Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease.

Electron microscopy (EM) following immunofluorescence (IF) imaging is a vital tool for the diagnosis of human glomerular diseases, but the implementation of EM is limited to specialised institutions and it is not available in many countries. Recent progress in fluorescence microscopy now enables conventional widefield fluorescence microscopes to be adapted at modest cost to provide resolution below 50 nm in biological specimens. We show that stochastically switched single-molecule localisation microscopy can be applied to clinical histological sections stained with standard IF techniques and that such super-resolved IF may provide an alternative means to resolve ultrastructure to aid the diagnosis of kidney disease where EM is not available. We have implemented the direct stochastic optical reconstruction microscopy technique with human kidney biopsy frozen sections stained with clinically approved immunofluorescent probes for the basal laminae and immunoglobulin G deposits. Using cases of membranous glomerulonephritis, thin basement membrane lesion, and lupus nephritis, we compare this approach to clinical EM images and demonstrate enhanced imaging compared to conventional IF microscopy. With minor modifications in established IF protocols of clinical frozen renal biopsies, we believe the cost-effective adaptation of conventional widefield microscopes can be widely implemented to provide super-resolved image information to aid diagnosis of human glomerular disease.

Predicting long-term renal and patient survival by clinicopathological features in elderly patients undergoing a renal biopsy in a UK cohort.

BACKGROUND: Several publications have demonstrated the use of renal biopsy in elderly patients in establishing a diagnosis and enabling directed therapy. However, evidence on the long-term outcomes following biopsies is lacking. The aim of this study is to describe the renal and patient outcomes in elderly patients according to indication for biopsy, clinical parameters and the histological diagnosis. METHODS: We performed a retrospective cohort study of 463 patients >70 years old who underwent a renal biopsy at our centre between 2006 and 2015. RESULTS: The median age of the patients was 74.8 (range 70.0-89.6) years. The most frequent primary diagnoses were pauci-immune crescentic glomerulonephritis (GN; 12%), acute interstitial nephritis (10.8%) and membranous GN (7.1%). Death-censored renal survival at 1 and 5 years following the index biopsy was 85.2 and 75.9%, respectively, and patient survival at 1 and 5 years was 92.2 and 71.6%, respectively. Patients who progressed to end-stage renal disease (ESRD) were at higher risk of dying compared with patients who did not require dialysis [hazard ratio 2.41 (95% confidence interval 1.58-3.68; P < 0.001]. On multivariate analysis, factors associated with the risk of progression to ESRD were creatinine (P < 0.001), heavy proteinuria (P = 0.002) and a non-chronic kidney disease (CKD) biopsy indication (P = 0.006). A histological diagnosis of primary GN (P = 0.001) or tubulointerstitial nephritis (P = 0.008) was associated with a favourable renal outcome, while patients with vasculitis and paraprotein-related renal disease (PPRD) had the highest risk of requiring dialysis (P = 0.0002 and P = 0.003, respectively). PPRD was also an independent risk factor for death. CONCLUSIONS: This study demonstrates that renal biopsies in the elderly not only enable directed therapy, but also provide prognostic information on renal and patient survival.

Development of pancreatic machine perfusion: translational steps from porcine to human models.

