Norbert Gretz

Mutations in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and hepatic fibrosis.

Nephronophthisis (NPHP), a group of autosomal recessive cystic kidney disorders, is the most common genetic cause of progressive renal failure in children and young adults. NPHP may be associated with Leber congenital amaurosis, tapeto-retinal degeneration, cerebellar ataxia, cone-shaped epiphyses, congenital oculomotor apraxia and hepatic fibrosis. Loci associated with an infantile type of NPHP on 9q22–q31 (NPHP2), juvenile types of NPHP on chromosomes 2q12–q13 (NPHP1) and 1p36 (NPHP4) and an adolescent type of NPHP on 3q21–q22 (NPHP3) have been mapped. NPHP1 and NPHP4 have been identified, and interaction of the respective encoded proteins nephrocystin and nephrocystin-4 has been shown. Here we report the identification of NPHP3, encoding a novel 1,330-amino acid protein that interacts with nephrocystin. We describe mutations in NPHP3 in families with isolated NPHP and in families with NPHP with associated hepatic fibrosis or tapeto-retinal degeneration. We show that the mouse ortholog Nphp3 is expressed in the node, kidney tubules, retina, respiratory epithelium, liver, biliary tract and neural tissues. In addition, we show that a homozygous missense mutation in Nphp3 is probably responsible for the polycystic kidney disease (pcy) mouse phenotype. Interventional studies in the pcy mouse have shown beneficial effects by modification of protein intake and administration of methylprednisolone, suggesting therapeutic strategies for treating individuals with NPHP3.

Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia.

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.

Angiotensin II Type 1 Receptor Overexpression in Podocytes Induces Glomerulosclerosis in Transgenic Rats

Angiotensin II (AngII) is a critical determinant of glomerular function involving both hemodynamic and pressure-independent effects that are insufficiently understood. A novel transgenic rat (TGR) model with overexpression of the human AngII type 1 receptor (hAT1) in podocytes was developed to study the consequences of an increased AT1 signaling on the structure and function of the glomerular filter. Use of the nephrin promoter to target the podocytes resulted in an expression of the hAT1 at a level roughly two times higher than the endogenous AT1 throughout life. All male TGR developed significant albuminuria starting at 8 to 15 wk of age; systolic BP was not elevated. More or less concurrently, structural changes at the glomerulus were encountered, starting with ubiquitous formation of pseudocysts at podocytes, followed by foot process effacement and local detachments. This damage progressed to nephron loss via the well known pathway typical for classic focal segmental glomerulosclerosis. The structural changes significantly correlated with age (r(2) = 0.76) and urinary albumin excretion (r(2) = 0.70). The data provide direct evidence that increased AT1 signaling in podocytes leads to protein leakage and structural podocyte damage progressing to focal segmental glomerulosclerosis.

Group testing for pathway analysis improves comparability of different microarray datasets

MOTIVATION The wide use of DNA microarrays for the investigation of the cell transcriptome triggered the invention of numerous methods for the processing of microarray data and lead to a growing number of microarray studies that examine the same biological conditions. However, comparisons made on the level of gene lists obtained by different statistical methods or from different datasets hardly converge. We aimed at examining such discrepancies on the level of apparently affected biologically related groups of genes, e.g. metabolic or signalling pathways. This can be achieved by group testing procedures, e.g. over-representation analysis, functional class scoring (FCS), or global tests. RESULTS Three public prostate cancer datasets obtained with the same microarray platform (HGU95A/HGU95Av2) were analyzed. Each dataset was subjected to normalization by either variance stabilizing normalization (vsn) or mixed model normalization (MMN). Then, statistical analysis of microarrays was applied to the vsn-normalized data and mixed model analysis to the data normalized by MMN. For multiple testing adjustment the false discovery rate was calculated and the threshold was set to 0.05. Gene lists from the same method applied to different datasets showed overlaps between 42 and 52%, while lists from different methods applied to the same dataset had between 63 and 85% of genes in common. A number of six gene lists obtained by the two statistical methods applied to the three datasets was then subjected to group testing by Fishers exact test. Group testing by GSEA and global test was applied to the three datasets, as well. Fishers exact test followed by global test showed more consistent results with respect to the concordance between analyses on gene lists obtained by different methods and different datasets than the GSEA. However, all group testing methods identified pathways that had already been described to be involved in the pathogenesis of prostate cancer. Moreover, pathways recurrently identified in these analyses are more likely to be reliable than those from a single analysis on a single dataset.

Yes-Associated Protein Up-regulates Jagged-1 and Activates the NOTCH Pathway in Human Hepatocellular Carcinoma

BACKGROUND & AIMS Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear. METHODS By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. RESULTS Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. CONCLUSIONS The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).

