The association between symmetric dimethylarginine concentrations and various neoplasms in dogs and cats.

Following the introduction of the symmetric dimethylarginine (SDMA) immunoassay, cases were reported where the SDMA concentration was markedly increased above the reference interval (RI) with neither concurrent increases in serum creatinine (Cr) concentrations nor clinical signs of kidney disease. Many of these animals were also concurrently diagnosed with cancer, most commonly lymphoma. The purpose of the study was to evaluate the association of increased SDMA in dogs and cats with lymphoma and other cancers as compared with age- and breed-matched non-tumour controls. In this retrospective case-control study, serum chemistry results from 1804 tumour cases, and age- and breed-matched non-tumour control animals were used. Matched-pair odds ratios between animals diagnosed with neoplasms and non-tumour controls for dichotomized SDMA values were determined by tumour type. SDMA concentrations were significantly higher in dogs and cats with lymphoma (p < .0001) compared with non-tumour controls. The odds ratio for increased SDMA concentrations in dogs with lymphoma was 10.0 (95% CI, 5.98-16.72) and for cats with lymphoma was 3.04 (95% CI 1.95-4.73). A significant number of canine and feline lymphoma cases had an increased SDMA concentration not associated with an increased Cr concentration (p < .001). Canine and feline lymphoma patients have an increased odds of having a SDMA concentration above the RI at diagnosis. Further characterization and evaluation of dogs and cats with lymphoma is required to help understand the mechanism(s) and the clinical significance of these alterations.

Evaluation of renal injury and function biomarkers, including symmetric dimethylarginine (SDMA), in the rat passive Heymann nephritis (PHN) model.

Symmetric dimethylarginine (SDMA) is a serum biomarker of excretory renal function which consistently correlates with glomerular filtration rate (GFR) across multiple species including rats, dogs, and humans. In human and veterinary clinical settings SDMA demonstrates enhanced sensitivity for detection of declining renal function as compared to other serum biomarkers, but application in preclinical study designs thus far has been limited. The purpose of this study was to determine the performance of serum SDMA in a rat passive Heyman nephritis model of glomerulopathy. In addition to SDMA other biomarkers of excretory renal function were measured including serum creatinine (sCr), blood urea nitrogen (BUN), and cystatin C along with creatinine clearance. Urinary renal biomarkers including microalbumin (µALB), clusterin (CLU), cystatin C, kidney injury marker-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin (OPN) were also measured. PHN was induced using commercial sheep anti-Fx1A serum. Tissue, serum, and urine were collected from groups of control and anti-Fx1A-treated animals for biomarker evaluation, hematology, urinalysis, serum biochemistry, and histologic examination of kidney. Over the course of a 28-day study, concentrations of the urinary biomarkers µALB, CLU, cystatin C, NGAL, KIM-1 and the serum biomarker cystatin C increased significantly in anti-Fx1A-treated rats as compared to controls but no significant increase in serum SDMA, sCr, BUN, or creatinine clearance were noted in anti-Fx1A-treated rats. Given lack of direct GFR measurement or significant change in the renal function biomarkers sCr, BUN, and creatinine clearance, it is unclear if GFR differed significantly between control and anti-Fx1A-treated rats in this study, though urinary biomarkers and histopathologic findings supported renal injury in anti-Fx1A-treated rats over the time course investigated. This study is among the first to investigate serum SDMA in a rat model relevant to preclinical safety assessment and serves to inform future experimental designs and biomarker selection when evaluation of glomerular injury is of priority.

Evaluation of Renal Biomarkers, Including Symmetric Dimethylarginine, following Gentamicin-Induced Proximal Tubular Injury in the Rat.

Symmetric dimethylarginine (SDMA) is an excretory renal function biomarker shown to correlate well with glomerular filtration rate in dogs, cats, humans, and rats. The objectives of this study were to determine utility of serum SDMA as a renal biomarker in a rat model of gentamicin-induced renal injury and to provide validation of a commercially available SDMA immunoassay for rat serum. Rats were randomly assigned to one of three dose levels of gentamicin (20, 50, or 100 mg/kg) or a vehicle control group and dosed once daily by subcutaneous injection for either four or ten days. Serum and urine renal biomarker evaluation, including serum SDMA, hematologic and serum biochemical analysis, urinalysis, and histologic examination of kidney, were performed. Before biologic validation, analytic validation of the SDMA immunoassay for rat serum was performed, including assessment of assay accuracy, precision, analytical sensitivity, linearity, analyte stability, and interference testing. Among markers of excretory renal function, SDMA and serum creatinine increased earliest and at the lowest gentamicin concentrations and were significantly increased in both the 50- and 100- mg/kg dose levels in the four- and ten-dose treatment groups compared with controls. Time- and dose-dependent increases were noted for all urinary biomarkers investigated in this study, with microalbumin being most responsive and osteopontin least responsive for detection of gentamicin-induced injury across dose levels and schedules investigated. The SDMA immunoassay met all set quality requirements assessed in analytical validation. This study is the first to investigate performance of serum SDMA compared with other excretory renal function markers in a rat gentamicin acute toxicity model. In this study, serum SDMA was an earlier biomarker for detection of gentamicin-induced toxicity than serum cystatin C, BUN, and creatinine clearance. The SDMA immunoassay provides a reliable commercially available assay for future renal investigations in rat models.

