Keep cool and carry on...

Delayed graft function (DGF) after kidney transplant is associated with long-term allograft dysfunction. Niemann et al. (NEJM 2015;373:405) reported that therapeutic hypothermia reduced DGF (defined here as the requirement of dialysis in the recipient within 7 days after renal transplantation).

Study group enrolled 370 kidney donors after neurological determination of death (DNDDs), randomized to either normothermia (36.5 to 37.5 deg., n= 280) vs. mild hypothermia (34-35 deg. either by allowing to spontaneously reach the temperature, or by passive-cooling devices or forced-air systems, n=286). Incidence of DGF was significantly lower in hypothermia group (28.2% vs 39.2%, p=0.008), and study was terminated early.  

Therapeutic hypothermia (targeted temperature management) is an established intervention for outside-hospital cardiac arrest to protect neurological function.

In transplant field, in contrast, current organ procurement protocol (e.g. one from NATCO) has a stipulation that normal body temperature should be maintained in donors, frequently requiring active warming with warm blankets.

 What is the protective mechanism?  Authors discussed possible contribution of ischemia-reperfusion injury. Is this procedure applicable to other organs? Many questions arise and there is a nice open forum with NEJM group to discuss these (til August 7^th EDT).

Naoka Murakami, MD PhD

Remote ischemic preconditioning (RIPC)— Preparing your kidneys for surgery.

Prevention of perioperative AKI, mostly from ATN by ischemic insult, has been a major challenge in nephrology. Recent study (Zarbock et al. JAMA) showed a simple, non-invasive method—remote ischemic preconditioning (RIPC), could be a promising strategy.

This multicenter, double-blind study in Germany randomized 240 patients undergoing on-pump cardiac surgery, who had high risk for AKI (based on Cleveland Clinic Foundation score). Exclusion criteria included eGFR below 30 ml/min or transplant recipient. Intervention consisted of: 3 cycles of 5-minute inflation of a blood pressure cuff to 200 mmHg (or 50 mmHg higher than the systolic pressure) to one upper arm, followed by 5-minute reperfusion with the cuff deflated.

Primary endpoint was the incidence of AKI within 72-hour post-op (KDIGO criteria). They found significantly fewer patients in RIPC group developed AKI (37.5% vs 52.5%; p=0.02; RR, 71% 95% CI, 54-95%). Use of renal replacement therapy was also lower (5.8% vs 15.8%; p=0.01). Of note, the authors examined urine biomarkers: tissue inhibitor of metaloproteinases 2 (TIMP-2) x insulin-like growth factor-binding protein 7 (IGFBP7), as well as urine neutrophil gelatinase-associated lipocalin (NGAL) and high-mobility group box (HMGB). TIMP-2 and IGFBP were measured by Nephrocheck® (see previous blog entry for details). Biproduct of TIMP2 x IGFBP7 and HMGB increased right after RIPC, suggestive of cell cycle arrest as an epithelial defensive mechanism against ischemic insult, however, in 4-24 hours post-op, TIMP2 x IGFBP7 and NGAL were lower in RIPC group compared to control (schematic figure above from eSupplement), indicating less kidney damage.

 RIPC has been studied in kidney transplant field as well. The REPAIR trial recruited 406 live donor-recipient pairs and looked into 12-month post-transplant eGFR with or without early or late RIPC for both donors and recipients. The result was not significant, but as Zarbock et al. pointed out in their article, it could be due to differences in study protocols, confounding comorbidities, or surgical technique. Further studies to explain the mechanism of protection is warranted and it might be no exaggeration to say that RIPC would be a standard pre-op protocol for cardiac surgery in the near future. Are your kidneys ready for surgery?

Naoka Murakami

FSGS biomarker updates—suPAR, B7-1, CD40 and more…

FSGS is the most common glomerular disorder causing end-stage kidney disease in the USA with a high post-transplant recurrence rate of 20-50%. Furthermore, the treatment of post-transplant recurrent FSGS is extremely challenging. While reading a recent article on biomarkers predicting post-transplant recurrent FSGS by Delville et al. (discussed below), it seemed a good idea to cover recent advancement in FSGS research. 

 suPAR (soluble plasminogen activator receptor) 
Since the first report by Wei et al. of suPAR as a circulating permeability factor causing FSGS, there has been extensive research to elucidate its role in FSGS. Although administration of suPAR molecule to mice was initially thought to be sufficient to cause proteinuria mimicking FSGS, via activation of integrin beta 3 and derangement of actin cytoskeleton, the situation does not seem that straightforward. As Reiser et al. discussed, there are challenges to overcome: existence of different suPAR isoforms with different disease modifying effects; heterogeneity of FSGS itself, which make it complicated to interpret correlation between levels of suPAR and disease activity; involvement of suPAR in other disease process, including cardiovascular disease (KI 2014). However, suPAR remains an intriguing molecule and potential biomarker in FSGS disease process.

