Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection.

Precise diagnosis of antibody-mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin-fixed, paraffin-embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor-specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti-HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy-based molecular assessment of antibody-mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.

The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions.

The 13th Banff Conference on Allograft Pathology was held in Vancouver, British Columbia, Canada from October 5 to 10, 2015. The cardiac session was devoted to current diagnostic issues in heart transplantation with a focus on antibody-mediated rejection (AMR) and small vessel arteriopathy. Specific topics included the strengths and limitations of the current rejection grading system, the central role of microvascular injury in AMR and approaches to semiquantitative assessment of histopathologic and immunophenotypic indicators, the role of AMR in the development of cardiac allograft vasculopathy, the important role of serologic antibody detection in the management of transplant recipients, and the potential application of new molecular approaches to the elucidation of the pathophysiology of AMR and potential for improving the current diagnostic system. Herein we summarize the key points from the presentations, the comprehensive, open and wide-ranging multidisciplinary discussion that was generated, and considerations for future endeavors.

Control of Immune Response to Allogeneic Embryonic Stem Cells by CD3 Antibody-Mediated Operational Tolerance Induction.

Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC-derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20-25 days. Recipients treated with CD3 antibody showed long-term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self-limited. Regulatory CD4(+)FoxP3(+) T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF-ß and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression.

Late Failing Heart Allografts: Pathology of Cardiac Allograft Vasculopathy and Association With Antibody-Mediated Rejection.

In heart transplantation, there is a lack of robust evidence of the specific causes of late allograft failure. We hypothesized that a substantial fraction of failing heart allografts may be associated with antibody-mediated injury and immune-mediated coronary arteriosclerosis. We included all patients undergoing a retransplantation for late terminal heart allograft failure in three referral centers. We performed an integrative strategy of heart allograft phenotyping by assessing the heart vascular tree including histopathology and immunohistochemistry together with circulating donor-specific antibodies. The main analysis included 40 explanted heart allografts patients and 402 endomyocardial biopsies performed before allograft loss. Overall, antibody-mediated rejection was observed in 19 (47.5%) failing heart allografts including 16 patients (40%) in whom unrecognized previous episodes of subclinical antibody-mediated rejection occurred 4.5 ± 3.5 years before allograft loss. Explanted allografts with evidence of antibody-mediated rejection demonstrated higher endothelitis and microvascular inflammation scores (0.89 ± 0.26 and 2.25 ± 0.28, respectively) compared with explanted allografts without antibody-mediated rejection (0.42 ± 0.11 and 0.36 ± 0.09, p = 0.046 and p < 0.0001, respectively). Antibody-mediated injury was observed in 62.1% of failing allografts with pure coronary arteriosclerosis and mixed (arteriosclerosis and atherosclerosis) pattern, while it was not observed in patients with pure coronary atherosclerosis (p = 0.0076). We demonstrate that antibody-mediated rejection is operating in a substantial fraction of failing heart allografts and is associated with severe coronary arteriosclerosis. Unrecognized subclinical antibody-mediated rejection episodes may be observed years before allograft failure.

Transcatheter heart valve crimping and the protecting effects of a polyester cuff.

INTRODUCTION: Prior to deployment, the percutaneous heart valves must be crimped and loaded into sheaths of diameters that can be as low as 6mm for a 23mm diameter valve. However, as the valve leaflets are fragile, any damage caused during this crimping process may contribute to reducing its long-term durability in vivo. MATERIAL AND METHOD: Bovine pericardium percutaneous valves were manufactured as follows. The leaflets were sutured on a nitinol frame. A polyester cuff fabric served as a buffer between the pericardium and the stent. Two valves were crimped and one valve was used as control. The valves were examined in gross observation and micro-CT scan and then the leaflets were processed for histology and analyzed in scanning electron microscopy, light microscopy and transmission electron microscopy. RESULT: Crimping of the valves resulted in the increase thickness of the leaflets and there was no evidence of additional delamination. The heavy prints of the stents were irregularly distributed on the outflow surface in the crimped devices and were shallow and did not penetrate throughout the thickness of the leaflets. However, the wavy microscopy of collagen fiber bundles was well preserved. They were found to remain individualized without any agglutination as shown by the regular banding appearance. CONCLUSION: Crimping of self-deployable valves per se caused only minor damages to the leaflets. However, the procedure could be refined in order to minimize areas of high pressure and swelling of the tissue that can be accompanied with flow surface disruption and increase of the hydraulic conductance. The incorporation of a polyester buffer serves to prevent the deleterious effects that may be caused if the pericardium tissue were in direct contact with the nitinol stent.

