Publications by authors named "Michael S Wiesener"

43 Publications

Kidney-transplant patients receiving living- or dead-donor organs have similar psychological outcomes (findings from the study).

Ment Illn 2020 20;12(1):17-22. Epub 2020 Feb 20.

Department of Psychiatry and Psychotherapy, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Germany.

Purpose: Kidney transplantation (KT) is the treatment of choice for end-stage chronic kidney disease (CKD) and is well known to improve the clinical outcome of patients. However, the impact of KT on comorbid psychological symptoms, particularly depression and anxiety, is less clear, and recipients of living-donor (LD) organs may have a different psychological outcome from recipients of dead-donor (DD) organs.

Design/methodology/approach: In total, 152 patients were included and analyzed using a cross-sectional design. Of these patients, 25 were pre-KT, 13 were post-KT with a LD transplant and 114 were post-KT with a DD transplant. The patients were tested for a variety of psychometric outcomes using the Hospital Anxiety and Depression Scale, the 12-Item Short Form Health Survey (assessing physical and mental health-related quality of life), the Resilience Scale, the Coping Self-Questionnaire and the Social Support Questionnaire.

Findings: The mean age of the patients was 51.25 years and 40 per cent of the patients were female. As expected, the post-KT patients had significantly better scores on the physical component of the Short Form Health Survey than the pre-KT patients, and there were no significant differences between the two post-KT groups. There were no significant differences among the groups in any of the other psychometric outcome parameters tested, including anxiety, depression and the mental component of health-related quality of life.

Research Limitations/implications: KT and the origin of the donor organ do not appear to have a significant impact on the psychological well-being of transplant patients with CKD. Although the diagnosis and early treatment of psychological symptoms, such as depression and anxiety, remain important for these patients, decisions regarding KT, including the mode of transplantation, should not be fundamentally influenced by concerns about psychological impairments at the population level.

Originality/value: CKD is a serious condition involving profound impairment of the physical and psychological well-being of patients. KT is considered the treatment of choice for most of these patients. KT has notable advantages over dialysis with regard to the long-term physical functioning of the renal and cardiovascular system and increases the life expectancy of patients. However, the data on the improvement of psychological impairments after KT are less conclusive.
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http://dx.doi.org/10.1108/MIJ-10-2019-0002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370952PMC
February 2020

cfNOMe - A single assay for comprehensive epigenetic analyses of cell-free DNA.

Genome Med 2020 06 24;12(1):54. Epub 2020 Jun 24.

Cologne Center for Genomics, University of Cologne, Cologne, Germany.

Cell-free DNA (cfDNA) analysis has become essential in cancer diagnostics and prenatal testing. We present cfNOMe, a two-in-one method of measuring cfDNA cytosine methylation and nucleosome occupancy in a single assay using non-disruptive enzymatic cytosine conversion and a custom bioinformatic pipeline. We show that enzymatic cytosine conversion better preserves cfDNA fragmentation information than does bisulfite conversion. Whereas previously separate experiments were required to study either epigenetic marking, cfNOMe delivers reliable results for both, enabling more comprehensive and inexpensive epigenetic cfDNA profiling. cfNOMe has the potential to advance biomarker discovery and diagnostic usage in diseases with systemic perturbations of cfDNA composition.
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http://dx.doi.org/10.1186/s13073-020-00750-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315486PMC
June 2020

Molecular diagnosis of kidney transplant failure based on urine.

Am J Transplant 2020 05 20;20(5):1410-1416. Epub 2020 Jan 20.

Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.

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.
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http://dx.doi.org/10.1111/ajt.15738DOI Listing
May 2020

Tuberculous granulomatous interstitial nephritis in a renal allograft.

Kidney Int 2019 11;96(5):1243

Department of Nephrology and Hypertension, Universitätsklinikum Erlangen, University of Erlangen-Nürnberg, Erlangen, Germany.

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http://dx.doi.org/10.1016/j.kint.2019.06.005DOI Listing
November 2019

Suspected colonic cancer turns out to be disseminated tuberculosis in a kidney transplant recipient: A case report.

Medicine (Baltimore) 2019 Sep;98(36):e16995

Department of Nephrology and Hypertension.

Rationale: Active tuberculosis constitutes a relevant risk for kidney transplant recipients. In contrast to immunocompetent hosts, kidney transplant recipients often show atypical presentation and course of the disease impeding diagnosis. Especially extrapulmonary or disseminated infection is more frequent and can resemble malignant processes. However, reactivation of tuberculosis mostly develops within the early post-transplant course, whereas malignancies are predominantly long-term complications. We report a case of disseminated abdominal tuberculosis developing 10 years after kidney transplantation and review the underlying literature.

Patient Concerns And Diagnoses: A 51-year-old lady presented with epigastric pain, diarrhea, weight loss and night sweats 10 years after deceased-donor kidney transplantation. An epigastric as well as multiple peritoneal masses were found suspicious of a cancer of unknown primary. Colonoscopy revealed a colon tumor with the biopsy showing no dysplasia but histiocytic and granulomatous infiltration with acid-fast bacilli. Mycobacterium tuberculosis was detected in the biopsy and stool and disseminated abdominal tuberculosi was diagnosed.