 B7-1 (CD80)
A case series in the NEJM from Peter Mundel’s group in the end of 2013 brought us to an excitement for personalized treatment of a subgroup of B7-1 positive FSGS patients. They found strong B7-1 stain in primary FSGS as well as post-transplant recurrent FSGS. These patients were successfully treated with abatacept (CTLA4-Ig), resulting in complete remission of proteinuria. The hypothetical pathophysiological mechanism was via direct interaction of B7-1 and integrin beta 1 causing podocyte actin cytoskeleton changes. However, it was followed by comments and larger case series (AJKD 2014) questioning specificity of the immunostaining and treatment effect of abatacept. Exploration of this costimulatory molecule in FSGS was just started and further research is needed. 

Micro RNA(s) as biomarkers for FSGS disease activity 
A group from China suggested that certain micro RNAs are associated with disease activity (level of proteinuria) and progression (Zhang et al. CJASN and AJKD 2014). Potential candidates are miR-186 (involved in cell cycle control, AKT and insulin signaling etc.) and miR-125b (involved in NFkB signaling etc.). However, their contribution to pathogenesis is not clear so far.

 Pre-transplant antibody panel (including anti-CD40) to predict post transplant recurrent FSGS
Expanding potential FSGS biomarkers is the publication by Delville et al. The authors did a beautiful translational work using human serum in protein arrays, validation of specific auto-antibodies and further experimentation using cell cultures and animals models. The authors elegantly showed that pre-transplant antibody panel, especially anti-CD40 antibody, can predict risk of post-transplant recurrent FSGS.
In more detail, they started by comparing pre-transplant sera of non-recurrent FSGS (nrFSGS) vs recurrent FSGS (rFSGS), identifying 789 autoantibodies upregulated only in rFSGS but not in nrFSGS. Then those antibodies were enriched for those Ab with antigen targets expressed in kidney (151 autoAbs) and more specifically in glomeruli (10 autoAbs). They validated the 7-antibody panel (CD40, CGB5 (chorionic gonadotropin b), PTPRO (protein tyrosine phosphatase receptor O), FAS (TNF receptor superfamily member 6), P2RY11 (P2Y purinoceptor 11), SNRPB2 (small nuclear retinoid X receptor a), and APOL2 (Apolipoprotein 2)) in rFSGS vs nrFSGS cohort, and obtained ROC AUC of 0.92. Surprisingly, anti-CD40 itself had a high ROC AUC (0.77). Then, the involvement of rFSGS-anti-CD40 IgG to enhance FSGS recurrence was confirmed in vitro—rFSGS-anti-CD40 IgG caused actin cytoskeleton derangement in podocyte cell culture—, as well as in vivo—co-injection of rFSGS-anti-CD40 IgG and suPAR molecule markedly enhanced proteinuria (in a suPAR-dependent manner), which was inhibited by CD40-blocking antibody or in CD40 knockout mice. This suggests that by checking pre-transplant anti-CD40 antibody, we may be able to identify a high risk FSGS recurrence group. To manage these patients, current options are: peritransplant plasmaphresis or rituximab. Interesting to see if CD40 antagonist or blocking antibody can play a role in preventing/treating rFSGS in the clinic...

Naoka Murakami

NephroCheck®—can we predict AKI in the ICU? And then what?

Nephrologists have been looking for sensitive biomarkers to predict AKI. Efforts have been made and the idea of “renal angina” was proposed by Goldstein and Chawla in 2010, but still there has been no reliable biomarker commercially available to detect AKI early enough. 
Well, the FDA has just approved a point-of-care biomarker assay, NephroCheck®, for predicting risk of AKI. Interestingly, none of the most studied biomarkers such as KIM1 and NGAL are included. NephroCheck® uses two urinary biomarkers : insulin-like growth factor binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases (TIMP-2).

