Are bioengineered kidneys becoming reality?

The lack of transplantable kidneys is an unavoidable problem for those of us who practice Nephrology. Whilst government policies and extended criteria for donor organs have a role to play in solving this problem, it has always seemed to me that science is going to have to serve us up a definitive solution.

Although many of us appreciate that induced pluripotent stem cells (iPSCs) will likely form part of the answer to this question, it is more difficult to visualise how iPSCs could be used to reconstitute a complex multi-cell organ such as the kidney. Additionally, since the emergence of 3D printing we have had media friendly stories of printed kidneys without much information about what will be used as the 'ink' in the printer.  

A paper in this month's Cell Stem Cell may hint at the future. Firstly, the authors worked out the developmental origins of the metanephric mesenchyme (MM), the embryonic tissue that generates most elements of a functioning kidney (see diagram). The group then used this information to derive MM from PSCs and found that this derived MM successfully reconstituted glomeruli and proximal and distal tubules in vitro. Furthermore, when these generated nephrons were transplanted beneath the renal capsule of mice they showed new vascularisation suggesting they may be functional.

So, is the answer to our transplantation problem to use these authors' protocol to derive lots of MM which can then be 3D printed around a small core of functioning renal tissue to make a working organ? Well, a number of issues remain. Firstly, as can be seen from the diagram, the collecting duct is derived from the separate ureteric bud. Therefore, it is not included in this model and successful combination of these structures would likely be needed to make a truly functional nephron. Perhaps most glaring is that the Cell Stem Cell paper makes no assessment of the excretory function of these derived nephrons.

In conclusion this is probably only "a good start" but I think it represents significantly more progress than any number of eye catching 3D printer stories.

Insert sensationalist headline here...

A recent BMJ Minerva column alerted me to an interesting paper in the journal Transplantation. The authors used collaborative databases to compare overall and age-specific graft survival in first deceased donor transplants carried out in the US & Europe. They found that although there was broad similarity in 1-year graft survival, 5 and 10 year graft survival was considerably higher in Europe than in the US. The tendency towards worse graft survival in the US persisted across all ethnic groups and was largest for children and young adults. The gap between European and US survival was greater beyond 3 to 4 years post engraftment.

I am British and have only ever practiced in the UK NHS. I am therefore in no position to comment meaningfully on the following points made in the paper’s discussion section: "it is necessary to recall…the 3-year restriction in medication coverage for immunosuppression in the United States by Medicare…A policy change may contribute to improving graft survival and ultimately saving lives and also help to reduce health care spending." I imagine that other readers and contributors to this site may have informed and/or deeply held opinions.

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?

When IgA-ttacks!

To many of us IgA nephropathy (IgAN) is a disease to be watched and monitored.  Most cases of IgAN in which the clinical course is aggressive occur with one of two atypical presentations:

1. Acute kidney injury (AKI) and macroscopic haematuria or

2. Nephritic syndrome or RPGN with a crescentic nephritis on biopsy

The first thing to say is that both of these presentations are rare. Whilst visible haematuria coinciding with mucosal infections is a well-known feature of IgAN, development of coincident acute kidney injury is uncommon.  In fact, the largest published case series with this presentation I could find included only 38 patients.  With regards to crescentic nephritis, it is instructive that the recently produced Oxford classification of IgAN study found so few patients with a severe crescentic nephritis (median % gloms with crescents 9%) that the authors were unable to include crescents as having “independent value in predicting renal outcome.”

The AKI seen with visible haematuria is often due to tubular injury from intra-tubular erythrocyte casts and a possible direct nephrotoxic effect of haemoglobin.  The key issue in these patients is not so much treatment, which is through general supportive measures, but ensuring that protracted or repeated AKI is not due to a crescentic IgAN.  As a result, the recently published KDIGO glomerulonephritis guidelines recommend that any patients with known IgA exhibiting AKI and visible haematuria who fail to show improvement of kidney function after 5 days should undergo repeat renal biopsy.