Interventions And Outcomes: With anti-tuberculosis therapy, the masses regressed, and all cultures became sterile, sparing graft function.

Lessons: This case emphasizes how variable and unspecific the presentation of tuberculosis in kidney transplant recipients may be and that tuberculosis constitutes a relevant risk also in the long-term post-transplant course.
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http://dx.doi.org/10.1097/MD.0000000000016995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738975PMC
September 2019

Mutations in PIK3C2A cause syndromic short stature, skeletal abnormalities, and cataracts associated with ciliary dysfunction.

PLoS Genet 2019 04 29;15(4):e1008088. Epub 2019 Apr 29.

Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, United States of America.

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.
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http://dx.doi.org/10.1371/journal.pgen.1008088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508738PMC
April 2019

Biallelic Expression of Mucin-1 in Autosomal Dominant Tubulointerstitial Kidney Disease: Implications for Nongenetic Disease Recognition.

J Am Soc Nephrol 2018 09 26;29(9):2298-2309. Epub 2018 Jul 26.

Department of Nephrology and Hypertension, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Background: Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by mutations (ADTKD-) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD- and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique.

Methods: We re-evaluated detection of mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction microsatellite analysis in three independent ADTKD- families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens.

Results: The detection of mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD- kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients.

Conclusions: Diagnosing ADTKD- by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD- and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.
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http://dx.doi.org/10.1681/ASN.2018030245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115663PMC
September 2018

HIF is not essential for suppression of experimental tumor growth by mTOR inhibition.

J Cancer 2017 1;8(10):1809-1817. Epub 2017 Jul 1.

Department of Nephrology and Hypertension, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

The Hypoxia Inducible Transcription Factor (HIF) is the master regulator of cellular response to hypoxic adaptation. Solid tumors inevitably harbour hypoxic regions with subsequent stabilization and activation of HIF and HIF target genes due to poor vascularization and rapid growth. The mammalian target of rapamycin (mTOR) is a global regulator of cellular growth and proliferation, which can also regulate HIF expression independantly of hypoxia via specific activation of cellular translation and transcription. An effective blockade of mTOR results in attenuation of HIF under hypoxic conditions . This mechanism could enable a simultaneous inhibition of both the mTOR- and the HIF-pathway, resulting in an effective tool for cancer targeting. We set out to analyze the effect of mTOR inhibition and the involvement of mTOR regulation on HIF in a subcutaneous xenograft model in nude mice. Our results demonstrate that mTOR inhibition in our model leads to a clear reduction in tumor growth of various cellular origins, most likely due to inhibition of cellular proliferation. Moreover, these effects can also be achieved independently of the HIF status of the tumor cells. The HIF levels seem to remain unaffected by mTOR inhibition, probably due to the profound hypoxic environment in these threedimensional structures, consequently leading to a strong HIF stabillization. Therefore, treatment of these experimental tumors with mTOR inhibitors is an effective tool to achieve size regression. The involvement of and the effect on HIF in this setting is nevertheless negligible.
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http://dx.doi.org/10.7150/jca.16486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556644PMC
July 2017

Mutations in mitochondrial DNA causing tubulointerstitial kidney disease.

PLoS Genet 2017 03 7;13(3):e1006620. Epub 2017 Mar 7.

School of Clinical Medicine, Cambridge University, Cambridge, United Kingdom.

Tubulointerstitial kidney disease is an important cause of progressive renal failure whose aetiology is incompletely understood. We analysed a large pedigree with maternally inherited tubulointerstitial kidney disease and identified a homoplasmic substitution in the control region of the mitochondrial genome (m.547A>T). While mutations in mtDNA coding sequence are a well recognised cause of disease affecting multiple organs, mutations in the control region have never been shown to cause disease. Strikingly, our patients did not have classical features of mitochondrial disease. Patient fibroblasts showed reduced levels of mitochondrial tRNAPhe, tRNALeu1 and reduced mitochondrial protein translation and respiration. Mitochondrial transfer demonstrated mitochondrial transmission of the defect and in vitro assays showed reduced activity of the heavy strand promoter. We also identified further kindreds with the same phenotype carrying a homoplasmic mutation in mitochondrial tRNAPhe (m.616T>C). Thus mutations in mitochondrial DNA can cause maternally inherited renal disease, likely mediated through reduced function of mitochondrial tRNAPhe.
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http://dx.doi.org/10.1371/journal.pgen.1006620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360345PMC
March 2017

Depression, Anxiety, Resilience and Coping Pre and Post Kidney Transplantation - Initial Findings from the Psychiatric Impairments in Kidney Transplantation (PI-KT)-Study.

PLoS One 2015 11;10(11):e0140706. Epub 2015 Nov 11.

Department of Psychiatry and Psychotherapy, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.