IGFBP7 and TIMP-2 were selected as biomarkers to predict AKI using a 522-patient cohort (median age 64, 91% Caucasian) of critically ill patients admitted with sepsis, shock, major surgery and trauma—though inclusion criteria differed based on the facilities (Crit Care 2013). Over 340 biomarkers were screened, including urine kidney injury molecule-1 (KIM-1), plasma and urine neutrophil gelatinase-associated lipocalin (NGAL), plasma cystatin-C, urine interleukin-18 (IL-18), urine pi-glutathione S-transferase (pi-GST) and urine liver fatty acid-binding protein (LFABP).

IGFBP7 and TIMP-2 are both inducers of G1 cell cycle arrest and showed the highest AUCs on the above cohort (0.76 and 0.79 respectively, and 0.80 when combined). The results were then validated using another 722-patient cohort, without evidence of AKI on admission. Primary outcome was AKI stage 2-3 (KDIGO) within 12-18 hours post-test. There was also another validation study published earlier this year (AJRCCM 2014), using 420 patient from 23 facilities in the US, demonstrating sensitivity of 92% (95% CI 85-98), and specificity of 46% (95% CI 41-52) with cut-off value of 0.3 (ng/ml)2/1000. AUC was 0.82 (0.76-0.88).

Although NephroCheck may have potential advantage to rapid response to developing AKI, still there is substantial limitations of the study including: heterogeneity of the validating cohorts, Caucasian racial predominance, focusing on septic AKI and reflecting more ischemic/hemodynamic-related AKI/ATN.
 So, what should we do next, based on the early detection/ risk prediction of AKI? What interventions or drugs could we use to prevent AKI development in those high-risk patients? Specific treatment strategies for AKI are now warranted for this biomarker to be in full use in the fight against AKI.
Figure from Crit Care 2013 paper: Proposed mechanistic involvement of the novel biomarkers in AKI.

Naoka Murakami

mTOR Pathway in Anti-phospholipid Syndrome

Antiphospholipid syndrome (APS) is an autoimmune hypercoagulable disorder characterized by small-to-large vascular (both arterial and venous) thrombosis with end-organ damages, in presence of circulating antibodies against phospholipid binding proteins.

Kidney transplantation in patients with APS is challenging because  post-transplant thrombosis, vascular complications and requirement of anticoagulation during peripoperative period. Let’s start with a brief review of recent advances in transplantation in APS.

For post-transplant TMA due to recurrent APS nephropathy, Canaud et al. recently explored the use of eculizumab. Eculizumab, a humanized mAb that binds C5, prevents cleavage of C5 into C5a and C5b, thereby preventing generation of the membrane attack complex (MAC). At a molecular level, the pathogenesis of endothelial damage in APS is in part via complement activation; C5b-9 MAC deposition on endothelium, leading to cell lysis and/or activation of other proinflammatory pathways, so the use of eculizumab is reasonable. Three patients, maintained with steroids, CNI and MMF, were treated with eculizumab for posttransplant TMA with robust improvement of allograft functions after several doses, and all three patients were successfully withdrawn from maintenance eculizumab treatment after 3-12 months of initial dose. Interestingly, although biopsy showed improved TMA lesions, C5b-9 depositions were persistent for as long as 3 months as “foot prints”. The authors also noticed that eculizumab treatment did not prevent the chronic vascular lesions seen in 12-month protocol biopsies.

Preemptive use of eculizumab in kidney transplant in APS-related ESRD was also attempted in another case series. Three patients, two with CAPS (catastrophic APS), received 1,200 mg of Eculizumab on day 0, 900 mg on POD 1, and weekly thereafter until week 4. After week 5, they received 1200 mg every 2 week. Despite one  biopsy proven cellular rejection successfully treated with pulse steroid, graft function and survival was acceptable without recurrence of APS during follow-up of 6 months to 4 years. In the setting of no specific treatment other than systemic anticoagulation, eculizumab seems potentially promising treatment, however, the sufficient treatment length of this drug needs to be optimized, especially due to high cost. Also, there is no description of immunosuppressive regimen either for induction nor maintenance, and it is unclear these patients were on sirolimus or not.

