Alport Syndrome

I recently saw a patient in clinic with long-standing hematuria with numerous family members on her mother’s side with hematuria.  She now presented with proteinuria but stable renal function.  Collagen IVa disease was highly suspected and genetic sequencing identified a heterozygous carrier for a previously characterized pathogenic mutation in Col4a5.

In the process of taking care of this patient who was heterozygous for X-linked Alport’s syndrome, I wondered, “Who is Dr. Alport?”.

Dr. Arthur Cecil Alport was a physician originally from South Africa who attained his medical training in Edinburgh, Scotland. He had many different interests initially studying malaria abroad and then practicing medicine in London before becoming a Professor in Egypt where he fought for the care of poor patients.  He showed that with careful observation one can provide valuable insights into a specific disease. 

Dr. Alport was not the first to identify the entity of hereditary hemorrhagic nephritis.  Initially, William Howship Dickinson described a family with 11 out of 16 members with albuminuria in 1875.  Subsequent studies by Guthrie and Hurst identified families with hematuria and kidney disease of varying severity.  Dr. Alport saw a patient from the Guthrie/Hurst cohort which he further studied and published with the title, “Hereditary Familial Congenital Haemorrhagic Nephritis”, in the British Medical Journal in 1927 which led to the identification of the disease as Alport’s syndrome. 

In this paper, he found a number of female members of the family had hematuria but did not develop edema, heart failure, and kidney failure, a fate reserved for a selected few male members of the family.  He also noted numerous female members with profound deafness that at time was not associated with hematuria.  Though he acknowledged the hereditary nature of this disease, he also found hematuria and albuminuria were exacerbated by streptococcal infection which he had limited success in recreating in rabbits. 

The history of medicine often provides an interesting context for our current understanding of human disease.  By observing the association of deafness in families with hereditary hematuria, Dr. Alport brought to light a key identifier of the disease entity.  This identification ultimately led to the disease to be associated with his name though the renal phenotype of familial hematuria was discovered by prior investigators.

Posted by Ankit Patel, Nephrology Fellow, Joint BWH/MGH Fellowship Program

Precision Nephrology

One of our attendings, Dr. Sylvia Betcher, PhD, MD gave an excellent presentation at our renal conference about genetic testing in renal diseases that she learned about at #KidneyWeek2015. There were so many good things I liked about her talk, that I want to share what I learned. On January 30th 2015, President Obama announced in his State of the Union address a Precision Medicine initiative. This will provide researchers in the biomedical field with the necessary tools to define preventive measures and treatment of disease by examining variability in genes, environment, and lifestyle of each patient. Precision Medicine relies on specific molecular and genetic information to classify a certain disease into subsets that allow consideration of focused therapies, which is currently being accomplished through GWAS (Check the #NephMadness genetic nephrology region bracket on GWAS here), whole gene sequence analysis via next generation sequencing (NGS) and VAAST (Variant Annotation, Analysis and Search Tool) which is used to identify damaged genes and their disease-causing variants in genome sequences. The president’s 2016 budget will provide $215 million to the various agencies including the NIH and FDA to accomplish his goals. The objectives of such initiative can be found here.

Most of the budget will go to cancer research and there is no mention of rare diseases or any particular hereditary or genetic disease. In the Nephrology world, are there any diseases in particular that need to be addressed? Yes, for instance Steroid Resistance Nephrotic Syndrome (SRNS), among many others. In this paper, a single-gene cause of SRNS was detected in 526 out of 1,783 families (29.5%), by examining 21 genes. The authors mentioned that screening of these genes is cost-effective and may avoid the undesirable side effects of steroids when a mutation is detected and potentially offer targeted therapy (for example with Coenzyme Q10 in cases of COQ2 nephropathy)

According to the NCBI Genetic Testing Registry as of August 2014, there were approximately 4,500 conditions for which genetic testing is available, many of which will have renal manifestations. Advantages to testing include providing specific therapies, allowing family counseling and to evaluate kidney donors in family members. Diseases that are being considered in Nephrology for genetic testing include: rare autosomal dominant interstitial nephropathies (UMOD, MUC1, REN), Syndromic and Polycystic Kidney Disease, Alport Syndrome and Congenital Anomalities of the Kidney and Urinary Tract (CAKUT). Additionally, I want to emphasize the fact that Genomics England (a company owned by the UK Department of Health) and Illumina (an American biotechnology company based in San Diego, California) have launched a $524M project to create a large genome database. Their plan is to sequence 100,000 whole genomes by 2017 focusing on rare diseases, cancer and infectious diseases. A nephrology consortium has been set up to provide renal patients for this ambitious project.

Let’s say we have screened our patients for congenital kidney diseases. Now what? We have to consider whether the results will influence any change in management, or perhaps we need to screen for extra-renal manifestations. I think that providing family counseling will definitively be helpful. It will also influence the decision on safety of kidney donation. Urine could be an excellent source of genetic information through DNA fragmentation which is a normal process in apoptotic cells to eliminate mutated, damaged or infected cells and is usually highly fragmented whereas in cancer cells the DNA maintains its integrity. So in conclusion, these are exciting times in Nephrology with precise genetic testing now a diagnostic option. Do you see yourself practicing Precision Nephrology in 10-20 years? Let us know what you think!