Purpose: Depression/anxiety, impaired Health-Related Quality of Life (HRQoL) and coping and resilience structures, are associated with increased mortality/poor outcome in chronic kidney disease (CKD) patients before (CKD/pre-KT) and after kidney (CKD-T) transplantation. Less is known about prevalence rates of psychiatric symptoms and impaired HRQoL of non-transplanted compared with transplanted patients.

Methods: In a cross-sectional study comparing 101 CKD/pre-KT patients with 151 cadaveric-transplanted (CKD-T) patients, we examined prevalence of depression/anxiety (HADS questionnaire) and coping, resilience and HRQoL (SF-12, Resilience-Scale and FKV-questionnaire).

Results: The prevalence of both depressive and anxiety symptoms was not significantly different between different pre-/and CKD-T patient groups. In CKD-T no significant relations of coping strategies with kidney function were identified. Furthermore, the Resilience Scales for acceptance and competence did not suggest any differences between the CKD/pre-KT and CKD-T subgroup. In the CKD/pre-KT patients, significant correlations were identified between the acceptance subscale and partnership, as well as between the competence subscale and older age/partnership.

Conclusions: Both the CKD/pre-KT and CKD-T patients exhibited notable impairments in the HRQoL which which showed a comparable pattern of results. KT itself does not appear to be the main risk factor for the development of mental impairments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140706PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641724PMC
June 2016

Association of a coding polymorphism in Fc gamma receptor 2A and graft survival in re-transplant candidates.

Hum Immunol 2015 Oct 30;76(10):759-64. Epub 2015 Sep 30.

Department of Internal Medicine 5, Hematology and Oncology, Friedrich-Alexander University Erlangen-Nuremberg, Germany.

The family of Fc gamma receptors (FcγRs) is involved in mediating immunological effector functions. FcγRs are differentially expressed on immune cells and can act either activating or inhibitory, with FcγR2A belonging to the first group. The polymorphism H131R (rs1801274) in FCGR2A has been associated with acute rejection and can shift the overall balance between activating and inhibitory FcγRs. Anti-HLA allo-antibodies in transplant recipients have been identified as risk factor for organ survival after transplantation. In this study we genotyped FCGR2A H131R in 200 patients who had undergone kidney transplantation and experienced loss of graft function. FCGR2A polymorphism was related to graft survival and anti-HLA antibodies. Graft survival was calculated as the time interval between transplantation and return to chronic dialysis after transplantation. The gene frequency of FCGR2A R/R131 was found significantly more often in patients with earlier (⩽60months) compared to patients with later (>60months) graft failure. Overall patients homozygous for R/R131 had a significantly shorter graft survival, compared to H/H131 or H/R131 which is even more pronounced, when anti-HLA antibodies were present. These data suggest, that FCGR2A polymorphisms constitute a risk factor for graft loss following kidney transplantation and that this effect is related to anti-HLA antibodies.
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http://dx.doi.org/10.1016/j.humimm.2015.09.034DOI Listing
October 2015

Discordant Clinical Course of Vitamin-D-Hydroxylase (CYP24A1) Associated Hypercalcemia in Two Adult Brothers With Nephrocalcinosis.

Kidney Blood Press Res 2015 22;40(5):443-51. Epub 2015 Aug 22.

Department of Nephrology and Hypertension, Friedrich-Alexander Universitx00E4;t Erlangen-Nx00FC;rnberg, Erlangen-Nx00FC;rnberg, Germany.

Background/aims: Hypercalcemia can result in nephrocalcinosis/nephrolithiasis and may lead to renal failure. Idiopathic infantile hypercalcemia is caused by mutations of the CYP24A1 gene, which regulates vitamin D activity. Classically infants present with hypercalcemia. Recently, a number of individuals have been reported with late onset clinical manifestation or late diagnosis in adulthood. All these patients are believed to show hypercalciuria.

Methods: We report a 24 year old patient of healthy consanguine parents. Genetic analysis was performed by Sanger sequencing of the CYP24A1 gene in the index patient and targeted exon 2 analysis of all other family members.

Results: The patient was hospitalized with severe malaise during an acute EBV-infection. He showed hypercalcemia > 3mmol/l and acute, hypovolemic renal failure with profound nephrocalcinosis, but no hypercalciuria. Genetic workup revealed a homozygous loss-of-function mutation p.E143del in the CYP24A1 gene. His clinically asymptomatic brother showed nephrocalcinosis of lesser degree. Repeatedly, low parathyroid hormone levels were detected in both brothers.

Conclusion: This family displays the highly variable phenotype of CYP24A1 biallelic mutation carriers. CYP24A1 associated disease is an important differential diagnosis for the workup and counseling of infants as well as adults with hypercalcemia since a proper genetic diagnosis may result in therapeutic consequences.
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http://dx.doi.org/10.1159/000368520DOI Listing
August 2016

Hypoxia inhibits nephrogenesis through paracrine Vegfa despite the ability to enhance tubulogenesis.

Kidney Int 2015 Dec 22;88(6):1283-1292. Epub 2015 Jul 22.

