Basement Membrane Of Tubules Research Articles

IgG expressed by renal tubular epithelial cells in epithelial mesenchymal transformation and interstitial fibrosis in diabetic kidney disease.

Studies have reported that immunoglobulin G (IgG) "deposited" in the basement membrane of renal tubules is associated with tubulointerstitial damage in patients with diabetic kidney disease (DKD). Our previous study found that renal tubular epithelial cells (RTECs) can express and secrete IgG (RTEC-IgG) which may be associated with fibrosis. The present study aimed to explore the role of RTEC-IgG in renal tubulointerstitial fibrosis (TIF) in DKD. The results showed that RTEC-IgG expression was up-regulated in the renal tubulointerstitium of DKD patients and was associated with worse kidney function, more severe anemia, and higher interstitial fibrosis and tubular atrophy (IFTA) scores, and positively correlated with tubular epithelial mesenchymal transition (EMT) and TIF. IgG expression was also enhanced in the renal tubulointerstitium of DKD mice, which was positively correlated with TIF. High glucose induced an over expression of IgG in human renal tubular epithelial cells, and knockdown of IgG with siRNA relieved the expression of α-smooth muscle actin (SMA), collagen IV, fibronectin, and transforming growth factor (TGF)-β1 under high glucose conditions. In conclusion, our study suggests that RTEC-IgG is involved in the development of DKD by promoting EMT and interstitial fibrosis via TGF-β1.

Mast Cells as a Component of Spermatogonial Stem Cells' Microenvironment.

The formation of mature spermatozoa originates from spermatogonial stem cells (SSCs) located near the basement membrane of the seminiferous tubules. This developmental process, known as spermatogenesis, is tightly regulated to ensure continuous sperm production. A critical aspect of this regulation is the balance between SSC differentiation and self-renewal, which is directed by various factors guiding SSCs in either of these two directions. The SSC niche, defined functionally rather than anatomically, includes all factors necessary for SSC maintenance. These factors are produced by cells surrounding the SSC niche, collectively creating the microenvironment of the seminiferous tubules. Coordination between the cells in this microenvironment is essential for the proper function of the SSC niche and successful spermatogenesis. Testicular mast cells (MCs) significantly influence the regulation of this niche, as they contain various biologically active substances that regulate a wide range of physiological processes and contribute to different pathological conditions affecting fertility. This review explores the effects of testicular MCs on SSCs, their role in regulating spermatogenesis under normal and pathological conditions, and their interactions with other components of the testicular microenvironment, with a focus on their potentially critical impact on spermatogenesis and male fertility.

HIV1-Nef perturbs the integrity of blood testis barrier in rat model

ABSTRACT The blood-testis barrier is a specialized feature within the mammalian testis, located in close proximity to the basement membrane of seminiferous tubules. This barrier serves to divide the seminiferous epithelium into distinct basal and adluminal (apical) compartments. The selectivity of the BTB to foreign particles makes it a safe haven for the virus, and the high affinity of HIV for testis might lead to the vertical transmission of the virus. In the present study, recombinant HIV1-Nef (rNef) protein was injected intravenously to examine the effect of rNef on BTB. SD male rats received 250 µg and 500 µg of rNef along with 2% Evans blue dye within 1 ml through the tail vein. After 1 hour of perfusion, the animals were sacrificed for analysis. The dye migration assay and ELISA confirmed a significant impairment in the blood-testis barrier (BTB) and the manifestation of rNef in testes tissues, respectively. Moreover, a decline in the expression of tight junction proteins, including ZO1 and Occludin, was observed during rNef-induced BTB disruption. Overall, our findings demonstrated that rNef induces BTB disruption through various signaling events. At the site of ectoplasmic specialization of the seminiferous epithelium, the localization of cadherins was found to be disrupted, making the testis a vulnerable site. In conclusion, rNef perturbs the integrity of the blood-testis barrier in rat models; hence, it can also serve as a suitable model for studying the dynamics of the blood-testis barrier.