SHROOM3, Making sense of GWAS risk alleles

In this months edition of JCI a group led by Dr B Murphy from Icahn School of Medicine at Mount Sinai describe a beautiful set of experiments that explain the mechanism by which the CKD and eGFR risk allele rs17319721 causes chronic kidney allograft damage.

The single nucleotide polymorphism rs17319721 is in intron 1 of a gene called SHROOM3. The risk allele A (major allele G) of rs17319721 was found in large GWAS studies of European ancestry to be associated with GFR (p=1*10−12), incident CKD (p=0.005) and GFR in type 2 diabetic patients (P= 3.18E-03). The risk allele, A, frequency is about 40% in caucasian populations but is less frequent in non-caucasian populations.

The authors decided to investigate the effect of rs17319721, the SHROOM3 risk genotype, on kidney allograft fibrosis and chronic allograft nephropathy (CAN). Furthermore, they sought to determine what role, if any, SHROOM3, plays in allograft fibrosis. The risk locus was genotyped in over 500 allograft recipients and 500 allograft donors from the GoCAR transplant cohort. Also, SHROOM3 transcript levels in 3month protocol allograft biopsies were recorded in some of these patients. Donor genotype carrying one risk allele A (A/A or A/G) was associated with higher 3month SHROOM3 expression levels compared to the normal donor G/G genotype. Interestingly, correlation occurred only in Caucasian donors when analyzed separately and there was no association with the recipient risk genotype.

The authors then looked at 12month allograft GFR and chronic allograft dysfunction index score at 12 months (CADI-12). 3month SHROOM3 expression levels were inversely related to 12 month GFR, predictive of CADI-12 and were predictive of worsening CADI score (3m to 12m)(termed ‘progressors’). These associations were not found in non-Caucasian donors.

To assess whether 3M SHROOM3 levels could predict CAN they generated logistic models that included recipient age, sex, race, AR and CIT with or without 3M SHROOM3 levels to predict 12M CADI ≥2 or 3-12M CADI change (ΔCADI) of ≥2. AUC for prediction of high CADI-12 and ΔCADI were improved in each subgroup when SHROOM3-3M level was added. For Caucasian donors AUC for ‘progression’ was 0.81 with SHROOM3 and 0.74 without SHROOM3. Furthermore, the A allele in the donor was associated with greater risk of CADI-12≥2 in all allografts (OR 1.98; CI, 1.10–3.59), indicating a higher risk of CAN with the risk allele.

This work demonstrates that the A risk allele (rs17319721) in donors is associated with higher 3M SHROOM3 levels and increase risk of CAN. Also, 3M SHROOM3 levels predict CAN and 12M GFR.

So what is the mechanistic consequence of having the A allele vs the G allele? rs17319721 is located in a transcription factor binding motif. The authors found that the transcription factor TCF7L2 binds more strongly to this motif when A is present vs when G is present. Wnt agonist increased TCF7L2/β-catenin complex binding to the A allele binding site but not the G allele site. TGF-β1 is a known key growth factor regulating renal fibrosis. The authors found that TGF-β1 induced increases in SHROOM3 expression via the Wnt/β-catenin/TGF-β1 pathway in renal tubular cells.

Then they showed SHROOM3, in turn, enhances the TGF-β1/SMAD3–induced expression of profibrotic genes including CTGF, Vimentin, and Collagen IV (downstream targets of TGF-β1/SMAD3 signaling) and these genes were significantly upregulated in allografts within the highest quartile of SHROOM3 expression. Finally the authors verified these data in a mouse model of fibrosis.

Taken together, this data suggest an increased profibrotic program in the presence of the enhancer function of the risk allele and/or increased SHROOM3 expression. This schema is illustrated below.

This paper nicely describes the mechanism of action conferred by a single risk allele found in large GWAS studies. Until recently there has been little data to explain the relevance of the many risk SNPs described in GWAS studies. Without an understanding of the mechanism through which these SNPs confer disease there can be no progress towards identifying potential therapeutic targets. This study has identified SHROOM3 as a potential therapeutic target for chronic allograft nephropathy.

Michelle P Winn Endowed Lectureship, ASN 2014

At this year's ASN Kidney Week in Philadelphia Andrey Shaw, MD, presented the inaugural Michelle P Winn Endowed Lectureship. Dr Shaw was not only a longtime collaborator of Michelle’s but also a very close personal friend making him the perfect choice for this inaugural lectureship. Dr Shaw delivered an excellent talk interweaving highlights from Michelle’s stellar career with examples of Michelle’s fun loving and genuine kindhearted nature. I was lucky enough to work in Michelle’s lab from 2012 to 2014. She cared greatly about all her mentees both professionally and personally. She was a huge inspiration and a friend.

Michelle did her undergraduate studies at the University of North Carolina before going to medical school at East Carolina University. She then entered Duke University for residency and fellowship before joining the Duke faculty. Despite spending most of her career at Duke she remained a true Tar Heel (UNC) fan!