BACKGROUND: Hypothermic machine perfusion (HMP) is increasingly being used for extended criteria kidney grafts. Pancreatic HMP is challenging because physiologically the pancreas is a low-flow organ susceptible to edema. We report the successful development of preclinical HMP models using porcine pancreases, as well as human pancreases unsuitable for clinical transplantation. METHODS: Ten porcine pancreases were used in the development of these perfusion models. Pancreases underwent 24 h of static cold storage (SCS, n = 3) and then viability assessment on an isolated oxygenated normothermic reperfusion (NRP) circuit or 24-h SCS, 5 h of HMP, and then NRP (SCS-HMP, n = 3). Human pancreases (n = 3) were used in the development of a preclinical model. RESULTS: Porcine HMP demonstrated stable perfusion indices at low pressures, with a weight gain of between 15.3% and 27.6%. During NRP, SCS-HMP pancreases demonstrated stable perfusion flow indices (PFIs) throughout reperfusion (area under the curve was in the range of 0.49-2.04 mL/min/100 g/mm Hg), whereas SCS-only pancreases had deteriorating PFI with a decline of between 19% and 46%. Human pancreas models demonstrated stable PFI between 0.18 and 0.69 mL/min/100 g/mm Hg during HMP with weight gain of between 3.9% and 14.7%. NRP perfusion in porcine and human models was stable, and functional assessment via insulin secretion demonstrated beta cell viability. Exocrine function was intact with production of pancreatic secretions only in human grafts. CONCLUSIONS: Application of machine perfusion in preclinical porcine and human pancreas models is feasible and successful; the development of these translational models could be beneficial in improving pancreas preservation before transplantation and allowing organ viability assessment and optimization.

An ex vivo comparison of adenosine and lidocaine solution and University of Wisconsin solution for hypothermic machine perfusion of porcine kidneys: potential for development.

BACKGROUND: The optimal hypothermic machine perfusion (HMP) solution has not yet been developed. An adenosine and lidocaine (AL) solution has been shown to be protective in cardiac preservation. The aim of the present study was to examine a modified AL solution with low Ca2+, 16 mM Mg2+, and 4% albumin on kidney preservation compared with University Wisconsin solution (UW). METHODS: Twenty donation of organs after cardiac death porcine kidneys underwent HMP for 10 h (AL, n = 10; UW, n = 10) and then 2 h of normothermic reperfusion. Perfusion dynamics, functional parameters, histology, and real-time microdialysis were used to assess kidney responses and viability. RESULTS: During HMP, modified AL-perfused kidneys maintained higher flow rates (21.5 versus 17.9 mL/min/100 g, P = 0.01), with perfusion flow index during the first 3 h 25% greater than with UW (AL = 0.50 ± 0.2, UW = 0.40 ± 0.17 mL/min/100 g/mmHg; P = 0.03), followed by an increase in UW kidneys which was not significantly different to AL over the remaining 7 h (0.54 versus 0.55 mL/min/100 g/mmHg, respectively). During warm reperfusion, there were no significant differences between the two HMP groups in creatinine clearance, oxygen, and glucose consumption between groups. Modified AL kidneys had significantly lower perfusate lactates (3.1 versus 4.1 mmol/L, P = 0.04) during reperfusion and lower cortical lactate levels (AL = 0.66 ± 0.31, UW = 0.89 ± 0.53 mM, P = 0.33). Histology showed similar degrees of reperfusion injury. CONCLUSIONS: We conclude that HMP with modified AL solution showed improved perfusion compared with UW and lower perfusate lactate levels during warm reperfusion. Further modification of the AL composition is warranted and may lead to more rapid kidney stabilization and improved graft viability assessment, potentially expanding donor pools.

Organ Pretreatment With Cytotopic Endothelial Localizing Peptides to Ameliorate Microvascular Thrombosis and Perfusion Deficits in Ex Vivo Renal Hemoreperfusion Models.