Crescentic IgAN (defined as RPGN with crescents in >50% of glomeruli seen in the biopsy) although rare has a poor prognosis: end-stage renal disease in 75% of one cohort at 10-year follow-up.  The KDIGO guidelines suggest on the basis of low quality evidence initial treatment as for ANCA vasculitis with steroids and cyclophosphamide.  Interestingly, no suggestion is made for maintenance therapy in these cases.

I have only ever seen a single case of severe crescentic IgAN and no cases of AKI with visible haematuria.  However, I think it’s important to know some details about these presentations.  In the near future I’ll look at Henoch-Schonlein nephritis and that most thorny of issues in IgAN; who to treat with immunosuppression outside of an RPGN presentation.

New Cases Site from the UK RA

The UK Renal Association (RA); the professional body for UK nephrologists and renal scientists, has recently started a website to foster discussion of interesting cases (There is also a link in the sidebar). The site remains embryonic whilst a development strategy is debated, but the RA would be very gratfeul for feedback and some comments/discussion of the cases.

Shedding some light on chronic inflammation

One of the most important concepts in nephrology is that of chronic systemic inflammation and its role in increased cardiovascular (CV) risk in patients with chronic kidney disease (CKD) and those on dialysis. Despite a significant research effort the precise drivers of this inflammation have remained difficult to pin down.

Recent work has implicated bacterial lipopolysaccharide (LPS or endotoxin), a glycolipid found in the outer membrane of gram negative bacteria, in this process. (The potency of LPS as a pro-inflammatory stimulus is detailed in this extraordinary case report from the NEJM archive)

Previous work in patients with congestive cardiac failure (reviewed here) has demonstrated that relative underperfusion of the gut results in leakage of LPS from the GI tract into the circulation. A group of UK investigators analogised this situation to the haemodynamic perturbations seen in haemodialysis sessions and used this as a starting point to investigate the concentration of circulating LPS in CKD patients.

They found that endotoxemia (i.e. circulating LPS concentrations) :

• tended to increase with worsening CKD stage

• showed a 6-fold increase in patients on dialysis

• tripled at the initiation of dialysis

Combining these findings with the fact that the study demonstrated correlations between LPS levels, the magnitude of hypotensive episodes during dialysis and dialysis-induced myocardial stunning, the authors concluded that haemodialysis associated circulatory stress leads to exposure to sustained endotoxemia. Importantly, they also found a significant association between this endotoxemia and reduced survival.

So should we be giving our most haemodynamically unstable dialysis patients antibiotic treatment, or selective gram negative gut decontamination or polymyxin B haemadsorption instead of dialysis? Well, my thought is that individually tailored dialysis prescriptions to reduce CV stress during dialysis sessions are likely to be a good start in reducing LPS translocation and potentially decreasing the sequelae of this. I also note that the same group have just published a paper suggesting that plain (and good) old aggressive anti-hypertensive therapy might be a good option too.

A matter of protocol

The use of the protocol transplant biopsy is a divisive topic: some units see a protocol program as necessary for proper management post-transplantation, whilst others deride the whole concept as unnecessary. So who is right?

Protocol biopsies are taken at predefined intervals after transplantation regardless of kidney function. Their use is based on the theoretical benefit of detecting acute rejection, chronic allograft injury, calcineurin inhibitor (CnI) toxicity, recurrent primary disease & BK virus infiltration where the presence of these diagnoses is not evident by a measurable decline in allograft function.

Most work to date has focused on sub-clinical rejection. I detect two relevant questions here: is sub-clinical rejection sufficiently common to be an issue and is detection and treatment beneficial? In answer to the first point; the KDIGO guidelines list seven studies (on pg S31) which have looked at prevalence of sub-clinical rejection providing estimates of 13-25% at 1-2 weeks, 11-43% at 1-2 months, 3-31% at 2-3 months and 4-50% at 1 year. A number of studies have shown that sub-clinical rejection is associated with reduced graft survival and chronic allograft injury and that treatment of sub-clinical rejection may improve long-term graft outcomes.