She received her training in classical human genetics from Drs Jeffery and Peggy Vance at the Duke Center for Human Genetics. In collaboration with another longtime friend and collaborator and early mentor at Duke, Dr Peter Conlon, Michelle began investigating the genetic heterogeneity of FSGS.

• Together Drs Winn and Conlon collected what is now one of the largest Familial FSGS datasets in the world.
• Michelle’s early work linked familial FSGS in one large family from New Zealand to a locus on chromosome 11.
• Following this she identified TRPC6 as the cause for FSGS in this family. This was a seminal paper published in Science and introduced an ion channel and calcium into the burgeoning field of podocyte biology. 
• Michelle’s further work on TRPC6 made a huge contribution to the understanding of the biology of TRPC6 in kidney disease. 

Michelle was also very interested in other inherited kidney diseases.

• She described linkage of a gene causing MPGN type III, 
• identified TNXB mutations causing vesicoureteral reflux, 
• was involved in studies of genetic factors influencing the development and progression of IgA nephropathy 
• a hybrid CFHR3-1 gene causing familial C3 glomerulopathy. 
• Her work also helped to define the disease burden and impact of other FSGS causing genes such as INF2, NPHS2 and PLCe1. 

Towards the end of her career and even while fighting her illness she remained very involved and continued to contribute in a huge way to the field we all love.

• She discovered Anillin a new gene causing FSGS, 
• a new mutation in the WT1 gene 
• added further insights into the function and regulation of TRPC6 in podocytes. 

Michelle was a leader in her field of podocyte biology and renal genetics. In 2007 Michelle won the ASN Young Investigator Award. I am sure that if her life had not been tragically cut short she would have been awarded the highest honors our specialty has to offer. The creation of the Michelle P Winn Endowed Lectureship is testament to this probability. Michelle was a beautiful person and will be missed by all who knew her.

Lessons from the Akita mouse

Diabetic nephropathy (DN) is a major cause of ESRD worldwide. While many attempts have been made to develop reliable animal models that mimic human disease—ob/ob, db/db obese diabetes type 2 diabetes models, NOD1 mice, streptozotocin (STZ)-induced diabetes model etc., current mouse models still do not display full spectrum of functional and pathological process of human DN (JASN 2009). In addition, it was revealed recently that genetic background has an important effect on the development and severity of diabetic nephropathy. Indeed, C57BL/6 strain, most commonly used for many experimental studies, is highly resistant to diabetic injury. Recently, Akita mouse gained great interest in research of DN. Let’s start with some history of Akita mouse.

Akita mouse was initially reported as a mouse model mimicking MODY (maturity onset diabetes of the young) in late 1990s (Diabetes 1997). Later, they were found to exhibit type 1 diabetes mellitus via a spontaneous point mutation (C96Y) in the Ins2 gene (Wang JCI 1999), which disrupts a disulphide bond between the insulin A and B chains in a dominant negative way, resulting in misfolding and accumulation of insulin molecules (a.k.a ER stress). Akita C57BL/6 develops spontaneous hyperglycemia at 400-500 mg/dl range, mild hypertension, and albuminuria approximately 50-100 microgram/ day, at around 3-4 weeks of age. Difference in disease susceptibility, especially in severity of albuminuria, depending on genetic backgrounds was reported. (Gurley, Am J physiol Renal Physiol 2010, Methods Mol Biol 2012)

Here are recent research updates on Akita mice:

 KKS (Kallikrein-kinin system)—bradikinin 1/2 receptor deficiency in Akita mouse
ACE inhibitors exhibit renoprotective effects via decrease in intra-glomerular pressure by dilating efferent arterioles. Another explanation of renoprotective effects of ACE inhibitors is via kallikrein-kinin system (KKS). B1R and B2R are the two bradikinin receptors; B2R expresses constitutively and, on the other hand, B1R is inducible by inflammatory stress and DN. Studies by Kakoki et al. (Kakoki PNAS 2004 and PNAS 2010) showed B1R and B2R KO mice developed more severe kidney albuminuria (1.7 x (B2R KO-Akita) and 3.0 x (BR double KO-Akita) compared to Akita WT) and glomerular pathology at 6 or 12 months of age, suggesting potential renoprotective effects of KKS. Hypothesis is that the lack of B2R/B1R enhances oxidative stress via reduction of eNOS and prostaglandins as well as mitochondorial dysfunctions. More details to follow.
 eNOS (endothelial Nitric Oxide) deficiency in Akita mice 
In humans, three variants in the endothelial NOS (eNOS) gene NOS3—G894T in exon 7, tandem repeats in intron 4, and C786T in the promoter—are associated with DN. Actually, the frequency of G894T is relatively common and 5-9% individuals are homozygous for TT and thus with less activity of eNOS. Wang et al. (Wang et al. PNAS 2011) developed eNOS-/-Akita mouse in B6-129 hybrid background (eNOS-/-Akita in C57BL/6 is lethal around 5 months before developing DN), and showed eNOS KO resulted in increase in glomerular filtration (increase at 3 mo, then decrease at 7 mo), basement membrane thickening, glomerulosclerosis and albuminuria, independent of blood glucose and blood pressure. 
 Other models in Akita mouse
ACE2 (anigiotensin-conversion enzyme 2) deficiency (Wong et al. Am J physiol 2007) and ketogenic diet (Poplawski et al. PlosONE 2011) in Akita mice have been explored and details to follow.

