Urinary neutrophil gelatinase-associated lipocalin as early biomarker for renal disease in dogs with leishmaniosis.

Canine leishmaniosis (CanL), caused by Leishmania sp., presents a wide array of symptoms; renal dysfunction is frequently observed in these dogs and is associated with a poor prognosis and increased mortality. The traditional biomarkers namely urea and creatinine can detect renal damage but only in advanced stages of the disease. However, it has been shown that the symmetric dimethylarginine assay (SDMA) or the protein/creatinine ratio (UPC) and are early biomarkers of renal dysfunction. Their elevation occurs earlier than that of creatinine, but other novel biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL) are currently under investigation. Our objective was to determine whether the urine NGAL-creatinine ratio (uNGAL/c) can provide very early diagnosis of kidney disease in CanL. In total, 68 dogs were included in the study: 15 healthy dogs and 53 dogs with CanL who were classified according to International Renal Interest Society (IRIS) classification: IRIS 1 (N= 34), IRIS 2 (N= 9) and IRIS 3/4 (N= 10). IRIS 1 was subdivided according to proteinuria in IRIS 1NP (13 dogs with UPC < 0.2), IRIS 1BL (8 dogs with UPC = 0.2-0.5) and IRIS 1 P (13 dogs with UPC > 0.5). Blood samples were collected for complete hematological and biochemistry analysis including plasma NGAL. Urinalysis included specific gravity, UPC, CysC and NGAL expressed as a ratio with creatinine. The mean concentrations of pCysC and SDMA in CanL, show a statistically significant increase from IRIS 1NP, not being statistically significant for pCysC in the IRIS 1BL group. The UPC show a statistically significant increase from IRIS 1NP. In all groups with CanL for uCysC/c and uNGAL/c was observed a statistically significant increase. The uNGAL/c in the group proteinuric animals, presents a positive correlation with all renal biomarkers studied. In the group of non-proteinuric animals, the uNGAL/c presents a positive correlation with SDMA and UPC. The uNGAL/c can be considered a reliable indicator of renal disease in dogs diagnosed with CanL who are non-azotemic and non-proteinuric.

Human sperm phosphoproteome reveals differential phosphoprotein signatures that regulate human sperm motility.

Human sperm motility is essential for fertilization and among pathologies underlying male infertility is asthenozoospermia. Nevertheless, mechanisms regulating sperm motility are not completely unraveled. This work investigates phosphoproteins underlying human sperm motility by using differential phosphoproteomic in two human sperm subpopulations: high (HM) and low (LM) motility, obtained by centrifugation in a density gradient. Phosphoproteomics (HPLC-MS/MS triple TOF), comparing human LM and HM phosphoproteomes, identified 210 phosphopeptides with different abundance that correspond with 119 sperm proteins. Analysis showed that 40% of phosphoproteins in LM spermatozoa are involved in metabolism, (catabolism, protein transport, lipid biosynthesis), 25% in spermatogenesis and sperm function, 8% in immune system and 6% in DNA repair. In HM spermatozoa, 48% of phosphoproteins are related to spermatogenesis and sperm function (motility), whereas 8% are associated to metabolism. GSK3α resulted one of the most abundant phosphoproteins in HM spermatozoa. Western blot confirmed that GSK3α phosphorylation is higher in HM spermatozoa. Summarizing, this study i) identified phosphoproteins in two human spermatozoa populations, ii) supports that human spermatozoa rely in protein phosphorylation, such as GSK3 α, to regulate sperm motility, iv) raises the challenge of using some identified human sperm phosphorylated proteins (GSK3α) as targets to develop into clinically relevant biomarkers. SIGNIFICANCE: Human sperm phosphoproteome analyzed by nano HPLC-MS/MS triple TOF identifies the differential abundance of sperm phosphoproteins in two human sperm populations exhibiting high motility (HM) and/or low motility (LM) that were isolated from normozoospermic healthy donors. Majority of human phosphoproteins found in LM spermatozoa are involved in sperm metabolism (40%), whereas those in HM spermatozoa are associated to spermatogenesis and sperm function, as motility (48%), and only 8% are associated to metabolism. One of the most abundant phosphoproteins found in HM spermatozoa is GSK3α, kinase directly involved in the regulation of sperm motility that was also validated by western blot. The biological relevance of this study is based in the fact that supports that mature human sperm cells rely in protein phosphorylation to efficiently regulate sperm motility and allows identifying those regulatory human sperm phosphoproteins. This work will clearly impacts the human reproductive field as it raises the challenge of consider identified human sperm phosphoproteins, such as GSK3α, as potential biological targets to develop into relevant biomarkers for the human clinic or assisted reproductive technology.