However, the majority of these studies have been performed with cyclosporine (CsA) and azathioprine (Aza) maintenance regimes. As a result, perhaps most relevant to current practice is an RCT that randomized 240 patients on tacrolimus & MMF to biopsies at 0,1,2,3,6 months or 0 & 6 months. Rejection rates were generally low with clinical episodes seen in 10% of biopsy arm patients and 7% in the control arm. Additionally, sub-clinical rejection was seen in only 4.6% of the biopsy arm and creatinine clearance at 6 months was identical between the two groups.

So, perhaps not surprisingly, KDIGO feels only able to state that 'based on very-low-quality evidence, the benefit of performing protocol biopsies in CsA/Aza patients without induction therapy may outweigh the harm' (pg S32). This strikes me as a vanishingly small proportion of incident transplants. Taking this together with the fact that the KDIGO authors could find NO data showing a benefit in detecting sub-clinical CnI toxicity, recurrent disease, BK nephropathy etc, I would suggest that perhaps the protocol biopsy has had its day.

Sepsis & AKI - an insoluble problem?

I expect very few medical users of this website went into nephrology to manage acute kidney injury (AKI) in the context of sepsis. It is hard to avoid pessimism when considering the state of play in this condition: 65% of patients with septic shock develop AKI; this AKI is an independent risk factor for death and up to 75% of patients with AKI and severe sepsis die.

It is, therefore, not surprising that research into this area continues. Much of which has focussed on the use of extra-corporeal blood purification techniques (EBP) to improve outcomes via immune modulation by removal of circulating inflammatory mediators (summarised in this recent review by Ricci et al.).

The most familiar EBP technique is standard CVVH delivered at high doses. Indeed, when I was training in ICU in 2008 we would regularly use high volume CVVH (often seeking treatments of up to 6 L/hr) in patients with co-existent severe sepsis and AKI. Although both the ATN and RENAL trials failed to demonstrate any benefit of this tactic in the management of severe AKI, many feel that the ‘high intensity’ groups in these trials were simply not high intensity enough to demonstrate a treatment effect in septic patients. This feeling largely stems from a favourable body of evidence (summarised in this review) from animal studies and small trials with soft end-points using CVVH doses of 45-115ml/kg/hr (in contrast to the ATN and RENAL CVVH doses of 35-40ml/kg/hr). The completed but unpublished IVOIRE trial prospectively randomised 139 patients with septic shock and AKI to 70ml/kg/hr or 35ml/kg/hr with a primary outcome of all-cause mortality and may help to settle this argument.

The counter view is that dose approaches are bound to fail because whilst inflammatory mediators are water-soluble, their molecular weight means that they are unlikely to be maximally cleared by standard haemofilters. This can be attacked in two ways: high-cut off membranes or haemadsorption.

High-cut off membranes have been evaluated in small studies and demonstrated reduction in vasopressor requirements in septic patients. However, when used in continuous treatments these filters are associated with a very large obligate loss of albumin, leading some to suggest such filters should only be used in intermittent treatments (i.e. dialysis; similar to strategies used in myeloma).

Haemadsorption passes blood across broadly interacting sorbents to attract larger molecules, which exceed the cut off of standard membranes, thus making this an attractive technique for EBP in sepsis. Use of this technology had previously been limited by poor biocompatibility and resultant haematological abnormalities but recent advances have eliminated this problem. The commonest sorbent is polymyxin B, a systemically toxic antibiotic which can bind lipopolysaccharide and damage gram-negative bacteria. Although a number of studies have evaluated polymyxin B haemadsorption significant further work is required to make a compelling case for this treatment (excellently summarised by Ricci et al.)

Furthermore, coupled plasma filtration and adsorption has been trialled. This allows initial separation of plasma from blood, followed by selective passage of the plasma across the sorbent and has demonstrated some efficacy in small initial studies.