In summary, Akita mouse is an excellent model of human DN, mimicking both pathophysiology (hyperfiltration and albuminuria) and pathology (GBM thickening and glomerular sclerosis) of kidneys exposed to high serum glucose.

Naoka Murakami

CFHR5 Nephropathy and Complement in Kidney Diseases.

It is fair to say that unless you a nephrologist practicing in Cyprus you are unlikely to ever see a case of CFHR5 (Complement factor H related protein 5) nephropathy. It does however give an insight into the growing appreciation of the role of complement in renal disease. The culprit mutation in CFHR5 is thought to affect around 1 in 6000 Cypriots and is associated with autosomal dominant glomerulonephritis and renal failure.

The disease seems to predominantly affects males (why is not well understood) and is characterised by microscopic haematuria with mild proteinuria. Macroscopic haematuria can occur, particularly during episodes of infection. Serum C3 and C4 levels are normal. Progressive decline in renal function leads to ESRD. Biopsy findings are of MPGN with C3, C5 and C9 deposition.

The alternative pathway is both one of the three complement pathways by which C3 can be generated and also provides an amplification loop for C3 generation by the other pathways. Complement factor H is a key regulator of alternative pathway activity. Inherited or acquired deficiencies in CFH can lead to the catastrophic consequences of uncontrolled complement act
ivation seen in aHUS. CFHR5 is also thought to be a regulator of the alternative pathway, though it is probably more important at membrane surfaces rather than in the fluid phase- hence the normal C3 and C4 levels. Defective CFHR5 fails to inhibit accumulation of C3 metabolites at the glomerular surfaces resulting in their accumulation.

CFHR5 nephropathy is one of a group of diseases recently included under the definition of “C3 glomerulonephritis”. These include dense deposit disease, C3 glomerulonephritis and CFHR5 nephropathy. An absence of immunoglobulin deposition differentiates them from immune-complex mediated GN. The C3 glomerulopathies, aHUS, Lupus nephritis, IgA nephropathy, ANCA-associated vasculitis and renal ischemia-reperfusion injury are just some of the diseases where complement is thought play an injurious role.

At present eculizimab –an anti-C5 mAb- is the only licensed complement inhibitor. A large number of other drugs are under development, many of which have other targets in the complement system.

Posted by Jonathan Dick

UMOD, Translational Nephrology! Contender for Top 10 of 2013

Genome-wide association studies (GWAS) are large population based collaborative studies seeking genetic patterns that would explain common complex phenotypes. These large studies have given us much insight into the genetic risk profile of patients with diseases such as chronic kidney disease and hypertension. Frequently the genetic markers described as risk genotypes are single nucleotide polymorphisms (SNPs) that lie in regions of the genome yet to be ascribed a functional role. Trudu et al in Nature Medicine this month describe a beautiful set of experiments that explain how risk variants for CKD and hypertension found by GWAS effect blood pressure regulation at the molecular level.

A number of GWAS have described risk SNPs in the promoter region of UMOD (1, 2, 3,4, 5, 6, 7). The UMOD gene codes for uromodulin or Tamm-Horsfall protein, which is secreted into the urine by cells of the thick ascending loop of Henle (TAL). Uromodulin has been shown to reduce UTIs and regulate NKCC2 and ROMK, the two main channels responsible for NaCl transport in the TAL. Furthermore, UMOD mutations cause dominantly inherited CKD (MCKD2). Susceptibility variants found in the UMOD gene are at high frequency in the general population and confer a 20% increased risk of CKD and 15% risk of hypertension.

This paper set out to uncover the biological mechanism that would explain the increased CKD and hypertension in patients with these risk genotypes. They looked specifically at the 2 lead variants located in the UMOD promotor region. Briefly, in human nephrectomy samples (removed due to RCC), those with the risk genotypes had higher uromodulin expression than those with non-risk genotypes. They confirmed this association of UMOD promotor risk variants and higher urinary uromodulin in large population-based cohort (SKIPOGH). In mouse-models, mice over-expressing UMOD had higher blood pressures and more LVH than controls and had more interstitial pathology (despite normal renal function) than controls. They then showed that mice over-expressing UMOD had more active NKCC2 (furosemide sensitive channels) than controls. They also showed that the higher BP in UMOD over-expressing mice could be dropped to baseline levels by furosemide (all mice had comparable levels of ENaC and NCC). More work was done to further elucidate the mechanism of NKCC2 phosphorylation by UMOD. Finally the authors bring their attention back to humans. They used a never-treated human hypertensive cohort (MI_HPT) and stratified them according to one of the risk variant genotypes (rs4293393). Patients homozygous for the risk genotype had statistically significant higher baseline diastolic BP. Some of these patients underwent furosemide testing. Patients with the homozygous risk allele had a higher natriuretic response and diastolic BP drop than others (both statistically significant). To summarize the authors conclusions; patients with risk alleles in the UMOD promotor had greater risk of hypertension and CKD. These patients made more uromodulin. Uromodulin increases NKCC2 activity and thus salt sensitive hypertension. Also, UMOD over-expression increases interstitial kidney damage and thus increases the risk of CKD. These risk genotypes for disease are at high frequency in all ethnicities tested. The authors suggest that selective pressure for disease related variants in UMOD is similar to the APOL1 story. The protective effect of uromodulin on UTIs and ability to raise blood pressure may have lead to its selective over-expression.

