in July 2010.

Nathan was most importantly a wonderful husband, father, son and brother. At work, he touched all of our lives with his warm heart and spirit, great sense of humor and remarkable intellect. He was an exceptional scientist, a talented and insightful clinician and a remarkably kind and humble human being. He is survived by his wife, Claire, his two children, Sophie and Max, his parents, Dr. and Mrs. Hellman, his two sisters, and their families.

A Memorial Service will be held to celebrate his achievements and commemorate his contributions to the MGH community at the O’Keefe Auditorium on Wednesday, February 17th at 2pm, followed by a reception in the Thier Conference Room at 3.30pm.

In lieu of flowers, memorials can be sent to:

Nathan Hellman Memorial Fund
PO Box 471044
Brookline Village, MA 02447

Tamm-Horsfall Protein and Kidney Disease

Tamm-Horsfall protein (THP), or uromodulin, is a glycoprotein secreted by the renal epithelium, best known as the proteinacious framework of all urinary casts. It’s function in health is an enigma, but prevention of bacterial colonization of the urinary tract, inhibition of stone formation and the maintenance of water impermeability in the thick ascending limb of Henle are all possible. Mutations in the gene UMOD, which encodes uromodulin, cause familial juvenile hyperuricemic nephropathy, which Albert reviewed last month. Recently, a large GWAS from the CHARGE consortium identified SNPs within the UMOD gene as being associated with an increased risk of CKD, also reviewed by Nate.


In this months issue of JASN, Kottgen et al. provide a nice proof of principle of the GWAS approach, by demonstrating urinary uromodulin levels predict incident CKD, in a prospective, case-control study of ten years duration. These findings were then externally replicated. In addition, they identify a protective SNP which confers a reduced risk of CKD via reduced urinary urimodulin concentration. Although the biology remains to be determined, this finding further supports the role of uromodulin in the pathogenesis of common variant CKD.

Did you know your endothelium is hairy?

Just heard an interesting talk on endothelial dysfunction and its relation to nephrology.  One thing I learned is that the vascular endothelium is actually "hairy", as demonstrated from this electron micrograph (taken from this website). These little hairs are the endothelial glycocalyx, a gel-like layer of negatively charged proteoglycans and membrane glycoproteins which helps serve as a protective barrier from blood flow for the endothelial cells. You can't see it on standard light microscopy since it apparently requires special preservation techniques not normally used.  There is some data to suggest that vasculopaths and diabetics have a less developed and overall thinner endothelial glycocalyx compared to healthy controls, suggesting the importance of this structure in maintaining a healthy vasculature. 

In the kidney, the glomerular endothelium is a little different than most other endothelia in that they contain fenestrae:  actual pores in the endothelial cells which are large enough to let most proteins pass through, but small enough to exclude circulating blood cells. The glomerular fenestrated endothelium makes up a component of the glomerular filtration barrier (along with the glomerular basement membrane and the podocyte layers), and the classic teaching is that the negatively charged proteoglycans on this layer help form part of the charge barrier keeping albumin from getting filtered (though this is now being debated by some). Here are some pretty pictures of the glomerular fenestrated endothelium:

World Series of Poker Celebrity Kidney Transplant Recipient

Meet Jennifer Harman. She is apparently one of the most famous female professional poker players in the world, having done quite well at the prestigious "World Series of Poker" tournament. It turns out she is also a kidney transplant recipient, having undergone 2 transplants. Admirably, she has become a real advocate for patients with kidney disease, seeking to raise awareness for living organ donation. Recently, she announced that she would be donating 1% of all profits to the NephCure foundation, which funds research relevant to nephrotic syndrome and FSGS in particular.

Super Bowl Sunday Contrast Nephropathy Post

How common is contrast nephropathy in the general outpatient setting? When you send one of your outpatients to get a CT scan with iv contrast, what is the real risk?  Much of the randomized, controlled trial data on contrast nephropathy comes from INPATIENTS undergoing angiography or other procedures, often focusing on those with the greatest perceived risk of contrast nephropathy (e.g., diabetics, those with underlying CKD to begin with) in order to generate enough meaningful datapoints for analysis. An article by Mitchell et al in this month's CJASN reports the risk of contrast nephropathy in a mixed OUTPATIENT group--and finds that the risk of contrast nephropathy may actually be higher than what has generally been appreciated.