Differential expression of NADPH oxidases in megakaryocytes and their role in polyploidy.

Megakaryocytes (MKs) undergo an endomitotic cell cycle, leading to polyploidy. We examined the expression of the flavoproteins and oxidative stress-promoting enzymes, NADPH oxidases (Nox's), in MKs because of their known role in promoting the cell cycle. Although the expression of Nox isoforms varies between cell types, they are induced at the mRNA level by mitogenic stimuli. Western blotting or reverse transcription-polymerase chain reaction of purified mouse MKs isolated from thrombopoietin (TPO)-treated bone marrow (BM) cultures indicated high expression of Nox1, a weak expression of Nox4, and no significant expression of Nox2. Immunofluorescence of freshly isolated MKs confirmed strong expression of Nox1 in one-third of MKs, whereas Nox1 staining was detected in nearly all MKs in TPO-stimulated BM cultures. Treatment of mouse BM cultures with Nox inhibitors resulted in accumulation of MKs with low DNA content levels and significant reduction of higher ploidy MKs. Purified, Nox-inhibited MKs showed a notable decrease in the level of the G(1) phase cyclin E, a cyclin associated with MK polyploidy, and its up-regulation restored most of the effect of Nox inhibitors. Hence, this study shows the expression of Nox isoforms in MKs and highlights a potential role of flavoproteins in promoting polyploidization in this lineage.

Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy.

Our recent reports indicated that polyploidization of aortic vascular smooth muscle cells (VSMC) serves as a biomarker for aging, and that the polyploid state is linked to a higher incidence of senescence in vivo. Here, we found that NADPH oxidase 4 (Nox4) expression is augmented in VSMC from aortas of old rats and that Nox4 levels are increased in polyploid VSMC in comparison to diploid cells in vivo. Seeking to determine if Nox4 upregulation plays a causal role in the accumulation of polyploid cells, we performed ploidy analysis on primary VSMC transduced with Nox4 adenovirus. We observed a consistent accumulation of polyploid cells and a concomitant decrease in the percentage of diploid cells in Nox4 overexpressing cells in comparison to controls or to cells overexpressing dominant negative Nox4. Further exploration of this phenomenon in VSMC cultures identified a Nox4-induced decrease in the chromosome passenger protein, survivin, whose absence and mislocalization during polyploidization was previously shown to induce VSMC polyploidy. Taken together, our study is the first to show increased Nox4 levels in VSMC during aging, and to demonstrate its role in induction of polyploidy in this lineage.

Survivin overexpression alone does not alter megakaryocyte ploidy nor interfere with erythroid/megakaryocytic lineage development in transgenic mice.

The level of survivin was reported to be scarce in mouse megakaryocytes (MKs) compared with erythroid cells. Considering this finding and previously reported in vitro data showing decreased MK ploidy upon retroviral-mediated overexpression of survivin, we sought to examine whether ectopic survivin expression in the MK lineage might alter ploidy level in vivo. Here we report the generation of 2 tissue specific hematopoietic transgenic mouse models, one expressing survivin in both the erythroid and MK lineages and the other expressing survivin solely in the MK lineage. Survivin protein overexpression was confirmed in MKs and erythrocytes. Surprisingly, analysis of both transgenic mouse lines showed no detectable changes in MK number, ploidy level, and platelet and erythrocyte counts, as compared with control mice. We conclude that elevated survivin expression does not alter MK/erythroid lineage development and that elevated survivin, alone, does not interfere with MK ploidy in vivo.

Vascular smooth muscle cell polyploidy: an adaptive or maladaptive response?

Polyploidy is a state in which a cell contains multiple copies of its entire genome, while a normal diploid cell contains only two sets of homologous chromosomes. Although widely studied and pervasive in nature, the signals and mechanisms of polyploidization and its accompanying operational consequences are still unclear. This review focuses on relevant questions in deciphering the regulation of polyploidization of vascular smooth muscle cells (VSMC) in mammals and the role of polyploidy in various vascular pathologies, such as hypertension and aging. Additionally, we will explore new investigations in polyploidization of VSMCs involving the rapidly expanding fields of oxidative stress and senescence. J. Cell. Physiol. 215: 588-592, 2008. (c) 2008 Wiley-Liss, Inc.

The A2b adenosine receptor protects against vascular injury.

The A2b adenosine receptor (A2bAR) is highly abundant in bone marrow macrophages and vascular smooth muscle cells (VSMC). To examine the functional significance of this receptor expression, we applied a femoral artery injury model to A2bAR knockout (KO) mice and showed that the A2bAR prevents vascular lesion formation in an injury model that resembles human restenosis after angioplasty. While considering related mechanisms, we noted higher levels of TNF-alpha, an up-regulator of CXCR4, and of VSMC proliferation in the injured KO mice. In accordance, CXCR4, which is known to attract progenitor cells during tissue regeneration, is up-regulated in lesions of the KO mice. In addition, aortic smooth muscle cells derived from A2bAR KO mice display greater proliferation in comparison with controls. Bone marrow transplantation experiments indicated that the majority of the signal for lesion formation in the null mice originates from bone marrow cells. Thus, this study highlights the significance of the A2bAR in regulating CXCR4 expression in vivo and in protecting against vascular lesion formation.

Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence.

We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16(INK4a) and senescence-associated beta-galactosidase activity are detected primarily in the old tissues. VSMC from p16(INK4a) knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.

Alphav beta6 integrin regulates renal fibrosis and inflammation in Alport mouse.

The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.

Vascular smooth muscle cell polyploidization involves changes in chromosome passenger proteins and an endomitotic cell cycle.

Vascular smooth muscle cell polyploidization occurs during normal development and is enhanced under physiologic stress, but the mechanism of this cell cycle has not been explored. We show via time-lapse video imaging and immunofluorescence analyses that primary vascular smooth muscle cells (VSMC) undergo an endomitotic-type cell cycle, including a normal progression through part of mitosis. Mononuclear polyploid cells are generated by defects in sister chromatid separation and/or segregation, and cellular binucleation occurs by reversal of cytokinesis. To obtain further leads to regulators involved, we examined the chromosomal passenger proteins, Aurora B, inner centromere protein and Survivin, and concluded that Aurora B and inner centromere protein are normally colocalized in centromeres, the midzone, and the midbody during mitosis. Survivin, however, is dim and diffused; it does not colocalize with either Aurora B or inner centromere protein in VSMC, which could account for defects in sister chromatid separation and/or segregation and reversal of cytokinesis. In accordance with the reported dependency of Aurora B activity on Survivin, the Aurora B substrate, vimentin, is not phosphorylated during cytokinesis. Finally, the data show that ectopically expressed Survivin inhibits polyploidization in vascular smooth muscle cells. Hence, aberrant chromosome passenger protein activity and endomitosis are associated with VSMC polyploidization.

Culture-based group B streptococcal screening. Adherence to current guidelines.

OBJECTIVE: To examine the ability of a universal screening strategy to identify and treat group B Streptococcus-positive women with intrapartum antibiotics. STUDY DESIGN: Charts were reviewed on all patients delivering at > or = 36 weeks of gestation as to the presence of culture results, whether or not antibiotics were ordered and when they were given relative to the time of delivery. RESULTS: Approximately 95% of patients presenting at > or = 36 weeks had group B Streptococcus results available. Overall, 84% of culture-positive patients received chemoprophylaxis. Removal of the elective cesareans from the study population increased the percentage of positive patients receiving antibiotics to 94%. Ideal chemoprophylaxis, defined as delivering > or = 2 hours after receiving the recommended antibiotics, occurred 87% of the time. Failure to order appropriate antibiotics was the most frequent reason for not receiving chemoprophylaxis. CONCLUSION: Successful universal culturing followed by intrapartum chemoprophylaxis can be accomplished in a majority of cases. Failure to treat can occur (6%), and chemoprophylaxis may be less than ideal (13%) due to rapid labor and human error.

Nonbilharzial bladder carcinoma complicating pregnancy: review of the literature.

UNLABELLED: The purpose of this review is to evaluate tumor presentation and characteristics, and maternal-fetal outcomes of pregnancies complicated by nonbilharzial bladder carcinoma. The mean age of the patients was 29.5 years (range = 18-40). Symptoms and diagnosis occurred after the first trimester in 20 (83%) and 22 (92%) cases, respectively. Presenting complaints included painless gross hematuria [N = 12 (50%)], vaginal bleeding [N = 7 (29%)], dysuria [N = 2 (8.4%)], abdominal pain [N = 2 (8.4%)], and 1 instance each of urgency, frequency, recurrent cystitis, and no symptoms. Tumors were initially identified by ultrasound [N = 12 (50%)], cystoscopy [N = 11 (46%)], and intravenous urography [N = 1 (4.5%)]. Transitional cell carcinoma was found in 17 (74%), adenocarcinoma in 5 (22%), and squamous cell carcinoma in 1 (4.5%) patient. Tumors did not favor a specific bladder location, tended to be low grade [8 (40%) = grade 1, 7 (35%) = grade 2; 5 (21%) = grade 3], and noninvasive [N = 19 (79%)]. Treatment was typically by transurethral resection (N = 18), but 3 women required radical cystectomy, 2 received radiation, 1 received chemotherapy, and 1 underwent partial cystectomy. Three (14%) women died of their disease and 3 (14%) fetuses were lost because of complications of cancer or its treatment. Bladder carcinoma in pregnancy can mimic cystitis or obstetric hemorrhage and should be considered when evaluations for these conditions are negative. Routine ultrasound evaluation of the bladder in these patients may improve the diagnostic yield. Pregnancy is not a contraindication to treating most forms of bladder cancer. TARGET AUDIENCE: Obstetricians and Gynecologists, Family Physicians. LEARNING OBJECTIVES: After completion of this article, the reader will be able to list the various types of bladder cancers, to describe the presenting symptoms in a patient with a bladder cancer, and to outline the work up and treatment strategies for bladder cancer.