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1.  Impaired Neovascularization and Reduced Capillary Supply in the Malignant vs. Non-malignant Course of Experimental Renovascular Hypertension 
Malignant hypertension develops in some cases of hypertension but not in others. We hypothesized that an impaired neovascularization and a reduced capillary supply characterizes the malignant course of experimental hypertension. Two-kidney, one-clip renovascular hypertension was induced in rats; controls (sham) were sham operated. To distinguish malignant hypertension from non-malignant hypertension, we considered two factors: weight loss, and the number of typical vascular lesions (onion skin lesions and fibrinoid necroses) per kidney section of the nonclipped kidney. Animals in the upper half for both criteria were defined as malignant hypertensives. After 5 weeks, mean arterial blood pressure was elevated to the same degree in malignant hypertension and non-malignant hypertension whereas plasma renin and aldosterone were significantly higher in malignant hypertensives. The expression of plasminogen activator inhibitor-1 was elevated (up to 14-fold) in non-malignant but significantly more increased (up to 36-fold) in malignant hypertensive rats, compared to sham. As a bioassay for neovascularization, the area of granulation tissue ingrowth in polyvinyl discs (implanted subcutaneously) was reduced in malignant hypertension compared to non-malignant hypertension and sham, while there was no difference between non-malignant hypertension and sham. The number of renal and left ventricular capillaries was significantly lower in malignant hypertension compared to non-malignant hypertension, as was the number of proliferating endothelial cells. We conclude that an impaired neovascularization and capillarization occurs in malignant renovascular hypertension but not in the non-malignant course of the disease despite comparable blood pressure levels. This might contribute to the unique vascular lesions and progressive target organ damage observed in malignant hypertension.
PMCID: PMC5003830  PMID: 27625610
angiogenesis; aldosterone; malignant hypertension; neovascularization; PAI-1; VEGF
2.  Comparison of different normalization strategies for the analysis of glomerular microRNAs in IgA nephropathy 
Scientific Reports  2016;6:31992.
Small nucleolar RNAs (snoRNAs) have been used for normalization in glomerular microRNA (miRNA) quantification without confirmation of validity. Our aim was to identify glomerular reference miRNAs in IgA nephropathy. We compared miRNAs in human paraffin-embedded renal biopsies from patients with cellular-crescentic IgA-GN (n = 5; crescentic IgA-GN) and non-crescentic IgA-GN (n = 5; IgA-GN) to mild interstitial nephritis without glomerular abnormalities (controls, n = 5). Laser-microdissected glomeruli were used for expression profiling of 762 miRNAs by low-density TaqMan arrays (cards A and B). The comparison of different normalization methods (GeNormPlus, NormFinder, global mean and snoRNAs) in crescentic IgA-GN, IgA-GN and controls yielded similar results. However, levels of significance and the range of relative expression differed. In median, two normalization methods demonstrated similar results. GeNormPlus and NormFinder gave different top ranked reference miRNAs. Stability ranking for snoRNAs varied between cards A and B. In conclusion, we suggest the geometric mean of the most stable reference miRNAs found in GeNormPlus (miR-26b-5p), NormFinder (miR-28-5p) and snoRNAs (RNU44) as reference. It should be considered that significant differences could be missed using one particular normalization method. As a starting point for glomerular miRNA studies in IgA nephropathy we provide a library of miRNAs.
PMCID: PMC4995590  PMID: 27553688
3.  CD101 inhibits the expansion of colitogenic T cells 
Mucosal immunology  2016;9(5):1205-1217.
CD101 exerts negative-costimulatory effects in vitro, but its function in vivo remains poorly defined. CD101 is abundantly expressed on lymphoid and myeloid cells in intestinal tissues, but absent from naïve splenic T cells. Here, we assessed the impact of CD101 on the course of inflammatory bowel disease (IBD). Using a T cell transfer model of chronic colitis, we found that in recipients of naïve T cells from CD101+/+ donors up to 30% of the recovered lymphocytes expressed CD101, correlating with an increased IL-2-mediated FoxP3-expression. Transfer of CD101−/− T cells caused more severe colitis and was associated with an expansion of IL-17-producing T cells and an enhanced expression of IL-2Rα/β independently of FoxP3. The co-transfer of naïve and regulatory T cells (Treg) protected most effectively from colitis, when both donor and recipient mice expressed CD101. While the expression of CD101 on T cells was sufficient for Treg-function and the inhibition of T cell proliferation, sustained IL-10-production required additional CD101-expression by myeloid cells. Finally, in patients with IBD a reduced CD101-expression on peripheral and intestinal monocytes and CD4+ T cells correlated with enhanced IL-17-production and disease activity. Thus, CD101-deficiency is a novel marker for progressive colitis and potential target for therapeutic intervention.