One big caveat, in my mind: even though the article is entitled, "Incidence of contrast-induced nephropathy after contrast-enhanced computed tomography in the outpatient setting," the phrase "outpatient setting" should be examined a little more closely. Their patient population wasn't exactly "outpatient" as we normally think of it; it was all the patients entering a large, academic emergency department who underwent CT scans with iv contrast. One could argue that by the very nature of their showing up in an emergency department (as opposed to their primary care physician office) they could be seen as "sicker" and therefore more susceptible to the effects of iv contrast use. Nonetheless, the researchers found that the incidence of contrast nephropathy (defined as an increase in serum creatinine greater than 0.5 mg/dL or greater than 25% of baseline between 2-7 days after contrast administration) was 11% (70 out of 633 patients receiving iv contrast), which is quite high. Not surprisingly, the development of CIN was also associated with an increased risk for developing severe renal failure and death.

There's still a lot of controversy about contrast nephropathy and just how significant it is--after all, the vast majority of patients see a return of their creatinine to baseline levels--but perhaps the knowledge that over 10% of patients in an emergency department show evidence of renal injury with a simple CT scan with iv contrast should make us think twice about ordering this test so frequently. 

Making Allograft Nephrectomies More Routine?

As much as kidney transplant outcomes have improved over the decades, we're not perfect: a sizable group of patients end up transitioning to dialysis therapies after the kidney transplant stops working. What should be done with the allograft once you've given up hope for any meaningful allograft function? In many transplant centers, the prevailing wisdom is simply to leave the allograft in place, as long as it is not causing any exceptional systemic inflammatory response as a result of prolonged rejection. If nothing else, it avoids an additional surgery in a dialysis population that is often plagued with comorbidities and the potential for surgical complications.

However, a recent study in this month's JASN by Ayus et al suggests otherwise, hinting that allograft nephrectomy might be considered as a more routine measure. The investigators probed the USRDS, looking at all transplant recipients who returned to long-term dialysis. A retrospective analysis demonstrated that those patients who had undergone an allograft nephrectomy had a 32% lower adjusted relative risk for all-cause death, even after controlling for factors such as age, comorbidities, and donor characteristics. The authors end the article by suggesting that "routine allograft nephrectomy in stable dialysis patients with a failed renal allograft should be evaluated against current management strategies in a randomized trial."

An accompanying editorial cautions against adopting this strategy too readily. They point out that patients in the allograft nephrectomy group tended to be younger and healthier, and even with appropriate adjustments to account for this, residual confounding and selection bias may well be altering the results. They also point to evidence that keeping the allograft in may actually be beneficial from an immune standpoint. Still, however, the retrospective by Ayus et al is intriguing and will doubtlessly be pursued further.

Warfarin and Vascular Calcification

Warfarin has recently been associated with an increased risk of stroke in ESRD patients with atrial fibrillation. This makes the decision whether to initiate warfarin therapy in dialysis patients with atrial fibrilltion anything but clear cut. Add to this the emerging evidence that it also promotes vascular calcification, and you have a real dilemma.

This latter side effect hinges on Matrix Gla protein, or MGP. In ESRD, vascular smooth muscle cells (VSMCs) undergo a phenotypic switch to take on the appearance of osteoblasts, in a well-described process thought to be driven by hyperphosphatemia. Once this has occurred, VSMCs lay down a protein matrix which can undergo calcification, similar to normal bone. MGP is an essential endogenous inhibitor of this process, mediated in part by it’s ability to bind calcium. This ability to bind calcium is a Vitamin K dependant process, explaining how warfarin may increase the risk of vascular calcification.

Warfarin has been linked to aortic valve calcification in both the general population and in ESRD, as well as calcific uremic arteriopathy in the latter. In fact, the odds ratio for severity of aortic valve calcification after 18 months of warfarin in ESRD was almost 4. Prospective trials are clearly needed, but for now, adopting a restrictive policy towards warfarin use in ESRD seems wise, such as reserving it for patients with a CHADS score > 2.