Department of Nephrology and Hypertension, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Reduced nephron number predisposes to hypertension and kidney disease. Interaction of the branching ureteric bud and surrounding mesenchymal cells determines nephron number. Since oxygen supply may be critical for intrauterine development, we tested whether hypoxia and hypoxia-inducible factor-1α (HIF-1α) influence nephrogenesis. We found that HIF-1α is required for branching of MDCK cells. In addition, culture of metanephric mouse kidneys with ureteric bud cell-specific stabilization or knockout of HIF-1α revealed a positive impact of HIF-1α on nephrogenesis. In contrast, widespread stabilization of HIF-1α in metanephric kidneys through hypoxia or HIF stabilizers impaired nephrogenesis, and pharmacological HIF inhibition enhanced nephrogenesis. Several lines of evidence suggest an inhibitory effect through the hypoxia response of mesenchymal cells. HIF-1α was expressed in mesenchymal cells during nephrogenesis. Expression of the anti-branching factors Bmp4 and Vegfa, secreted by mesenchymal cells, was increased upon HIF stabilization. The conditioned medium from hypoxic metanephric kidneys inhibited MDCK branching, which was partially rescued by Vegfa antibodies. Thus, the effect of HIF-1α on nephrogenesis appears context dependent. While HIF-1α in the ureteric bud is of importance for proper branching morphogenesis, the net effect of hypoxia-induced HIF activation in the embryonic kidney appears to be mesenchymal cell-dependent inhibition of ureter branching.
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http://dx.doi.org/10.1038/ki.2015.214DOI Listing
December 2015

Renal fibrosis is the common feature of autosomal dominant tubulointerstitial kidney diseases caused by mutations in mucin 1 or uromodulin.

Kidney Int 2014 Sep 26;86(3):589-99. Epub 2014 Mar 26.

1] Department of Nephrology and Hypertension, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany [2] Nikolaus-Fiebiger-Center of Molecular Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

For decades, ill-defined autosomal dominant renal diseases have been reported, which originate from tubular cells and lead to tubular atrophy and interstitial fibrosis. These diseases are clinically indistinguishable, but caused by mutations in at least four different genes: UMOD, HNF1B, REN, and, as recently described, MUC1. Affected family members show renal fibrosis in the biopsy and gradually declining renal function, with renal failure usually occurring between the third and sixth decade of life. Here we describe 10 families and define eligibility criteria to consider this type of inherited disease, as well as propose a practicable approach for diagnosis. In contrast to what the frequently used term 'Medullary Cystic Kidney Disease' implies, development of (medullary) cysts is neither an early nor a typical feature, as determined by MRI. In addition to Sanger and gene panel sequencing of the four genes, we established SNaPshot minisequencing for the predescribed cytosine duplication within a distinct repeat region of MUC1 causing a frameshift. A mutation was found in 7 of 9 families (3 in UMOD and 4 in MUC1), with one indeterminate (UMOD p.T62P). On the basis of clinical and pathological characteristics we propose the term 'Autosomal Dominant Tubulointerstitial Kidney Disease' as an improved terminology. This should enhance recognition and correct diagnosis of affected individuals, facilitate genetic counseling, and stimulate research into the underlying pathophysiology.
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http://dx.doi.org/10.1038/ki.2014.72DOI Listing
September 2014

Renal uptake of the antiapoptotic protein survivin is mediated by megalin at the apical membrane of the proximal tubule.

Am J Physiol Renal Physiol 2013 Sep 3;305(5):F734-44. Epub 2013 Jul 3.

Dept. of Nephrology and Hypertension, Friedrich-Alexander Univ. Erlangen-Nuremberg, Ulmenweg 18, 91054 Erlangen, Germany.

The inhibitor of apoptosis protein survivin is a bifunctional molecule that regulates cellular division and survival. We have previously shown that survivin protein can be found at high concentrations in the adult kidney, particularly in the proximal tubules. Here, survivin is localized primarily at the apical membrane, a pattern that may indicate absorption of the protein. Several proteins in primary urine are internalized by megalin, an endocytosis receptor, which is in principle found in the same localization as survivin. Immunolabeling for survivin in different species confirmed survivin signal localizing to the apical membrane of the proximal tubule. Immunoelectron microscopy also showed apical localization of survivin in human kidneys. Furthermore, in polarized human primary tubular cells endogenous as well as external recombinant survivin is stored in the apical region of the cells. Costaining of survivin and megalin by immunohistochemistry and immunoelectron microscopy confirmed colocalization. Finally, by surface plasmon resonance we were able to demonstrate that survivin binds megalin and cubilin and that megalin knockout mice lose survivin through the urine. Survivin accumulates at the apical membrane of the renal tubule by reuptake, which is achieved by the endocytic receptor megalin, collaborating with cubilin. For this to occur, survivin will have to circulate in the blood and be filtered into the primary urine. It is not known at this stage what the functional role of tubular survivin is. However, a small number of experimental and clinical reports implicate that renal survivin is important for functional integrity of the kidney.
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http://dx.doi.org/10.1152/ajprenal.00546.2012DOI Listing
September 2013

Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts.

PLoS One 2012 27;7(1):e31034. Epub 2012 Jan 27.

