A dual role of miR-22 in rhabdomyolysis-induced acute kidney injury.

AIM: In acute kidney injury (AKI), regions of the kidney are hypoxic. However, for reasons yet unknown, adaptation to hypoxia through hypoxia-inducible factor (HIF) is limited. Here, we studied miR-22, a potential HIF repressor, in normal kidneys, as well as in rhabdomyolysis-induced AKI, a condition where miR-22 is up-regulated. METHODS: AKI in mice was provoked by IM injection of glycerol. Tissue homogenates were processed to determine the levels of candidate RNAs and proteins, as well as global gene expression profiles. Reporter assays quantified in vitro miR-22 activity and its modulation by mimic or inhibitor molecules, under normoxia or hypoxia (1% O2 ) respectively. In vivo, anti-miR-22 molecules were applied to normal mice or prior to induction of AKI. Renal outcome was assessed by measuring plasma creatinine, plasma urea and the levels of the injury markers Kim-1 and Ngal. RESULTS: Renal miR-22 is inducible by hypoxia and represses hypoxia-inducible factor (HIF). Specific inhibition of miR-22 regulates 1913 gene transcripts in kidneys controls and 3386 in AKI, many of which are involved in development or carcinogenesis. Specific inhibition of miR-22 up-regulates tissue protective HIF target genes, yet renal function and injury markers are unchanged or worsened. CONCLUSIONS: miR-22 is a HIF repressor constitutively expressed in the adult kidney and up-regulated in AKI. Specific inhibition of miR-22 is efficient in vivo and profoundly affects renal gene expression in health and disease, including up-regulation of HIF. However, the net effect on rhabdomyolysis-induced AKI outcome is neutral or even negative.

Cyclosporine A induces endothelin-converting enzyme-1: Studies in vivo and in vitro.

AIM: Cyclosporine A (CsA) induces renal vasoconstriction and hypoxia and enhances the expression of endothelin-1 (ET-1) pro-hormone (pre-pro-ET-1), plausibly leading to a feed-forward loop of renal vasoconstriction, hypoxia and enhanced synthesis of the potent vasoconstrictor ET-1. Endothelin-converting enzyme (ECE)-1 cleaves big endothelin to generate endothelin (ET)-1 and is upregulated by hypoxia via hypoxia-inducible factor (HIF). We hypothesized that in addition to the direct induction of ET-1 synthesis, CsA might also intensify renal ECE-1 expression, thus contributing to enhanced ET-1 synthesis following CsA. METHODS: CsA was administered to Sprague Dawley rats (120 mg/kg/SC) for 4 days, and renal HIF and ECE-1 expression were assessed with Western blots and immunostaining. Human umbilical vein endothelial cells (HUVEC) and proximal tubular cell line (HK-2) were subjected to CsA, and ECE-1 induction was evaluated using real-time mRNA PCR and Western blots. RESULTS: Cyclosporine A intensified renal parenchymal ECE-1 expression in the rat kidney, particularly in distal nephron segments, along with renal hypoxia (detected by pimonidazole adducts) and HIF expression, in line with our recent observations showing episodic hypoxia in mice subjected to CsA. Furthermore, in cultured normoxic HUVEC and HK-2 cells, CsA dose-dependently induced both pre-pro-ET-1 and ECE-1 mRNA and protein expression, with enhanced ET-1 generation. CONCLUSION: CsA induces ECE-1 via both hypoxic and non-hypoxic pathways. ECE-1 may contribute to increased renal ET-1 generation following CsA, participating in a feed-forward loop of renal parenchymal hypoxia and ET synthesis.

Myoglobin facilitates angiotensin II-induced constriction of renal afferent arterioles.