#1115 TGFβ induced mechanosignaling controls the transcriptome and microenvironmental composition of proximal tubules in chronic kidney disease

Abstract Background and Aims Interstitial fibrosis of the renal parenchyma and progressive atrophy of proximal tubules (PT) are hallmark features of chronic kidney disease (CKD). The latter is characterized by pronounced thickening and multilamellation of the tubular basement membrane, whereas fibrosis is promoted by accumulation of extracellular matrix (ECM) translating into increased matrix rigidity. The role of mechanotransduction via the integrin adhesion complex (IAC) as well as the corresponding signaling programs activated in this context remain largely elusive. Here, we aimed to elucidate the functional role of the ILK-Pinch-Parvin (IPP)-complex as an essential part of the IAC in response to PT damage and CKD. Method Morphometric assessment of a CKD patient biopsy cohort as well as ischaemia-reperfusion injury (IRI) mouse models was performed. Transcriptome studies of human proximal tubular epithelial cells (hRPTECs) under pro-fibrotic and rigid matrix conditions were analyzed. An in vitro model, mimicking CKD, was established and functionally characterized. Reanalysis of single-cell RNA sequencing (scRNA-seq) from a human CKD cohort was employed for cross-correlation and validation. Knockout (KO) models for the IPP-complex were generated by CRISPR/Cas9 genome editing and combined with functional assays (e.g. ECM deposition and sensing). Results In IRI mice, we observed a strong recruitment of the ILK-Pinch-Parvin (IPP)-complex and related-IAC proteins to the tubular basement membrane of proximal tubules (PTs) showing features of tubular atrophy. These findings were translated to CKD patients further assessing markers of active mechanosignaling (YAP) as well as tubular damage (SOX9). Rigidity sensing assays in a hRPTEC model were employed to define the underlying mechanisms. Remarkably, extensive transcriptome analysis revealed that only under pro-fibrotic signaling conditions, hRPTECs sensed rigidity alterations in the underlying substratum, and translated these changes into regulation of matrisome- and adhesome-related gene signatures (including members of the IPP-complex). Further filtering and cross-correlation with available scRNA-seq data from CKD-patients highlighted differential expression of secreted signaling proteins including the matricellular molecule CCN1. Subsequent functional studies demonstrated the underlying pivotal role of the IPP-complex as a central signaling node required for active mechanosignaling in damaged hRPTECs. Conclusion In CKD, IPP and other IAC proteins are recruited to the massively thickened tubular basement membrane of atrophic tubules, indicating a critical role in disease progression. Our results demonstrate that hRPTECs do not express IAC proteins in healthy conditions. However, when inflammation processes (TGFβ treatment) occur, PTs undergo a phenotypic switch, resulting in altered IAC expression signatures. Our data indicate that IAC proteins are crucially involved in sensing of the microenvironmental composition of PTs and thereby reciprocally fine-tune auto-/paracrine signaling properties of epithelial cells.

Rosa26 Locus Is Inactive in Spermatogenesis of C57BL/6 Mice

ABSTRACT Background: The mouse Rosa26 locus demonstrates ubiquitous transcriptional activity, encoding long noncoding RNAs (LncRNAs) with no functional significance in various types of tissues and cells. It is widely employed in targeting transgene knock-in studies. However, the expression pattern of Rosa26 in the testes remains elusive. In this study, we conducted a detailed transcriptional analysis of the Rosa26 locus during mouse spermatogenesis. Methods: The transcriptional activity of the Rosa26 locus in the testes was assessed through immunohistochemistry or direct fluorescence observation in two Rosa26 locus-targeting knock-in mouse lines, Rosa26-Flag-Cas9 and Rosa26-mT/mG, both with a C57BL/6 background. The two mouse lines feature constitutively expressed Flag-tagged Cas9 and red fluorescent membrane-tethered tdTomato (mTomato) proteins. Results: In Rosa26-Flag-Cas9 mice, immunostaining of testis sections revealed robust expression of the Flag-Cas9 transgene in spermatogonia adjacent to the basement membrane of seminiferous tubules. However, its expression was absent in germ cells undergoing spermatogenesis in seminiferous tubules and in maturing spermatids in the epididymis. The transgene was also observed in intertubular Leydig cells and epididymal epithelia but was absent in Sertoli cells. Similarly, in Rosa26-mT/mG mice, fluorescence microscopy showed a complete absence of mTomato expression in the spermatogonial lineage and Sertoli cells. However, robust mTomato expression was observed in intertubular Leydig cells out of the seminiferous tubules and epididymal epithelia. Conclusion: Our findings suggest that the Rosa26 locus is inactive during spermatogenesis, indicating that functional gene studies by targeting knock-in transgenes at the Rosa26 locus in the spermatogonial lineage may not be applicable in C57BL/6 mice.