The proteoglycan biglycan regulates expression of the B cell chemoattractant CXCL13 and aggravates murine lupus nephritis

CXCL13 is a key B cell chemoattractant and marker of disease activity in patients with SLE; however, the mechanism of its induction has not been identified yet. Here, we have shown that the proteoglycan biglycan triggers CXCL13 expression via TLR2/4 in macrophages and dendritic cells. In vivo, levels of biglycan were markedly elevated in the plasma and kidneys of human SLE patients and lupus-prone (MRL/lpr) mice. Overexpression of soluble biglycan in MRL/lpr mice raised plasma and renal levels of CXCL13 and caused accumulation of B cells with an enhanced B1/B cell ratio in the kidney, worsening of organ damage, and albuminuria. Importantly, biglycan also triggered CXCL13 expression and B cell infiltration in the healthy kidney. Conversely, biglycan deficiency improved systemic and renal outcome in lupus-prone mice, with lower levels of autoantibodies, less enlargement of the spleen and lymph nodes, and reduction in renal damage and albuminuria. This correlated with a marked decline in circulating and renal CXCL13 and a reduction in the number of B cells in the kidney. Collectively, our results describe what we believe to be a novel mechanism for the regulation of CXCL13 by biglycan, a host-derived ligand for TLR2/4. Blocking biglycan-TLR2/4 interactions might be a promising strategy for the management of SLE and other B cell-mediated inflammatory disease entities.

Gene expression profiling of CD34+ cells identifies a molecular signature of chronic myeloid leukemia blast crisis.

Despite recent success in the treatment of early-stage disease, blastic phase (BP) of chronic myeloid leukemia (CML) that is characterized by rapid expansion of therapy-refractory and differentiation-arrested blasts, remains a therapeutic challenge. The development of resistance upon continuous administration of imatinib mesylate is associated with poor prognosis pointing to the need for alternative therapeutic strategies and a better understanding of the molecular mechanisms underlying disease progression. To identify transcriptional signatures that may explain pathological characteristics and aggressive behavior of BP blasts, we performed comparative gene expression profiling on CD34+ Ph+ cells purified from patients with untreated newly diagnosed chronic phase CML (CP, n=11) and from patients in BP (n=9) using Affymetrix oligonucleotide arrays. Supervised microarray data analysis revealed 114 differentially expressed genes (P<10−4), 34 genes displaying more than two-fold transcriptional changes when comparing CP and BP groups. While 24 of these genes were downregulated, 10 genes, especially suppressor of cytokine signalling 2 (SOCS2), CAMPATH-1 antigen (CD52), and four human leukocyte antigen-related genes were strongly overexpressed in BP. Expression of selected genes was validated by real-time-polymerase chain reaction and flow cytometry. Our data suggest the existence of a common gene expression profile of CML-BP and provide new insight into the molecular phenotype of blasts associated with disease progression and high malignancy.

Is gender a determinant for evolution of renal failure? A study in autosomal dominant polycystic kidney disease.

More males than females enter renal replacement therapy programs. This may reflect greater propensity of men to acquire renal disease, faster progression of renal disease, or a combination of both. In order to address this problem, autosomal dominant polycystic kidney disease (ADPKD), a well-defined genetically homogenous hereditary disorder, was studied. One hundred fifty-eight cases of the disease in adults were diagnosed by sonography and studied (73 men, 85 women); 58 of the patients had reached end-stage renal failure. Survival analysis of age at renal death revealed a significant gender difference (log-rank test, P = 0.0072): median age at renal death was 52.5 years in men and 58.0 years in women. In 64 patients with adequate sequential measurements of serum creatinine, progression of renal failure was followed retrospectively. When serum creatinine was greater than 3 mg/dL, the average rate of progression was similar in both sexes. In contrast to ADPKD, a sex difference for the age at renal death was not found in prepubertal individuals with hereditary renal diseases, ie, cystinosis or nephronophthisis. The data suggest that sex (hormones) influences evolution of renal failure.

Coactivator function of RIP140 for nf{kappa}b/rela-dependent cytokine gene expression

Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor kappaB (NFkappaB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that RIP140 suppresses the expression of metabolic gene networks, but we have found that genetic as well as acute deficiency of RIP140 leads to the inhibition of the proinflammatory program in macrophages. The ability of RIP140 to function as a coactivator for cytokine gene promoter activity relies on direct protein-protein interactions with the NFkappaB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP). RIP140-dependent control of proinflammatory gene expression via RelA/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.

Microarray-based approach identifies microRNAs and their target functional patterns in polycystic kidney disease.

BackgroundMicroRNAs (miRNAs) play key roles in mammalian gene expression and several cellular processes, including differentiation, development, apoptosis and cancer pathomechanisms. Recently the biological importance of primary cilia has been recognized in a number of human genetic diseases. Numerous disorders are related to cilia dysfunction, including polycystic kidney disease (PKD). Although involvement of certain genes and transcriptional networks in PKD development has been shown, not much is known how they are regulated molecularly.ResultsGiven the emerging role of miRNAs in gene expression, we explored the possibilities of miRNA-based regulations in PKD. Here, we analyzed the simultaneous expression changes of miRNAs and mRNAs by microarrays. 935 genes, classified into 24 functional categories, were differentially regulated between PKD and control animals. In parallel, 30 miRNAs were differentially regulated in PKD rats: our results suggest that several miRNAs might be involved in regulating genetic switches in PKD. Furthermore, we describe some newly detected miRNAs, miR-31 and miR-217, in the kidney which have not been reported previously. We determine functionally related gene sets, or pathways to reveal the functional correlation between differentially expressed mRNAs and miRNAs.ConclusionWe find that the functional patterns of predicted miRNA targets and differentially expressed mRNAs are similar. Our results suggest an important role of miRNAs in specific pathways underlying PKD.