Shared alloimmune responses against blood and transplant donors result in adverse clinical outcomes following blood transfusion post-renal transplantation.

De novo HLA donor-specific antibodies (DSA) following transplantation are associated with alloimmune injury and allograft failure. Blood transfusions are allogeneic, and when given posttransplant (PTBT) they may independently increase the risk of HLA antibody development. This study aims to analyze the development of HLA transfusion-specific antibodies (TSA) to blood donors of transfusions given posttransplant and examine the impact on clinical outcomes. A total of 244 blood donors of transfusions received by 86 transplant patients (46 who developed a DSA post transfusion and 40 who remained DSA negative) were HLA typed. De novo TSA developed against 150/244 (61.5%) blood donors. In 70/150 (46.7%) cases the TSA was of shared HLA antibody specificity with a DSA response in the recipient (DSA+ = TSA+). This occurred when there was a greater overall HLA match between the blood and transplant donor. DSA+ = TSA+ patients had increased risk of allograft failure (P = .0025) and AMR (P = .02) compared with the DSA+ ≠ TSA+ patients. To conclude, PTBT may elicit de novo HLA antibodies. Enhanced HLA matching between the blood and transplant donor is more likely to result in a DSA and TSA of shared antibody specificities. Transfusion avoidance or the use of HLA matched or selected blood may reduce this risk and improve outcomes.

The natural history of immunoglobulin M nephropathy in adults.

BACKGROUND: Immunoglobulin M (IgM) nephropathy is an idiopathic glomerulonephritis characterized by diffuse mesangial deposition of IgM. IgM nephropathy has been a controversial diagnosis since it was first reported, and there are few data identifying specific pathological features that predict the risk of progression of renal disease. METHODS: We identified 57 cases of IgM nephropathy among 3220 adults undergoing renal biopsy at our institution. Biopsies had to satisfy the following three criteria to meet the definition of IgM nephropathy in this study: (i) dominant mesangial staining for IgM, (ii) mesangial deposits on electron microscopy (EM) and (iii) exclusion of systemic disease. RESULTS: The median age was 42 years and 24 patients were male. Thirty-nine per cent of patients presented with the nephrotic syndrome, 49% presented with non-nephrotic proteinuria and 39% had eGFR <60 mL/min. The median post-biopsy follow-up was 40 months and serum creatinine had doubled in 31% by 5 years. Of histological parameters, glomerular sclerosis and tubular atrophy, but not mesangial proliferation, were risk factors for renal insufficiency. Thirty-nine per cent of nephrotic patients achieved complete remission, and outcome was significantly worse in those who did not respond to treatment. Focal segmental glomerulosclerosis was diagnosed in 80% of those undergoing repeat renal biopsy, despite ongoing mesangial IgM deposition. CONCLUSIONS: We propose criteria for a consensus definition of IgM nephropathy.

Incidence, Risk Factors, and Effect on Allograft Survival of Glomerulonephritis Post-transplantation in a United Kingdom Population: Cohort Study.

Background: Post-transplant glomerulonephritis (PTGN) has been associated with inferior long-term allograft survival, and its incidence varies widely in the literature. Methods: This is a cohort study of 7,623 patients transplanted between 2005 and 2016 at four major transplant UK centres. The diagnosis of glomerulonephritis (GN) in the allograft was extracted from histology reports aided by the use of text-mining software. The incidence of the four most common GN post-transplantation was calculated, and the risk factors for disease and allograft outcomes were analyzed. Results: In total, 214 patients (2.8%) presented with PTGN. IgA nephropathy (IgAN), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), and membranoproliferative/mesangiocapillary GN (MPGN/MCGN) were the four most common forms of post-transplant GN. Living donation, HLA DR match, mixed race, and other ethnic minority groups were associated with an increased risk of developing a PTGN. Patients with PTGN showed a similar allograft survival to those without in the first 8 years of post-transplantation, but the results suggest that they do less well after that timepoint. IgAN was associated with the best allograft survival and FSGS with the worst allograft survival. Conclusions: PTGN has an important impact on long-term allograft survival. Significant challenges can be encountered when attempting to analyze large-scale data involving unstructured or complex data points, and the use of computational analysis can assist.

Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice.

Aristolochic acid (AA) is the causative agent of urothelial tumors associated with AA nephropathy and is also implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. These tumors contain AA-characteristic TP53 mutations. We examined gene expression changes in Hupki (human TP53 knock-in) mice after treatment with aristolochic acid I (AAI) by gavage (5 mg/kg body weight). After 3, 12 and 21 days of treatment gene expression profiles were investigated using Agilent Whole Mouse 44K Genome Oligo Array. Expression profiles were significantly altered by AAI treatment in both target (kidney) and nontarget (liver) tissue. Renal pathology and DNA adduct analysis confirmed kidney as the target tissue of AAI-induced toxicity. Gene ontology for functional analysis revealed that processes related to apoptosis, cell cycle, stress response, immune system, inflammatory response and kidney development were altered in kidney. Canonical pathway analysis indicated Nfκb, aryl hydrocarbon receptor, Tp53 and cell cycle signaling as the most important pathways modulated in kidney. Expression of Nfκb1 and other Nfκb-target genes was confirmed by quantitative real-time PCR (qRT-PCR) and was consistent with the induction of Nfκb1 protein. Myc oncogene, frequently overexpressed in urothelial tumors, was upregulated by AAI on the microarrays and confirmed by qRT-PCR and protein induction. Collectively we found that microarray gene expression analysis is a useful tool to define tissue-specific responses in AAI-induced toxicity. Several genes identified such as TP53, Rb1, Mdm2, Cdkn2a and Myc are frequently affected in human urothelial cancer, and may be valuable prognostic markers in future clinical studies.