BACKGROUND: Hypothermic machine organ perfusion (HMP) offers opportunity to manipulate grafts with pharmacological agents prior to transplantation. Pretreating organs with novel cytotopic anticoagulant peptides that localize to endothelial cell membranes could ameliorate microvascular thrombotic sequelae posttransplantation. We describe experiments testing thrombalexin (TLN), a novel cell binding thrombin inhibitor, using porcine and unused human kidneys in a series of ex vivo normothermic hemoreperfusion models. METHODS: Thirty-eight porcine kidneys were used. Control kidneys underwent pretreatment via HMP with either unmodified perfusion solution (n = 15) or solution with inactive-TLN (absent anticoagulant effect, n = 4). Test kidneys were perfused with TLN-treated solution (n = 19). All kidneys then underwent hemoreperfusion. Two unused human kidneys underwent a similar protocol. RESULTS: Hypothermic machine perfusion pretreatment facilitated delivery and tethering of TLN in the organ microvasculature. Hemoreperfusion challenge demonstrated improved perfusion in TLN-treated kidneys compared with controls: 26.4% superior flow (30.6 vs. 23.1 mL/min per 100 g, P = 0.019) and 28.9% higher perfusion flow indices (0.43 vs. 0.32 mL/min per 100 g mm Hg, P = 0.049). Orthogonal polarization spectral imaging demonstrated superior microvascular capillary perfusion in TLN-treated organs versus controls (9.1 vs 2.8 pl/s per mm, P = 0.021). Rapid-sampling microdialysis for cortical [lactate] as a marker of tissue ischemia/metabolism detected lower levels in TLN-treated kidneys. Perfusate analysis demonstrated reduced fibrin generation in TLN-treated kidneys correlating with perfusion data. CONCLUSIONS: Our data suggest that HMP graft pretreatment with cytotopic anticoagulants is feasible and ameliorates perfusion deficits seen in ex vivo hemoreperfusion models. There is potential for further development and application of this translational strategy to deliver locally active anticoagulants directly within grafts and decrease microvascular thrombotic sequelae, while avoiding systemic anticoagulation and its associated risks.

Acute cellular rejection: impact of donor-specific antibodies and C4d.

BACKGROUND: Mixed rejection in kidney transplantation consists of histologic and/or serological evidence of both cellular and humoral components. As it is not confined to a distinct category in the Banff classification, how to best manage these patients is not clear. The aim of this study was to determine the incidence and outcome of morphological T-cell-mediated rejection (TCMR) with a humoral component, defined as the presence of either DSA or C4d, compared with the outcome of pure TCMR. METHODS: We retrospectively studied 922 consecutive renal transplant recipients and analyzed patients with TCMR according to the evidence of a humoral component. RESULTS: A total of 147 cases of morphological TCMR were analyzed. Of these, 92 (62.6%) had "pure" TCMR and 55 (37.4%) had "mixed" TCMR on the index biopsy. On univariant analysis, diffuse C4d (odds ratio [OR]=10.9, 95% confidence interval [CI]=1.8-66.9, P=0.01) and DSA positivity at the time of index biopsy (OR=2.8, 95% CI=1.2-6.6, P=0.02) were associated with allograft loss, whereas arteritis (OR=0.5, 95% CI=0.2-1.2, P=0.11) and glomerulitis (OR=0.9, 95% CI=0.4-2.1, P=0.8) were not. Arteritis was associated with subsequent antibody-mediated rejection (OR=4.9, 95% CI=1.1-20.8, P=0.03), and glomerulitis was associated with the development of transplant glomerulopathy (OR=10.7, 95% CI=3.1-37.1, P<0.01). On the multivariate analysis, only patients with C4d and DSA were at risk of graft failure (OR=4.9, 95% CI=2.0-12.0, P<0.01) in the medium term. CONCLUSION: TCMR with a humoral component has a worse prognosis when compared with pure TCMR. As such, it is important to test for alloantibody in cases of morphological TCMR to optimize patient management. Such cases might benefit from more aggressive immunotherapy.

Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal allografts.

BACKGROUND.: Donor-specific anti-HLA antibodies (DSA) are a major cause of alloimmune injury. Transplant recipients with negative complement-dependent cytotoxic crossmatch (CDC-XM) and donor cell-based flow cytometric crossmatch (flow-XM) but low level DSA (i.e., by Luminex) have worse outcomes compared with nonsensitized patients. The aim of this study was to establish whether complement-activating ability in this low-level DSA, present before transplantation, as determined by this technique is important in dictating pathogenicity. METHODS.: We retrospectively studied 52 patients with preformed DSA detected by single-antigen flow cytometric fluorescent beads (SAFBs). Patients were transplanted using a steroid-sparing regimen consisting of alemtuzumab induction, 1 week of corticosteroids and tacrolimus monotherapy.Fifteen (29%) of 52 patients experienced antibody-mediated rejection (AMR), whereas 37 (71%) patients did not. There were no demographic differences between patients with AMR and those without. Pretransplant sera were retested using a modified (SAFB) assay, which detects the presence of the complement fragment C4d as a result of DSA-induced complement activation. RESULTS.: C4d+DSA were detected in 10 (19%) of 52 patients. Biopsy-proven AMR occurred in 7 (70%) of the 10 patients with C4d+DSA and in 8 (19%) of 42 patients with C4d-DSA. AMR-free survival was worse in patients with C4d+DSA (P<0.001). CONCLUSIONS.: The ability of preformed, low-level, DSA to trigger C4d fixation in vitro in patients with negative conventional crossmatch tests is predictive for AMR. C4d SAFB is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression.