Evaluation of chemical castration using intra-testicular injection of zinc gluconate into the testis of the male donkey versus surgical castration: antimullerian hormone as an endpoint marker

BackgroundChemical castration of male animals is an alternative to surgical castration for inducing azoospermia, consequent sterility. Intra-testicular injection of zinc gluconate has been used for chemical castration in several animal species. However, its application to equine species, such as donkeys, has yet to be reported. This study aimed to evaluate the use of zinc gluconate for the chemical castration of male donkeys and to compare its effectiveness relative to routine surgical castration. For this purpose, investigations of serum testosterone and anti-Müllerian hormone levels, testicular ultrasonographic echogenicity, and histopathological findings were performed.MethodsFourteen clinically healthy adult male donkeys were randomly and equally divided into two groups. The donkeys in group I (n = 7) underwent surgical castration. The donkeys in group II (n = 7) received intra-testicular zinc gluconate injections. The donkeys were kept under close clinical observation for 60 days. Abnormalities in donkey behavior and gross alterations in the external genitalia were recorded daily. Serum testosterone and anti-Müllerian hormone (AMH) levels were measured 15 days before the start of the treatment and 15, 30, 45, and 60 days after treatment. The testicles of group II donkeys were evaluated ultrasonographically. At the end of the study, the testes were removed and histologically examined.ResultsSerum testosterone levels significantly declined compared to pre-castration levels in surgically castrated donkeys (group I), but donkeys exposed to chemical castration (group II) showed a non-significant reduction in testosterone levels. Donkeys in the surgical group had considerably lower serum AMH levels. In contrast, there was a non-significant (p > 0.05) increase in AMH levels in the chemical group compared with the pre-sterilization level. In addition, ultrasonographic examination revealed that the testicular echo-density had changed, as observed by a few scattered hyperechoic regions throughout the entire testis parenchyma. The histopathological investigation confirmed the presence of necrosis of the spermatogenic epithelium, increased thickness of the basement membrane of the seminiferous tubules, marked interstitial fibrosis, and shrinkage of the seminiferous tubules. Furthermore, syncytial giant cells were present in the lumen of seminiferous tubules and were associated with Sertoli cell vacuolation. Donkeys subjected to chemical castration (group II) had orchitis, as confirmed histopathologically.ConclusionIntra-testicular injection of zinc gluconate resulted in histopathological and ultrasonographic testicular changes in adult male donkeys, which may affect their reproductive potential. However, it did not significantly alter serum testosterone or AMH levels, indicating that it cannot be used as a substitute for surgical castration in male donkeys.

Androgen Receptor (AR) Depletion Underlies the Reproductive Dysfunctions in Male Rats Exposed to Alcohol and Combination Antiretroviral Therapy (cART)