Vasoconstriction plays an important role in the development of acute kidney injury in rhabdomyolysis. We hypothesized that myoglobin enhances the angiotensin II (ANG II) response in afferent arterioles by increasing superoxide and reducing nitric oxide (NO) bioavailability. Afferent arterioles of C57Bl6 mice were isolated perfused, and vasoreactivity was analyzed using video microscopy. NO bioavailability, superoxide concentration in the vessel wall, and changes in cytosolic calcium were measured using fluorescence techniques. Myoglobin treatment (10-5 M) did not change the basal arteriolar diameter during a 20-min period compared with control conditions. NG-nitro-l-arginine methyl ester (l-NAME, 10-4 M) and l-NAME + myoglobin reduced diameters to 94.7 and 97.9% of the initial diameter, respectively. Myoglobin or l-NAME enhanced the ANG II-induced constriction of arterioles compared with control (36.6 and 34.2%, respectively, vs. 65.9%). Norepinephrine responses were not influenced by myoglobin. Combined application of myoglobin and l-NAME further facilitated the ANG II response (7.0%). Myoglobin or l-NAME decreased the NO-related fluorescence in arterioles similarly. Myoglobin enhanced the superoxide-related fluorescence, and tempol prevented this enhancement. Tempol also partly prevented the myoglobin effect on the ANG II response. Myoglobin increased the fura 2 fluorescence ratio (cytosolic calcium) during ANG II application (10-12 to 10-6 M). The results suggest that the enhanced afferent arteriolar reactivity to ANG II is mainly due to a myoglobin-induced increase in superoxide and associated reduction in the NO bioavailability. Signaling pathways for the augmented ANG II response include enhanced cytosolic calcium transients. In conclusion, myoglobin may contribute to the afferent arteriolar vasoconstriction in this rhabdomyolysis model.

Cyclosporin a induces renal episodic hypoxia.

AIM: Cyclosporin A (CsA) causes renal toxicity. The underlying mechanisms are incompletely understood, but may involve renal hypoxia and hypoxia-inducible factors (Hifs). We sought for hypoxia and Hif in mouse kidneys with CsA-induced toxicity, assessed their time course, Hif-mediated responses and the impact of interventional Hif upregulation. METHODS: Mice received CsA or its solvent cremophore for up to 6 weeks. Low salt diet (Na+ ↓) was given in combination with CsA to enhance toxicity. We assessed fine morphology, renal function, blood oxygen level-dependent magnetic resonance imaging under room air and following changes in breathing gas composition which correlate with vascular reactivity, pimonidazole adducts (which indicate O2 tensions below 10 mmHg), Hif-α proteins, as well as expression of Hif target genes. Stable Hif upregulation was achieved by inducible, Pax8-rtTA-based knockout of von Hippel-Lindau protein (Vhl-KO), which is crucial for Hif-α degradation. RESULTS: Cyclosporin A transiently increased renal deoxyhaemoglobin (R2*). Augmented vascular reactivity was observed at 2 h, but decreased at 24 h after CsA treatment. Na+ ↓/CsA provoked chronic renal failure with tubular degeneration and interstitial fibrosis. Nephron segments at risk for injury accumulated pimonidazole adducts, as well as Hif-α proteins. Remarkably, Hif target gene expression remained unchanged, while factor-inhibiting Hif (Fih) was enhanced. Na+ ↓/CsA/Vhl-KO aggravated morpho-functional outcome of chronic renal CsA toxicity. CONCLUSIONS: Cyclosporin A provokes episodic hypoxia in nephron segments most susceptible to chronic CsA toxicity. Fih is upregulated and likely blocks further Hif activity. Continuous tubular Hif upregulation via Vhl-KO worsens the outcome of chronic CsA-induced renal toxicity.

[Psychological treatments for pain in cancer patients : A systematic review on the current state of research]. / Schmerzpsychologische Interventionen bei onkologischen Patienten : Ein systematisches Review zum aktuellen Stand der Forschung.

BACKGROUND: In cancer patients, pain is one of the main symptoms and especially in the late stages of disease, these symptoms can be associated with considerable suffering. In psycho-oncology, preliminary psychological therapies targeting cancer pain have been tested; however, a systematic review of available interventions is lacking, especially considering their dissemination, evidence base, study quality, and the comparison with established treatments. Therefore, the aim of the current study is to systematically review the current research on psychological treatments for pain in cancer patients. MATERIALS AND METHODS: During May 2014, MEDLINE, PsycINFO, PSYNDEX, and CENTRAL databases were searched. Psychological treatments for pain in adult cancer patients studied in randomized, controlled trials (RCTs) and referring to pain as primary or secondary outcome were included. After examination for inclusion, structured data extraction and assessment followed. Data were synthesized narratively. RESULTS: In the review, 32 RCTs were included. Studies mainly referred to patients with breast cancer or patients in earlier stages of the disease. The methodological quality of included studies was heterogeneous. Most commonly, short interventions were delivered by nurses in out-patient settings. Interventions including education and relaxation techniques were utilized most often, followed by interventions with behavioral or cognitive components. CONCLUSION: A need for research persists regarding efficacy of current psychotherapeutic interventions, or the role of mediator variables (e. g., coping) on pain perception in cancer patients. Studies with high methodological quality which comprehensively and transparently report on interventions and designs are lacking.