Antibody-mediated rejection after alemtuzumab induction: incidence, risk factors, and predictors of poor outcome.

BACKGROUND: Antibody-mediated rejection (AMR) is associated with allograft loss. Identification of factors associated with poor outcome has not been extensively studied. METHODS: We retrospectively studied 469 patients who received a negative crossmatch renal transplant with alemtuzumab induction. Forty-eight of 469 (10.2%) patients were treated for AMR. Thirty of 48 (62.5%) of the cases fulfilled the Banff criteria for definite AMR, whereas 18 of 48 (37.5%) were categorized as suspicious for AMR (tissue injury with C4d staining or donor-specific antibodies [DSAbs]). Sensitization, high human leukocyte antigen, and -DR mismatch were risk factors for the development of AMR (P = 0.0016, 0.001, and 0.012, respectively). RESULTS: Allograft survival was inferior in the AMR group (70.2%) compared with the nonrejector group (97.0%) (P<0.001). Forty-two of 48 (87.5%) of patients with acute AMR had DSAbs. Patients with CII DSAbs at the time of AMR, whether alone or in combination with CI DSAbs had the worst allograft survival (P = 0.014). Both the mean cumulative and immunodominant mean fluorescence index were higher in those patients who subsequently lost their grafts (P<0.001). Patients with diffuse C4d staining had inferior allograft survival than those with focal C4d or no staining (P = 0.02). There was no significant difference in survival by histological grade but a trend to inferior outcomes in those with vascular involvement (P = 0.06). Those patients who met the full Banff criteria had worse survival than those with suspicion for AMR only (P = 0.04). CONCLUSION: This study identifies patients at risk of graft failure from AMR. These patients may benefit from newer therapeutic strategies including the use of eculizumab or bortezomib.

Recombinant adeno-associated virus-mediated in utero gene transfer gives therapeutic transgene expression in the sheep.

Somatic in utero gene therapy aims to treat congenital diseases where pathology develops in perinatal life, thereby preventing permanent damage. The aim of this study was to determine whether delivery of self-complementary (sc) adeno-associated virus (AAV) vector in utero would provide therapeutic long-term transgene expression in a large animal model. We performed ultrasound-guided intraperitoneal injection of scAAV2/8-LP1-human Factor IX (hFIX)co (1 × 10(12) vector genomes/kg) in early (n = 4) or late (n = 2) gestation fetal sheep. The highest mean hFIX levels were detected 3 weeks after injection in late gestation (2,055 and 1,687.5 ng/ml, n = 2) and 3 days after injection in early gestation (435 ng/ml, n = 1). Plasma hFIX levels then dropped as fetal liver and lamb weights increased, although low levels were detected 6 months after late gestation injection (75 and 52.5 ng/ml, n = 2). The highest vector levels were detected in the fetal liver and other peritoneal organs; no vector was present in fetal gonads. hFIX mRNA was detectable only in hepatic tissues after early and late gestation injection. Liver function tests and bile acid levels were normal up to a year postnatal; there was no evidence of liver pathology. No functional antibodies to hFIX protein or AAV vector were detectable, although lambs mounted an antibody response after injection of hFIX protein and Freund's adjuvant. In conclusion, hFIX expression is detectable up to 6 months after delivery of scAAV vector to the fetal sheep using a clinically applicable method. This is the first study to show therapeutic long-term hFIX transgene expression after in utero gene transfer in a large animal model.