Interdisciplinary Centre for Clinical Research, University Erlangen-Nuremberg, Erlangen, Germany.

The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031034PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3267769PMC
July 2012

Hypoxia-inducible transcription factors stabilization in the thick ascending limb protects against ischemic acute kidney injury.

J Am Soc Nephrol 2011 Nov 15;22(11):2004-15. Epub 2011 Sep 15.

Department of Nephrology and Hypertension, Friedrich-Alexander-University, Erlangen-Nuremberg, Krankenhausstrasse 12, 91054 Erlangen, Germany.

Hypoxia-inducible transcription factors (HIF) protect cells against oxygen deprivation, and HIF stabilization before ischemia mitigates tissue injury. Because ischemic acute kidney injury (AKI) often involves the thick ascending limb (TAL), modulation of HIF in this segment may be protective. Here, we generated mice with targeted TAL deletion of the von Hippel-Lindau protein (Vhl), which mediates HIF degradation under normoxia, using Tamm-Horsfall protein (Thp)-driven Cre expression. These mice showed strong expression of HIF-1α in TALs but no changes in kidney morphology or function under control conditions. Deficiency of Vhl in the TAL markedly attenuated proximal tubular injury and preserved TAL function following ischemia-reperfusion, which may be partially a result of enhanced expression of glycolytic enzymes and lactate metabolism. These results highlight the importance of the thick ascending limb in the pathogenesis of AKI and suggest that pharmacologically targeting the HIF system may have potential to prevent and mitigate AKI.
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http://dx.doi.org/10.1681/ASN.2010121249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279994PMC
November 2011

Inhibition of prolyl hydroxylases increases erythropoietin production in ESRD.

J Am Soc Nephrol 2010 Dec 29;21(12):2151-6. Epub 2010 Nov 29.

Department of Nephrology and Hypertension, Friedrich-Alexander-University, Krankenhausstrasse 12, 91054 Erlangen, Germany. wanja.bernhardt@uk-erlangen

The reasons for inadequate production of erythropoietin (EPO) in patients with ESRD are poorly understood. A better understanding of EPO regulation, namely oxygen-dependent hydroxylation of the hypoxia-inducible transcription factor (HIF), may enable targeted pharmacological intervention. Here, we tested the ability of fibrotic kidneys and extrarenal tissues to produce EPO. In this phase 1 study, we used an orally active prolyl-hydroxylase inhibitor, FG-2216, to stabilize HIF independent of oxygen availability in 12 hemodialysis (HD) patients, six of whom were anephric, and in six healthy volunteers. FG-2216 increased plasma EPO levels 30.8-fold in HD patients with kidneys, 14.5-fold in anephric HD patients, and 12.7-fold in healthy volunteers. These data demonstrate that pharmacologic manipulation of the HIF system can stimulate endogenous EPO production. Furthermore, the data indicate that deranged oxygen sensing--not a loss of EPO production capacity--causes renal anemia.
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http://dx.doi.org/10.1681/ASN.2010010116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014028PMC
December 2010

The GTPase RAB20 is a HIF target with mitochondrial localization mediating apoptosis in hypoxia.

Biochim Biophys Acta 2011 Jan 5;1813(1):1-13. Epub 2010 Nov 5.

University of Erlangen-Nuremberg, Erlangen, Germany.

Hypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress.
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http://dx.doi.org/10.1016/j.bbamcr.2010.10.019DOI Listing
January 2011

Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia-inducible factor-1.

FASEB J 2010 Nov 12;24(11):4443-58. Epub 2010 Jul 12.

Department of Nephrology and Hypertension, University of Erlangen-Nuremberg, Erlangen, Germany.

Hypoxia-inducible protein 2 (HIG2) has been implicated in canonical Wnt signaling, both as target and activator. The potential link between hypoxia and an oncogenic signaling pathway might play a pivotal role in renal clear-cell carcinoma characterized by constitutive activation of hypoxia-inducible factors (HIFs), and hence prompted us to analyze HIG2 regulation and function in detail. HIG2 was up-regulated by hypoxia and HIF inducers in all cell types and mouse organs investigated and abundantly expressed in renal clear-cell carcinomas. Promoter analyses, gel shifts, and siRNA studies revealed that HIG2 is a direct and specific target of HIF-1, but not responsive to HIF-2. Surprisingly, HIG2 was not secreted, and HIG2 overexpression neither stimulated proliferation nor activated Wnt signaling. Instead, we show that HIG2 decorates the hemimembrane of lipid droplets, whose number and size increase on hypoxic inhibition of fatty acid β-oxidation, and colocalizes with the lipid droplet proteins adipophilin and TIP47. Normoxic overexpression of HIG2 was sufficient to increase neutral lipid deposition in HeLa cells and stimulated cytokine expression. HIG2 could be detected in atherosclerotic arteries and fatty liver disease, suggesting that this ubiquitously inducible HIF-1 target gene may play an important functional role in diseases associated with pathological lipid accumulation.
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http://dx.doi.org/10.1096/fj.10-159806DOI Listing
November 2010

Mutation analysis of hypoxia-inducible factors HIF1A and HIF2A in renal cell carcinoma.