The prevalence of alcohol abuse and HIV/AIDS is high in males of reproductive age; unfortunately, a high incidence of alcohol intake has been reported in HIV/AIDS patients including those on combination antiretroviral therapy (cART). Incidentally, alcohol and cART use have each been implicated in male reproductive dysfunction. Therefore, the interactive effects of alcohol and cART on reproductive hormone levels, testicular connective tissue, and androgen receptor and Ki-67 expression were evaluated in HIV naïve adult male Sprague Dawley rats. Adult male rats were divided into four groups: control, alcohol (A), cART, and A+cART. Animals were terminated after 90 days of treatment; then, the blood and testis were extracted for analysis. Special staining for testicular connective tissue, immunoassay for reproductive hormones, and immunohistochemistry for androgen receptor and Ki-67 were conducted. The study found significantly ( p < 0.05 ) increased thickness of seminiferous tubule basement membrane in the cART group and testicular capsule in the A+cART group. Collagen, reticulin, and elastin fibers decreased significantly in all the treated groups, except for reticulin in the testis of A group animals. With exception of luteinizing hormone in the cART group, all the treated groups had significantly elevated levels of luteinizing and follicle-stimulating hormones, but no significant ( p > 0.05 ) difference was found in their testosterone and inhibin B levels. The number of Sertoli and Leydig cells expressing androgen receptor reduced significantly in all the treated groups, except for A group’s number of Sertoli cells expressing androgen receptor. Further, in all the treated groups, Ki-67 expression significantly reduced relative to control. In this study, with exception of seminiferous tubule basement membrane, all study parameters were greatly altered in the A+cART group; we suggest that the exacerbated androgen receptor depletion recorded in the A+cART group might have led to the observed severe testicular structural alteration and spermatogenesis derangement.

Clinicopathological characteristics of Klinefelter syndrome: a testicular biopsy analysis of 87 cases

Objective: To investigate the clinicopathological characteristics of testicular biopsies from Klinefelter syndrome (KS) patients. Methods: The testicular biopsy specimens of 87 patients with KS (a total of 107 biopsy specimens) were collected from the Department of Pathology, Peking University Third Hospital, Beijing, China from January 2017 to July 2022. All patients were diagnosed as KS by peripheral blood karyotyping analysis. The testicular histopathologic features, testicular volume and hormone levels were evaluated retrospectively. The histopathologic analysis was used to assess the quantity and morphology of Leydig cells, the spermatogenic state of seminiferous tubules, the thickening of the basement membrane of seminiferous tubules and the changes of stroma. Results: Leydig cell proliferative nodules were seen in 95.3% (102/107) of KS testicular biopsy tissues. The eosinophilic inclusion bodies and lipofuscin in Leydig cells were found in 52.3% (56/107) and 57.9% (62/107) of specimens, respectively. The Sertoli cell only seminiferous tubules and the hyalinized tubules were found in 66.4% (71/107) and 76.6% (82/107) of the examined tissues, respectively. The tubules with complete spermatogenic arrest were found in 15.9% (17/107) of specimens, and 5.6% (6/107) of the specimens showed low spermatogenesis or incomplete spermatogenic arrest. In 85.0% (91/107) of the specimens, increased thick-walled small vessels with hyaline degeneration were identified. Conclusions: The most common features of KS testicular specimens are Leydig cell proliferative nodules, hyaline degeneration of seminiferous tubules and proliferation of thick-walled blood vessels. Testicular biopsy specimens of KS are rare. The pathologists can make a tentative diagnosis of KS based on the histological findings, combined with the ultrasound and laboratory results, which is helpful for further diagnosis and treatment of KS.

A Fundamental Research in In Vitro Spermatogonial Stem Cell Culturing: What Are Clump Cells?

Spermatogonial stem cells (SSCs) are a small group of testicular cells located in the basement membrane of seminiferous tubules and can balance self-renewal and differentiation during spermatogenesis. Our in vitro culture experiments of mouse SSCs indicated heterogeneity of cultured cells. Highly compact colonies were observed next to SSC colonies, which we call clump cells. We used immunocytochemical staining to identify SSCs and somatic cells with VASA and Vimentin antibodies. Subsequently, we compared mRNA expression levels of VASA, DAZL, PLZF, GFRA1, Lin28, Kit, Myc and Vimentin genes using Fluidigm real-time RT-polymerase chain reaction in clump cells, SSCs, and testicular stromal cells. To better understand the functions of selected genes, we created a protein-protein interaction network and performed an enrichment analysis using different databases. Based on the data collected, we state that clump cells do not express the molecular markers of SSCs, so we cannot consider them as SSCs; however, we claim that these cells are altered SSCs. The molecular mechanism of this conversion is still obscure. Therefore, this study can support the analysis of germ cell development both in vitro and in vivo. In addition, it can be effective in finding new and more efficient treatments for male infertility.