A report of succinate dehydrogenase B deficiency associated with metastatic papillary renal cell carcinoma: successful treatment with the multi-targeted tyrosine kinase inhibitor sunitinib.

UNLABELLED: We report a patient who initially presented with an abdominal paraganglioma and subsequently metastatic papillary cell renal cancer. Genetic analysis revealed a 141 G>A (exon 2) Trp47X mutation within the succinate dehydrogenase B gene. Treatment with the novel multi-targeted tyrosine kinase inhibitor sunitinib resulted in a sustained partial response and reduced the level of the angiogenic marker PIGF. TRIAL REGISTRATION NUMBER: a6181037.

International IgA nephropathy network clinico-pathological classification of IgA nephropathy.

There is no international consensus for the pathological or clinical classification of IgA nephropathy (IgAN). This has limited international comparisons between outcome studies which use different histological classifications, and made it more difficult to identify appropriate patients to enter into clinical intervention trials, as well as making it harder to choose appropriate therapy in individual patients. The International IgA Nephropathy Network (www.igan-world.org), working with members of the Renal Pathology Society, has established an international working group which is developing a consensus classification. Agreement has been reached on definitions of pathological features, and the consistency of scoring these features has been tested by pathologists around the world. Pathological features are now being scored in biopsies from large cohorts of patients from many parts of the world in whom sequential clinical information is available. From the integrated analysis of these clinical and pathological features, a clinico-pathological classification will be proposed which will be further refined and tested in additional cohorts of patients. The goal is to establish a reproducible and clinically effective classification which will gain worldwide acceptance for use in clinical practice and research.

Targeting the respiratory muscles of fetal sheep for prenatal gene therapy for Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a lethal degenerative muscular disease. Fetal gene therapy may correct the primary genetic defect. Our aim was to achieve expression of a reporter gene in the respiratory muscles of early gestation fetal sheep. STUDY DESIGN: An adenovirus vector containing the beta-galactosidase reporter gene (AdRSVbetagal) was injected into the thoracic musculature (n = 3) and pleural cavity (n = 6) of fetal sheep (61-67 days' gestation) under ultrasound guidance. Tissues were harvested after 48 hours and site and intensity of beta-galactosidase expression were assessed. RESULTS: Limited transgene expression observed after a single injection was improved by multiple injections, but remained localized. Ultrasound-guided creation of a hydrothorax led to an increase in the intensity of beta-galactosidase expression (ELISA). X-gal staining and immunohistochemistry showed that vector spread was confined to the innermost intercostal musculature. CONCLUSION: Ultrasound-guided injection can deliver gene therapy vectors to the fetal pleural cavity and achieve transduction of the respiratory muscles.

DNA adducts and p53 mutations in a patient with aristolochic acid-associated nephropathy.

BACKGROUND: Aristolochic acid-associated nephropathy (AAN) is a specific type of renal disease that predisposes patients to a high risk of urothelial carcinoma. The authors have analyzed DNA from a patient who had urothelial malignancy 6 years after presenting with AAN and later had a breast carcinoma that metastasized to the liver. METHODS AND RESULTS: DNA was isolated from the primary breast tumor, the liver tumor, and the original urothelial malignancy and assayed for aristolochic acid (AA)-DNA adducts and mutations in the p53 gene. The adduct detected was the adenosine adduct of aristolochic acid I 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI). In DNA from the breast and liver tumors the authors showed the same missense mutation in codon 245 (GGC-->GAC; Gly-->Asp) of exon 7 of p53. In contrast, DNA extracted from the urothelial tumor showed an AAG to TAG mutation in codon 139 (Lys-->Stop) of exon 5. CONCLUSION: A to T transversions, as observed here, are the typical mutations observed in the H-ras gene of tumors induced when rodents are treated with AA and correspond with DNA adduct formation at adenosine residues. These data indicate the probable molecular mechanism whereby AA causes urothelial malignancy.