Urinary angiotensin-converting enzyme 2 and its activity in cats with chronic kidney disease.

Introduction: Angiotensin-converting enzyme 2 (ACE2) played an important role in the renin-angiotensin-aldosterone system (RAAS) and it was proved to be renoprotective in renal disease. Urinary angiotensin-converting enzyme 2 (uACE2) has been shown to reflect renal injury in human and experimental studies, but its role in feline kidney disease remains unknown. Aims: Our objectives involve comparing uACE2 concentrations and activities in cats across CKD stages with healthy controls, investigating the relationship between uACE2 concentrations, activities, and clinicopathological data in feline CKD patients, and assessing the predictive abilities of both for CKD progression. Methods: A retrospective, case-control study. The concentration and activity of uACE2 were measured by commercial ELISA and fluorometric assay kits, respectively. The concentration was adjusted to give uACE2 concentration-to-creatinine ratios (UACCRs). Results: In total, 67 cats consisting of 24 control and 43 chronic kidney disease (CKD), including 24 early-stage CKD and 19 late-stage CKD, were enrolled in this study. UACCR values were significantly higher in both early-stage (2.100 [1.142-4.242] x 10-6) and late-stage feline CKD (4.343 [2.992-5.0.71] x 10-6) compared to healthy controls (0.894 [0.610-1.076] x 10-6; p < 0.001), and there was also significant difference between-early stage group and late-stage group (p = 0.026). Urinary ACE2 activity (UAA) was significantly lower in CKD cats (1.338 [0.644-2.755] x pmol/min/ml) compared to the healthy cats (7.989 [3.711-15.903] x pmol/min/ml; p < 0.001). UACCR demonstrated an independent, positive correlation with BUN (p < 0.001), and UAA exhibited an independent, negative correlation with plasma creatinine (p < 0.001). Both UACCR and UAA did not yield significant results in predicting CKD progression based on the ROC curve analysis. Conclusion and clinical importance: uACE2 concentration and activity exhibit varying changes as renal function declines, particularly in advanced CKD cats.

A mouse seminal vesicle-secreted lysozyme c-like protein modulates sperm capacitation.

Lysozyme (LYZ) c-like proteins are primarily present in the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c-type LYZ family, the seminal vesicle-secreted LYZ c-like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N-linked glycans. SVLLP is structurally similar to c-type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c-type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin-induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm-oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor.

Urine hemojuvelin in cats with naturally occurring kidney disease.

BACKGROUND: Soluble-type hemojuvelin in serum and urine has been shown to be a biomarker in humans for chronic kidney disease (CKD) and acute kidney injury (AKI). No similar research has been conducted on cats. OBJECTIVE: Urine hemojuvelin (u-hemojuvelin) can be used as a clinical indicator for cats with various renal diseases. ANIMALS: Eighteen healthy cats, 10 cats with AKI, 21 cats with acute-on-chronic kidney injury (ACKI), and 45 cats with CKD were enrolled. METHODS: The expression profile of u-hemojuvelin was assessed by Western blot analysis, whereas the u-hemojuvelin concentration was measured using an in-house sandwich ELISA. Each cat's u-hemojuvelin-to-creatinine ratio (UHCR) also was determined. RESULTS: Significant differences were found in both u-hemojuvelin concentration and UHCR between the control cats and the other cats (AKI, CKD, ACKI). Both u-hemojuvelin and UHCR had high areas under the receiver operator curve (AUROC) for diagnoses of AKI (u-hemojuvelin, 0.885; UHCR, 0.982), CKD (hemojuvelin, 0.869; UHCR, 0.959), and ACKI (hemojuvelin, 0.910; UHCR, 1). Late stage (International Renal Interest Society, IRIS stages 3 and 4) CKD cats had significantly higher u-hemojuvelin concentration and UHCR than did early stage cats (IRIS stages 1 and 2). Both u-hemojuvelin and UHCR were significantly correlated with high blood urea nitrogen, plasma creatinine, and plasma phosphate concentrations and with low hematocrit (Hct), red blood cell (RBC) count, and plasma albumin concentration. The UHCR values were also significantly correlated with white blood cell count in blood. CONCLUSION: Both u-hemojuvelin and UHCR potentially can serve as diagnostic indicators for a range of renal diseases in cats.