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.

Pathological predictors of prognosis in immunoglobulin A nephropathy: a review.

PURPOSE OF REVIEW: To provide a review and discussion of histological prognostic indicators in immunoglobulin A (IgA) nephropathy (IgAN). RECENT FINDINGS: A variety of histological parameters and classifications have been used to attempt to predict prognosis in patients with IgAN. Grading systems used thus far do not consistently provide a useful adjunct to clinical prognostic parameters. This may be due to the variety of grading systems used, to inconsistent patient recruitment processes, and to the use of actuarial renal survival as an end point. This has led to the development of a new IgAN classification proposal by the International IgA Nephropathy Network in conjunction with the Renal Pathological Society. Additional potential markers of disease progression currently under investigation include glomerular parameters such as number and size, markers of podocyte function and of complement activation, inflammatory infiltrates and mediators of tubulointerstitial fibrosis. SUMMARY: There is a need for an internationally accepted, reproducible and clinically meaningful pathological classification of IgAN. Such a classification is currently being developed. By using only reproducible, single parameters and validating them on a large group of cases from all over the world, it is hoped that a useful clinically predictive tool will be developed.

Pulmonary Inflammation Impacts on CYP1A1-Mediated Respiratory Tract DNA Damage Induced by the Carcinogenic Air Pollutant Benzo[a]pyrene.

Pulmonary inflammation can contribute to the development of lung cancer in humans. We investigated whether pulmonary inflammation alters the genotoxicity of polycyclic aromatic hydrocarbons (PAHs) in the lungs of mice and what mechanisms are involved. To model nonallergic acute inflammation, mice were exposed intranasally to lipopolysaccharide (LPS; 20 µg/mouse) and then instilled intratracheally with benzo[a]pyrene (BaP; 0.5 mg/mouse). BaP-DNA adduct levels, measured by (32)P-postlabeling analysis, were approximately 3-fold higher in the lungs of LPS/BaP-treated mice than in mice treated with BaP alone. Pulmonary Cyp1a1 enzyme activity was decreased in LPS/BaP-treated mice relative to BaP-treated mice suggesting that pulmonary inflammation impacted on BaP-induced Cyp1a1 activity in the lung. Our results showed that Cyp1a1 appears to be important for BaP detoxification in vivo and that the decrease of pulmonary Cyp1a1 activity in LPS/BaP-treated mice results in a decrease of pulmonary BaP detoxification, thereby enhancing BaP genotoxicity (ie, DNA adduct formation) in the lung. Because less BaP was detoxified by Cyp1a1 in the lungs of LPS/BaP-treated mice, more BaP circulated via the blood to extrapulmonary tissues relative to mice treated with BaP only. Indeed, we observed higher BaP-DNA adduct levels in livers of LPS/BaP-treated mice compared with BaP-treated mice. Our results indicate that pulmonary inflammation could be a critical determinant in the induction of genotoxicity in the lung by PAHs like BaP. Cyp1a1 appears to be involved in both BaP bioactivation and detoxification although the contribution of other enzymes to BaP-DNA adduct formation in lung and liver under inflammatory conditions remains to be explored.

Value of antibodies to free light chains in immunoperoxidase studies of renal biopsies.

AIMS: Because immunoglobulin abnormalities may affect the kidney, investigation of renal biopsies requires immunohistological study of light chains. A problem is that most antibodies to light chains react with whole immunoglobulins as well as free light chains, and there are generally many more whole immunoglobulins than free light chains. The usefulness of antibodies that only detected free light chains was investigated. METHODS: Antibodies to free light chains were used in an immunoperoxidase method on paraffin sections of 198 renal biopsies, and compared with conventional antibodies against light chains examined by immunofluorescence on 13 frozen sections and by immunoperoxidase on 46 paraffin sections. RESULTS: Immunofluorescence and immunoperoxidase were concordant on 10 of 13 biopsies. Immunofluorescence detected slight deposition of light chains in three biopsies not shown by immunoperoxidase, of undetermined clinical significance. Using immunoperoxidase, the free light chain antibodies were more sensitive than conventional antibodies, giving much cleaner staining and better detection of deposits in AL amyloid, light chain deposition disease and cryoglobulinaemic glomerulonephritis. The free light chain antibodies showed discordance or ambiguity between immunohistological and clinical findings in seven (4%) of 185 patients with known immunoglobulin status. These included two of 28 cases of AL amyloid that showed no light chain deposition. The method was not designed for detection of light chain restriction in neoplastic plasma or lymphoplasmacytic cells. CONCLUSIONS: Polyclonal antibodies to free light chains are an improvement on conventional antibodies in immunoperoxidase study of paraffin sections of renal biopsies and are useful in everyday practice.

