Anlaysis of complementary expression profiles following WT1 induction versus repression reveals the cholesterol/fatty acid synthetic pathways as a possible major target of WT1

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and other malignancies. WT1 is one of the earliest genes expressed during kidney development. WT1 proteins can activate and repress putative target genes in vitro, although the in vivo relevance of such target genes often remains unverified. To better understand the role of WT1 in tumorigenesis and kidney development, we need to identify downstream target genes. In this study, we have expression pro. led human embryonic kidney 293 cells stably transfected to allow inducible WT1 expression and mouse mesonephric M15 cells transfected with a WT1 antisense construct to abolish endogenous expression of all WT1 isoforms to identify WT1-responsive genes. The complementary overlap between the two cell lines revealed a pronounced repression of genes involved in cholesterol biosynthesis by WT1. This pathway is transcriptionally regulated by the sterol responsive element-binding proteins (SREBPs). Here, we provide evidence that the C-terminal end of the WT1 protein can directly interact with SREBP, suggesting that WT1 may modify the transcriptional function of SREBPs via a direct protein-protein interaction. Therefore, the tumour suppressor activities of WT1 may be achieved by repressing the mevalonate pathway, thereby controlling cellular proliferation and promoting terminal differentiation.

Establishment of metanephros transplantation in mice highlights contributions by both nephrectomy and pregnancy to developmental progression

Background: It has been demonstrated that embryonic kidneys (metanephroi) xenotransplanted into the omentum of adult recipients continue to develop and display immune protection due to their more nave immune presentation. To date, this has been achieved using rat, pig and human metanephroi, with unilateral nephrectomy (UNX) of recipient rats a requisite of renal development. The aim of this study was to adapt this approach for use in mice and examine the parameters affecting successful onward development in this species. Methods: Metanephroi at embryonic age (E) 13.5 were transplanted either onto the body wall, abdominal fat pads or omentum of recipient isogenic C57/Bl6 mice using either sutures or polyglycolic acid mesh. Having established greatest success with polyglycolic acid mesh on the body wall, E12.5 and 15.5 days metanephroi from C57/Bl6 mice were then transplanted onto the body wall of control (non-pregnant non-UNX), UNX or 12.5 days post-coitum pregnant isogenic recipients. After 7 days, implanted tissue was harvested and examined using histology and immunohistochemistry for markers of renal maturation. The mean number of S-shaped bodies and glomeruli per section were recorded and statistically analysed for significant differences between all recipient groups and untransplanted metanephroi. The degree of development was scored qualitatively. Results: Transplanted E12.5 metanephroi developed S-shaped bodies and glomeruli in all recipient groups, although there were statistically higher numbers of S-shaped bodies in UNX (n = 2) and pregnant recipients (n = 9) than in control recipients (n = 4). Continued development, as indicated by mature vascularized glomeruli, was only observed in those E15.5 metanephroi transplanted into pregnant recipients (n = 11) with a 15.5-fold increase in S-shaped bodies and 4-fold increase in glomeruli compared with control transplants (n = 12). Conclusions: We have successfully established metanephros transplantation in mice and demonstrated enhancement of onward development of E12.5 metanephroi in response to both pregnancy and UNX. Using E15.5 metanephroi, continued development only occurred in pregnant recipients, implying pregnancy provides an environment conducive to continued organogenesis. This murine assay, when coupled with transgenically-tagged strains of mice, will allow the investigation of the relative contribution of donor and recipient cells to this process. Copyright (C) 2005 S. Karger AG, Basel.

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter. (c) 2006 Elsevier B.V. All rights reserved.

Development of a rapid screening test for veterinary sedatives and the beta-blocker carazolol in porcine kidney by ELISA

Sedatives and tranquillisers are frequently used to reduce stress during the transportation of food producing animals. The most widely used classes of sedatives include the butyrophenone azaperone, the phenothiazines acepromazine, propionylpromazine, chlorpromazine and the beta-blocker, carazolol. For regulatory control purposes, tolerances for azaperone and carazolol have been set by the European Union as 100 and 25 mug kg(-1), respectively. Furthermore, the use of the phenothiazines is prohibited and therefore has a zero tolerance. A method for the detection of residues of five tranquillisers and one beta-blocker using a single ELISA plate has been developed. Kidney samples (2.5 g) were extracted with dichloromethane and applied to a competitive enzyme immunoassay using three polyclonal antibodies raised in rabbits against azaperol, propionylpromazine and carazolol conjugates. In sample matrix, the azaperol antibody cross-reacted 28.0% with azaperone and the propionylpromazine antibody cross-reacted 24.9% with acepromazine and 11.7% with chlorpromazine. In the ELISA, the detection capabilities of the six sedatives, azaperol, azaperone, carazolol, acepromazine, chlorpromazine, and propionylpromazine are 5, 15, 5, 5, 20 and 5 mug kg(-1), respectively. The proposed method is a sensitive and rapid multi-residue technique that offers a cost effective alternative to current published procedures, without any concession on the ability to detect sedative misuse.