Autologous mitochondrial microinjection; a strategy to improve the oocyte quality and subsequent reproductive outcome during aging.

Along with the decline in oocyte quality, numerous defects such as mitochondrial insufficiency and the increase of mutation and deletion have been reported in oocyte mitochondrial DNA (mtDNA) following aging. Any impairments in oocyte mitochondrial function have negative effects on the reproduction and pregnancy outcome. It has been stated that infertility problems caused by poor quality oocytes in women with in vitro fertilization (IVF) and repeated pregnancy failures are associated with aging and could be overcome by transferring large amounts of healthy mitochondria. Hence, researches on biology, disease, and the therapeutic use of mitochondria continue to introduce some clinical approaches such as autologous mitochondrial transfer techniques. Following mitochondrial transfer, the amount of ATP required for aged-oocyte during fertilization, blastocyst formation, and subsequent embryonic development could be an alternative modality. These modulations improve the pregnancy outcome in women of high reproductive aging as well. In addition to overview the clinical studies using mitochondrial microinjection, this study provides a framework for future approaches to develop effective treatments and preventions of congenital transmission of mitochondrial DNA mutations/diseases to offspring. Mitochondrial transfer from ovarian cells and healthy oocytes could lead to improved fertility outcome in low-quality oocytes. The modulation of mitochondrial bioactivity seems to regulate basal metabolism inside target oocytes and thereby potentiate physiological activity of these cells while overcoming age-related infertility in female germ cells.

Identification of urine neutrophil gelatinase-associated lipocalin molecular forms and their association with different urinary diseases in cats.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL), a promising renal biomarker, can exists as a monomer, a dimer and/or in a NGAL/matrix metalloproteinase-9 (MMP-9) complex form when associated with different urinary diseases in humans and dogs. In this study, the presence of the various different molecular forms of NGAL in cat urine (uNGAL) was examined and whether these forms are correlated with different urinary diseases was explored. RESULTS: One hundred and fifty-nine urine samples from cats with various different diseases, including acute kidney injury (AKI, 22 cats), chronic kidney disease (CKD, 55 cats), pyuria (44 cats) and other non-renal and non-pyuria diseases (non-RP, 26 cats), as well as healthy animals (12 cats), were collected. The molecular forms of and concentrations of urinary NGAL in these cats were analyzed, and their uNGAL-to-creatinine ratio (UNCR) were determined. The cats with AKI had the highest UNCR (median: 2.92 × 10- 6), which was followed by pyuria (median: 1.43 × 10- 6) and CKD (median: 0.56 × 10- 6); all of the above were significantly higher than the healthy controls (median: 0.17 × 10- 6) (p < 0.05). Three different NGAL molecular forms as well as the MMP-9 monomer were able to be detected in the cat urine samples. Moreover, the cases where urine NGAL monomer were present also had significantly higher levels of BUN (median: 18.9 vs 9.6 mmol/L) and creatinine (327.1 vs 168 umol/L). The presence of dimeric NGAL was found to be associated with urinary tract infections. Most cats in the present study (126/159, 79.2%) and more than half of healthy cats (7/12, 58.3%) had detectable NGAL/MMP-9 complex present in their urine. CONCLUSIONS: The monomeric and dimeric molecular forms of uNGAL suggest upper and lower urinary tract origins of disease, respectively, whereas the presence of the uNGAL/MMP-9 complex is able to be detected in most cats, including seemingly healthy ones.

Toxic effects of VCD on kidneys and liver tissues: a histopathological and biochemical study.

OBJECTIVE: We explored detrimental effects of VCD on non-ovarian tissues such as kidneys and liver 14 days post-drug administration. Twelve rats were randomly assigned into two groups. In VCD group, rats received 160 mg/kgbw VCD intraperitoneally for 15 consequent days. Control rats were injected with VCD-free normal saline. At the respective time point, rats were euthanized, blood and tissue samples were collected. H&E staining was performed to evaluate pathological changes. Serum level of ALT, AST, creatinine and urea were also measured. RESULTS: Histological analysis revealed hyperemia and follicular atresia in the ovaries, indicating successful POF induction in rats. In renal tissue, extensive tubular necrosis, focal hemorrhage, hyaline casts, and interstitial nephritis were observed. Analysis of hepatic tissue showed numerous hemorrhagic foci, chronic cholangitis, and hepatocyte necrosis, indicating apparent VCD toxicity of both hepatic and renal tissues. The biochemical evaluation revealed a tendency of increase in ALT, AST, creatinine, and Urea in VCD-treated rats; however, the values did not reach significant level. In conclusion, the induction of POF in rats by VCD correlates with renal and hepatic damages. Commensurate with data from this study, any conclusions from experiments based on VCD-induced premature ovarian failure rats should be reported with caution.