Anticancer Res 2009 Nov;29(11):4337-43

Department of Medical and Molecular Genetics, University of Birmingham, Institute of Biomedical Research, Edgbaston, Birmingham, B15 2TT, UK.

Background: Inactivation of the Von Hippel-Lindau (VHL) tumour suppressor gene leading to overexpression of hypoxia-inducible transcription factors (HIF)-1alpha and -2alpha is a critical event in the pathogenesis of most clear cell renal cell carcinomas (RCC). HIF-1alpha and HIF-2alpha share significant homology and regulate overlapping repertoires of hypoxia-inducible target genes but may have differing effects on RCC cell growth. Loss of HIF-1alpha expression has been described in RCC cell lines and primary tumours. Whether mutations in the alpha-subunits of HIF-1alpha and HIF-2alpha contribute to renal tumourigenesis was investigated here.

Materials And Methods: Mutation analysis of the complete coding sequence of HIF-1alpha and HIF-2alpha was carried out in primary RCC (n=40).

Results: The analysis revealed a somatic HIF1A missense substitution, p.Val116Glu, in a single RCC. Functional studies demonstrated that p.Val116Glu impaired HIF-1alpha transcriptional activity. Genotyping of HIF1A variants p.Pro582Ser and p.Ala588Thr demonstrated no significant differences between RCC patients and controls.

Conclusion: The detection of a loss-of-function HIF1A mutation in a primary RCC is consistent with HIF-1 and HIF-2 having different roles in renal tumourigenesis, However, somatic mutations of HIF1A are not frequently implicated in the pathogenesis of RCC.
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November 2009

The lysyl oxidases LOX and LOXL2 are necessary and sufficient to repress E-cadherin in hypoxia: insights into cellular transformation processes mediated by HIF-1.

J Biol Chem 2010 Feb 21;285(9):6658-69. Epub 2009 Dec 21.

Interdisciplinary Centre for Clinical Research, University of Erlangen-Nuremberg, 91054 Erlangen, Germany.

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.
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http://dx.doi.org/10.1074/jbc.M109.042424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825461PMC
February 2010

Novel insights into the role of the tumor suppressor von Hippel Lindau in cellular differentiation, ciliary biology, and cyst repression.

J Mol Med (Berl) 2009 Sep 24;87(9):871-7. Epub 2009 Jul 24.

Department of Nephrology and Hypertension, University of Erlangen-Nuremberg, Erlangen, Germany.

The growth and differentiation of renal tubular epithelial cells is normally tightly controlled. Disturbances can lead to the development of renal cysts or renal cell carcinomas, clinically relevant disease entities, which have so far been considered as being caused by entirely distinct mechanisms. Clear cell renal carcinoma, the most frequent type of renal cancer is associated with inactivation of the von Hippel Lindau (VHL) protein. Genetic defects leading to cystic kidney disease usually affect proteins that play a role in structure or function of primary cilia of renal epithelial cells. Accumulating evidence suggests that the VHL protein also controls cilia function and that its inactivation may result in both malignant or nonmalignant growth of epithelial cells and that this effect is in part mediated through the accumulation of hypoxia-inducible factors. Unraveling the complex role of VHL in renal epithelial cells is likely to shed further insight into mechanism of epithelial growth control, epithelial-mesenchymal transformation, and tumor development.
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http://dx.doi.org/10.1007/s00109-009-0504-xDOI Listing
September 2009

Mutual regulation of hypoxia-inducible factor and mammalian target of rapamycin as a function of oxygen availability.

Mol Cancer Res 2009 Jan;7(1):88-98

Interdisciplinary Centre for Clinical Research, Friedrich Alexander Universität Erlangen-Nürnberg, Nuremberg, Germany.

The mammalian target of rapamycin (mTOR) regulates cellular growth and proliferation, mainly by controlling cellular translation. Most tumors show constitutive activation of the mTOR pathway. In hypoxia, mTOR is inactivated, which is believed to be part of the program of the cell to maintain energy homeostasis. However, certain proteins are believed to be preferentially translated during hypoxia via 5' terminal oligopyrimidine tract mechanisms with controversial discussion about the involvement of the mTOR-dependent ribosomal protein S6 (rpS6). The hypoxia-inducible transcription factor (HIF) is the master regulator of hypoxic adaptation and itself strongly implicated in tumor growth. HIF is translationally regulated by mTOR. The regulatory features and the involvement of molecular oxygen itself in this regulation of HIF by mTOR are poorly understood. mTOR inhibition leads to profound attenuation of HIFalpha protein in the majority of primary and cancer cells studied. Under severe hypoxia, no influence of mTOR inhibitors was observed; thus, stimulation of HIFalpha by mTOR may only be relevant under mild hypoxia or even normoxia. HIF expression and phosphorylated rpS6 negatively correlate in experimental tumors. In cell culture, prolonged hypoxia abolishes rpS6 phosphorylation, which seems to be partly independent of the upstream p70S6 kinase. We show that hypoxic repression of rpS6 is largely dependent on HIF, implicating a negative feedback loop, which may influence cellular translational rates and metabolic homeostasis. These data implicate that the hypoxic microenvironment renders tumor cells resistant to mTOR inhibition, at least concerning hypoxic gene activation, which would add to the difficulties of other established therapeutic strategies in hypoxic cancer tissues.
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http://dx.doi.org/10.1158/1541-7786.MCR-08-0288DOI Listing
January 2009

The specific contribution of hypoxia-inducible factor-2alpha to hypoxic gene expression in vitro is limited and modulated by cell type-specific and exogenous factors.