PMCID: PMC4963314  PMID: 26813346
4.  Knockdown of ApoL1 in Zebrafish Larvae Affects the Glomerular Filtration Barrier and the Expression of Nephrin 
PLoS ONE  2016;11(5):e0153768.
APOL1, a secreted high-density lipoprotein, is expressed in different human tissues. Genetic variants of APOL1 are described to be associated with the development of end stage renal diseases in African Americans. In human kidney, APOL1 is mainly expressed in podocytes that are responsible for proper blood filtration. Since mice do not express ApoL1, the zebrafish is an ideal model to study the role of ApoL1. Injection of morpholinos against zApoL1 into zebrafish eggs and larvae, respectively, induces severe edema indicating a leakage of the filtration barrier. This was demonstrated in zApoL1 knockdown larvae by intravascular injection of fluorescently-labeled 10- and 500-kDa dextrans and by clearance of the vitamin D-binding protein from the circulation. Immunohistochemistry and RT-PCR revealed the reduction of nephrin, a podocyte-specific protein essential for blood filtration. Coinjection of human nephrin mRNA rescued the zApoL1 knockdown induced phenotype. Reduced APOL1 and nephrin levels were also found in biopsies of patients suffering from end stage renal diseases. Our results demonstrate that zApoL1 is essential for proper blood filtration in the zebrafish glomerulus and that zApoL1 affects the expression of nephrin.
PMCID: PMC4854397  PMID: 27138898
5.  Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation 
Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing the transcriptome of IECs from IBD patients using a genome-wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBDs, and inflammation was associated with suppressed Rho-A activation due to reduced expression of the Rho-A prenylation enzyme geranylgeranyltransferase-I (GGTase-I). Functionally, we found that mice with conditional loss of Rhoa or the gene encoding GGTase-I, Pggt1b, in IECs exhibit spontaneous chronic intestinal inflammation with accumulation of granulocytes and CD4+ T cells. This phenotype was associated with cytoskeleton rearrangement and aberrant cell shedding, ultimately leading to loss of epithelial integrity and subsequent inflammation. These findings uncover deficient prenylation of Rho-A as a key player in the pathogenesis of IBDs. As therapeutic triggering of Rho-A signaling suppressed intestinal inflammation in mice with GGTase-I–deficient IECs, our findings suggest new avenues for treatment of epithelial injury and mucosal inflammation in IBD patients.
PMCID: PMC4731169  PMID: 26752649
6.  CD163+ M2c-like macrophages predominate in renal biopsies from patients with lupus nephritis 
The role of macrophages in the pathogenesis of lupus nephritis, in particular their differentiation to a certain subtype (e.g., M1- or M2-like) modulating the inflammatory reaction, is unknown. Here we investigated whether the differentiation in M1- or M2-like macrophages depends on the stage of lupus nephritis and whether this correlates with clinical parameters.
Using immunohistochemical analysis we analyzed renal biopsies from 68 patients with lupus nephritis (ISN/RPS classes II–V) for infiltration with M1-like (iNOS+/CD68+), M2a-like (CD206+/CD68+), M2c-like macrophages (CD163+/CD68+), and FoxP3+ regulatory T-cells. In addition, clinical parameters at the time of renal biopsy, i.e., blood pressure, proteinuria and serum urea were correlated with the macrophage infiltration using the Spearman test.
The mean number of CD68+ macrophages was related to the diagnosed ISN/RPS class, showing the highest macrophage infiltration in biopsies with diffuse class IV and the lowest number in ISN/RPS class V. In all ISN/RPS classes we detected more M2c-like CD163+/CD68+ than M2a-like CD206+/CD68+ cells, while M1-macrophages played only a minor role. Cluster analysis using macrophage subtype numbers in different renal compartments revealed three main clusters showing cluster 1 dominated by class V. Clusters 2 and 3 were dominated by lupus class IV indicating that this class can be further differentiated by its macrophage population. The number of tubulointerstitial FoxP3+ cells correlated with all investigated macrophage subtypes showing the strongest association to numbers of M2a-like macrophages. Kidney function, as assessed by serum creatinine and serum urea, correlated positively with the number of total CD68+, M2a-like and M2c-like macrophages in the tubulointerstitium. In addition, total CD68+ and M2c-like macrophage numbers highly correlated with Austin activity score. Interestingly, in hypertensive lupus patients only the number of M2a-like macrophages was significantly increased compared to biopsies from normotensive lupus patients.