A rare case of monoclonal immunoglobulin deposition disease identified on renal biopsy

Monoclonal light chain immunoglobulin deposition disease (MIDD) is a rare disease disorders in which monoclonal light chains are deposited within the basement membranes of renal structures, including glomeruli, tubules and vessel walls.1 The vast majority of patients diagnosed with MIDD will have renal insufficiency (80%) and half of MIDD patients have myeloma.2 We present the case of a 52-year-old female with a subacute history of impaired renal function and recent creatinine of 220. A renal biopsy performed at Flinders Medical Centre showed 50% renal cortical scarring with prominent tubular atrophy and peritubular basement membrane thickening on light microscopy. Regions containing periglomerular eosinophilic material were seen that labelled with kappa, favouring periglomerular light chain deposition. Direct immunofluorescence revealed linear labelling with kappa along the tubular basement membranes and electron microscopy showed punctate electron-dense deposits in areas, including the glomeruli and tubular basement membranes. The combined light microscopy, immunofluorescence, and electron microscopy results confirmed the diagnosis of MIDD. A discussion of MIDD is presented.

Protective efficacy of dietary natural antioxidants on microplastic particles-induced histopathological lesions in African catfish (Clarias gariepinus)

Microplastic particles (MPs) are a common environmental pollutant easily ingested by fish in aquaculture. The current study evaluated the protective efficacies of some antioxidant, e.g., lycopene, citric acid, and chlorella, against the toxic effects of MP ingestion by Clarias gariepinus using histopathological biomarkers. Five experimental groups were established, a control group receiving only a standard diet, a group exposed to 500 mg/kg MP concomitant with the standard diet, and three antioxidant groups exposed to MPs plus either lycopene (500 mg/kg), citric acid (30 g/kg), or chlorella (50 g/kg) in the standard diet. After 15 days, fish were sacrificed for histological and histochemical examinations. Histological analysis of the kidney for group 2 (fed 500 mg/kg MPs alone) revealed distributed tissue dissociation, regional glomerular hypertrophy or shrinkage, melanomacrophage accumulation, and expansion of Bowman’s space, while liver tissue exhibited dilation and rupture of the central vein wall, hemorrhage, cytoplasmic vacuolation, and cellular necrosis or apoptosis. Fish exposed to MPs also exhibited connective tissue fiber accumulation around renal blood vessels, renal tubules, the central hepatic vein, hepatic blood sinusoids, and serosal, muscle, and submucosal layers of the intestine. In addition, MP exposure reduced carbohydrate (mainly glycogen) contents in the brush borders and basement membranes of renal tubules, glomeruli, and intestinal tissues as well as in the cytoplasm of hepatocytes. These signs of renal, hepatic, and intestinal histopathology were fully or partially reversed by dietary lycopene, chlorella, or citric acid. Enhancing dietary antioxidants is an effective strategy for preventing MP toxicity in Clarias gariepinus in aquaculture.

KAJIAN HISTOKIMIA DISTRIBUSI KARBOHIDRAT PADA TESTIS SAPI SUMBA ONGOLE (Bos indicus)

Testis is the male reproductive organ that performs both endocrine and exocrine functions. This study aimed to determine the distribution of carbohydrates in the testis of sumba ongole cattle. The sample consisted of six testis were collected from East Sumba Slaughter House, fixed in formalin 10 %, processed histologically, and continued with AB pH 2,5 - PAS staining. The result showed a weak alcian blue intensity (+) in the tunica albuginea and moderate intensity (++) in the interstitial connective tissue, basement membrane, and connective tissue in the lumen of the seminiferous tubules. A strong intensity (+++) to PAS staining was seen in spermatogonia, spermatocytes, and myoid cells. Spermatids, spermatozoa, and sertoli cells showed a negative reaction (-). In the intertubular area, Leydig cells and fibroblasts showed a strong PAS reaction (+++), connective tissue showed a moderate reaction (++). The intensity of the weak reaction (+) is indicated by the basement membrane of the seminiferous tubules. The variation in the distribution of acidic and neutral carbohydrates in the testis of sumba ongole cattle is correlated with the proliferation of different cell types and steroidogenic activity at various stages of testicular development.