I find it hard to see where this field is going to end up; only high volume haemofiltration strategies seem to have a consensus behind them and these will always now be open to the charge that randomised controlled trials have shown them to be ineffective. Adsorptive techniques are currently relatively heterogeneous and therefore, the large multicentre trials required to demonstrate their applicability seem a long way off. It appears that septic AKI may continue to be a feared condition for some time yet.

Whole Lot of Pressure

The maintenance of mean arterial pressure to prevent tissue dysoxia and conserve organ function is central to the management of the critically ill. In patients with increased capillary permeability this is often achieved by administration of large volumes of IV fluids. However, resuscitation volumes of >5 litres in the first 24 hours are associated with raised intra-abdominal pressure (IAP). Although Conall & Nate have mentioned it before, a recent review of intra-abdominal hypertension (IAH) and abdominal compartment syndrome (ACS) by Mohmand & Goldfarb presents an excellent opportunity to review the topic.

IAH is defined as sustained or repeated elevation of intra-abdominal pressure >12mmHg, and ACS as an IAP >20mmHg associated with new organ dysfunction. IAP is easy to measure, either through the transduction of pressure in the bladder via an indwelling urinary catheter, or using an NG tube in patients whom bladder pressure measurement is not feasible.

Estimates of the prevalence of IAH and ACS suggest figures of approx. 60 and 10% respectively in non-selected ICU populations. As well as large volume fluid resuscitation, risk factors for IAH/ACS include trauma, abdominal surgery, mechanical ventilation, increased abdominal contents due to ileus or ascites, and increased capillary leak secondary to sepsis, pancreatitis, coagulopathy etc.

The review of Mohmand & Goldfarb collates a number of studies showing that IAH is an independent predictor of both mortality and the development of AKI. Indeed, they report on one study, which found that IAH was the best single predictor of the development of AKI after shock.

As regards management; severe ACS requires abdominal decompression by laparotomy, whereas medical strategies to reduce IAH include drainage of intra or extra-luminal contents, reduction of capillary leak and improvement of abdominal wall compliance. Given the link with volume expansion it is tempting to suggest a role for renal replacement therapy and ultrafiltration. However, good quality data is currently in short supply and probably all that can be surmised from existing studies is that aggressive continuous venovenous haemodiafiltration can be used to reduce IAP.

Clearly, IAP is extremely important to bear in mind when approaching AKI in the ICU. However, whether renal replacement therapy will be able to offer improvements in outcomes requires significant further study.

To ATG or not to ATG?

KDIGO (page S6 of PDF) and the UK renal association have recently published guidelines for the clinical care of kidney transplant recipients. Both of these documents suggest that recipients at ‘higher immunological risk’ should be considered for induction therapy with lymphocyte depleting antibodies (LDAs) such as anti-thymoctye globulin (ATG,)  anti-lymphocyte globulin (ALG) and anti-T cell (CD3) antibody (OKT3).  The US scientific registry of transplant recipients (SRTR) annual report for 2008 states that 44.8% of patients received ATG as induction therapy in that year. As a nephrologist practicing in the UK, I find this surprising. None of the 3 major transplantation centres I have worked at have used LDAs for induction under any circumstances and I have found only a single unit using the drug for induction in the UK; and then only in recipients receiving non-hearting beating kidneys.

This is an intriguing discrepancy and seems to invite the question; which side of the Atlantic is right? I think the question has two parts:

1. Are LDAs and IL-2 receptor antibodies, the other main class of induction agents (IL2-RAs; such as basiliximab and daclizumab), of comparable efficacy and safety? 
2. What data justifies preferential use of LDAs in high immunological risk patients as recommended in the guidelines?