M2-like macrophages are the dominant subpopulation in human lupus nephritis and particularly, M2a subpopulations were associated with disease progression, but their role in disease progression remains unclear.
PMCID: PMC4835936  PMID: 27091114
Lupus nephritis; Macrophages; T-reg-cells; Macrophage subtypes
8.  Marine alkaloid Monanchocidin a overcomes drug resistance by induction of autophagy and lysosomal membrane permeabilization 
Oncotarget  2015;6(19):17328-17341.
Monanchocidin A (MonA) is a novel alkaloid recently isolated from the marine sponge Monanchora pulchra. The compound reveals cytotoxic activity in genitourinary cancers including cisplatin-sensitive and -resistant germ cell tumor (GCT) cell lines, hormone-sensitive and castration-resistant prostate carcinoma cell lines and different bladder carcinoma cell lines. In contrast, non-malignant cells were significantly less sensitive. MonA is highly synergistic with cisplatin in GCT cells. Induction of autophagy at lower and lysosomal membrane permeabilization (LMP) at higher concentrations were identified as the dominating modes of action. Cytotoxicity and protein degradation could be inhibited by 3-methyladenine, an inhibitor of autophagy. LMP was confirmed by loss of acridine orange staining of lysosoms and by release of cathepsin B. In conclusion, MonA exerts cytotoxiс activity by mechanisms different from “classical” apoptosis, and could be a promising new compound to overcome resistance to standard therapies in genitourinary malignancies.
PMCID: PMC4627311  PMID: 26093146
Monanchocidin A; autophagy; lysosomal membrane permeabilization; germ cell tumor cells; cisplatin resistance
9.  Oxidative DNA Damage in Kidneys and Heart of Hypertensive Mice Is Prevented by Blocking Angiotensin II and Aldosterone Receptors 
PLoS ONE  2014;9(12):e115715.
Recently, we could show that angiotensin II, the reactive peptide of the blood pressure-regulating renin-angiotensin-aldosterone-system, causes the formation of reactive oxygen species and DNA damage in kidneys and hearts of hypertensive mice. To further investigate on the one hand the mechanism of DNA damage caused by angiotensin II, and on the other hand possible intervention strategies against end-organ damage, the effects of substances interfering with the renin-angiotensin-aldosterone-system on angiotensin II-induced genomic damage were studied.
In C57BL/6-mice, hypertension was induced by infusion of 600 ng/kg • min angiotensin II. The animals were additionally treated with the angiotensin II type 1 receptor blocker candesartan, the mineralocorticoid receptor blocker eplerenone and the antioxidant tempol. DNA damage and the activation of transcription factors were studied by immunohistochemistry and protein expression analysis.
Administration of angiotensin II led to a significant increase of blood pressure, decreased only by candesartan. In kidneys and hearts of angiotensin II-treated animals, significant oxidative stress could be detected (1.5-fold over control). The redox-sensitive transcription factors Nrf2 and NF-κB were activated in the kidney by angiotensin II-treatment (4- and 3-fold over control, respectively) and reduced by all interventions. In kidneys and hearts an increase of DNA damage (3- and 2-fold over control, respectively) and of DNA repair (3-fold over control) was found. These effects were ameliorated by all interventions in both organs. Consistently, candesartan and tempol were more effective than eplerenone.
Angiotensin II-induced DNA damage is caused by angiotensin II type 1 receptor-mediated formation of oxidative stress in vivo. The angiotensin II-mediated physiological increase of aldosterone adds to the DNA-damaging effects. Blocking angiotensin II and mineralocorticoid receptors therefore has beneficial effects on end-organ damage independent of blood pressure normalization.
PMCID: PMC4297153  PMID: 25551569
10.  G Protein-coupled Estrogen Receptor Protects from Atherosclerosis 
Scientific Reports  2014;4:7564.
Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.
PMCID: PMC4274506  PMID: 25532911
11.  Successful Antiviral Triple Therapy in a Longstanding Refractory Hepatitis C Virus Infection with an Acute Kidney Injury 
Case Reports in Nephrology  2014;2014:308729.
