A noninvasive diagnostic approach to retrospective donor HLA typing in kidney transplant patients using urine.

Antibody-mediated rejection (AMR) is a major obstacle to long-term kidney transplantation. AMR is mostly caused by donor specific HLA antibodies, which can arise before or any time after transplantation. Incomplete donor HLA typing and unavailability of donor DNA regularly preclude the assessment of donor-specificity of circulating anti-HLA antibodies. In our centre, this problem arises in approximately 20% of all post-transplant HLA-antibody assessments. We demonstrate that this diagnostic challenge can be resolved by establishing donor renal tubular cell cultures from recipient´s urine as a source of high-quality donor DNA. DNA was then verified for genetic origin and purity by fluorescence in situ hybridization and short tandem repeat analysis. Two representative cases highlight the diagnostic value of this approach which is corroborated by analysis of ten additional patients. The latter were randomly sampled from routine clinical care patients with available donor DNA as controls. In all 12 cases, we were able to perform full HLA typing of the respective donors confirmed by cross-comparison to results from the stored 10 donor DNAs. We propose that this noninvasive diagnostic approach for HLA typing in kidney transplant patients is valuable to determine donor specificity of HLA antibodies, which is important in clinical assessment of suspected AMR.

Molecular diagnosis of kidney transplant failure based on urine.

In light of the organ shortage, there is a great responsibility to assess postmortal organs for which procurement has been consented and to increase the life span of transplanted organs. The former responsibility has moved many centers to accept extended criteria organs. The latter responsibility requires an exact diagnosis and, if possible, omission of the harmful influence on the transplant. We report the course of a kidney transplant that showed a steady decline of function over a decade, displaying numerous cysts of different sizes. Clinical workup excluded the most frequent causes of chronic transplant failure. The filed allocation documents mentioned the donor's disease of oral-facial-digital syndrome, a rare ciliopathy, which can also affect the kidney. Molecular diagnosis was performed by culturing donor tubular cells from the recipient´s urine more than 10 years after transplantation. Next-generation panel sequencing with DNA from tubular urinary cells revealed a novel truncating mutation in OFD1, which sufficiently explains the features of the kidney transplants, also found in the second kidney allograft. Despite this severe donor disease, lifesaving transplantation with good long-term outcome was enabled for 5 recipients.

Increase in CGRP- and nNOS-immunoreactive neurons in the rat trigeminal ganglion after infusion of an NO donor.

BACKGROUND: Nitrovasodilators, such as glyceroltrinitrate (GTN), which produce nitric oxide (NO) in the organism, are known to cause delayed headaches in migraineurs, accompanied by increased plasma levels of calcitonin gene-related peptide (CGRP) in the cranial venous outflow. Increases in plasma CGRP and NO metabolites have also been found in spontaneous migraine attacks. In a rat model of meningeal nociception, infusion of NO donors induced activity of neurons in the spinal trigeminal nucleus. METHODS: Isoflurane-anaesthetised rats were intravenously infused with GTN (250 µg/kg) or saline for two hours and fixed by perfusion after a further four hours. Cryosections of dissected trigeminal ganglia were immunostained for detection of CGRP and neuronal NO synthase (nNOS). The ganglion neurons showing immunofluorescence for either of these proteins were counted. RESULTS: The proportions of CGRP- and nNOS- as well as double-immunopositive neurons were increased after GTN infusion compared to saline treatment in all parts of the trigeminal ganglion (CGRP) or restricted to the ophthalmic region (nNOS). The size of immunopositive neurons was not significantly different compared to controls. CONCLUSION: High levels of NO may induce the expression or availability of CGRP and nNOS. Similar changes may be involved in nitrovasodilator-induced and spontaneous headache attacks in migraineurs.

Release of calcitonin gene-related peptide from the jugular-nodose ganglion complex in rats--a new model to examine the role of cardiac peptidergic and nitrergic innervation.

OBJECTIVE: Afferent information from the heart and the lung is conveyed to the brainstem by primary afferent fibers originating from vagal sensory neurons (jugular-nodose ganglion complex, JNC). The present study was made to evaluate if release of the sensory neuropeptide calcitonin gene-related peptide (CGRP) from the JNC can be used as a model for future studies on changes in neuropeptide release under pathological conditions of the heart. METHODS: Freshly isolated rat JNC's were passed through a series of solutions based on oxygenated synthetic interstitial fluid (SIF). Substances such as the TRPV1 receptor agonist capsaicin and the nitric oxide (NO) donor sodium nitroprusside (SNP) were added as excitatory test stimuli. The eluates were processed using an enzyme immuno-assay (EIA) for measurement of CGRP concentrations. Immunohistochemistry was used to visualize CGRP containing and NO producing neurons in the JNC. RESULTS: Both SNP and capsaicin caused significant increases in CGRP release. CGRP-immunoreactive neurons (somata) were preferentially found in the jugular ganglion, whereas neurons immunoreactive for neuronal NO synthase were mostly localized in the nodose ganglion. CONCLUSION: The present study demonstrates an easily reproducible model for measuring stimulated CGRP release from vagal afferents arising from the JNC. Nitric oxide produced by vagal afferents may stimulate CGRP release upon afferent activation.

Stimulated release of calcitonin gene-related peptide from the human right atrium in patients with and without diabetes mellitus.

Calcitonin gene-related peptide (CGRP), a potent vasodilator released during activation from a subset of sensory Adelta- and C-fiber afferents, has been suggested to play a beneficial role in myocardial ischemia. Variations in CGRP release can possibly be correlated with diseases that involve changes in activity or degeneration of cardiac afferent fibers. The aim of the present study was to examine basal and stimulated CGRP release from cardiac tissue in patients who underwent cardiopulmonary bypass surgery and to compare patients with and without known history of diabetes mellitus. Small pieces of the right atrium routinely excised during the bypass operations were passed through series of oxygenated solutions. The TRPV1 receptor agonist capsaicin and the nitric oxide donor NONOate were added for stimulation of cardiac afferent fibers. The eluates were processed using an enzyme immuno-assay (EIA) for measurement of CGRP concentrations. Both capsaicin and NONOate caused significant increases in CGRP release. No significant differences in CGRP release between patients with and without diabetes mellitus were examined. The present study evaluates a simple and reproducible model for measuring stimulated CGRP release from the human right atrium.