This paper demonstrates the mutual importance of large-scale studies such as GWAS and studies of rare monogenetic diseases such as Bartter and Gitelman syndromes. This paper is a worthy contender of a top ten listing this year with a truly translational set of studies combining modern basic science with clinical studies.

I also want to give a shout out for stories on new targets for ADPKD and the new AHA Cholesterol guidelines.

Electrolyte Channels and Aldosteronism

Over the past few years, it has become apparent that hyperaldosteronism is far commoner than was once suspected and screening of unselected patients with hypertension reveals that about 5-10% of patients have primary hyperaldosteronism. In patients with resistant hypertension, that percentage increases to 15-20%. About 30% of hyperaldosteronism is caused by aldosterone producing adrenal adenomas (APA). Most of the rest is related to bilateral adrenal hyperplasia with less than 5% of cases being familial. The secretion of aldosterone in adrenal cells is dependent on the intracellular calcium concentration and increases in response to higher plasma calcium. Entry of calcium into the cells is in turn dependent on voltage-gated membrane calcium channels (which allow calcium influx when the cells are depolarized) and a calcium ATPase which removes calcium from the cells. Under normal circumstances, adrenal cells are hyperpolarized thus keeping these calcium channels closed. Cell polarization is maintained by a combination of the action of the Na-K-ATPase (which exchanges 3 intracellular Na for 2 extracellular K) and membrane K channels lead to K loss from the cells.

Angiotensin II inhibits the Na-K-ATPase leading to cell depolarization, calcium influx into cells and aldosterone secretion. Similar effects are seen when cells are treated with oubain, a specific Na-K-ATPase inhibitor that also leads to hyperaldosteronism.

In 2011 in a seminal paper in Science, Choi et al reported finding somatic mutations in KCNJ5, a membrane potassium channel in patients with APA. These were identified by sequencing tissue from the tumors and comparing with the surrounding tissue. Subsequently, it has been found that about 30-40% of patients with APA have somatic mutations in KCNJ5. These mutations are believed to reduce the ion selectivity of the channels, allowing Na to move into the cell and reduce the resting membrane potential. A number of families have been identified with KCNJ5 mutations resulting in bilateral hyperplasia - now called Familial Hyperaldosteronism type III.

Recently, a paper was published in Nature Genetics which attempted to determine if there were other somatic mutations in patients with APA. In this study, they took KCNJ5-normal patients and sequenced the exons of the tumors and the surrounding tissue. There were very few mutations identified but 5/9 patients had mutations in ATP1A1, a component of the Na-K-ATPase or ATP2B3, a component of the calcium ATPase that removes calcium from adrenal cells. Follow-up targeted sequencing of 300 patients with APA revealed that about 7% had mutations in one of these two genes. Patients with these mutations had higher aldosterone levels, lower minimum potassium levels and higher systolic BP, all indicators of more severe disease. Notably, no families have been identified with these mutations. In vitro studies revealed that cells with these mutations have very low membrane potentials and it is speculated that if this was a germline mutation, it would likely not be compatible with life. This is a fascinating insight into how very small changes in electrolyte channels can have far-reaching consequences and shows a great progression from exome sequencing to the bench and to clinical investigation.

The images in this post are taken from the recent paper in Nature Genetics. One would wonder if somatic mutations explain some of the missing heritability that were are seeing in genetic studies of common diseases. See this previous post by Lisa on the genetic causes of hypertension.

New potential drug targets in ADPKD

Currently there is no good treatment for the most common inherited cause of ESRD, adult polycystic kidney disease. There have been a number of high profile trials in ADPKD in recent years. These trials have endeavored to show a reduction in cyst growth and GFR decline with everolimus, sirolimus) and most recently Tolvaptan (TEMPO). The longer (2years) and larger (433 patients) of the two mTOR inhibitor trials (everolimus) did show a significant reduction in cyst growth at one year but not in GFR reduction. The shorter sirolimus trial failed to show a reduction in cyst growth or GFR decline. The TEMPO trial was over 3 years, had 1445 patients and did show that the V2 antagonist Tolvaptan slowed GFR decline (reciprocal of the serum creatinine level, −2.61 [mg per milliliter]−1 per year vs. −3.81 [mg per milliliter]−1 per year; p=0.001) and cyst growth, 2.8% per year (95% confidence interval [CI], 2.5 to 3.1), versus 5.5% per year in the placebo group (95% CI, 5.1 to 6.0). The jury is still out about the clinical applicability of these drugs and there have been criticisms. For example, tolvaptan is very expensive and would need to be used long term. In the mTOR inhibitor trials some argue doses could have been higher and the lack of hard end points speaks for itself.