Introduction. The HCV infection is a common disease with many chronically infected patients worldwide. So far, the standard therapy of a chronic HCV infection consisted of interferon as single therapy or in combination with ribavirin. After approval of the two protease inhibitors, boceprevir and telaprevir, the standard therapy for patients with genotype 1 changed. In patients with acute kidney injury (AKI) these therapies are not approved and have so far not been evaluated in studies. Case Report. In April 2012, a 58-year-old female was admitted due to a cryoglobulin-positive chronic HCV infection which had been treated with interferon and ribavirin. Currently, the patient was admitted because of severe complications with an acute kidney injury. We treated our patient successfully with a boceprevir based triple therapy. Conclusion. Limited data suggests that a therapy with ribavirin in patients with AKI seems to be safe under close monitoring. Our patient was treated successfully with a protease inhibitor based triple therapy. Nevertheless, it is necessary to plan an interventional study to evaluate the exact risk-benefit profile of triple therapy regimens in patients with AKI and hepatitis C.
PMCID: PMC4147195  PMID: 25197586
12.  Increased malignancy of oral squamous cell carcinomas (oscc) is associated with macrophage polarization in regional lymph nodes – an immunohistochemical study 
BMC Cancer  2014;14:522.
It is largely accepted that specific immunological parameters in solid malignancies are associated with patient’s prognosis. Recently a correlation of macrophage polarization with histomorphological parameters could also be shown in oral squamous cell carcinoma (oscc). The observed tumor derived peripheral immune tolerance could be associated with the macrophage polarization in regional tumor draining lymph nodes.
So far there are no studies analyzing the macrophage polarization in cervical lymph nodes of oscc patients. In the present study we aimed to correlate macrophage polarization in different anatomical lymph node compartments of patients diagnosed with oscc with histopathologic parameters of the primary tumor (T-, N-, L-, V-, Pn-status, grading).
Tumor free (n = 37) and metastatic (n = 17) lymph nodes of T1 and T2 oscc patients were processed for immunohistochemistry to detect CD68, CD11c, CD163 and MRC1 positive cells. Samples were digitized using whole slide imaging and the number of cells expressing the aforementioned markers in the region of interest quantitatively analyzed.
The malignancy of the primary tumor (defined by T-, L-, Pn-status, grading) correlated with the lymph node macrophage polarization. L1 and Pn1 tumor cases displayed a significantly (p < 0.05) decreased M1 and increased M2 polarization in the sinus of the lymph nodes. G3 cases presented a significantly (p < 0.05) increased M2 polarization in the sinus compared to G2 cases. T2 tumors had significantly (p < 0.05) increased M2 polarization in the interfollicular zone of regional lymph nodes compared to T1 tumors. Metastatic and non-metastatic lymph nodes did not differ regarding their macrophage polarization.
The current study revealed for the first time an influence of oscc on the macrophage polarization in regional lymph nodes. Markers of malignant behavior in the primary tumor were associated with a shift of macrophage polarization in lymph nodes from the anti-tumoral M1 type to the tumor-promoting M2 type. As tumor free and metastatic lymph nodes did not differ in terms of their macrophage polarization pattern, there must be other factors influencing the location for lymph node metastasis formation.
PMCID: PMC4223559  PMID: 25042135
Oral squamous cell carcinoma; Oral cancer; Lymph node; Macrophage polarization; Peripheral tolerance; oscc; M1; M2
13.  Darbepoetin Alpha Reduces Oxidative Stress and Chronic Inflammation in Atherosclerotic Lesions of Apo E Deficient Mice in Experimental Renal Failure 
PLoS ONE  2014;9(2):e88601.
Cardiovascular morbidity and mortality is very important in patients with chronic renal failure. This occurs even in mild impairment of renal function and may be related to oxidative stress and chronic inflammation. The nephrectomized apo E knockout mouse is an accepted model for evaluating atherosclerosis in renal dysfunction. Erythropoietin derivates showed anti-oxidative and anti-inflammatory effects. Therefore, this study evaluates the effects of Darbepoetin on markers of oxidative stress and chronic inflammation in atherosclerotic lesions in apo E knockout mice with renal dysfunction.