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler(R) real-time PCR hybridization probe-based assay that detects a conserved region of the 16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n = 180) and aborted pig foetuses (n = 24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n = 7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n = 30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

The murine Fgfrl1 receptor is essential for the development of the metanephric kidney

Fgfrl1 is a novel member of the fibroblast growth factor receptor family. Its extracellular domain resembles the four conventional Fgfrs, while its intracellular domain lacks the tyrosine kinase domain necessary for Fgf mediated signal transduction. During embryonic development Fgfrl1 is expressed in the musculoskeletal system, in the lung, the pancreas and the metanephric kidney. Targeted disruption of the Fgfrl1 gene leads to the perinatal death of the mice due to a hypoplastic diaphragm, which is unable to inflate the lungs. Here we show that Fgfrl1-/- embryos also fail to develop the metanephric kidney. While the rest of the urogenital system, including bladder, ureter and sexual organs, develops normally, a dramatic reduction of ureteric branching morphogenesis and a lack of mesenchymal-to-epithelial transition in the nephrogenic mesenchyme result in severe renal dysgenesis. The failure of nephron induction might be explained by the absence of the tubulogenic markers Wnt4, Fgf8, Pax8 and Lim1 at E12.5 of the mutant animals. We also observed a loss of Pax2 positive nephron precursor cells and an increase of apoptosis in the cortical zone of the remnant kidney. Fgfrl1 is therefore essential for mesenchymal differentiation in the early steps of nephrogenesis.

Association of oliguria with the development of acute kidney injury in the critically ill

Urine output (UO) criterion may increase the sensitivity of the definition of acute kidney injury (AKI). We determined whether the empirically derived definition for oliguria(<0.5 ml/kg/h) is independently associated with adverse outcome. Data analysis included hourly recorded UO from the prospective, multicenter FINNAKI study conducted in 16 Finnish intensive care units. Confounder-adjusted association of oliguria of different severity and duration primarily with the development of AKI defined by creatinine criterion (Cr-AKI) or renal replacement therapy(RRT) was assessed. Secondarily, we determined the association of oliguria with 90-day mortality. Of the 1966 patients analyzed for the development of AKI, 454 (23.1%) reached this endpoint. Within this AKI cohort, 312 (68.7%)developed Cr-AKI, 21 (4.6%) commenced RRT without Cr-AKI, and 121 (26.7%) commenced RRT with Cr-AKI. Episodes of severe oliguria (<0.1 ml/kg/h) for more than 3 h were independently associated with the development of Cr-AKI or RRT. The shortest periods of consecutive oliguria independently associated with an increased risk for 90-day mortality were 6–12 h of oliguria from 0.3 to <0.5 ml/kg/h, over 6 h of oliguria from 0.1 to <0.3 ml/kg/h, and severe oliguria lasting over 3 h.Thus, our findings underlie the importance of hourly UO measurements.

Adherence to fish oil intervention in patients with chronic kidney disease

Objective: With growing recognition of the role of inflammation in the development of chronic and acute disease, fish oil is increasingly used as a therapeutic agent, but the nature of the intervention may pose barriers to adherence in clinical populations. Our objective was to investigate the feasibility of using a fish oil supplement in hemodialysis patients. ---------- Design: This was a nonrandomized intervention study.---------- Setting: Eligible patients were recruited at the Hemodialysis Unit of Wesley Hospital, Brisbane, Queensland, Australia. Patients The sample included 28 maintenance hemodialysis patients out of 43 eligible patients in the unit. Exclusion criteria included patients regularly taking a fish oil supplement at baseline, receiving hemodialysis for less than 3 months, or being unable to give informed consent.---------- Intervention: Eicosapentaenoic acid (EPA) was administered at 2000 mg/day (4 capsules) for 12 weeks. Adherence was measured at baseline and weekly throughout the study according to changes in plasma EPA, and was further measured subjectively by self-report.---------- Results: Twenty patients (74%) adhered to the prescription based on changes in plasma EPA, whereas an additional two patients self-reported good adherence. There was a positive relationship between fish oil intake and change in plasma EPA. Most patients did not report problems with taking the fish oil. Using the baseline data, it was not possible to characterize adherent patients.---------- Conclusions: Despite potential barriers, including the need to take a large number of prescribed medications already, 74% of hemodialysis patients adhered to the intervention. This study demonstrated the feasibility of using fish oil in a clinical population.