Mouse quiescin sulfhydryl oxidases exhibit distinct epididymal luminal distribution with segment-specific sperm surface associations.

Sulfhydryl oxidation is part of the sperm maturation process essential for the acquisition of sperm fertilization competency and its structural stabilization; however, the specific sulfhydryl oxidases that fulfill these roles have yet to be identified. In this study, we investigate the potential involvement of one atypical thiol oxidase family called quiescin Q6/sulfhydryl oxidase (QSOX) using the mouse epididymis as our model system. With multidisciplinary approaches, we show that QSOX isoform 1 and 2 exhibit complementary distribution throughout the epididymal duct, but that each variant possesses distinct subcellular localization within the epididymal principal cells. While QSOX2 was exclusively present in the Golgi apparatus of the caput and corpus epididymis, QSOX1c, the most profusely express QSOX1 variant, was abundantly present in the cauda luminal fluids. Moreover, immunohistochemistry studies together with proteomic identification in isolated epididymosomes provided evidence substantiating the release of QSOX2, but not QSOX1c, via an apocrine secretory pathway. Furthermore, we demonstrate for the first time, distinct association of QSOX1c and QSOX2 with the sperm acrosome and implantation fossa, during different stages of their epididymal maturation. In conclusion, our study provides the first comprehensive comparisons between QSOX1 and QSOX2 in the mouse epididymis, revealing their distinct epididymal distribution, cellular localization, mechanisms of secretion and sperm membrane association. Together, these data suggest that QSOX1 and QSOX2 have discrete biological functions in male germ cell development.

Identification, characterization and purification of porcine Quiescin Q6-Sulfydryl Oxidase 2 protein.

BACKGROUND: Post-spermiogenesis membrane surface modifications rely on molecules present in the reproductive tracts. Two isoforms (isoform 1 and 2) from Quiescin Q6-Sulfydryl Oxidase protein family have been identified in the male reproductive tract of rodent species. However, unlike isoform 1, scarce information is available for isoform 2, likely due to its lower expression level and lack of proper purification methods to obtain sufficient protein quantity for further assays. RESULTS: This study demonstrated the presence of short and long forms of Quiescin Q6-Sulfydryl Oxidase 2 in boar, likely representing the secretory (short form) and transmembrane (long form) forms of Quiescin Q6-Sulfydryl Oxidase 2. Immunohistochemistry studies revealed the presence of Quiescin Q6-Sulfydryl Oxidase 2 in a broad range of porcine tissues; the pronounced vesicle-contained Quiescin Q6-Sulfydryl Oxidase 2 at the apical region of epididymis and seminal vesicles epithelium suggested its involvement in sperm physiology and its participation in semen formation. The majority of porcine Quiescin Q6-Sulfydryl Oxidase 2 could be purified via either antibody affinity column or be salted out using 10%-40% ammonium sulfate. Higher amount of low molecular weight Quiescin Q6-Sulfydryl Oxidase 2 observed in the seminal vesicle likely represents the secretory form of Quiescin Q6-Sulfydryl Oxidase 2 and reflects an exuberant secretory activity in this organ. CONCLUSIONS: We demonstrated for the first time, the presence of Quiescin Q6-Sulfydryl Oxidase 2 in porcine species; moreover, two forms of Quiescin Q6-Sulfydryl Oxidase 2 were identified and exhibited distinct molecular weights and properties during protein purification processes. This study also provided feasible Quiescin Q6-Sulfydryl Oxidase 2 purification methods from slaughterhouse materials that could potentially allow obtaining sufficient amount of Quiescin Q6-Sulfydryl Oxidase 2 for future functional investigations.