The protective effect of rabeprazole on cisplatin-induced apoptosis and necroptosis of renal proximal tubular cells

Nephrotoxicity is a major adverse reaction of cisplatin-based chemotherapy. Organic cation transporter 2 (OCT2) which is located on the basement membrane of human proximal renal tubules is responsible for the renal accumulation of cisplatin and its nephrotoxicity. This study aimed to investigate the protective effect of PPIs to CP-induced nephrotoxicity. Three kinds of PPIs including lansoprazole, omeprazole and rabeprazole (Rab) were co-administrated with CP to mice. In addition, OCT2-overexpressed HEK293, HK-2 and A549 cells were co-incubated with CP and PPIs. The results showed that PPIs can attenuate CP-induced increase of CRE, BUN and histological damage of kidney. Among the three PPIs, Rab was found with a superior protective effect. It significantly reduced the accumulation of CP in OCT2-overexpressed HEK293 cells and in the renal cortex tissues of mice, but not in HK-2 cells. Moreover, Rab reduced the expression levels of cleaved-caspase-3, RIPK1, RIPK3, MLKL and p-MLKL and the apoptosis rate of renal tubular cells induced by CP in vivo, but not in HK-2 cells. However, Rab increased the viability of CP-treated cells in a concentration-dependent manner and attenuated CP-induced apoptosis and necroptosis in OCT2 over-expressed HEK293 cells. Finally, we demonstrated that Rab have no influence on the antitumor effect of CP. In conclusion, Rab attenuate CP-induced nephrotoxicity mainly through inhibiting OCT2-mediated CP uptake, without interfering with its anti-tumor property of inducing apoptosis and necroptosis.

Macro and Micro Anatomical Observation on the Testes of Local Dog (Canis lupusfamiliaris) of Assam

Background: The study on testes of local dog of Assam is of great value in regard to germplasm conservation. The aim of the study was to evaluate the gross and histomorphological examination of testes of male reproductive system. Methods: The testes were collected at the time of castration from Department of Surgery and Radiology, College of Veterinary Science, Assam Agricultural University, Khanapra, Guwahati, Assam, India. The research was carried out for a period of one year in Department of Anatomy and Histology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam. Then gross anatomical studies were made on it and the tissue samples were fixed in 10% neutral buffered formalin solution and were processed as per the standard technique of procedure (Luna, 1968). The paraffin blocks were sectioned in Shandon Finesse microtome at 5 µm thickness and the sections were stained with Mayer’s Haematoxylin and Eosin staining technique for Cellular details as per the method of Luna (1968). Result: Grossly, the testes of local dog consisted of two surface viz., lateral and medial and two ends i.e. upper end and lower end. The upper end of the testes was occupied by the head of the epididymis and the lower end of the testes was occupied by the tail of the epididymis. Mediastinum testis was observed in the centre of testes of local dog. Histologically, the testes were covered by serous layer (Tunica vaginalis), connective tissue layer (Tunica albugenia) and vascular layer (Tunica vasculosa) from outside to inwards. Spermatogenic cells like spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa, and sertoli cells were observed in seminiferous tubules. The sertoli cells were attached to the basement membrane of seminiferous tubules. Cluster of Leyding cells were found between the semineniferous tubules and it contained large spherical nuclei. The epididymides were lined by pseudo stratified ciliated columnar epithelium.

Protective effect of resveratrol on acrylamide-induced renal impairment.