Apo E knockout mice underwent unilateral (Unx, n = 20) or subtotal (Snx, n = 26) nephrectomy or sham operation (Sham, n = 16). Mice of each group were either treated with Darbepoetin or saline solution, a part of Snx mice received a tenfold higher dose of Darbepoetin. The aortic plaques were measured and morphologically characterized. Additional immunhistochemical analyses were performed on tissue samples taken from the heart and the aorta.
Both Unx and Snx mice showed increased expression of markers of oxidative stress and chronic inflammation. While aortic plaque size was not different, Snx mice showed advanced plaque stages when compared to Unx mice. Darbepoetin treatment elevated hematocrit and lowered Nitrotyrosin as one marker of oxidative stress, inflammation in heart and aorta, plaque stage and in the high dose even plaque cholesterol content. In contrast, there was no influence of Darbepoetin on aortic plaque size; high dose Darbepoetin treatment resulted in elevated renal serum parameters.
Darbepoetin showed some protective cardiovascular effects irrespective of renal function, i.e. it improved plaque structure and reduced some signs of oxidative stress and chronic inflammation without affecting plaque size. Nevertheless, the dose dependent adverse effects must be considered as high Darbepoetin treatment elevated serum urea. Elevation of hematocrit might be a favorable effect in anemic Snx animals but a thrombogenic risk in Sham animals.
PMCID: PMC3938414  PMID: 24586350
14.  Osteo-radio-necrosis (ORN) and bisphosphonate-related osteonecrosis of the jaws (BRONJ): the histopathological differences under the clinical similarities 
Objectives: Both Osteoradionecrosis (ORN) and Bisphosphonate associated osteonecrosis of the jaws (BRONJ) present clinically as regions of exposed necrotic bone. The study aimed to demonstrate the histopathological differences behind the observed clinical similarities. Study Design: Ten ORN specimens and ten BRONJ specimens were used, as well as ten samples of normal mandibular bone as control. Two bone-specific stainings were used, i.e. Sirius Red for the study of the relative presence of collagen types I and III and toluidine blue for the study the osteon density. Results: The Red Green Blue (RGB)-analysis of the specimens stained with Sirius Red identified significant differences between the chromatic patterns observed in bone preparations of patients suffering from ORN when compared to both BRONJ and control samples. Moreover, the osteon density of the BRONJ samples was significantly lower when compared to ORN and normal bone samples. Conclusions: The demonstrated differences in the bone architecture and in the bone collagen content between the two pathological conditions most likely reflect underlying pathophysiological differences.
PMCID: PMC3925894  PMID: 24551270
Osteoradionecrosis; bisphosphonate-associated osteonecrosis; bone structure; collagen; toluidine blue; Sirius red
15.  Complement factor H–related hybrid protein deregulates complement in dense deposit disease 
The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H–related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H–mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies.
PMCID: PMC3871254  PMID: 24334459
16.  Activation of Rac-1 and RhoA Contributes to Podocyte Injury in Chronic Kidney Disease 
PLoS ONE  2013;8(11):e80328.
Rho-family GTPases like RhoA and Rac-1 are potent regulators of cellular signaling that control gene expression, migration and inflammation. Activation of Rho-GTPases has been linked to podocyte dysfunction, a feature of chronic kidney diseases (CKD). We investigated the effect of Rac-1 and Rho kinase (ROCK) inhibition on progressive renal failure in mice and studied the underlying mechanisms in podocytes. SV129 mice were subjected to 5/6-nephrectomy which resulted in arterial hypertension and albuminuria. Subgroups of animals were treated with the Rac-1 inhibitor EHT1846, the ROCK inhibitor SAR407899 and the ACE inhibitor Ramipril. Only Ramipril reduced hypertension. In contrast, all inhibitors markedly attenuated albumin excretion as well as glomerular and tubulo-interstitial damage. The combination of SAR407899 and Ramipril was more effective in preventing albuminuria than Ramipril alone. To study the involved mechanisms, podocytes were cultured from SV129 mice and exposed to static stretch in the Flexcell device. This activated RhoA and Rac-1 and led via TGFβ to apoptosis and a switch of the cells into a more mesenchymal phenotype, as evident from loss of WT-1 and nephrin and induction of α-SMA and fibronectin expression. Rac-1 and ROCK inhibition as well as blockade of TGFβ dramatically attenuated all these responses. This suggests that Rac-1 and RhoA are mediators of podocyte dysfunction in CKD. Inhibition of Rho-GTPases may be a novel approach for the treatment of CKD.