A plasma calcium-phosphorus product can be used to predict the lifespan of dogs with chronic kidney disease.

A serum calcium-phosphorus (sCaPP) product was assessed for prediction of survival in dogs affected with chronic kidney disease (CKD). Dogs (N = 150) were retrospectively studied and followed up to determine their lifespan using 25 healthy dogs as controls. Blood and urine analyses were performed and blood pressure was measured. The dogs were divided into groups according to sCaPP (higher or lower than 70 mg2/dL2) and International Renal Interest Society (IRIS) stage (IRIS 1-4). Shorter survival was observed with sCaPP > 70 mg2/dL2 compared to dogs with sCaPP < 70 mg2/dL2 [45.48 days (range: 5.8 to 149 days) versus 505.40 days (range: 113.31 to 539.52 days), mean (95% confidence interval); P ≤ 0.001 respectively]. Similarly, dogs with advanced IRIS stages showed higher levels of sCaPP [mean (95% confidence interval) in mg2/dL2; IRIS 1: 42.83 (range: 29.58 to 62.10); IRIS 2: 63.18 (range: 46.34 to 90.09); IRIS 3: 95.57 (range: 88.34 to 127.19); IRIS 4: 130.38 (range: 125.16 to 153.52)], accompanied by lower survival rates. Therefore, sCaPP could represent a valuable tool in the prognosis of canine CKD.

Un produit plasmatique calcium-phosphore peut être utilisé pour prédire la durée de vie de chiens avec une maladie rénale chronique. Un produit sérique calcium-phosphore (sCaPP) fut évalué pour prédire la survie de chiens souffrant de maladie rénale chronique (CKD). Des chiens (N = 150) furent étudiés rétrospectivement et suivis pour déterminer leur survie en utilisant 25 chiens en santé comme témoins. Des analyses urinaires et sanguines furent effectuées et la pression sanguine fut mesurée. Les chiens furent divisés en groupes en fonction de leur sCaPP (plus élevé ou plus faible que 70 mg2/dL2) et de leurs stages selon l'International Renal Interest Society (IRIS) (IRIS 1­4). Un temps de survie plus court fut observé avec une sCaPP > 70 mg2/dL2 comparativement aux chiens avec une sCaPP < 70 mg2/dL2 [45,48 jours (varie de 5,8 à 149 jours) versus 505,40 jours (varie de 113,31 à 539,52 jours), moyenne (intervalle de confiance 95 %); P ≤ 0,001 respectivement]. De manière similaire, les chiens avec un stages IRIS avancé avaient des niveaux de sCaPP plus élevés [moyenne (intervalle de confiance 95 %) en mg2/dL2; IRIS 1 : 42,83 (varie de 29,58 à 62,10); IRIS 2 : 63,18 (varie de 46,34 à 90,09); IRIS 3 : 95,57 (varie de 88,34 à 127,19); IRIS 4 : 130,38 (varie de 125,16 à 153,52], accompagnés de taux de survie plus bas. Ainsi, la valeur de sCaPP pourrait représenter un outil utile dans le pronostic des maladies rénales chroniques chez le chien.(Traduit par Dr Serge Messier).

Effect of exercise on serum markers of muscle inflammation in Spanish Greyhounds.