 In a recent NEJM article, Canaud et al.  indicated the beneficial effect of sirolimus in proliferative vascular changes associated with APS and CAPS, which were not reversed by eculizumab in their previous study. They demonstrated that the chronic vascular changes in APS patients were induced by activation of mTORC via phosphorylation of Akt-S6K pathway, using immunohistochemistry of renal biopsy samples and in vitro signaling studies with HIMEC-1, a human microvascular endothelial cell line, as well as autopsy samples of CAPS. Furthermore, using a cohort of kidney transplant patients with APS (10 treated with steroids+sirolimus+purine inhibitor and 27 with steroids+CNI+purine inhibitor), their nested-case-control study demonstrated that posttransplant allograft functions were better preserved at 144 months post transplant in the sirolimus group compared with CNI group (7 of 10 vs 3 of 27 patients with functioning grafts) and this effect was observed only in patients with APS and not in patients without APS. Other variables including cold ischemia time and immunologic risk profile were comparable between sirolimus and CNI groups. Although this is a relatively small case-controlled study, the use of mTOR inhibitors for the prevention of APS post-transplant seems very promising.

Naoka Murakami

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

“Liquid Biopsy” – measuring cell-free circulating donor DNA to predict allograft rejection

Transplant physicians face the challenge to detect allograft rejection using non-sensitive biomarkers and clinical signs/symptoms. 

For kidney transplant, serum creatinine, urinalysis for proteinuria or hematuria, are so far used for screening for rejection and/or assessment of graft function. In a recent article in Sci Transl Med, De Vlaminck et al. investigated the use of cell-free circulating DNA (cfdDNA) as a marker of allograft rejection in adult heart transplants. 

Authors used shotgun sequence approach to detect donor-specific DNA fragments (SNP genotyping), and correlated the level of cfdDNA with cardiac biopsy results, the diagnostic “gold standard” for rejection. 

The idea of a “liquid biopsy” —using plasma circulating DNA fragments—as a diagnostic tool has been explored in the Oncology field, to detect early and late stage cancers. In transplant field, the presence of donor-derived DNA fragments in recipient’s plasma (or urine), called microchimerism, has been known since the end of 1990s  and using them as a measure of transplant injury has been tried. For example, Sidgel et al. used Y-specific DNA fragments as a marker in male-to-female transplants. 

 In this current article, De Vlaminck et al. further improved this approach to overcome dependency on donor-recipient pair. Authors concluded that using the cutoff value of 0.25%, cfdDNA method is as sensitive (0.58) and specific (0.87) as biopsy (0.58 and 0.96, respectively). Furthermore, cfdDNA approach was able to detect rejection as early as 5 months prior to tissue diagnosis. 

There are, of course, some limitations to overcome: 1) cfdDNA cannot differentiate ACR vs AMR, 2) cannot rule out other source of DNA fragments, such as endothelial damage without rejection 3) exhibit still substantial discordance with biopsy results. Although currently using the shotgun approach, the technique can be refined by using targeted-sequencing approach, to be less laborious process. 

Based on the same underlying principles, it is very likely that similar test would have application in kidney transplantation and potentially help to detect earlier ongoing graft injury. We will still need the biopsy though to clarify the type of injury...

Naoka Murakami

Transplantation of HIV+ organs: from ban to HOPE

Organ donation from HIV positive patients has been prohibited in the USA since 1988. Pre-transplant screening for HIV is mandatory using nuclear amplification test (NAT), though false negative results during “window period” are a potential concern in donor selection.

Elmi Muller spoke at our Transplant Grand Rounds yesterday and told us about the challenge she faces in South Africa, where HIV infection affects more than 15% of the population, HIV patients frequently develop HIV-associated nephropathy (HIVAN) and progress to ESRD. Moreover, the availability of renal replacement therapy (hemodialysis) is limited.

In the early 90's, there were a number of uncertainties related to transplanting HIV positive patients including the worrisome concern about introducing a donor-derived virus that could lead to out of control HIV infection (resistant strain?); the effect on the immune system of HIV infection in combination with immunosuppression (worse immunodeficiency?); and the financial burden of expanding the services of transplantation to HIV+ patients in a country with limited health budget.

Against all the odds, Elmi performed four cases of HIV positive kidney donors to HIV positive recipients in South Africa in 2008. No IRB approval... Elmi reported having some intuition that it would work and she was in touch with other physicians around the world who shared her view. In her side, HIV resistance rate is very low in South Africa.