However, all is not lost. A potential new drug target in ADPKD was reported by Rowe et al. in Nature Medicine last month. A good overview of the topic can also be found in the same issue.

Using MEF cells from pkd-/- and pkd+/+ mouse littermates they found that growth medium from the pkd-/- cells was more acidic and that the pkd-/- cells had a higher ATP content. To investigate which metabolic pathways might be causing this difference they used NMR spectroscopy and found lower glucose and higher lactate levels in the knock out cells. They then used a mitochondrial ATPase inhibitor to determine the source of higher ATP and found only wt cell had a reduction in ATP with this treatment.  Then the investigators did a real-time PCR analysis on the pkd-/- cells and found an upregulated glycolysis signature. They thus concluded that the pkd-/- cells rely on aerobic glycolysis for their energy demands. This is known as the Warburg effect described in cancer cells (Otto Warburg, a physician-scientist, received the Nobel Prize in Physiology or Medicine in 1931). To see if these in vitro findings translated into in vivo they used Ksp-Cre; Pkd1flox/− mice, which develop early and severe PKD and measured 13C-glucose or 13C-lactate using 13C-NMR. The findings were the same. The authors then used 2-deoxyglucose (2-DG) an analogue of glucose that is unmetabolised. They treated wt and pkd deficient mice this compound and found that the pkd deficient mice had a lower cyst index and lower 13C-glucose consumption as measured using 13C-NMR.

This interesting study proposes that the use of drugs targeting this pathway in combination with other drugs may reduce cystogenesis and progression of CKD in ADPKD. The authors do stress that their summary with regard to human treatments is speculative. In all I think the future is not so gloomy for ADPKD.

Posted by Andrew Malone

Bad Odor

Cystinosis is an autosomal recessive disease caused by a mutation in CTNS, which encodes the lysosomal transporter of cystine. This leads to intracellular cystine accumulation which leads to renal, neurological and cardiac damage. The treatment for this condition is life-long cysteamine. Back in 2011, we reported on a new formulation of cysteamine that is given just twice daily (rather than q6 hours) and is associated with a lower incidence of side effects including halitosis and body odor. There was a higher incidence of GI side effects in patients treated with the new drug. The halitosis occurs because a proportion of the drug is converted to dimethylsulfide and this can appear in expired air. The reason why the new formulation is associated with less halitosis is because, although serum drug levels are the same as with the older drug, the total dose is reduced so there is less overall conversion to the offending metabolites. This is welcome news for patients given that the drug should be started before age 5 and the halitosis and body odor cause major social problems for patients which can result in non-compliance.

Yesterday, the FDA approved cysteamine bitartrate ER (Procysbi) for the treatment of cystinosis. Essentially, this is an enteric-coated delayed-release version of the drug that is largely absorbed in the small intestine. The major issue, of course, is cost. The traditional form of the drug costs about $8,000/year while the new formulation will cost approximately $250,000. That is an enormous difference for a drug which does no more than reduce side effects. However, when you read this New York Times article on procysbi, you can better understand why the parents of these children think that this is entirely worth it.

According to the same article, it is estimated that by 2018, spending on orphan drugs will account for about 16% of overall spending on prescription drugs. This is bound to lead to conflict between insurers, patients and the manufacturers when trying to decide who to treat and who will pay. Is it worth $242,000 every year for a drug to be given twice, rather than four times daily? The problem, of course, is that this is the only way that the company can recoup the cost of development because there are so few patients with the disease - approximately 300 in the US. It should be remembered, of course, that the ulitmate cost of non-compliance in this case is very high - dialysis, transplantation and loss of future earnings so the cost calculation is not as simple as I suggested above. This is a debate that is certainly going to continue over the next few years.

Does nephrology need personalized medicine?

Systems biology is one of science’s growth areas. Sequencing technologies and software tools developed on the back of the human genome project have reduced the cost of, and therefore increased access to, large and complex datasets (ending in -ome) of genome sequences (genomics), gene expression (transcriptomics) and proteins and metabolites (proteomics and metabolomics). Systems biological techniques integrate these datasets and provide insights into how phenotypes may emerge from interacting biological processes rather than isolated genes or proteins.

A recent editorial in the journal Nephrology Dialysis Transplantation examined this field in general and its relevance to nephrology. The authors mention that –omic datasets have been useful in modeling “self-organized highly interconnected networks”, and that such networks have implicated unexpected candidates in disease pathogenesis (see for example, this paper on cardiac hypertrophy). 

The review goes on to suggest that using the tools of systems biology to finely phenotype individuals will usher in an era of truly personalized medicine. However, it is not clear to me that a definite sequel to this type of analysis will be the personalization of treatment or even that the concept of personalized medicine is particularly suited to our current view of what constitutes clinical evidence.