Inflammatory cell burden and phenotype in endomyocardial biopsies with antibody-mediated rejection (AMR): a multicenter pilot study from the AECVP.

This multicenter case-controlled pilot study evaluated myocardial inflammatory burden (IB) and phenotype in endomyocardial biopsies (EMBs) with and without pathologic antibody-mediated rejection (pAMR). Sixty-five EMBs from five European heart transplant centers were centrally reviewed as positive (grade 2, n = 28), suspicious (grade 1, n = 7) or negative (n = 30) for pAMR. Absolute counts of total, intravascular (IV) and extravascular (EV) immunophenotyped mononuclear cells were correlated with pAMR grade, capillary C4d deposition, donor specific antibody (DSA) status and acute cellular rejection (ACR). In pAMR+ biopsies, equivalent number of IV CD3+ T lymphocytes (23 ± 4/0.225 mm(2) ) and CD68+ macrophages (21 ± 4/0.225 mm(2) ) were seen. IB and cell phenotype correlated with pAMR grade, C4d positivity and DSA positivity (p < 0.0001). High numbers of IV T lymphocytes were associated with low grade ACR (p = 0.002). In late-occurring AMR EV plasma cells occurring in 34% of pAMR+ EMBs were associated with higher IB. The IB in AMR correlated with pAMR+, C4d positivity and DSA positivity. In pAMR+ equivalent numbers of IV T lymphocytes and macrophages were found. The presence of plasma cells was associated with a higher IB and occurrence of pAMR late after transplantation.

First reported long-term two- and three-dimensional echocardiographic follow-up with histopathological analysis of a transcatheter pulmonary valve implantation in a pet dog.

Transcatheter pulmonary valve implantation (TPVI) is indicated for use in the management of failing pulmonary valves in humans. We report here the long-term follow-up of the first documented transcatheter pulmonary valve implanted in a client-owned dog. A one-year-old Beagle dog with severe congenital type A valvular pulmonic stenosis first underwent percutaneous balloon pulmonary valvuloplasty, leading two years later to severe pulmonary regurgitation. A TPVI using a Melody™ bioprosthetic valve was then successfully performed, with normalization of the right heart cavities. Repeated two- and three-dimensional transthoracic echocardiographic examinations combined with Doppler modes confirmed the appropriate position and function of the valve for four years. Mitral myxomatous valvular degeneration led to refractory left-sided congestive heart failure, and the dog was humanely euthanized. After postmortem examination, X-ray imaging and histopathological evaluation of the stent and the valve were performed. Ex-vivo imaging of the implanted valve using a Faxitron® Path radiography system and microscopic evaluation of the implanted stent and bioprosthetic leaflets did not show any relevant leaflet or stent alterations. This case provides a proof of concept in interventional veterinary cardiology, showing that TPVI can be performed in dogs with subsequent long-term maintaining normal pulmonary valve function.

Expression and mutational status of treatment-relevant targets and key oncogenes in 123 malignant salivary gland tumours.

BACKGROUND: Malignant tumours of the salivary glands (MSGT) are rare and pleomorphic entities. Patients with advanced disease may benefit from targeted therapy; however, specific targets for optimising and personalising treatments are yet to be identified. DESIGN: Immunohistochemistry for C-KIT, EGFR, HER2, MUC1, phospho-mTOR, androgen/estrogens/progesterone receptors and Ki67 was carried out and evaluated in terms of progression-free and overall survival. High throughput molecular screening of key oncogenes was done in 107 patients using routine diagnostic methods and Sequenom technology. RESULTS: Several therapy leads were identified, including high levels of HER2 and androgen receptors in salivary duct carcinomas, C-KIT in myoepithelial carcinomas and EGFR in mucoepidermoid carcinomas. Recurrent mutations involving downstream elements of the EGFR pathway were found in HRAS, notably in tumours with a myoepithelial component, and in other key oncogenes (KRAS/NRAS/PI3KCA/BRAF/MAP2K). On the other hand, <1% of samples had EGFR or HER2 mutations. CONCLUSION: Several tumour subtypes overexpressed targets of directed therapies suggesting potential therapy leads. Genotyping results suggest activation downstream of EGFR in 18 of the 107 samples that could be associated with low efficacy of EGFR inhibitors. Other molecules, such as PI3K/MEK or mTOR inhibitors, may have anti-tumour activity in this subgroup. The high mutation rate in HRAS highlights a novel key oncogenic event in MSGT.