After performing those transplant, her colleagues and the hospital prosecuted her and banned her from performing surgery for more than 1 year. This past week, the HIV Organ Policy Equity (HOPE) Act was approved by the US Congress, permitting donation from HIV-positive organs to HIV-positive recipients. It took time but she is now recognized as a pioneer in the field and her courage to perform those surgeries were remarkable. The law that passed will help expand the availability of organ donors to HIV positive patients and will help with organ shortage.

Some challenges though still remain. HIV resistance is much higher in the States (~19%) and HIV+ transplant recipients experience a higher rate of rejection and significant difficulties with drug-drug interactions (P450 inhibitors). Details about a recent trial can be reviewed on this prior blog. In contrary to the idea of over-immunosuppression, recent paper suggests that ATG may be a better induction therapy choice for these patients. Closely monitoring of these patients will be essential as we learn more about HIV and transplantation. By bending rules, Elmi changed a transplant policy and made history.

Naoka Murakami
Leo Riella

Renal transplant across an ABO barrier

A 24 year-old ESRD patient secondary to IgA nephropathy has been on HD for almost one year. His mother is a potential donor for living related kidney transplant. The condition seemed perfect—zero mismatch on HLA, no PRA, no DSA—except for ABO-incompatibility. Type O in the recipient and type A in the donor.

What are ABO antigens, and where are they expressed?
 ABO antigens, discovered by Karl Landsteiner in 1901, are glycoprotein antigens, expressed not only on the surface of red blood cells, but also on endothelial cells, and epithelial cells.

Why is ABO incompatibility a relative contraindication to transplantation? 
As noted above, ABO antigens are expressed on the donor kidney, and can be a target of antibody-mediated rejection. The first ABO-incompatible kidney transplant (ABOi KT) was performed in mid 1950s and ended up with hyperacute rejection (HAR). Therefore, this procedure was considered as a contraindication until recently. The concept of depleting anti-AB Abs was introduced in early 1980s, leading to successful ABOi KT by Alexandre et al., followed by great effort in Japan to improve outcomes, where ABOi KT now consists of 30% of living donor KT. Nowadays, ABOi KT is becoming a reasonable option in the US as well.

What is the usual immunosuppressive protocol to transplant these patients? Are ABO antibody titers important for determining the feasibility of transplant? 
Unfortunately, there are no randomized controlled trials for the pre-transplant conditioning regimens for ABOi KT. However, the basic idea of overcoming ABO-incompatibility is decreasing the circulating ABO antibodies via combination of (1) antibody depletion (e.g. plasmaphresis) (2) IVIG, (3) Rituximab or splenectomy. Pre-transplant ABO antibody titers should be taken into consideration, which generally should be 1:16 or lower, although the number is exclusively based on empirical evidence. Interestingly, there may be no correlation with baseline antibody titers and graft survivals. For maintenance immunosuppressive regimen, therapy, there is no need for higher intensity of immunosuppression compared to ABO compatible KT.

What are the potential complications?
Initially, HAR was the largest concern for ABOi KT, but is not major concern with the use of current desensitization protocols . Antibody-mediated hemolysis and delayed antibody-mediated rejection are potential complications. Therefore, post-transplant monitoring of AB antibody titers is usually recommended. Of note, however, in the setting of ABOi KT, peritubular capillary C4d deposition alone is not diagnostic as this can be observed even in 80% of fully functioning grafts, called “accommodation”, the mechanism of which is not fully understood.

How good are the long-term outcomes of transplants crossing an ABO barrier? 
Reports showed long-term graft survival of ABO-incompatible renal transplants are the same as those of ABO-matched transplants. One report from Japan showed that the overall patient survival rate was 97% for the first year and 95%, 93% and 90% for 3, 5 and 9 years after surgery, respectively. The 1-year graft survival rate was 93%, and that for 3, 5 and 9 years was 89%, 84%, and 72%, respectively. Of note, Montgomery et al.  reported no difference in long-term patient survival but suggested more graft loss in the first 14 days after transplantation.

Going back to our case, the patient went through plasmapheresis and two doses of rituximab injections (150 mg/m2, on POD #-14 and -1) as a pre-conditioning regimen. This was followed by a successful ABOi KT using Basiliximab as induction therapy, FK/MMF/Prednisone as maintenance regimen with excellent graft function months after transplant.

Naoka Murakami
Leonardo V. Riella