Adaptive response to hypoxia and remote ischaemia pre-conditioning: a new hypoxia-inducible factors era in clinical medicine.

Transient ischaemia leads to tolerance to subsequent protracted ischaemia. This 'ischaemia pre-conditioning' results from the induction of numerous protective genes, involved in cell metabolism, proliferation and survival, in antioxidant capacity, angiogenesis, vascular tone and erythropoiesis. Hypoxia-inducible factors (HIF) play a pivotal role in this transcriptional adaptive response. HIF prolyl hydroxylases (PHDs), serving as oxygen sensors, control HIFα degradation. HIF-mediated ischaemic pre-conditioning can be achieved with the administration of PHD inhibitors, with the attenuation of organ injury under various hypoxic and toxic insults. Clinical trials are currently under way, evaluating PHD inhibitors as inducers of erythropoietin. Once their safety is established, their potential use might be further tested in clinical trials in various forms of acute ischaemic and toxic organ damage. Repeated transient limb ischaemia was also found to attenuate ischaemic injury in remote organs. This 'remote ischaemic pre-conditioning' phenomenon (RIP) has been extensively studied recently in small clinical trials, preceding, or in parallel with an abrupt insult, such as myocardial infarction, cardiac surgery or radiocontrast administration. Initial results are promising, suggesting organ protection. Large-scale multi-centre studies are currently under way, evaluating the protective potential of RIP in cardiac surgery, in the management of myocardial infarction and in organ transplantation. The mechanisms of organ protection provided by RIP are poorly understood, but HIF seemingly play a role as well. Thus, Inhibition of HIF degradation with PHD inhibitors, as well as RIP (in part through HIF), might develop into novel clinical interventions in organ protection in the near future.

Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein.

AIM: Von Hippel-Lindau protein (VHL) provides the degradation of hypoxia-inducible factor (HIF). Tetracycline-induced, Pax8-rtTA-based knockout of VHL (VHL-KO) affects all renal tubules and periportal hepatocytes and leads to sustained upregulation of HIF. Here, we study the phenotype of VHL-KO in both organs, the time course of changes, and long-term morpho-functional outcome. METHODS: Mice with doxycycline-induced VHL-KO and controls (CON) were followed for up to 9 months. Systemic and tissue parameters were evaluated using clinical chemistry, histology, immunohistochemistry, RT-PCR and in situ hybridisation. RESULTS: At day 3 following VHL-KO, substantial abundance of HIF-1α and -2α was detected in the nuclei of hepatocytes and renal tubular epithelia. Hypoxia, induced by bleeding anaemia, did not further augment HIF signal. Erythropoietin mRNA was detectable in hepatocytes but not in the kidney. Vascular endothelial growth factor mRNA was upregulated in kidney but not in liver. At day 7 following VHL-KO, the renal capillary density was enhanced, reaching its maximum at day 14. Blood haemoglobin increased constantly up to day 28 (23.3 vs. 15.8 g dL(-1) , VHL-KO vs. CON). Thereafter, it was kept within the normal range by weekly blood collections. Pathological changes were absent from kidney and liver 9 months after VHL-KO. CONCLUSIONS: Inducible, Pax8-rtTA-based deletion of VHL leads to organ-specific expression of epithelial HIF and erythropoietin in liver and kidney without causing pathological changes. Uniform, maximal and sustained HIF activation along the renal tubule may serve to study the potential benefits of hypoxia adaptation in experimental renal injury.

Chronic activation of vasopressin V2 receptor signalling lowers renal medullary oxygen levels in rats.