OBJECTIVE: To investigate effects of exercise on hematologic and biochemical values (especially markers of inflammation and muscle damage) in Spanish Greyhounds used for hunting without previous training. ANIMALS: 32 Spanish Greyhounds and 31 dogs of other breeds. PROCEDURES: Hematologic variables and concentrations of C-reactive protein (CRP) and other biochemical markers were compared in samples obtained from Spanish Greyhounds 24 hours after exercise (eg, a hunting race) and 2 months after exercise (ie, at rest) and from non-Spanish Greyhounds at rest. All dogs were healthy. Hematologic and biochemical analyses were performed within 24 hours after samples were obtained, and results were compared by means of a Student t test. RESULTS: CRP concentration and muscle enzyme (creatine kinase, lactate dehydrogenase, and aspartate aminotransferase) activities were significantly higher and serum iron concentration was significantly lower for Spanish Greyhounds after exercise than at rest. The WBC and neutrophil counts were significantly higher after exercise then at rest. Plasma alanine transaminase activity and total protein, calcium, and phosphorus concentrations were significantly higher after exercise than at rest. Spanish Greyhounds at rest had higher RBC counts, PCVs, and hemoglobin concentrations and lower WBC, neutrophil, and lymphocyte counts, compared with values for non-Spanish Greyhounds at rest. CONCLUSIONS AND CLINICAL RELEVANCE: Exercise of Spanish Greyhounds without prior training activated an acute-phase response represented by an increase in serum CRP concentration and decrease in serum albumin and iron concentrations. These changes, along with leukocytosis and neutrophilia, were indicative of a subclinical inflammatory state in Spanish Greyhounds.

Accuracy of ultrasonographic measurements of kidney dog for clinical use

The aim of this study was to determine the accuracy of ultrasonographic measurement of canine kidneys. A method agreement analysis comparing pairs of measurements (ultrasonographic kidney dimensions and direct kidney dimensions) was performed. Nineteen dogs aging from one to thirteen years were used. The ultrasonographic and anatomic linear parameters obtained from the kidneys were the following: width, length and height; the kidney volume was estimated from these measures using the formula of an ellipsoid. The statistical method performed (method agreement) demonstrated a satisfactory agreement. It can be concluded that ultrasound measurement is sufficiently accurate for clinical use, assuming a careful scanning technique.

El objetivo de este trabajo fue determinar la fiabilidad de las medidas ecográficas de los riñones del perro. Se llevó a cabo un análisis de concordancia entre métodos (medidas ecográficas del riñón y medidas anatómicas directas). Se utilizaron perros cuyas edades iban de uno a trece años. Las medidas ecográficas y anatómicas lineales que se obtuvieron de los riñones fueron las siguientes: anchura, longitud y altura. El volumen renal se estimó aplicando estas medidas en la fórmula de un elipsoide. A través de un análisis de concordancia estadística se observó un nivel de acuerdo satisfactorio. Se puede concluir que las medidas ecográficas son suficientemente fiables para su uso en clínica, si se lleva a cabo una cuidadosa técnica ecográfica.

Assessing Circle of Willis blood circulation in dogs with transcranial color-coded duplex sonography.

Insonation of Circle of Willis by transcranial Doppler duplex color sonography is described in 30 healthy dogs with 15 weighing < 33 lb and 15 weighing >33 lb. Imaging was via a temporal window to explore the rostral, middle, and caudal cerebral arteries on both the left and right-hand sides; and through an suboccipital window to study the basilar artery. Normal mean values of the peak systolic velocity (PSV), end diastolic velocity, mean velocity, resistance index (RI), and pulsatility index (PI) were characterized and compared with those obtained in previous studies. There was significant differences in the PSV, RI, and PI in the rostral cerebral artery between dogs weighing < 33 vs. > 33 lb. Mean PSV was higher in weighing over 33 lb, whereas the mean resistive index and mean PI were lower in these dogs.

Relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat: could autophagy be a "brake" in paraquat-induced apoptotic death?

Paraquat (PQ) (1, 1'-dimethyl-4, 4'-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson's disease (PD). In neurons from patients with PD display characteristics of autophagy, a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Low concentrations of paraquat have been recently found to induce autophagy in human neuroblastoma cells, and ultimately the neurons succumb to apoptotic death. Whereas caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by paraquat. These findings suggest a relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat and open a new line of investigation to advance our knowledge regarding the origin of PD.

