Peter R. Mertens

The Outcome of Neutrophil Gelatinase-Associated Lipocalin-Positive Subclinical Acute Kidney Injury: A Multicenter Pooled Analysis of Prospective Studies

OBJECTIVES The aim of this study was to test the hypothesis that, without diagnostic changes in serum creatinine, increased neutrophil gelatinase-associated lipocalin (NGAL) levels identify patients with subclinical acute kidney injury (AKI) and therefore worse prognosis. BACKGROUND Neutrophil gelatinase-associated lipocalin detects subclinical AKI hours to days before increases in serum creatinine indicate manifest loss of renal function. METHODS We analyzed pooled data from 2,322 critically ill patients with predominantly cardiorenal syndrome from 10 prospective observational studies of NGAL. We used the terms NGAL(-) or NGAL(+) according to study-specific NGAL cutoff for optimal AKI prediction and the terms sCREA(-) or sCREA(+) according to consensus diagnostic increases in serum creatinine defining AKI. A priori-defined outcomes included need for renal replacement therapy (primary endpoint), hospital mortality, their combination, and duration of stay in intensive care and in-hospital. RESULTS Of study patients, 1,296 (55.8%) were NGAL(-)/sCREA(-), 445 (19.2%) were NGAL(+)/sCREA(-), 107 (4.6%) were NGAL(-)/sCREA(+), and 474 (20.4%) were NGAL(+)/sCREA(+). According to the 4 study groups, there was a stepwise increase in subsequent renal replacement therapy initiation-NGAL(-)/sCREA(-): 0.0015% versus NGAL(+)/sCREA(-): 2.5% (odds ratio: 16.4, 95% confidence interval: 3.6 to 76.9, p < 0.001), NGAL(-)/sCREA(+): 7.5%, and NGAL(+)/sCREA(+): 8.0%, respectively, hospital mortality (4.8%, 12.4%, 8.4%, 14.7%, respectively) and their combination (4-group comparisons: all p < 0.001). There was a similar and consistent progressive increase in median number of intensive care and in-hospital days with increasing biomarker positivity: NGAL(-)/sCREA(-): 4.2 and 8.8 days; NGAL(+)/sCREA(-): 7.1 and 17.0 days; NGAL(-)/sCREA(+): 6.5 and 17.8 days; NGAL(+)/sCREA(+): 9.0 and 21.9 days; 4-group comparisons: p = 0.003 and p = 0.040, respectively. Urine and plasma NGAL indicated a similar outcome pattern. CONCLUSIONS In the absence of diagnostic increases in serum creatinine, NGAL detects patients with likely subclinical AKI who have an increased risk of adverse outcomes. The concept and definition of AKI might need re-assessment.

Hepatocyte-Specific Smad7 Expression Attenuates TGF-β–Mediated Fibrogenesis and Protects Against Liver Damage

BACKGROUND & AIMS The profibrogenic role of transforming growth factor (TGF)-beta in liver has mostly been attributed to hepatic stellate cell activation and excess matrix synthesis. Hepatocytes are believed to contribute to increased rates of apoptosis. METHODS Primary hepatocyte outgrowths and AML12 cells were used as an in vitro model to detect TGF-beta effects on the cellular phenotype and expression profile. Furthermore, a transgenic mouse model was used to determine the outcome of hepatocyte-specific Smad7 expression on fibrogenesis following CCl(4)-dependent damage. Samples from patients with chronic liver diseases were assessed for (partial) epithelial-to-mesenchymal transition (EMT) in hepatocytes. RESULTS In primary cell cultures and in vivo, the majority of hepatocytes survive despite activated TGF-beta signaling. These cells display phenotypic changes and express proteins characteristic for (partial) EMT and fibrogenesis. Experimental expression of Smad7 in hepatocytes of mice attenuated TGF-beta signaling and EMT, resulted in less accumulation of interstitial collagens, and improved CCl(4)-provoked liver damage and fibrosis scores compared with controls. CONCLUSIONS The data indicate that hepatocytes undergo TGF-beta-dependent EMT-like phenotypic changes and actively participate in fibrogenesis. Furthermore, ablation of TGF-beta signaling specifically in this cell type is sufficient to blunt the fibrogenic response.