Ultrafast and whole-body cooling with total liquid ventilation induces favorable neurological and cardiac outcomes after cardiac arrest in rabbits.

BACKGROUND: In animal models of cardiac arrest, the benefit afforded by hypothermia is closely linked to the rapidity of the decrease in body temperature after resuscitation. Because total liquid ventilation (TLV) with temperature-controlled perfluorocarbons induces a very rapid and generalized cooling, we aimed to determine whether this could limit the post-cardiac arrest syndrome in a rabbit model. We especially focused on neurological, cardiac, pulmonary, liver and kidney dysfunctions. METHODS AND RESULTS: Anesthetized rabbits were submitted to either 5 or 10 minutes of untreated ventricular fibrillation. After cardiopulmonary resuscitation and resumption of a spontaneous circulation, the animals underwent either normothermic life support (control) or therapeutic hypothermia induced by TLV. The latter procedure decreased esophageal and tympanic temperatures to 32°C to 33°C within only 10 minutes. After rewarming, the animals submitted to TLV exhibited an attenuated neurological dysfunction and decreased mortality 7 days later compared with control. The neuroprotective effect of TLV was confirmed by a significant reduction in brain histological damages. We also observed limitation of myocardial necrosis, along with a decrease in troponin I release and a reduced myocardial caspase 3 activity, with TLV. The beneficial effects of TLV were directly related to the rapidity of hypothermia induction because neither conventional cooling (cold saline infusion plus external cooling) nor normothermic TLV elicited a similar protection. CONCLUSIONS: Ultrafast cooling instituted by TLV exerts potent neurological and cardiac protection in an experimental model of cardiac arrest in rabbits. This could be a relevant approach to provide a global and protective hypothermia against the post-cardiac arrest syndrome.

Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA).

The significance of C4d-Banff scores in protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA) has not been determined. We reviewed 157 protocol biopsies from 80 DSA+ patients obtained at 3 months and 1 year post-transplant. The C4d Banff scores (1,2,3) were associated with significant increments of microcirculation inflammation (MI) at both 3 months and 1 year post-transplant, worse transplant glomerulopathy and higher class II DSA-MFI (p < 0.01). Minimal-C4d had injury intermediate between negative and focal, while focal and diffuse-C4d had the same degree of microvascular injury. A total of 54% of patients had variation of C4d score between 3 months and 1 year post-transplant. Cumulative (3 month + 1 year) C4d scores correlated with long-term renal function worsening (p = 0.006). However, C4d staining was not a sensitive indicator of parenchymal disease, 55% of C4d-negative biopsies having evidence of concomitant MI. Multivariate analysis demonstrated that the presence of MI and class II DSA at 3 months were associated with a fourfold increased risk of progression to chronic antibody-mediated rejection independently of C4d (p < 0.05). In conclusion, the substantial fluctuation of C4d status in the first year post-transplant reflects a dynamic humoral process. However, C4d may not be a sufficiently sensitive indicator of activity, MI and DSA being more robust predictors of bad outcome.

Very late heart transplant rejection is associated with microvascular injury, complement deposition and progression to cardiac allograft vasculopathy.

In heart transplants, the significance of very late rejection (after 7 years post-transplant, VLR) detected by routine endomyocardial biopsies (EMB) remains uncertain. Here, we assessed the prevalence, histopathological and immunological phenotype, and outcome of VLR in clinically stable patients. Between 1985 and 2009, 10 662 protocol EMB were performed at our institution in 398 consecutive heart transplants recipients. Among the 196 patients with >7-year follow-up, 20 (10.2%) presented subclinical ≥3A/2R-ISHLT rejection. The VLR group was compared to a matched control group of patients without rejection. All biopsies were stained for C4d/C3d/CD68 with sera screened for the presence of donor-specific antibodies (DSAs). In addition to cellular infiltrates with myocyte damage, 60% of VLR patients had evidence of intravascular macrophages. C4d and/or C3d-capillary deposition was found in 55% VLR EMB. All cases of VLR associated with microcirculation injury had DSAs (mean DSA(max) -MFI = 1751 ± 583). This entity was absent from the control group (p < 0.0001). Finally, after a similar follow-up postreference EMB of 6.4 ± 1 years, the mean of CAV grade was 0.76 ± 0.18 in the control group compared to 2.06 ± 0.26 in the VLR group respectively, p = 0.001). There was no difference in patient survival between study and control groups. In conclusion, VLR is frequently associated with complement-cascade activation, microvascular injury and DSA, suggesting an antibody-mediated process. VLR is associated with a dramatic progression to severe CAV in long-term follow-up.