A reasonable body of evidence addresses the first question and was summarised in a recent Cochrane review.  With regards to efficacy, patients induced with ATG as compared to IL2-RAs showed less episodes of biopsy-proven rejection at one year but no benefit to rates of graft loss or clinically diagnosed or steroid-resistant rejection. Concerning safety, the review found significant reductions in CMV disease and malignancy when IL2-RAs were used over LDAs. The authors derived a number needed to treat of 16 to avoid one case of CMV disease and of 58 to prevent one case of cancer.

The evidence base for question 2 has been accumulating since at least 1998 when a meta-analysis showed that LDA induction mediated reduction in graft loss (compared to placebo) was greater in patients with high panel-reactive antibody. Since then, two recent high-quality randomised controlled trials (Brennan et al. and Noel et al.) have been performed specifically in high risk recipients. The trials addressed diverse patient populations and used different IL2-RAs and maintenance regimes but produced similar findings of significant reductions in rejection rates at one year without differences in graft or patient survival (although Brennan et al. used a composite end point).

So, the recommendations about the use of ATG in high-risk patients are based on decreased rates of rejection. However, decreased rejection has not translated into increased graft survival in a number of studies looking at ATG vs IL2-RAs (summarised in this editorial).

Having looked through the evidence I feel that almost 45% ATG induction is too high, and the UK’s avoidance of ATG probably unwise too. I think the next challenge would be to produce an evidence based decision algorithm for assigning patients into high and low risk groups. Anybody else have any thoughts?

Tom Oates, MD

High Maintenance

Many nephrologists will have faced “interesting specialty, shame about the evidence-base” jokes in the past. However, following a profusion of randomised controlled trials in lupus nephritis, we are now entering an era where we can integrate data to make truly evidence-based treatment plans.

The latest randomised trial to appear is the MAINTAIN study. This was designed to assess whether MMF is superior to azathioprine (aza) in the maintenance phase of treatment of biopsy-proven proliferative lupus nephritis. For the most part, recent studies in lupus have focused on induction treatment, with an emerging consensus that MMF and IV cyclophosphamide (IVC) are at least equivalently efficacious with a favourable avoidance of ovarian failure associated with MMF.

MAINTAIN was an open label study of 105 patients who were followed-up for 48 months. All patients received induction treatment with steroids and IVC before being randomised to either MMF or aza regardless of renal response to induction. The primary endpoint was time to renal flare (defined as either development of nephritic syndrome, a 33% climb in serum creatinine or 3 fold increases in urinary protein); secondary end points were set as the numbers of severe systemic and benign flares, those withdrawing steroids and those achieving renal remission.

The headline result was that whilst fewer renal flares (25% v 19%) were seen in those treated with MMF this, and all secondary end points, did not differ significantly.

So what is important and original about MAINTAIN? Well, recent publication of the 10 year results of EUROLUPUS has shown the effectiveness of aza as a maintenance therapy. Therefore, given that MMF is about 15 times more expensive than aza and carries a reasonable burden of side effects, some feel it should be demonstrated to be superior to justify its widespread usage.

The trial was performed in Europe with a huge Caucasian predominance. This is likely to be relevant given the racial disparity in MMF response demonstrated during the induction phase of the ALMS trial. ALMS was designed to compare MMF and IVC as induction therapy for 24 weeks and then saw re-randomisation of patients to MMF and aza. Black and Hispanic patients responded better to MMF. The maintenance phase results of ALMS are now available in abstract form and show that MMF was superior to aza in preventing a composite end point of death, renal damage, and renal relapse. This difference between MAINTAIN and ALMS may be a result of the fact that in ALMS only patients who had achieved a renal response at 6 months were included in the maintenance phase, or because a composite end point was used in ALMS, or, more simply, because ALMS was larger (227 randomised patients v 105 in MAINTAIN).

So what more do we need for trial evidence in lupus? Some long term data focused on ESRD and death would be helpful. Especially if it focused on those patients both induced and maintained with MMF. One thing is for sure though, the days of expert opinion guiding treatment of glomerular diseases is receding fast.

Tom Oates M.D.

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