Intensive Supportive Care plus Immunosuppression in IgA Nephropathy

BACKGROUND The outcomes of immunosuppressive therapy, when added to supportive care, in patients with IgA nephropathy are uncertain. METHODS We conducted a multicenter, open-label, randomized, controlled trial with a two-group, parallel, group-sequential design. During a 6-month run-in phase, supportive care (in particular, blockade of the renin-angiotensin system) was adjusted on the basis of proteinuria. Patients who had persistent proteinuria with urinary protein excretion of at least 0.75 g per day were randomly assigned to receive supportive care alone (supportive-care group) or supportive care plus immunosuppressive therapy (immunosuppression group) for 3 years. The primary end points in hierarchical order were full clinical remission at the end of the trial (protein-to-creatinine ratio <0.2 [with both protein and creatinine measured in grams] and a decrease in the estimated glomerular filtration rate [eGFR] of <5 ml per minute per 1.73 m(2) of body-surface area from baseline) and a decrease in the eGFR of at least 15 ml per minute per 1.73 m(2) at the end of the trial. The primary end points were analyzed with the use of logistic-regression models. RESULTS The run-in phase was completed by 309 of 337 patients. The proteinuria level decreased to less than 0.75 g of urinary protein excretion per day in 94 patients. Of the remaining 162 patients who consented to undergo randomization, 80 were assigned to the supportive-care group, and 82 to the immunosuppression group. After 3 years, 4 patients (5%) in the supportive-care group, as compared with 14 (17%) in the immunosuppression group, had a full clinical remission (P=0.01). A total of 22 patients (28%) in the supportive-care group and 21 (26%) in the immunosuppression group had a decrease in the eGFR of at least 15 ml per minute per 1.73 m(2) (P=0.75). There was no significant difference in the annual decline in eGFR between the two groups. More patients in the immunosuppression group than in the supportive-care group had severe infections, impaired glucose tolerance, and weight gain of more than 5 kg in the first year of treatment. One patient in the immunosuppression group died of sepsis. CONCLUSIONS The addition of immunosuppressive therapy to intensive supportive care in patients with high-risk IgA nephropathy did not significantly improve the outcome, and during the 3-year study phase, more adverse effects were observed among the patients who received immunosuppressive therapy, with no change in the rate of decrease in the eGFR. (Funded by the German Federal Ministry of Education and Research; STOP-IgAN ClinicalTrials.gov number, NCT00554502.).

Disruption of the Y-box binding protein-1 results in suppression of the epidermal growth factor receptor and HER-2.

The overexpression of the epidermal growth factor receptor (EGFR) and HER-2 underpin the growth of aggressive breast cancer; still, it is unclear what governs the regulation of these receptors. Our laboratories recently determined that the Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, induced breast tumor cell growth in monolayer and in soft agar. Importantly, mutating YB-1 at Ser(102), which resides in the DNA-binding domain, prevented growth induction. We reasoned that the underlying cause for growth attenuation by YB-1(Ser(102)) is through the regulation of EGFR and/or HER-2. The initial link between YB-1 and these receptors was sought by screening primary tumor tissue microarrays. We determined that YB-1 (n = 389 cases) was positively associated with EGFR (P < 0.001, r = 0.213), HER-2 (P = 0.008, r = 0.157), and Ki67 (P < 0.0002, r = 0.219). It was inversely linked to the estrogen receptor (P < 0.001, r = -0.291). Overexpression of YB-1 in a breast cancer cell line increased HER-2 and EGFR. Alternatively, mutation of YB-1 at Ser(102) > Ala(102) prevented the induction of these receptors and rendered the cells less responsive to EGF. The mutant YB-1 protein was also unable to optimally bind to the EGFR and HER-2 promoters based on chromatin immunoprecipitation. Furthermore, knocking down YB-1 with small interfering RNA suppressed the expression of EGFR and HER-2. This was coupled with a decrease in tumor cell growth. In conclusion, YB-1(Ser(102)) is a point of molecular vulnerability for maintaining the expression of EGFR and HER-2. Targeting YB-1 or more specifically YB-1(Ser(102)) are novel approaches to inhibiting the expression of these receptors to ultimately suppress tumor cell growth.

Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a potential target for therapy