AIM: In the present study, we aimed to elucidate the effects of chronic vasopressin administration on renal medullary oxygen levels. METHODS: Adult Sprague Dawley or vasopressin-deficient Brattleboro rats were treated with the vasopressin V2 receptor agonist, desmopressin (5 ng/h; 3d), or its vehicle via osmotic minipumps. Immunostaining for pimonidazole and the transcription factor HIF-1α (hypoxia-inducible factor-1α) were used to identify hypoxic areas. Activation of HIF-target gene expression following desmopressin treatment was studied by microarray analysis. RESULTS: Pimonidazole staining was detected in the outer and inner medulla of desmopressin-treated rats, whereas staining in control animals was weak or absent. HIF-1α immunostaining demonstrated nuclear accumulation in the papilla of desmopressin-treated animals, whereas no staining was observed in the controls. Gene expression analysis revealed significant enrichment of HIF-target genes in the group of desmopressin-regulated gene products (P = 2.6*10(-21) ). Regulated products included insulin-like growth factor binding proteins 1 and 3, angiopoietin 2, fibronectin, cathepsin D, hexokinase 2 and cyclooxygenase 2. CONCLUSION: Our results demonstrate that an activation of the renal urine concentrating mechanism by desmopressin causes renal medullary hypoxia and an upregulation of hypoxia-inducible gene expression.

Behavioural and neural correlates of visceral pain sensitivity in healthy men and women: does sex matter?

INTRODUCTION: We assessed sex differences in behavioural and neural responses to rectal pain stimuli in healthy subjects. METHODS: In age- and body mass index-matched healthy subjects (n = 15 men, 15 women), rectal sensory and pain thresholds were assessed with a pressure-controlled barostat device. The blood oxygen level-dependent response during cued anticipation and painful stimulation was measured using functional magnetic resonance imaging (fMRI). Retrospective pain evaluations were accomplished with visual analogue scales. For fMRI data, region-of-interest (ROI) analyses and additional whole-brain analyses were carried out. RESULTS: There were no sex differences in rectal thresholds or pain ratings. ROI analyses revealed comparable distension-induced activation of the thalamus, somatosensory cortex, insula and dorsolateral prefrontal cortex (DLPFC). Only in additional whole-brain analyses did we find increased activation in women in DLPFC and middle temporal gyrus during pain anticipation and in the cerebellum and medial frontal gyrus during pain. A significant inverse association between rectal pain threshold and distension-induced activation in virtually all ROIs was found in women. In men, pain thresholds and insula activation were positively correlated, as were pain ratings and anterior cingulate cortex activation. CONCLUSIONS: Healthy men and women do not differ in behavioural measures of visceral pain sensitivity. The pattern of neural activation is comparable in the majority of pain-processing brain regions, although women may differ in the activation of DLPFC which could reflect sex differences in cognitive-emotional pain regulation. Women with lower pain thresholds showed greater neural responses, which may be relevant in the pathophysiology of visceral hyperalgesia.

Affective disturbances modulate the neural processing of visceral pain stimuli in irritable bowel syndrome: an fMRI study.

OBJECTIVE: To address the role of anxiety and depression symptoms in altered pain processing in irritable bowel syndrome (IBS). DESIGN: In this functional magnetic resonance imaging study, the blood oxygen level-dependent (BOLD) response to rectal distensions delivered at previously determined individual discomfort thresholds was assessed. PATIENTS: 15 female patients with irritable bowel syndrome (IBS) and with normal rectal pain thresholds, and 12 healthy women. MEASURES: The correlation of anxiety and depression symptoms, measured with the Hospital Anxiety and Depression Scale (HADS), with subjective pain ratings and the BOLD response during distension-induced brain activation were analysed within IBS. Group differences in pain-induced brain activation with and without controlling for HADS scores were evaluated. RESULTS: Patients with IBS experienced significantly more pain and discomfort upon rectal distensions in the scanner, despite unaltered rectal sensory thresholds. Anxiety and depression scores were associated with these subjective stimulus ratings, but not with rectal sensory thresholds. Anxiety symptoms in IBS were significantly associated with pain-induced activation of the anterior midcingulate cortex and pregenual anterior cingulate cortex. Depression scores correlated with activation of the prefrontal cortex (PFC) and cerebellar areas within IBS. Group comparisons with the two-sample t test revealed significant activation in the IBS versus controls contrast in the anterior insular cortex and PFC. Inclusion of anxiety and depression scores, respectively, as confounding variables led to a loss of significant group differences. CONCLUSIONS: Altered central processing of visceral stimuli in IBS is at least in part mediated by symptoms of anxiety and depression, which may modulate the affective-motivational aspects of the pain response.

Effects of psychological stress on the cerebral processing of visceral stimuli in healthy women.