Expression of the familial cardiac valvular dystrophy gene, filamin-A, during heart morphogenesis.

Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X-linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin-A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin-A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin-A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio-temporal distribution of filamin-A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin-A is robustly expressed in non-myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin-A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin-A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin-A result in clinically relevant valve disease.

Specific Follicular Helper T Cell Signature in Takayasu Arteritis.

OBJECTIVE: Our aim was to compare transcriptome and phenotype profiles of CD4+ T cells and CD19+ B cells in patients with Takayasu arteritis (TAK), patients with giant cell arteritis (GCA), and healthy donors. METHODS: Gene expression analyses, flow cytometry immunophenotyping, T cell receptor (TCR) gene sequencing, and functional assessments of cells from peripheral blood and arterial lesions from TAK patients, GCA patients, and healthy donors were performed. RESULTS: Among the most significantly dysregulated genes in CD4+ T cells of TAK patients compared to GCA patients (n = 720 genes) and in CD4+ T cells of TAK patients compared to healthy donors (n = 1,447 genes), we identified a follicular helper T (Tfh) cell signature, which included CXCR5, CCR6, and CCL20 genes, that was transcriptionally up-regulated in TAK patients. Phenotypically, there was an increase in CD4+CXCR5+CCR6+CXCR3- Tfh17 cells in TAK patients that was associated with a significant enrichment of CD19+ B cell activation. Functionally, Tfh cells helped B cells to proliferate, differentiate into memory cells, and secrete IgG antibodies. Maturation of B cells was inhibited by JAK inhibitors. Locally, in areas of arterial inflammation, we found a higher proportion of tertiary lymphoid structures comprised CD4+, CXCR5+, programmed death 1+, and CD20+ cells in TAK patients compared to GCA patients. CD4+CXCR5+ T cells in the aortas of TAK patients had an oligoclonal α/ß TCR repertoire. CONCLUSION: We established the presence of a specific Tfh cell signature in both circulating and aorta-infiltrating CD4+ T cells from TAK patients. The cooperation of Tfh cells and B cells might be critical in the occurrence of vascular inflammation in patients with TAK.

[Fighting against unexplained sudden death]. / Mort subite, en quête d'une explication.

Sudden cardiac death, mostly related to ventricular arrhythmia, is a major public health issue, with still very poor survival at hospital discharge. Although coronary artery disease remains the leading cause, other etiologies should be systematically investigated. Exhaustive and standardized exploration is required to eventually offer specific therapeutics and management to the patient as well as his/her family members in case of inherited cardiac disease. Identification and establishing direct causality of the detected cardiac anomaly may remain challenging, underlying the need for a multidisciplinary and experimented team.

Outcome of subclinical antibody-mediated rejection in kidney transplant recipients with preformed donor-specific antibodies.

This study describes clinical relevance of subclinical antibody-mediated rejection (SAMR) in a cohort of 54 DSA-positive kidney transplant recipients receiving a deceased donor. In 3 months screening biopsies, 31.1% of patients met the criteria of SAMR. A total of 48.9% had an incomplete form of SAMR (g+/ptc+/C4d-negative) whereas 20% had no humoral lesions. Patients with SAMR at 3 months had at 1 year: a higher C4d score, ptc score, and arteriosclerosis score, higher rate of IFTA (100% vs. 33.3%, p < 0.01) and a higher rate of transplant glomerulopathy (43% vs. 0%, p = 0.02) compared to patients without 3-month SAMR. Patients with SAMR at 3 months exhibited at 1 year a higher class II MFImax-DSA and a lower mGFR compared to patients without SAMR (39.2 +/- 13.9 vs. 61.9 +/- 19.2 mL/min/1.73 m(2) respectively, p < 0.01). The group of patients with C4d-negative SAMR at 3 months developed more ptc and IFTA lesions, and lower GFR at 1 year in comparison to biopsies without humoral lesions. SAMR is a frequent entity in KTR with preexisting DSAs and promotes subsequent GFR impairment and development of chronic AMR. C4d-negative SAMR patients displayed an intermediate course between the no-SAMR group and the C4d+ SAMR group. Screening biopsies may be useful to recognize patients more likely to develop SAMR.