Inhibition of paraquat-induced autophagy accelerates the apoptotic cell death in neuroblastoma SH-SY5Y cells.

Autophagy is a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. This phenomenon of autophagy has been observed in neurons from patients with Parkinson's disease (PD), suggesting a functional role for autophagy in neuronal cell death. On the other hand, it has been demonstrated that exposure to pesticides can be a risk factor in the incidence of PD. In this sense, paraquat (PQ) (1,1'-dimethyl-4,4'-bipyridinium dichloride), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPP(+) (1-methyl-4-phenyl-pyridine), has been suggested as a potential etiologic factor for the development of PD. The current study shows, for the first time, that low concentrations of PQ induce several characteristics of autophagy in human neuroblastoma SH-SY5Y cells. In this way, PQ induced the accumulation of autophagic vacuoles (AVs) in the cytoplasm and the recruitment of a LC3-GFP fusion protein to AVs. Furthermore, the cells treated with PQ showed an increase of the long-lived protein degradation which is blocked in the presence of the autophagy inhibitor 3-methyladenine and regulated by the mammalian target of rapamycin (mTOR) signaling. Finally, the cells succumbed to cell death with hallmarks of apoptosis such as phosphatidylserine exposure, caspase activation, and chromatin condensation. While caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by PQ. Altogether, these findings show the relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with PQ.

Squirrel monkeys support replication of BK virus more efficiently than simian virus 40: an animal model for human BK virus infection.

We performed experiments to test the suitability of squirrel monkeys (Saimiri sciureus) as an experimental model for BK virus (BKV) and simian virus 40 (SV40) infection. Four squirrel monkeys received intravenous inoculation with BKV Gardner strain, and six squirrel monkeys received intravenous inoculation with SV40 777 strain. Eight of 10 monkeys received immunosuppression therapy, namely, cyclophosphamide subcutaneously either before or both before and after viral inoculation. The presence of viral infection was assessed by quantitative real-time PCR amplification of viral DNA from blood, urine, and 10 tissues. We found that squirrel monkeys were susceptible to infection with BKV, with high viral copy number detected in blood and viral genome detected in all tissues examined. BKV genome was detected in urine from only one monkey, while three monkeys manifested focal interstitial nephritis. BKV T antigen was expressed in renal peritubular capillary endothelial cells. By contrast, SV40 was detected at very low copy numbers in only a few tissues and was not detected in blood. We conclude that the squirrel monkey is a suitable animal for studies of experimental BKV infection and may facilitate studies of viral entry, pathogenesis, and therapy.

Inducible podocyte-specific gene expression in transgenic mice.

The podocyte plays a key role in glomerular function and glomerular disease. To facilitate studies of podocyte function, we have developed a transgenic mouse model with inducible expression in the podocyte. The tetracycline-inducible transgenic system facilitates gene expression with restricted cellular distribution and tight temporal control. Recently, Bujard and colleagues have developed a functionally improved reverse tetracycline-controlled transcriptional activator (rtTA) with substantially lower background in the off state (the absence of tetracycline) and greater inducibility in the on state (the presence of tetracycline). We used the human podocin (NPHS2) gene promoter to control expression of the rtTA cassette and bred these mice with a reporter mouse line that contains the cytomegalovirus minimal promoter and tetO promoter elements together with LacZ, encoding beta-galactosidase. Dual transgenic mice, bearing both podocin-rtTA and tetO-LacZ transgenes, had no detectable expression in kidney or other organs in the absence of tetracycline. Administration of tetracycline in the drinking water was associated with podocyte expression of beta-galactosidase, in a fashion that was time dependent (maximal at 1 wk) and dose-dependent (maximal at 2 mg/ml). Podocyte expression was confirmed in two ways: histochemical staining for beta-galactosidase and double-immunostaining using the podocyte marker WT-1 and beta-galactosidase. This transgenic system should aid future investigations of podocyte function.

SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis.

Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.