Drug Trials

Last week's poll results:  who should provide funding for large, randomized controlled drug trials?  Most respondents (58%) chose the diplomatic answer and said that it should be a combination between government and pharmaceutical companies.  I was somewhat surprised to note that more people felt that conducting RCTs should be the responsibility of the government (24%) rather than a responsibility of the pharmaceutical industry (only 7%), one of the most profitable industries within the U.S. currently. 

Check out the new poll question of the week in the right margin.

The trials of a lab rat...

I was on the phone to one of my friends the other day. He's a renal fellow currently doing lab-based research in Boston. He's been having a difficult time of it lately, with none of his experiments working out, and has understandably been getting a bit frustrated and disillusioned.

Me: "So, how's your research going? Any major breakthroughs?"

G: "Well, I've had an epiphany!"

Me: "An epiphany, that's fantastic! About time!"

G:"What do you mean fantastic, I said a rat pissed on me! I think I'm going to switch careers..."

Well, I thought it was funny...

The actin cytoskeleton of the podocyte

Peter Mundel, a well-known researcher of podocyte biology, gave our Renal Grand Rounds today.  Here's what I took away from this morning's talk:

-regulation of the podocyte's actin cytoskeleton is postulated to be the final common pathway in most instances of nephrotic syndrome/proteinuria.

-in some ways it is better to think of the podocyte as a modified smooth muscle cell rather than a modified epithelial cell based on its intricate network of actin & myosin which mediate contractility.

-the medication cyclosporine (used in the treatment of some refractory forms of nephrotic syndrome) acts NOT on modulation of immune cell function as previously assumed, but rather on direct effects on the podocyte actin cytoskeleton, as detailed in a prior post.

-a key event in generating abnormal, protein-leaking podocytes is turning them from a stationary cell to a motile one.  This key transition is mediated by three distinct Rho GTPase proteins, already established to mediate motility in several cell types. In general, Cdc42 and Rac1 promote podocyte motility, whereas RhoA promotes podocyte stabilization. It is possible that targeting these Rho GTPase pathways could lead to novel therapies for podocytopathies, and this is being investigated.  

Check out a good 2009 Kidney International review of the topic by Mundel and Reiser.

How Luminex Beads Work

You may have come across the phrase "Luminex beads" while doing your transplant nephrology rotation, or heard the term while hanging out in the Tissue Typing Lab. This post is intended to briefly describe the Luminex technology and how it works. A good review by Tait et al can be found here.  

Acute cellular rejection in kidney transplant recipients comes from the presence of recipient anti-HLA antibodies directed against donor antigens. How do we detect such antibodies and prevent donor-recipient mismatches from occurring? Traditionally--and still one of the most practical tests--we use the complement-dependent cytotoxicity (CDC) "cross-match" assay. While useful, however, more recent "solid-phase" technologies have allowed a more sensitive detection of anti-HLA antibodies in recipient serum, and the Luminex system is one of them.  

Briefly, the Luminex technology consists of a series of differently-colored polysterene beads, populations of which contain distinct HLA molecules attached to them. The lab incubates an aliquot of the kidney transplant recipient's serum with an aliquot of beads, and if there are any anti-HLA antibodies present they will bind to the beads. A second phycoerythrin-labelled anti-human IgG antibody (aka, a "secondary antibody") is then added, and after washing off unbound antibody, the beads are passed through a machine which works like a flow cytometer, dropping beads one-by-one through a narrow chamber. As the beads pass through the chamber, they are hit with lasers of a specific wavelength, exciting both the fluorescently-labeled secondary antibody AND the fluorochrome within the bead itself--thus allowing the detection and identification of specific recipient antibodies.  

There are different types of beads:  for example, some beads are coated with many different Class I or Class II molecules, designed to detect a wide range of recipient antibodies. Some beads are designed to be like a single cell, containing HLA antigens encoded by two different alleles from each of the Class I (HLA-A, B, & C) and Class II (HLA-DP, DQ, DR) loci. Finally, there are the "single antigen beads" (SAG), a bead coated with one sole antigen. This can be particularly useful in patients with a high PRA (panel reactive antibody)--you can actually identify the specific HLA antigens to which the individual is sensitized.  

While potentially quite useful, it is important to also realize the limitations of this technology. While much more sensitive than the standard CDC cross-match, it is not always clear what a positive antibody result means--though in most instances, the CDC cross-match is still the primary determinant as to whether a given kidney donor-recipient pairing is deemed acceptable.