PMCID: PMC3820652  PMID: 24244677
17.  BK viremia and polyomavirus nephropathy in 352 kidney transplants; risk factors and potential role of mTOR inhibition 
BMC Nephrology  2013;14:207.
Polyomavirus BK nephropathy (PyVAN) remains an important cause of early graft dysfunction and graft loss in kidney transplantation.
In this retrospective, single centre cohort study we studied the incidence and outcome of BK viral infection in 352 patients transplanted in 2008–2011.
During follow-up viral replication was detected in 48 patients (13.6%); 22 patients (6.2%) had biopsy proven PyVAN.
In multivariate logistic regression analyses risk factors for BK-viremia were lack of enrolment into randomized controlled trials (RCTs), biopsy proven acute rejections, cytomegaly virus (CMV) serostatus of both donor and recipient and previous transplantation.
In patients without PyVAN reduction or switch of immunosuppression was associated with rapid viral clearance and stable graft function. In contrast, in most patients with PyVAN graft function deteriorated and 5 patients prematurely lost their allograft. Switch of immunosuppression to a low dose cyclosporine plus mTOR inhibitor based regimen in patients with PyVAN was safe, well tolerated and tended to be associated with a better short-term outcome in terms of graft function compared to reduction of existing immunosuppression alone.
With the lack of licensed anti-polyoma viral drugs reduction or conversion of immunosuppression remains the mainstay of therapy in patients with PyVAN. The combination of low dose cyclosporine plus mTOR inhibition appears to be safe and warrants further investigation.
PMCID: PMC3850699  PMID: 24088187
Polyomavirus BK nephropathy; PyVAN; mTOR inhibition
18.  Granulomatous Lung Disease Requiring Mechanical Ventilation Induced by a Single Application of Oxaliplatin-Based Chemotherapy for Colorectal Cancer: A Case Report 
Combined chemotherapeutic regimens in conjunction with oxaliplatin are considered safe and effective treatment options in the clinical management of metastatic colorectal cancer. A 62-year-old male patient with a metastatic rectal carcinoma developed a pulmonary reaction after the first application of the combined standard chemotherapy regimen (5-fluorouracil and sodium folinic acid as a 24 h infusion and oxaliplatin). Following the first dose of chemotherapy, the patient developed acute dyspnoea and fever. A computerised scan of the chest revealed bilateral pulmonary patchy consolidation. Despite high-dose empiric antibiotic and antimycotic treatment, no clinical improvement was seen. The patient's condition deteriorated, and he required invasive mechanical ventilation. Diagnostic thoracoscopic wedge resections were performed for further diagnosis. The histological workup revealed distinct granulomatous inflammation, but no microbial pathogens were to be found. Thereupon, a drug-induced reaction to chemotherapy was suspected and high-dose steroid treatment initiated. Subsequently, the patient's respiratory condition improved and he was extubated. The present case exemplifies the rare course of a bilateral pneumonia-like, drug-induced granulomatous reaction following a single application of oxaliplatin. In addition to the known side effects of oxaliplatin-containing combination chemotherapy, unexpected serious adverse events in the form of pulmonary toxicities should also be taken into account.
PMCID: PMC3638573  PMID: 23691384
19.  Oncofertility: combination of ovarian stimulation with subsequent ovarian tissue extraction on the day of oocyte retrieval 
New anticancer treatments have increased survival rates for cancer patients, but often at the cost of sterility. Several strategies are currently available for preserving fertility. However, the chances of achieving a pregnancy with one technique are still limited. A combination of methods is therefore recommended in order to maximize women’s chances of future fertility. In this retrospective study, ovarian stimulation with subsequent ovarian tissue extraction on the day of oocyte retrieval were combined and the quality of the ovarian tissue, the numbers and quality of oocytes, time requirements, and the safety of the strategy were examined.
Fourteen female patients suffering from malignant diseases underwent one in vitro fertilization cycle. Different stimulation protocols were used, depending on the menstrual cycle. Transvaginal oocyte retrieval was scheduled 34–36 h after human chorionic gonadotropin administration. Immediately afterwards, ovarian tissue was extracted laparoscopically.
A mean of 10 oocytes were retrieved per patient, and 67% of the oocytes were successfully fertilized using intracytoplasmic sperm injection. No periprocedural complications and no complications leading to postponement of the start of chemotherapy occurred. The ovarian tissues were of good quality, with a normal age-related follicular distribution and without carcinoma cell invasion.