The aim of the study was to analyse effects of psychological stress on the neural processing of visceral stimuli in healthy women. The brain functional magnetic resonance imaging blood oxygen level-dependent response to non-painful and painful rectal distensions was recorded from 14 healthy women during acute psychological stress and a control condition. Acute stress was induced with a modified public speaking stress paradigm. State anxiety was assessed with the State-Trait-Anxiety Inventory; chronic stress was measured with the Perceived Stress Questionnaire. During non-painful distensions, activation was observed in the right posterior insular cortex (IC) and right S1. Painful stimuli revealed activation of the bilateral anterior IC, right S1, and right pregenual anterior cingulate cortex. Chronic stress score was correlated with activation of the bilateral amygdala, right posterior IC (post-IC), left periaqueductal grey (PAG), and right dorsal posterior cingulate gyrus (dPCC) during non-painful stimulation, and with activation of the right post-IC, right PAG, left thalamus (THA), and right dPCC during painful distensions. During acute stress, state anxiety was significantly higher and the acute stress - control contrast revealed activation of the right dPCC, left THA and right S1 during painful stimulation. This is the first study to demonstrate effects of acute stress on cerebral activation patterns during visceral pain in healthy women. Together with our finding that chronic stress was correlated wit the neural response to visceral stimuli, these results provide a framework for further studies addressing the role of chronic stress and emotional disturbances in the pathophysiology of visceral hyperalgesia.

Diabetes and radiocontrast media increase endothelin converting enzyme-1 in the kidney.

Plasma endothelin-1 levels rise in diabetes and after exposure to contrast media suggesting a role in progressive diabetic and acute radiocontrast nephropathies. Here we studied individual and combined effects of streptozotocin-induced diabetes and contrast media on renal endothelin converting enzyme-1 levels in the rat. In vivo, medullary (but not cortical) endothelin converting enzyme protein gradually increased 4 to 5-fold following the induction of diabetes or after the administration of contrast media but rose 15-fold when diabetic rats were given contrast media. Changes in mRNA expression paralleled those of the protein. Immunohistochemistry confirmed that increased tubular and endothelial cell endothelin converting enzyme-1 were most pronounced in the medulla. In vitro, endothelin-1 levels increased 3-fold following incubation of endothelial cells with media high in glucose or with contrast and 4-fold with their combination. Endothelin converting enzyme-1 protein and mRNA expression changed in a similar pattern while prepro endothelin-1 mRNA increased with each insult but not in an additive way. Our study shows that diabetes and contrast media up-regulate renal medullary endothelin converting enzyme-1 expression and synthesis.

Adaptation to hypoxia in the diabetic rat kidney.

Hypoxia of the kidney in diabetes could predispose it to develop acute and chronic renal failure. To examine the relationship between renal hypoxia and renal failure, we measured hypoxia (as a pimonidazole adducts), hypoxia-inducible factors (HIFs), and a hypoxia target gene heme oxygenase-1. The studies were performed in rats with streptozotocin (STZ)-induced diabetes, Cohen diabetes sensitive rats, and during short-term artificial hyperglycemia in rats induced by intravenous glucose and octreotide. STZ-treated rats received insulin, the superoxide dismutase mimetic tempol, or contrast medium. Radiocontrast media causes hypoxia and HIF induction. Hypoxia, HIFs, and heme oxygenase were undetectable in controls, but transiently activated in STZ-treated and the Cohen diabetes sensitive rats. Different patterns of HIFs and pimonidazole were observed between the three models. Insulin abolished pimonidazole and HIF induction, whereas tempol lead to increased HIFs and heme oxygenase induction at similar levels of pimonidazole. When compared with control rats, STZ-treated rats exhibited more intense and protracted renal pimonidazole, with augmented hypoxia inducible factor production and reduced GFR following contrast media. Our data suggest that both regional hypoxia and hypoxia adaptation transiently occur in early stages of experimental diabetes, largely dependent on hyperglycemia or after contrast media. Tempol may augment the HIF response in diabetes.

Development of Kaposi's sarcoma under sirolimus-based immunosuppression and successful treatment with imiquimod.

Kaposi's sarcoma (KS) is a vascular neoplasm typically observed in the immunocompromised patient populations, such as acquired immunodeficiency syndrome or transplant patients. KS can appear simultaneously at multiple sites as red to purple, maculo-papular or nodular cutaneous lesions sometimes showing a visceral extension. Sirolimus, an immunosuppressive agent with potent antitumor activity, has been effective in combating post-transplant KS. However, an aggressive regimen of immunosuppression for therapy of severe acute rejection episodes may abolish the antitumor effects of sirolimus. The following is a description of KS development under immunosuppressive therapy with sirolimus, and the successful treatment of KS lesions utilizing the topical application of imiquimod 5% cream, an immune response modifier.