Acrylamide (ACR) has a wide range of uses. It possesses a renal impairment effect. The work aimed to study the possible protecting role of resveratrol (RVS) over the ACR-mediated renal impairment in rats. The suggested underlying mechanisms participating in such protection were investigated. Thirty Sprague-Dawley adult albino rats were divided into three groups: control, ACR, and RVS. After 4 weeks, the kidney was removed and prepared for histological, immunohistochemical, and biochemical studies. The activity of tissue oxidative (malondialdehyde [MDA]) and anti-oxidative (glutathione [GSH]) markers were assessed. Acrylamide induced glomerular renal affection in the form of shrinkage and distortion of the glomeruli with wrinkling of their basement membranes and widening of the urinary spaces. Degenerative tubular changes were markedly present in the proximal convoluted tubules. The necrotic tubular cells exhibited cytoplasmic vacuolation with desquamated epithelial cells within the tubular lumen. ACR increases the deposition of collagen fibres in the basement membrane of the glomerular capillaries and induced thickening of the basement membranes of the renal corpuscles and renal tubules. The administration of RVS affords high protection to the kidney. The glomeruli and renal tubules were nearly normal. The content of collagen fibres and the periodic acid Schiff reaction of the basement membrane of the renal tubules were 70% and 19% lower linked to the ACR group. The creatinine and urea levels decreased by 51% and 47%. RVS induced such a protective role through its antioxidant effect as the MDA level decreased by 45%, while the GSH level increased by 83% compared with the ACR group. Acrylamide causes structural and functional disorders of the kidney. It induces kidney damage through oxidative stress and apoptosis. With the use of RVS, normal kidney architecture was preserved with little structural changes. Adding, functional kidney test became normal. RVS exerts its protective effect through its anti-apoptotic and antioxidant features.

Subcellular distribution of α2-adrenoceptor subtypes in the rodent kidney.

Renal α2-adrenoceptors have been reported to play a role in the regulation of urinary output, renin secretion, and water and sodium excretion in the kidneys. However, the distribution of α2-adrenoceptor subtypes in the kidneys remains unclear. In this study, we aimed to investigate the localization of α2-adrenoceptor subtypes in rat kidneys using 8-week-old Sprague-Dawley rats. Immunofluorescence imaging revealed that both α2A- and α2B-adrenoceptors were expressed in the basolateral, but not apical, membrane of the epithelial cells of the proximal tubules. We also found that α2A- and α2B-adrenoceptors were not expressed in the glomeruli, collecting ducts, or the descending limb of the loop of Henle and vasa recta. In contrast, α2C-adrenoceptors were found to be localized in the glomeruli and lumen of the cortical and medullary collecting ducts. These results suggest that noradrenaline may act on the basement membrane of the proximal tubules through α2A- and α2B-adrenoceptors. Moreover, noradrenaline may be involved in the regulation of glomerular filtration and proteinuria through the induction of morphological changes in mesangial cells and podocytes via α2C-adrenoceptors. In the collecting ducts, urinary noradrenaline may regulate morphological changes of the microvilli through α2C-adrenoceptors. Our findings provide an immunohistochemical basis for understanding the cellular targets of α2-adrenergic regulation in the kidneys. This may be used to devise therapeutic strategies targeting α2-adrenoceptors.

Histological evaluation of the effects of rapamycin and 3-methyladenine on cisplatin-induced epididymal injury in rats

Purpose: In this study, we aimed to determine the effects of autophagy inhibitor and activator on Cisplatin (Cis)-induced tissue damage. 
 Materials and Methods: A total of 24 male Wistar albino rats were divided into 4 groups including 6 rats per group in this study. Groups are as follows; Control, Cisplatin (Cis) (8 mg/kg), Rapamycin (Rapa) (2 mg/kg), 3-methyladenine (3-MA) (15 mg/kg). Rapa and 3-MA were given intraperitoneally for 15 days. Cis was administered as a single dose on the 7th day of the experimental period. At the end of the experimental procedure, epididymis tissues were extracted. Hematoxylin and eosin staining and Heat shock protein-70 (HSP70) immunohistochemistry were applied to the sections taken after histological techniques. 
 Results: Dispersion in the tubule basement membrane and vacuolization in the tubule was observed in the Cis group. It was also observed that some epithelial cells were more eosinophilic in the Cis group. Tissue sections of Rapa and 3-MA had a more regular appearance in terms of epithelization and tubule basement membrane. HSP70 immunoreactivity was observed in the intertubuler connective tissue of all groups. 
 Conclusion: The epididymis was affected by agents such as Cis in terms of the protection of semen quality and potency of spermatozoa. Rapa may be more effective than 3-MA in the epididymis against Cis toxicity.

UPEC kidney infection triggers neuro-immune communication leading to modulation of local renal inflammation by splenic IFNγ.