Renin-producing Tumour in the Kidney of a Cat.

Clinical and post-mortem examination of an adult neutered male cat with immune-mediated haemolytic anaemia revealed suspected nodules of tumour tissue in the cortex of the right kidney. Cytology and histopathology indicated a malignant renal tumour of undetermined type. Immunohistochemistry confirmed renin production by a proportion of the tumour cells. The lesion may represent a renal adenocarcinoma producing renin or a tumour of juxtaglomerular cells ('reninoma').

Unmixing multi-spectral photoacoustic sources in human carotid plaques using non-negative independent component analysis.

Multi-spectral photoacoustic imaging (MSPAI) is promising for morphology assessment of carotid plaques; however, obtaining unique spectral characteristics of chromophores is cumbersome. We used MSPAI and non-negative independent component analysis (ICA) to unmix distinct signal sources in human carotid plaques blindly. The feasibility of the method was demonstrated on a plaque phantom with hemorrhage and cholesterol inclusions, and plaque endarterectomy samples ex vivo. Furthermore, the results were verified with histology using Masson's trichrome staining. Results showed that ICA could separate recent hemorrhages from old hemorrhages. Additionally, the signatures of cholesterol inclusion were also captured for the phantom experiment. Artifacts were successfully removed from signal sources. Histologic examinations showed high resemblance with the unmixed components and confirmed the morphologic distinction between recent and mature hemorrhages. In future pre-clinical studies, unmixing could be used for morphology assessment of intact human plaque samples.

Combined heart and kidney transplantation in a patient with Fabry disease in the enzyme replacement therapy era.

Fabry disease (FD) is an X-linked genetic disease, resulting from the deficiency of alpha-galactosidase A, a lysosomal enzyme responsible for the cleavage of glycosphingolipids. In absence of enzyme replacement therapy (ERT), globotriaosylceramide (Gb3) accumulates in tissue, leading to progressive organ damage with severe renal, cardiac and central nervous system complications. We herein describe the first case of successful combined and simultaneous heart and kidney transplantation in a young male patient with FD complicated by end-stage renal disease and severe heart failure not responding to late-onset ERT. Combined heart and kidney transplantation can be recommended for Fabry patients with end-stage renal disease and overt hypertrophic cardiomyopathy, severe ischemic or valvular heart disease.

Vascular damage without hypertension in transgenic rats expressing prorenin exclusively in the liver.

We have developed a transgenic animal model to investigate the effects of overexpression of rat prorenin on the cardiovascular system. Two transgenic rat lines were generated in which rat prorenin expression was directed to the liver by a human alpha1-antitrypsin promoter. Liver-specific expression was confirmed by RNase protection assay. Plasma prorenin concentrations in transgenic rats were increased 400-fold in the males of both lines but were increased only two- to threefold in the females. Thus, transgene expression exhibited sexual dimorphism. Blood pressures were not significantly higher in transgenic rats than in nontransgenic controls. The ratio of heart weight to body weight was greater in male transgenic rats than in the nontransgenic controls. Histological analysis revealed severe renal lesions and hypertrophic cardiomyocytes in transgenic males only. This transgenic model demonstrates a likely role of prorenin in the development of cardiac and renal pathology independent of hypertension. These animals will facilitate studies of the effects of blockade of the renin-angiotensin system and other pharmacological interventions on the development and treatment of cardiac, vascular, and renal lesions induced by changes in this system in the absence of chronic hypertension.

Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney.

Erythropoietin (Epo)-producing cells were identified in the murine hypoxic kidney by in situ hybridization. Profound anemia was induced in order to greatly increase Epo production. This resulted in high levels of Epo mRNA in the kidney. 35S-labeled DNA fragments of the murine Epo gene were used as probes for in situ hybridization. Control experiments conducted in parallel included kidneys of nonanemic mice, RNase-treated hypoxic kidney sections, and 35S-labeled non-Epo-related DNA. The Epo probe gave a specific hybridization signal in the hypoxic kidney in the cortex and to a lesser extent in the outer medulla. Glomerular and tubular cells were not labeled. All positive cells were identified as peritubular cells. Using immunofluorescence, we showed that cells with the same topography contained Factor VIII-related antigen. These data demonstrated that peritubular cells, most likely endothelial cells, constitute the major site of Epo production in the murine hypoxic kidney.