Heightened expression of the cytotoxicity receptor NKG2D correlates with acute and chronic nephropathy after kidney transplantation.

The activating cytotoxicity receptor NKG2D binds to stress-regulated molecules encoded by the major histocompatibility complex class I chain-related (MIC) and UL-16-binding protein (ULBP)/retinoic acid early transcript (RAET) gene family. To assess whether acute allograft rejection leads to an induction of these inducible ligands and their receptor NKG2D, we examined the mRNA profiles in kidney transplant biopsies. Expression levels were correlated with the incidence of acute rejection (aRx) episodes and chronic allograft nephropathy (CAN) proven by histology. Whereas MICA, ULBP1/3 and RAET1-E did not display heightened gene expression, elevated levels of NKG2D mRNA could be associated with aRx (p < 0.001). Immunohistology of kidney biopsies diagnosed with aRx revealed NKG2D+ cells in tubulointerstitial areas positive for CD8+ cells. Most importantly, elevated levels of NKG2D mRNA were associated with restricted long-term graft function assessed by the glomerular filtration rate at 6, 12 and 18 months posttransplantation. Induced NKG2D mRNA expression was still observable in biopsies diagnosed with CAN (p < 0.001), demonstrating a higher sensitivity and specificity compared to CD3, granzyme B and granulysin mRNA measurement. Significant elevated levels of NKG2D mRNA could be further detected in urine sediment prior to aRx, suggesting this receptor as a new candidate marker for the diagnosis of acute and chronic allograft rejection.

Hypoxia-inducible factors and tubular cell survival in isolated perfused kidneys.

Adaptation to hypoxic environment is conferred through hypoxia-inducible transcription factors (HIFs). We have previously shown that the HIF system is transiently activated in vivo in radiocontrast-induced acute renal failure, associated with profound hypoxia in the renal medulla. Medullary thick ascending limbs (mTALs), the most affected nephron segments in this model, were virtually unable to mount an adaptive HIF response. Here, we study correlations between oxygenation, HIF activation, and cell viability in a related ex vivo model, the isolated perfused rat kidney (IPK). In IPKs perfused with cell-free oxygenated medium, severe medullary hypoxic damage developed, affecting 42+/-9% of mTALs in the mid-inner stripe. HIF-1alpha tubular immunostaining was noted with a zonal and tubular pattern largely similar to our findings in vivo: in 34+/-3% of collecting ducts (CDs) within the mid-inner stripe and extensively in the papillary tip, whereas mTALs were all HIF-negative. In IPKs supplemented with RBCs (improved oxygen supply), mTAL damage was totally prevented and CDs' HIF expression was attenuated (22+/-4%). By contrast, although measures designed to reduce medullary hypoxia by decreasing tubular reabsorptive activity (furosemide, ouabain, or high-albumin-non-filtering system) reduced mTAL damage, all paradoxically resulted in increased HIF expression in CDs (51+/-4%), and 17+/-3% of mTALs became immunostained as well. Our data confirm that CDs and mTALs have markedly different HIF responses, which correlate with their viability under hypoxic stress. mTALs transcriptional adaptation occurs within a narrow hypoxic range, and it appears that workload reduction can shift mTALs into this window of opportunity for HIF activation and survival.

HLA type-independent method to monitor polyoma BK virus-specific CD4 and CD8 T-cell immunity.

(Re)activation of quiescent viral diseases is a major problem in immunosuppressed transplant patients. Polyoma BK virus-associated nephropathy (PVAN) caused by active polyoma BK virus (BKV) infection became a main reason for graft loss in kidney transplantation. After diagnosis, most transplant centers react by reducing immunosuppression (IS) to allow the immune system to control the infection. However, the impact of reduced IS on BKV immunity is not well researched. Here we present an HLA type-independent method to monitor BKV-specific T-cell immunity. Applying our method, viral protein 1-specific CD4+ and CD8+ T-cell responses were detected in patients with serum BKV-DNA levels >250 000 copies/mL. In addition, specific T-cell responses were also found in allograft-infiltrating cells. The method can be used to assess the impact of decreased immunosuppression on BKV immunity and to clarify the role of specific T cells in the pathogenesis of PVAN. We strongly recommend its implementation in future clinical studies.