An approach using ovarian stimulation first, followed by laparoscopic collection of ovarian tissue, is a useful strategy for increasing the efficacy of fertility preservation techniques. The ovarian tissue is not affected by prior ovarian stimulation.
PMCID: PMC3599192  PMID: 23510640
Fertility preservation; Ovarian tissue; Ovarian stimulation; IVF; Oocyte cryopreservation; Ovarian tissue cryopreservation
20.  Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells 
PLoS ONE  2013;8(2):e56650.
Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca2+ concentration ([Ca2+]i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca2+]i by Ca2+ ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7−/−) than in erythrocytes from wild type mice (anxA7+/+). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7−/− and anx7+/+-mice following increase of [Ca2+]i by Ca2+ ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7−/− than in anx7+/+-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7−/− than in anx7+/+-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.
PMCID: PMC3577872  PMID: 23437197
21.  The Mitogen-Activated Protein Kinase p38α Regulates Tubular Damage in Murine Anti-Glomerular Basement Membrane Nephritis 
PLoS ONE  2013;8(2):e56316.
p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus examined expression and activation of p38MAPK isoforms during anti-glomerular basement membrane (GBM) nephritis. Therefore, p38α conditional knockout mice (MxCre-p38αΔ/Δ) were used to examine the role of p38α in anti-GBM induced nephritis. Both wild type and MxCre-p38αΔ/Δ mice developed acute renal failure over time. Histological examinations revealed a reduced monocyte influx and less tubular damage in MxCre-p38αΔ/Δ mice, whereas glomerular crescent formation and renal fibrosis was similar. Likewise, the levels of pro- and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar, but IL-8 was even up-regulated in MxCre-p38αΔ/Δ mice. In contrast, we could detect strong down-regulation of chemotactic cytokines such as CCL-2, -5 and -7, in the kidneys of MxCre-p38αΔ/Δ mice. In conclusion, p38α is the primary p38MAPK isoform expressed in anti-GBM nephritis and selectively affects inflammatory cell influx and tubular damage. Full protection from nephritis is however not achieved as renal failure and structural damage still occurs.
PMCID: PMC3575386  PMID: 23441175
22.  FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair 
Nature genetics  2012;44(8):910-915.
Chronic kidney disease (CKD) represents a major health burden1. Its central feature of renal fibrosis is not well understood. By whole exome resequencing in a model disorder for renal fibrosis, nephronophthisis (NPHP), we identified mutations of Fanconi anemia-associated nuclease 1 (FAN1) as causing karyomegalic interstitial nephritis (KIN). Renal histology of KIN is indistinguishable from NPHP except for the presence of karyomegaly2. FAN1 has nuclease activity, acting in DNA interstrand crosslinking (ICL) repair within the Fanconi anemia pathway of DNA damage response (DDR)3–6. We demonstrate that cells from individuals with FAN1 mutations exhibit sensitivity to the ICL agent mitomycin C. However, they do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from patients with Fanconi anemia. We complement ICL sensitivity with wild type FAN1 but not mutant cDNA from individuals with KIN. Depletion of fan1 in zebrafish revealed increased DDR, apoptosis, and kidney cysts akin to NPHP. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms of renal fibrosis and CKD.
PMCID: PMC3412140  PMID: 22772369
Hypertension  2011;59(2):507-512.
Endogenous estrogens mediate protective effects in the cardiovascular system, affecting both endothelium-dependent and -independent mechanisms. Previous studies have suggested that nonselective estrogen receptor agonists such as endogenous estrogens inhibit endothelium-dependent vasoconstriction; however, the role of estrogen receptors in this response has not yet been clarified. This study investigated whether the novel intracellular G protein-coupled estrogen receptor GPER regulates vascular reactivity in mice. Effects of chronic deficiency (using mice lacking the GPER gene) and acute inhibition (using the GPER-selective antagonist G15) on endothelium-dependent and -independent vascular reactivity, and the effects of GPER deficiency on vascular gene expression and structure were investigated. We found that chronic GPER deficiency is associated with increased endothelial prostanoid-mediated vasoconstriction, but had no effect on endothelial nitric oxide (NO) bioactivity, gene expression of endothelial NO synthase and thromboxane prostanoid (TP) receptor, or vascular structure. GPER deletion also increased TP receptor-mediated contraction. Acute GPER blockade enhanced endothelium-dependent contractions, and reduced endothelial NO bioactivity. Contractions to TP receptor activation were unaffected by G15. In conclusion, this study has identified GPER as the first estrogen receptor with inhibitory activity on endothelium-dependent contractility. These findings may be important for understanding and treating diseases associated with increased endothelial vasoconstrictor prostanoid activity such as hypertension and obesity.