Diseases such as the ANCA-associated vasculitides (AAV) are now known to exhibit genomic variability. Randomised controlled trials (RCTs) in AAV (such as here and here) have been hampered by: 

1. Short follow-up times 
2. Inter-group heterogeneity which may have affected outcomes. These factors have contributed to ongoing debate about the applicability of the results of these trials (see correspondence here). 
3. Additionally a recent trial in membranous nephropathy, likely to represent another disease with distinct –omic subsets, was marked by slow recruitment. 

 

All these points together suggest that it may be difficult to conduct meaningful clinical studies of distinct –omic subtypes in nephrological diseases. Currently, primacy is given to RCTs when evaluating the efficacy of new treatments; and in nephrology the community is finally beginning to produce the RCTs which have been absent historically. 

If the focus is to switch away from RCTs with their large, well-matched study groups and towards splitting groups up by some -omic fingerprint I am able to envisage a time when one has to choose between giving more credence to the results of larger, “non-personalised” trials or smaller studies featuring –omic data but lacking the controlled element of RCTs.  Would this represent progress?

Cystinosis

Cystinosis is an autosomal recessive disease caused by a mutation in CTNS, which encodes the lysosomal transporter of cystine. Without this, cystine gradually accumulates in cells causing progressive damage. The commonest kind is “nephropathic cystinosis” which is the juvenile form and presents in infancy although there are milder adolescent and adult forms. It is a rare disease with only 6,000 sufferers worldwide.

It is characterized by the early development of fanconi syndrome with progressive loss of tubular function leading to ESRD by the age of 10. Most affected children are blond and have marked growth retardation. Corneal deposits are common and can cause blindness later in life. Patients may also have hepatosplenomegaly, muscle weakness, delayed puberty and eventual neurological disease. The adult form is more benign and usually presents with visual problems although it can lead to ESRD in some patients.

There is an effective treatment for cystinosis – cysteamine, given orally, enters the lysosomes and reacts with cystine forming a complex that can exit the lysosome and prevent build-up. It should be started as soon as the disease is diagnosed but even with this, the tubular defects often persist. However, there is a definite improvement in overall renal prognosis with this drug. So, if there is an effective treatment, why do many children with this disease still go on to require renal transplantation? There are huge issues surrounding compliance with the medication. It needs to be taken four times daily at equal intervals. More importantly, it causes severe halitosis and body odor that, for a young child, can be devastating. I had a patient a number of years ago who told me that he and his brother had to be removed from class because the problems with bad odors while on the drug and that he preferred going on dialysis and getting a transplant than remaining on the treatment. Unfortunately, at that time, he was developing severe visual problems related to build-up of cystine in his corneas.

There may be some more light at the end of the tunnel. Recently, phase III trials were completed on a new formulation of cysteamine. This can be given twice daily, has a lower total cumulative dose than the standard formulation and appears to cause less halitosis and body odor. According to the company, 40 of 41 patients enrolled in the study decided to continue the drug after the phase III period had ended. This is certainly encouraging.

As Nate mentioned previously, this should not be confused with cystinuria, a disease of amino acid transport in the kidney leading to an increased susceptibility to kidney stones.

Fabry's Disease – modes of inheritance

Just a quick piece to review the inheritance pattern of Fabry's disease, a relatively rare, but under-recognized cause of End-Stage Kidney Disease in adults.

Fabry’s disease is an X-linked recessive disease, caused by a defect in the gene coding for the alpha-galactosidase enzyme on the X chromosome. This enzyme has an important role in intracellular trafficking and metabolism of glycosphingolipids, with deficiency leading to accumulation of globotriaosylceramide in particular.

A wide variety of mutations have been identified (over 300 so far) and can present with a corresponding wide range of disease severity. The spectrum of disease is possibly related to how severely a particular mutation affects the production, transport and breakdown of the enzyme. This knowledge has potential importance in treatment, as those with no enzyme activity will be much more reliant on enzyme replacement therapy than those with some residual activity.

As an X-linked disease, it is generally fully expressed in males, while females have characteristically been labeled as ‘carriers’. Thankfully this term is becoming less common, as it really does an injustice to those females who have quite severe disease.

How do women become affected if they have two X chromosomes, with only one of them carrying the Fabry-related mutation. Well, remember back to reproductive biology and the fact that there is random (though perhaps not as random as we think) inactivation of one X chromosome in every somatic cell in a female’s body. Depending on the ratio of inactive ‘normal’ X chromosomes to ‘abnormal’ X chromosomes, this will determine the net tissue expression, or lack of expression, of an abnormal alpha-galactosidase enzyme. This concept is called mosaicism – every female is truly a mosaic, with each somatic cell having either the paternal or maternal X chromosome inactivated.

Consider this: what about an affected male, with a known sequenced genetic mutation, who has two daughters. We have genetic evidence that non-paternity is not an issue. Both daughters are tested for the father’s mutation by genetic sequencing, but only one carries the mutation. How can this happen – as the father only has one X chromosome, surely he must transmit this to all his daughters?