Development and validation of an expedited 10g protein counter (EP-10) for dietary protein intake quantification

Precise protein quantification is essential in clinical dietetics, particularly in the management of renal, burn and malnourished patients. The EP-10 was developed to expedite the estimation of dietary protein for nutritional assessment and recommendation. The main objective of this study was to compare the validity and efficacy of the EP-10 with the American Dietetic Association’s “Exchange List for Meal Planning” (ADA-7g) in quantifying dietary protein intake, against computerised nutrient analysis (CNA). Protein intake of 197 food records kept by healthy adult subjects in Singapore was determined thrice using three different methods – (1) EP-10, (2) ADA-7g and (3) CNA using SERVE program (Version 4.0). Assessments using the EP-10 and ADA-7g were performed by two assessors in a blind crossover manner while a third assessor performed the CNA. All assessors were blind to each other’s results. Time taken to assess a subsample (n=165) using the EP-10 and ADA-7g was also recorded. Mean difference in protein intake quantification when compared to the CNA was statistically non-significant for the EP-10 (1.4 ± 16.3 g, P = .239) and statistically significant for the ADA-7g (-2.2 ± 15.6 g, P = .046). Both the EP-10 and ADA-7g had clinically acceptable agreement with the CNA as determined via Bland-Altman plots, although it was found that EP-10 had a tendency to overestimate with protein intakes above 150 g. The EP-10 required significantly less time for protein intake quantification than the ADA-7g (mean time of 65 ± 36 seconds vs. 111 ± 40 seconds, P < .001). The EP-10 and ADA-7g are valid clinical tools for protein intake quantification in an Asian context, with EP-10 being more time efficient. However, a dietician’s discretion is needed when the EP-10 is used on protein intakes above 150g.

COX-derived prostanoid pathways in gastrointestinal cancer development and progression : novel targets for prevention and intervention

Arachidonic acid metabolism through cyclooxygenase (COX) pathways leads to the generation of biologically active eicosanoids. Eicosanoid expression levels vary during development and progression of gastrointestinal (GI) malignancies. COX-2 is the major COX-isoform responsible for G.I. cancer development/progression. COX-2 expression increases during progression from a normal to cancerous state. Evidence from observational studies has demonstrated that chronic NSAID use reduces the risk of cancer development, while both incidence and risk of death due to G.I. cancers were significantly reduced by daily aspirin intake. A number of randomized controlled trials (APC trial, Prevention of Sporadic Adenomatous Polyps trial, APPROVe trial) have also shown a significant protective effect in patients receiving selective COX-2 inhibitors. However, chronic use of selective COX-2 inhibitors at high doses was associated with increased cardiovascular risk, while NSAIDs have also been associated with increased risk. More recently, downstream effectors of COX-signaling have been investigated in cancer development/progression. PGE 2, which binds to both EP and PPAR receptors, is the major prostanoid implicated in the carcinogenesis of G.I. cancers. The role of TXA 2 in G.I. cancers has also been examined, although further studies are required to uncover its role in carcinogenesis. Other prostanoids investigated include PGD 2 and its metabolite 15d-PGJ2, PGF 1α and PGI 2. Targeting these prostanoids in G.I. cancers has the promise of avoiding cardiovascular toxicity associated with chronic selective COX-2 inhibition, while maintaining anti-tumor reactivity.A progressive sequence from normal to pre-malignant to a malignant state has been identified in G.I. cancers. In this review, we will discuss the role of the COX-derived prostanoids in G.I. cancer development and progression. Targeting these downstream prostanoids for chemoprevention and/or treatment of G.I. cancers will also be discussed. Finally, we will highlight the latest pre-clinical technologies as well as avenues for future investigation in this highly topical research field. © 2011 Elsevier B.V.

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI2/TXA2 ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer. © 2010 Elsevier B.V. All rights reserved.

Increased tubulointerstitial recruitment of human CD141(hi) CLEC9A(+) and CD1c(+) myeloid dendritic cell subsets in renal fibrosis and chronic kidney disease

Dendritic cells (DCs) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated, and phenotyped them by multicolor flow cytometry. We detected significantly greater numbers of total DCs as well as CD141(hi) and CD1c(+) myeloid DC (mDCs) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy kidney tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared with healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141(hi) DCs expressed C-type lectin domain family 9 member A (CLEC9A), whereas the majority of CD1c(+) DCs lacked the expression of CD1a and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), suggesting these mDC subsets may be circulating CD141(hi) and CD1c(+) blood DCs infiltrating kidney tissue. Our analysis revealed CLEC9A(+) and CD1c(+) cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared with healthy tissue. Transforming growth factor-β levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared with nonfibrotic biopsies, with mDCs identified as a major source of this profibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and, thus, progression to chronic kidney disease.