PMCID: PMC3266468  PMID: 22203741
EDCF; Endothelium; Estrogen; GPER; GPR30; Nitric Oxide; Prostanoid
24.  Membranoproliferative glomerulonephritis complicating Waldenström’s macroglobulinemia 
BMC Nephrology  2012;13:172.
Lymphoproliferative disorders causing paraproteinemia can be associated with various kidney injuries including the deposition of monoclonal immunoglobulins (Ig). A known glomerular manifestation of Waldenström’s macroglobulinemia is characterized by prominent intracapillary hyaline thrombi and lack of conspicuous glomerular proliferation. The present case was special in 2 aspects: 1. the diagnosis of glomerulonephritis was unexpected before renal biopsy, 2. the prominent glomerular proliferation paired with large intracapillary hyaline thrombi is uncommon in Waldenström’s macroglobulinemia-associated glomerulonephritis.
Case presentation
A 73-year-old Caucasian woman with a long-standing history of rheumatoid arthritis and Waldenström’s macroglobulinemia was admitted for acute renal failure (ARF), which initially was presumed to be the consequence of extrarenal causes. Proteinuria and hematuria were only mild. In renal core biopsy, a membranoproliferative glomerulonephritis (MPGN) and prominent intracapillary hyaline monoclonal IgM thrombi were found in addition to acute tubular necrosis. Of note, the patient’s history was positive for purpuric skin changes, suspicious for cryoglobulinemia. However, serological tests for cryoglobulins were repeatedly negative. The ARF resolved before the start of immunomodulatory therapy for Waldenström’s macroglobulinemia.
The presence of MPGN with prominent hyaline thrombi in the context of Waldenström’s macroglobulinemia is uncommon and can be oligosymptomatic. We discuss this case in the context of previous literature and classifications suggested for monoclonal Ig-related renal pathologies.
PMCID: PMC3541200  PMID: 23259458
Waldenström’s macroglobulinemia; MPGN; Hyaline thrombi
25.  Guanylate-binding protein 1 expression from embryonal endothelial progenitor cells reduces blood vessel density and cellular apoptosis in an axially vascularised tissue-engineered construct 
BMC Biotechnology  2012;12:94.
Guanylate binding protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells. It is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. We previously demonstrated that stable expression of GBP-1 in murine endothelial progenitor cells (EPC) induces their premature differentiation and decreases their migration capacity in vitro and in vivo. The goal of the present study was to assess the antiangiogenic capacity of EPC expressing GBP-1 (GBP-1-EPC) and their impact on blood vessel formation in an axially vascularized 3-D bioartificial construct in vivo.
Functional in vitro testing demonstrated a significant increase in VEGF secretion by GBP-1-EPC after induction of cell differentiation. Undifferentiated GBP-1-EPC, however, did not secrete increased levels of VEGF compared to undifferentiated control EPC expressing an empty vector (EV-EPC). In our In vivo experiments, we generated axially vascularized tissue-engineered 3-D constructs. The new vascular network arises from an arterio-venous loop (AVL) embedded in a fibrin matrix inside a separation chamber. Total surface area of the construct as calculated from cross sections was larger after transplantation of GBP-1-EPC compared to control EV-EPC. This indicated reduced formation of fibrovascular tissue and less resorption of fibrin matrix compared to constructs containing EV-EPC. Most notably, the ratio of blood vessel surface area over total construct surface area in construct cross sections was significantly reduced in the presence of GBP-1-EPC. This indicates a significant reduction of blood vessel density and thereby inhibition of blood vessel formation from the AVL constructs caused by GBP-1. In addition, GBP-1 expressed from EPC significantly reduced cell apoptosis compared to GBP-1-negative controls.
Transgenic EPC expressing the proinflammatory antiangiogenic GTPase GBP-1 can reduce blood vessel density and inhibit apoptosis in a developing bioartificial vascular network and may become a new powerful tool to manipulate angiogenetic processes in tissue engineering and other pathological conditions such as tumour angiogenesis.
PMCID: PMC3610105  PMID: 23217187
Angiogenesis; Endothelial progenitor cells; Guanylate-binding protein 1; In vivo tissue engineering

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