Exp Cell Res 2008 Jun 18;314(10):2016-27. Epub 2008 Mar 18.

Department of Nephrology and Hypertension, University Clinic Erlangen, Germany.

Cellular integrity in hypoxia is dependent on molecular adaptations dominated by the heterodimeric transcription factor hypoxia-inducible factor (HIF). The HIF complex contains one of two alternative oxygen-regulated alpha-subunits considered to play distinct roles in the hypoxia response. Although HIF-2alpha may be more important in tumour biology and erythropoiesis, the spectrum of individual target genes is still insufficiently characterized. We therefore performed an Affymetrix gene array on Hep3B cells stimulated with a hypoxia-mimetic and transfected with either HIF-1alpha or HIF-2alpha siRNA. 271 transcripts were found to be induced HIF-dependently, including most previously identified HIF targets and a number of novel genes. Most were influenced by HIF-1alpha knock-down, whereas a smaller number were regulated by HIF-2alpha. Validation of a selection of genes by RNase protection confirmed the hypoxic regulation and HIF-1alpha- or HIF-2alpha-dependency in most cases, with the latter showing a lower amplitude. Many HIF-2alpha targets also responded to HIF-1alpha knock-down. Interestingly, regulation by HIF-2alpha was markedly influenced not only by cell type, but also by cell culture conditions, features that were not shared with HIF-1alpha-regulated genes. Thus, HIF-2alpha effects are modulated by a number of intrinsic and extrinsic factors which may be most relevant in tumour cells.
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http://dx.doi.org/10.1016/j.yexcr.2008.03.003DOI Listing
June 2008

Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function.

J Immunol 2008 Apr;180(7):4697-705

Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany.

Dendritic cells (DC) play a key role in linking innate and adaptive immunity. In inflamed tissues, where DC become activated, oxygen tensions are usually low. Although hypoxia is increasingly recognized as an important determinant of cellular functions, the consequences of hypoxia and the role of one of the key players in hypoxic gene regulation, the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), are largely unknown. Thus, we investigated the effects of hypoxia and HIF-1alpha on murine DC activation and function in the presence or absence of an exogenous inflammatory stimulus. Hypoxia alone did not activate murine DC, but hypoxia combined with LPS led to marked increases in expression of costimulatory molecules, proinflammatory cytokine synthesis, and induction of allogeneic lymphocyte proliferation compared with LPS alone. This DC activation was accompanied by accumulation of HIF-1alpha protein levels, induction of glycolytic HIF target genes, and enhanced glycolytic activity. Using RNA interference techniques, knockdown of HIF-1alpha significantly reduced glucose use in DC, inhibited maturation, and led to an impaired capability to stimulate allogeneic T cells. Alltogether, our data indicate that HIF-1alpha and hypoxia play a crucial role for DC activation in inflammatory states, which is highly dependent on glycolysis even in the presence of oxygen.
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http://dx.doi.org/10.4049/jimmunol.180.7.4697DOI Listing
April 2008

HIF activation protects from acute kidney injury.

J Am Soc Nephrol 2008 Mar 6;19(3):486-94. Epub 2008 Feb 6.

Medical Clinic 4, Department of Nephrology and Hypertension, Friedrich-Alexander-University Erlangen Nuremberg, Krankenhausstrasse 12, D - 91054 Erlangen, Germany.

The contribution of hypoxia to cisplatin-induced renal tubular injury is controversial. Because the hypoxia-inducible factor (HIF) pathway is a master regulator of adaptation to hypoxia, we measured the effects of cisplatin on HIF accumulation in vitro and in vivo, and tested whether hypoxic preconditioning is protective against cisplatin-induced injury. We found that cisplatin did not stabilize HIF-1alpha protein in vitro or in vivo under normoxic conditions. However, hypoxic preconditioning of cisplatin-treated proximal tubular cells in culture reduced apoptosis in an HIF-1alpha-dependent fashion and increased cell proliferation as measured by BrdU incorporation. In vivo, rats preconditioned with carbon monoxide before cisplatin administration had significantly better renal function than rats kept in normoxic conditions throughout. Moreover, the histomorphological extent of renal damage and tubular apoptosis was reduced by the preconditional treatment. Therefore, development of pharmacologic agents to induce renal HIF might provide a new approach to ameliorate cisplatin-induced nephrotoxicity.
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http://dx.doi.org/10.1681/ASN.2007040419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2391048PMC
March 2008

Organ protection by hypoxia and hypoxia-inducible factors.