ACB-PCR measurement of H-ras codon 61 CAA→CTA mutation provides an early indication of aristolochic acid I carcinogenic effect in tumor target tissues.

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen, which induces forestomach and kidney tumors in mice and is associated with development of urothelial cancer in humans. This study sought to gain mechanistic insight into AAI-induced carcinogenesis through analysis of a tumor-relevant endpoint. Female Hupki mice were treated daily with 5 mg AAI/kg body weight by gavage for 3, 12, or 21 days. Histopathology and DNA adduct analysis confirmed kidney and forestomach as target tissues for AAI-induced toxicity. H-ras codon 61 CAA→CTA mutations were measured in mouse kidney and forestomach, as well as liver and glandular stomach (nontarget organs) by allele-specific competitive blocker-PCR (ACB-PCR), because A→T transversion is the predominant mutation induced by AA and this particular mutation was found previously in AA-induced rodent forestomach tumors. Treatment-related differences were observed, with the H-ras mutant fraction (MF) of mouse kidney and forestomach exposed to 5 mg AAI/kg body weight for 21 days significantly higher than that of vehicle-treated controls (Fisher's exact test, P < 0.05). Statistically significant correlations between dA-AAI adduct levels (measured previously in the same animals) and induced H-ras MFs were evident in forestomach of mice treated for 21 days (linear regression, P < 0.05). The significant increase in H-ras MF in kidney and forestomach, along with the correlation between DNA adducts, histopathology, and oncogene mutation, provide definitive evidence that AA induces tumors through a directly mutagenic mode of action. Thus, measurement of tumor-associated mutations is a useful tool for elucidating the mechanisms underlying the tissue specificity of carcinogenesis.

Peritubular capillary basement membrane multilayering on electron microscopy: a useful marker of early chronic antibody-mediated damage.

BACKGROUND: Chronic antibody-mediated rejection is an important cause of late graft failure. Developing an early marker of the disease may allow diagnosis and treatment before irreversible graft damage has occurred. The aim of this study was to assess whether, on electron microscopy examination, peritubular capillary (PTC) basement membrane multilayering precedes and predicts the development of transplant glomerulopathy (TG). METHODS: We used a vintage matched case-control method. Sixteen case-control pairs were created among all renal transplant patients from October 2005. Cases were patients who developed TG, and controls were patients with a late (>36 months) posttransplant (indication or surveillance) biopsy without TG. Electron microscopy was carried out on a biopsy taken earlier in the posttransplantation period for both cases and controls. RESULTS: For every additional PTC of 25 examined with three or more layers in the early biopsy, the risk of having TG in the later biopsy was increased by 1.4-fold (95% confidence interval, 1.1-1.9; P=0.015). For every PTC of 25 with five or more layers, the risk was increased by 1.6-fold (95% confidence interval, 1.0-2.7; P=0.063). Thus, the risk of future TG increased substantially with every additional PTC of 25 showing multilayering in the early biopsy. CONCLUSIONS: Peritubular capillary basement membrane multilayering on electron microscopy is a useful marker of early chronic antibody-mediated damage, and information can be obtained by assessing PTC with three to four layers of basement membrane in addition to those with five or more layers. This finding must be validated in a prospective study.

Bone marrow-derived cells contribute to podocyte regeneration and amelioration of renal disease in a mouse model of Alport syndrome.

In a model of autosomally recessive Alport syndrome, mice that lack the alpha3 chain of collagen IV (Col4alpha3(-/-)) develop progressive glomerular damage leading to renal failure. The proposed mechanism is that podocytes fail to synthesize normal glomerular basement membrane, so the collagen IV network is unstable and easily degraded. We used this model to study whether bone marrow (BM) transplantation can rectify this podocyte defect by correcting the deficiency in Col4alpha3. Female C57BL/6 Col4alpha3(-/-) (-/-) mice were transplanted with whole BM from male wild-type (+/+) mice. Control female -/- mice received BM from male -/- littermates. Serum urea and creatinine levels were significantly lower in recipients of +/+ BM compared with those of -/- BM 20 weeks post-transplant. Glomerular scarring and interstitial fibrosis were also significantly decreased. Donor-derived cells were detected by in situ hybridization (ISH) for the Y chromosome, and fluorescence and confocal microscopy indicated that some showed an apparent podocyte phenotype in mice transplanted with +/+ BM. Glomeruli of these mice showed small foci of staining for alpha3(IV) protein by immunofluorescence. alpha3(IV) mRNA was detectable by reverse transcription-polymerase chain reaction and ISH in some mice transplanted with +/+ BM but not -/- BM. However, a single injection of mesenchymal stem cells from +/+ mice to irradiated -/- recipients did not improve renal disease. Our data show that improved renal function in Col4alpha3(-/-) mice results from BM transplantation from wild-type donors, and the mechanism by which this occurs may in part involve generation of podocytes without the gene defect.