The answer is interesting – during the father’s early embryonic cell division, some time after the two-cell stage, there must have been a spontaneous mutation that gave rise to the Fabry genetic defect. However, the other cell line did not undergo this mutation. Now we have two distinct cell lines that make up the developing male, similar in all things, except the presence of a new Fabry mutation. Therefore, we have essentially a male mosaic. We must presume that one of his sperm had a ‘normal’ X, which produced the unaffected daughter and the other sperm had an ‘abnormal’ X chromosome, which produced the affected daughter.

If ANCA cause vasculitis, what causes ANCA?

Over the past decade, controversy about whether ANCA is itself directly pathogenic has largely abated, primarily based on convincing animal models of small vessel vasculitis in mice and rats mediated by MPO ANCA. But as this issue is laid to rest, a new burning question has emerged in the field: if ANCA cause disease, what causes ANCA? Specifically, why are the ANCA antigens PR3 and MPO turned on in the neutrophils of ANCA patients, and not in their normally silenced state (as in healthy controls). This question hints at the regulation of gene expression itself, and brings us to the rapidly expanding field of epigenetics.

Epigenetics, the study of changes in phenotype and/or gene expression that are independent of the underlying DNA sequence, is one of the hottest areas of basic science research at the moment. Recently, the Jennette and Falk group in Chapel Hill examined whether high expression of the major auto antigens in ANCA patients, MPO and PR3, might have an epigenetic basis. The reason this is a good bet is that, although mature neutrophils from healthy subjects do not express MPO and PR3, these proteins are expressed in developing neutrophils. This suggests that there is transcriptional silencing of MPO and PR3 during development, which is the hallmark of epigenetic control.

They showed that levels of a specific histone methylation (H3K27me3) were reduced at the MPO and PR3 loci in ANCA neutrophils compared to those from healthy controls. H3K27me3 is associated with the formation of heterochromatin, which is inaccessible to transcription and often causes gene silencing. They went on to show that Jmjd3, the demethylase responsible for removing this particular histone methylation is upregulated in ANCA patients. This suggests that Jmjd3-mediated changes in histone methylation status may be responsible for aberrant expression of MPO and PR3 in mature ANCA neutrophils. This study throws up a number of areas for further research. For example, elucidation of what causes the initial failure of gene silencing mechanisms will give further insight into the pathogenesis of the disease, whilst insight into the pathways which both establish and maintain silence of MPO and PR3 in healthy patients may suggest new therapeutic approaches.

Thomas Oates MD

This isn’t meant to be happening…

A paradigm of modern genetic studies, such as GWAS, is that there is a natural balance to allelic variation, with common variation in the population conferring mild disease risk and, conversely, genes of strong disease effect being rare. This phenomenon is formally described by the ‘common disease/ common variant’ (CDCV) and ‘multiple rare variant’ (MRV) models. The CDCV model predicts the existence of common genetic variants (present in 5–50%) that confer low to modest disease risk (e.g relative risks of 1.1–1.5). Complementing this is the MRV model, which holds that complex traits result from many different mutations, each of which is individually rare (a few percent at most and perhaps orders of magnitude less common), but with very strong effect (for example, relative risks 5 – 10, or even more; see figure). This phenomenon is thought to explain why the search for causative genes derived from GWA studies has been relatively unsuccessful, where only a handful of causative genes have been discovered in follow-up sequencing studies. It is assumed that this difficulty in finding culprit genes is due to these modest effects making them difficult to recognize. Undiscovered common genes of strong effect are simply not thought to exist…

So what is going on in Nephrology? Within the space of a few short weeks there have been 2 separate, high quality studies identifying common disease-causing gene polymorphisms of very strong effect. First, there was the identification of 2 independent APOL1 variants, present in over 30% of African-American chromosomes, that carry odds ratios of 10.5 in idiopathic FSGS and 7.3 in hypertension-attributed ESRD. Analagous to HgbS-mediated protection from malaria, these APOL1 risk variants appear to have risen to very high frequency in Africa as they cause resistance to trypanosomal infection, thus protecting from sleeping sickness. Although the mechanism by which APOL1 variants cause kidney disease are not known, the mechanism of trypanosomal resistance has been described, and offers the hope for a new treatment of this life-threatening infection.

And now this morning, investigators from Hong Kong, reporting in JAMA, describe finding another set of common gene polymorphisms of strong effect. This candidate gene study of Chinese patients with type 2 diabetes, identified several variants of the PRKCß 1 gene as being associated with incident ESRD and, in a follow-up analysis, CKD. The adjusted risk for ESRD was 6.04 (95% CI, 2.00-18.31) for individuals with 4 risk alleles compared with those with 0 or 1, and allele frequencies were high (7-12.2%). PRKCß 1 is an excellent candidate for kidney disease risk in diabetes, with a prior RCT of blockade of its gene-product, PKC-ß, demonstrating slowed disease progression and reduced proteinuria in diabetic patients already on maximal medical therapy.


So, recent studies in Nephrology are bucking the trend in genetic epidemiology, and challenging one of its most basic hypotheses. Whether this is because the assumptions themselves are flawed, or that Nephrology research is just catching up with other fields, remains to be seen.