IntroductionBasal-like breast cancers (BLBCs) are very aggressive, and present serious clinical challenges as there are currently no targeted therapies available. We determined the regulatory role of Y-box binding protein-1 (YB-1) on epidermal growth factor receptor (EGFR) overexpression in BLBC, and the therapeutic potential of inhibiting EGFR. We pursued this in light of our recent work showing that YB-1 induces the expression of EGFR, a new BLBC marker.MethodsPrimary tumour tissues were evaluated for YB1 protein expression by immunostaining tissue microarrays, while copy number changes were assessed by comparative genomic hybridization (CGH). The ability of YB-1 to regulate EGFR was evaluated using luciferase reporter, chromatin immunoprecipitation (ChIP) and gel shift assays. The impact of Iressa on monolayer cell growth was measured using an ArrayScan VTI high-throughput analyser to count cell number, and colony formation in soft agar was used to measure anchorage-independent growth.ResultsYB-1 (27/37 or 73% of cases, P = 3.899 × 10-4) and EGFR (20/37 or 57.1% of cases, P = 9.206 × 10-12) are expressed in most cases of BLBC. However, they are not typically amplified in primary BLBC, suggesting overexpression owing to transcriptional activation. In support of this, we demonstrate that YB-1 promotes EGFR reporter activity. YB-1 specifically binds the EGFR promoter at two different YB-1-responsive elements (YREs) located at -940 and -968 using ChIP and gel shift assays in a manner that is dependent on the phosphorylation of S102 on YB-1. Inhibiting EGFR with Iressa suppressed the growth of SUM149 cells by ~40% in monolayer, independent of mutations in the receptor. More importantly anchorage-independent growth of BLBC cell lines was inhibited with combinations of Iressa and YB-1 suppression.ConclusionWe have identified for the first time a causal link for the expression of EGFR in BLBC through the induction by YB-1 where it binds specifically to two distinguished YREs. Finally, inhibition of EGFR in combination with suppression of YB-1 presents a potential opportunity for therapy in BLBC.

Id1 is a critical mediator in TGF‐β–induced transdifferentiation of rat hepatic stellate cells

Transforming growth factor (TGF)‐β is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF‐β antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)‐induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down‐regulated collagen and α‐smooth muscle actin (α‐SMA) expression. The aim of this study was to further characterize the molecular details of TGF‐β pathways that control the transdifferentiation process. In an attempt to elucidate TGF‐β target genes responsible for fibrogenesis, an analysis of Smad7‐dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7‐dependent inhibition of transdifferentiation. Moreover, knock‐down of Id1 in HSCs interfered with α‐SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF‐β1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co‐induced during fibrogenesis. In conclusion, Id1 is identified as TGF‐β/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. (HEPATOLOGY 2006;43:1032–1041.)

Effect of mean arterial pressure, haemoglobin and blood transfusion during cardiopulmonary bypass on post-operative acute kidney Injury

BACKGROUND Acute kidney injury (AKI) after cardiac surgery is a common and serious condition carrying significant costs. During cardiopulmonary bypass (CPB) surgery, modifiable factors may contribute to post-operative AKI. Their avoidance might be a potential target for nephroprotection. METHODS The objective of the present study was to identify and determine whether intraoperative hypotension, anaemia, or their combination, red blood cell transfusion or vasopressor use are independent risk factors for post-operative AKI defined by the RIFLE (renal Risk, Injury, Failure, Loss of renal function and End-stage renal disease) classification and other thresholds using a mixed logistic multivariate model. RESULTS We analysed 381 468 mean arterial pressure (MAP) measurements from 920 consecutive on-pump cardiac surgery patients. Overall, 19.5% developed AKI which was associated with an 8.2-fold increase in-hospital mortality. Haemoglobin concentration was an independent risk factor for AKI {odds ratio [OR] 1.16 per g/dL decrease [95% confidence interval (CI) 1.05-1.31]; P = 0.018} with systemic arterial oxygen saturation and pressure values not adding further strength to such an association. MAP alone or vasopressor administration was not independently associated with AKI but volume of red blood cell transfusion was, with its effect being apparent at a haemoglobin level of >8 g/dL (>5 mmol/L). In patients with severe anaemia (<25th percentile of lowest haemoglobin), the independent effect of hypotension (>75th percentile of area under the curve MAP <50 mmHg) on AKI was more pronounced [OR 3.36 (95% CI 1.34-8.41); P = 0.010]. CONCLUSION Intraoperative avoidance of the extremes of anaemia, especially during severe hypotension and avoidance of transfusion in patients with haemoglobin levels >8 g/dL (>5 mmol/L) may help decrease AKI in patients undergoing cardiac surgery and represent targets for future controlled interventions.