Bacterial infection results in a veritable cascade of host responses, both local and systemic. To study the initial stages of host-pathogen interaction in living tissue we use spatially-temporally controlled in vivo models. Using this approach, we show here that within 4 h of a uropathogenic Escherichia coli (UPEC) infection in the kidney, an IFNγ response is triggered in the spleen. This rapid infection-mediated inter-organ communication was found to be transmitted via nerve signalling. Bacterial expression of the toxin α-hemolysin directly and indirectly activated sensory neurons, which were identified in the basement membrane of renal tubules. Nerve activation was transmitted via the splenic nerve, inducing upregulation of IFNγ in the marginal zones of the spleen that led to increasing concentrations of IFNγ in the circulation. We found that IFNγ modulated the inflammatory signalling generated by renal epithelia cells in response to UPEC infection. This demonstrates a new concept in the host response to kidney infection; the role of nerves in sensing infection and rapidly triggering a systemic response which can modulate inflammation at the site of infection. The interplay between the nervous and immune systems is an exciting, developing field with the appealing prospect of non-pharmaceutical interventions. Our study identifies an important role for systemic neuro-immune communication in modulating inflammation during the very first hours of a local bacterial infection in vivo.

Cryopreservation of grey wolf (Canis lupus) testicular tissue

Development of genomic preservation technologies for canids, especially for seasonally breeding species like the grey wolf (Canis lupus), is needed in advance of growing species conservation concerns. Here, we evaluated the efficacy of two cryopreservation protocols – needle immersion vitrification (NIV) and slow freezing (SF) on grey wolf (n = 7) testicular tissue morphology. NIV samples were equilibrated in a 7.5% v/v dimethyl sulfoxide (DMSO or Me2SO) + 7.5% ethylene glycol (EG) solution in minimum essential medium with 20% FBS for 10 min at 4 °C, then exposed to 15% DMSO + 15% EG + 0.5 M sucrose for 10 min at 4 °C before plunging into liquid nitrogen. For slow freezing, we assessed two cryoprotectant (CPA) strategies, DMSO, 15% v/v alone (SF-D) or 7.5% EG + 7.5% DMSO (SF-ED). Following thawing, there were no significant differences in seminiferous tubule area among treatment groups, although all cryopreserved tissues displayed reduced tubule size compared with fresh controls and increased apoptosis, the latter reaching significance for SF-D treated tissues. Slow freezing improved maintenance of testis architecture, with minimal detachment of seminiferous tubule basement membranes post-thaw. Spermatogonia densities were reduced in NIV tissues compared with fresh, with no differences in spermatocyte, spermatid, or Sertoli cell counts, or germ cell marker DDX4+ cell densities among groups. In sum, we conclude that slow freezing better maintained morphology of cryopreserved testicular tissues compared with needle vitrification with 15% each DMSO and EG and 0.5 M sucrose, and that DMSO + EG combination SF supports cell viability. This represents a first step in the development of male gonadal tissue preservation strategies for the grey wolf.

Characterization of DDX4 Gene Expression in Human Cases with Non-Obstructive Azoospermia and in Sterile and Fertile Mice.

Background:In mammals, spermatogenesis is the main process for male fertility that is initiated by spermatogonial stem cells (SSCs) proliferation. SSCs are unipotent progenitor cells accountable for transferring the genetic information to the following generation by differentiating to haploid cells during spermato-and spermiogenesis. DEAD-box helicase 4 (DDX4) is a specific germ cell marker and its expression pattern is localized to, spermatocytes, and spermatids. The expression in the SSCs on the basement membrane of the seminiferous tubules is low.Methods:Immunohistochemistry (IHC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze the expression of DDX4 in testis tissue of fertile and sterile mice and human cases with non-obstructive azoospermia.Results:Our immunohistochemical findings of fertile and busulfan-treated mice showed expression of DDX4 in the basal and luminal compartment of seminiferous tubules of fertile mice whereas no expression was detected in busulfan-treated mice. The immunohistochemical analysis of two human cases with different levels of non-obstructive azoospermia revealed more luminal DDX4 positive cells.Conclusion:Our findings indicate that DDX4 might be a valuable germ cell marker for analyzing the pathology of germ cell tumors and infertility as global urological problems.