Methods Enzymol 2007 ;435:221-45

Department of Nephrology and Hypertension, Friedrich-Alexander University, Erlangen, Nürnberg, Germany.

Since the first description of a protective effect of hypoxic preconditioning in the heart, the principle of reducing tissue injury in response to ischemia by prior exposure to hypoxia was confirmed in a number of cells and organs. However, despite impressive preclinical results, hypoxic preconditioning has so far failed to reach clinical application. Nevertheless, it remains of significant interest to induce genes that are normally activated during hypoxia and ischemia as part of an endogenous escape mechanism prior to or during the early phase of an ischemic insult. This approach has recently been greatly facilitated by the identification of hypoxia-inducible factors (HIFs), transcription factors that operate as a master switch in the cellular response to hypoxia. Far more than 100 target genes are regulated by HIF, including genes such as erythropoietin and hemoxygenase-1, which have been shown to be tissue-protective. The identification of small molecule inhibitors of the oxygen-sensing HIF-prolyl hydroxlases now offers the possibility to mimic the hypoxic response by pharmacological stabilization of HIF in order to achieve organ protection. Oxygen-independent activation of HIF is therefore a promising therapeutic strategy for the prevention of organ injury and failure.
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http://dx.doi.org/10.1016/S0076-6879(07)35012-XDOI Listing
February 2008

Hypoxia, via stabilization of the hypoxia-inducible factor HIF-1alpha, is a direct and sufficient stimulus for brain-type natriuretic peptide induction.

Biochem J 2008 Jan;409(1):233-42

Department of Nephrology and Hypertension, University of Erlangen-Nuremberg, Krankenhausstr. 12, 91054 Erlangen, Germany.

BNP (brain-type natriuretic peptide) is a cardiac hormone with systemic haemodynamic effects as well as local cytoprotective and antiproliferative properties. It is induced under a variety of pathophysiological conditions, including decompensated heart failure and myocardial infarction. Since regional hypoxia is a potential common denominator of increased wall stretch and myocardial hypoperfusion, we investigated the direct effects of hypoxia on BNP expression, and the role of the HIF (hypoxia-inducible transcription factor) in BNP regulation. Using an RNase protection assay we found a strong hypoxic induction of BNP mRNA expression in different cell lines and in cultured adult rat cardiomyocytes. Systemic hypoxia and exposure to 0.1% CO induced BNP expression in the rodent myocardium in vivo, although this was at a lower amplitude. BNP promoter-driven luciferase expression increased 10-fold after hypoxic stimulation in transient transfections. Inactivation of four putative HREs (hypoxia-response elements) in the promoter by site-directed mutagenesis revealed that the HRE at -466 nt was responsible for hypoxic promoter activation. A functional CACAG motif was identified upstream of this HRE. The HIF-1 complex bound specifically and inducibly only to the HRE at -466 nt, as shown by EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation). siRNA (small interfering RNA)-mediated knockdown of HIF-1alpha, but not HIF-2alpha, interfered with hypoxic BNP mRNA induction and BNP promoter activation, confirming that BNP is a specific HIF-1alpha target gene. In conclusion, BNP appears to be part of the protective program steered by HIF-1 in response to oxygen deprivation. Induction of BNP may therefore contribute to the potential benefits of pharmacological HIF inducers in the treatment of ischaemic heart disease and heart failure.
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http://dx.doi.org/10.1042/BJ20070629DOI Listing
January 2008

Erythropoietin gene expression in renal carcinoma is considerably more frequent than paraneoplastic polycythemia.

Int J Cancer 2007 Dec;121(11):2434-42

Interdisciplinary Centre for Clinical Research (IZKF), Friedrich-Alexander University of Erlangen-Nuremburg, Erlangen, Germany.

Signalling by erythropoietin (EPO) is increasingly recognised as a relevant mechanism in tumour biology, potentially leading to enhanced proliferation, angiogenesis and therapy resistance. Paraneoplastic polycythemia by cancerous overproduction of EPO is a rare event, but most frequently seen in patients with renal cell carcinoma (RCC). The majority of clear cell RCC displays a strong activation of the transcription factor regulating EPO, the Hypoxia-inducible Factor (HIF). Therefore, it is unclear why only a small minority of patients develop polycythemia. We studied 70 RCC for EPO gene and HIFalpha isoform expression. 34% of all RCC showed expression of EPO mRNA in RNase protection assays, which were almost exclusively of the clear cell type. Only 1 patient presented with polycythemia. In situ hybridisation revealed that expression of EPO was in the tumour cells. Expression of EPO mRNA was always associated with activation of HIF, which could involve HIF-1alpha and/or HIF-2alpha. The frequency of EPO gene expression in RCC is therefore much higher than the prevalence of polycythemia. Furthermore, activation of HIF appears necessary for EPO gene expression in RCC, but is clearly not the only determinant. Further to the reported expression of EPO receptors in tumour tissues, the finding of widespread expression of EPO in RCC supports the recent notion of an involvement of this system in paracrine or autocrine effects of tumour cells.
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http://dx.doi.org/10.1002/ijc.22961DOI Listing
December 2007