The etiology of liver damage imparts cytokines transforming growth factor β1 or interleukin-13 as driving forces in fibrogenesis†

It is unknown whether transforming growth factor β1 (TGF‐β1) signaling uniformly participates in fibrogenic chronic liver diseases, irrespective of the underlying origin, or if other cytokines such as interleukin (IL)‐13 share in fibrogenesis (e.g., due to regulatory effects on type I pro‐collagen expression). TGF‐β1 signaling events were scored in 396 liver tissue samples from patients with diverse chronic liver diseases, including hepatitis B virus (HBV), hepatitis C virus (HCV), Schistosoma japonicum infection, and steatosis/steatohepatitis. Phospho‐Smad2 staining correlated significantly with fibrotic stage in patients with HBV infection (n = 112, P < 0.001) and steatosis/steatohepatitis (n = 120, P < 0.01), but not in patients with HCV infection (n = 77, P > 0.05). In tissue with HBx protein expression, phospho‐Smad2 was detectable, suggesting a functional link between viral protein expression and TGF‐β1 signaling. For IL‐13, immunostaining correlated with fibrotic stage in patients with HCV infection and steatosis/steatohepatitis. IL‐13 protein was more abundant in liver tissue lysates from three HCV patients compared with controls, as were IL‐13 serum levels in 68 patients with chronic HCV infection compared with 20 healthy volunteers (72.87 ± 26.38 versus 45.41 ± 3.73, P < 0.001). Immunohistochemistry results suggest that IL‐13–mediated liver fibrogenesis may take place in the absence of phospho–signal transducer and activator of transcription protein 6 signaling. In a subgroup of patients with advanced liver fibrosis (stage ≥3), neither TGF‐β nor IL‐13 signaling was detectable. Conclusion: Depending on the cause of liver damage, a predominance of TGF‐β or IL‐13 signaling is found. TGF‐β1 predominance is detected in HBV‐related liver fibrogenesis and IL‐13 predominance in chronic HCV infection. In some instances, the underlying fibrogenic mediator remains enigmatic. (HEPATOLOGY 2009.)

Y-Box Binding Protein-1 Controls CC Chemokine Ligand-5 (CCL5) Expression in Smooth Muscle Cells and Contributes to Neointima Formation in Atherosclerosis-Prone Mice

Background— The CC chemokine CCL5/Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) is upregulated in mononuclear cells or deposited by activated platelets during inflammation and has been implicated in atherosclerosis and neointimal hyperplasia. We investigated the influence of the transcriptional regulator Y-box binding protein (YB)-1 on CCL5 expression and wire-induced neointimal hyperplasia. Methods and Results— Analysis of the CCL5 promoter revealed potential binding sites for YB-1, and interaction of YB-1 with a sequence at position −204/−173 was confirmed by DNA binding assays. Both YB-1 expression and CC chemokine ligand-5 (CCL5) mRNA expression were increased in neointimal versus medial smooth muscle cells, as analyzed by real-time polymerase chain reaction. Overexpression of YB-1 in smooth muscle cells (but not macrophages) enhanced CCL5 transcriptional activity in reporter assays, mRNA and protein expression, and CCL5-mediated monocyte arrest. Carotid arteries of hyperlipidemic apolipoprotein E-deficient mice were subjected to intraluminal transfection with a lentivirus encoding YB-1 short hairpin RNA or empty vector directly after wire injury. Double immunofluorescence revealed YB-1 expression in neointimal smooth muscle cells but not macrophages and colocalization with neointimal CCL5, which was downregulated by YB-1 short hairpin RNA. Neointima formation was decreased significantly after YB-1 knockdown compared with controls and was associated with a diminished content of lesional macrophages. A reduction of lesion formation by YB-1 knockdown was not observed in apolipoprotein E-deficient mice deficient in the CCL5 receptor CCR5 or after treatment with the CCL5 antagonist Met-RANTES, which indicates that YB-1 effects were dependent on CCL5. Conclusions— The transcriptional regulator YB-1 mediates CCL5 expression in smooth muscle cells and thereby contributes to neointimal hyperplasia, thus representing a novel target with which to limit vascular remodeling.

Y-box Protein-1 Is the Crucial Mediator of Antifibrotic Interferon-γ Effects

Y-box protein-1 (YB-1) is a known negative regulator of collagen (Col) expression by two different mechanisms, acting directly through binding to an interferon-γ response element within the col1A2 promoter and/or by physically interacting with p300/Smad3, thereby abrogating the stimulatory effect of transforming growth factor-β (TGF-β). Here, we report that YB-1 activation via the Jak1 signaling pathway is required and sufficient to confer interferon-γ-dependent activation of the smad7 gene. By binding to a bona fide recognition site within the smad7 promoter, YB-1 up-regulates smad7 transcription, which was additively enhanced by autoinhibitory TGF-β signaling. Importantly, the anti-TGF-β effect was not only supplied by induced Smad7 expression but was recapitulated in the context of the col1A2 promoter, where YB-1 overexpression abolished the trans-stimulatory TGF-β effect in a dominant fashion. In conclusion, YB-1 is the main target of interferon-γ signaling via Jak1 that exerts antifibrotic action by both interference with TGF-β signaling and direct down-regulation of collagen expression.