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Background Kidneys with chronic inflammation develop tertiary lymphoid structures (TLSs). Infectious pyelonephritis is characterized by renal pelvis (RP) inflammation. However, the pathologic features of TLSs, including their formation and association with non-infectious nephritis, are unclear. Methods RPs from humans and mice that were healthy or had non-infectious chronic nephritis were analyzed for TLS development, and the mechanism of TLS formation investigated using urothelium or lymphoid structure cultures. Results Regardless of infection, TLSs in the RP, termed urinary tract-associated lymphoid structures (UTALSs), formed in humans and mice with chronic nephritis. Moreover, urine played a unique role in UTALS formation. Specifically, we identified urinary IFN-gamma as a candidate factor affecting urothelial barrier integrity because it alters occludin expression. In a nephritis mouse model, urine leaked from the lumen of the RP into the parenchyma. In addition, urine immunologically stimulated UTALS-forming cells via cytokine (IFN-gamma, TNF-alpha) and chemokine (CXCL9, CXCL13) production. CXCL9 and CXCL13 were expressed in UTALS stromal cells and urine stimulation specifically induced CXCL13 in cultured fibroblasts. Characteristically, type XVII collagen (BP180), a candidate autoantigen of bullous pemphigoid, was ectopically localized in the urothelium covering UTALSs and associated with UTALS development by stimulating CXCL9 or IL-22 induction via the TNF-alpha/ FOS/JUN pathway. Notably, UTALS development indices were positively correlated with chronic nephritis development. Conclusions TLS formation in the RP is possible and altered urine-urothelium barrier-based UTALS formation may represent a novel mechanism underlying the pathogenesis of chronic nephritis, regardless of urinary tract infection.

The interleukin (IL) 36 subfamily belongs to the IL-1 family and is comprised of agonists (IL-36α, IL-36β, IL-36γ) and antagonists (IL-36Ra, IL-38). We previously reported IL-36α overexpression in renal tubules of chronic nephritis mice. To understand the localization status and biological relationships among each member of the IL-36 subfamily in the kidneys, MRL/MpJ-Faslpr/lpr mice were investigated as autoimmune nephritis models using pathology-based techniques. MRL/MpJ-Faslpr/lpr mice exhibited disease onset from 3 months and severe nephritis at 6–7 months (early and late stages, respectively). Briefly, IL-36γ and IL-36Ra were constitutively expressed in murine kidneys, while the expression of IL-36α, IL-36β, IL-36Ra, and IL-38 was induced in MRL/MpJ-Faslpr/lpr mice. IL-36α expression was significantly increased and localized to injured tubular epithelial cells (TECs). CD44+-activated parietal epithelial cells (PECs) also exhibited higher IL-36α-positive rates, particularly in males. IL-36β and IL-38 are expressed in interstitial plasma cells. Quantitative indices for IL-36α and IL-38 positively correlated with nephritis severity. Similar to IL-36α, IL-36Ra localized to TECs and PECs at the late stage; however, MRL/MpJ-Faslpr/lpr and healthy MRL/MpJ mice possessed IL-36Ra+ smooth muscle cells in kidney arterial tunica media at both stages. IL-36γ was constitutively expressed in renal sympathetic axons regardless of strain and stage. IL-36 receptor gene was ubiquitously expressed in the kidneys and was induced proportional to disease severity. MRL/MpJ-Faslpr/lpr mice kidneys possessed significantly upregulated IL-36 downstream candidates, including NF-κB- or MAPK-pathway organizing molecules. Thus, the IL-36 subfamily contributes to homeostasis and inflammation in the kidneys, and especially, an IL-36α-dominant imbalance could strongly impact nephritis deterioration.

Cotton rats (Sigmodon hispidus, CRs) are commonly used as animal models in biomedical research. However, the reproductive characteristics and ovarian development in the CRs has not been widely investigated. We have previously shown that female CRs, in particular, show several unique phenotypes associated with the urogenital system, such as chronic kidney disease and pyometra. Our investigation revealed unique morphologies in CR ovaries, particularly in oocytes. Cotton rat ovaries at 6-8 weeks of age were obtained from the Hokkaido Institute of Public Health, and their sections analyzed by light microscopy and transmission electron microscopy. Although the general histology and folliculogenesis of CR ovaries were similar to those of other experimental rodents, multi-oocyte follicles (MOFs) and double nucleated oocytes (DNOs) were also observed. Although MOFs were found at all stages of follicular development, a greater frequency of MOFs was observed in the primary and secondary stages. However, DNOs tended to be frequently observed in primordial follicles. Almost all MOF oocytes and a few DNOs possessed a clear zona pellucida, expressed DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 and Forkhead box protein 2, a representative marker of oocytes and follicular epithelial cells. Thus, our investigations revealed the unique phenotypes of the CR ovary. As MOFs and DNOs are occasionally observed in human patients with infertility, the CR would be a useful animal model to study for gaining a better understanding of folliculogenesis and oocytogenesis, as well as their abnormalities in humans and other animals.

The ovarian bursa is a small peritoneal cavity enclosed by the mesovarium and mesosalpinx, which surrounds the ovaries and oviductal infundibulum in mammals. The ovarian bursa is considered as the structure facilitating the transport of ovulated oocytes into the oviduct. Our previous study revealed reduced oocyte pick‐up function in the oviduct of lupus‐prone MRL/MpJ‐Fas(lpr) (/) (lpr) mouse, suggesting the possibility of an escape of ovulated oocytes into the peritoneal cavity, despite the presence of an almost complete ovarian bursa in the mouse. In this study, we revealed anatomical and histological characteristics of the ovarian bursa in C57BL/6 N, MRL/MpJ, and MRL/MpJ‐Fas(lpr) (/) (lpr) mice. All strains had the foramen of ovarian bursa (FOB), with a size of approximately 0.04 to 0.12 cm(2), surrounded by the ligament of ovarian bursa (LOB), which is part of the mesosalpinx. The LOB was partially lined with the cuboidal mesothelial cells and consisted of a thick smooth muscle layer in all strains. In 6‐month‐old MRL/MpJ‐Fas(lpr) (/) (lpr) mice, in which the systemic autoimmune abnormality deteriorated and oocyte pick‐up function was impaired, the size of the FOB tended to be larger than that of other strains. Additionally, in MRL/MpJ‐Fas(lpr) (/) (lpr) mice at 6 months of age, there was infiltration by numerous immune cells in the mesosalpinx suspending the isthmus; however, the LOB prevented severe inflammation and showed deposition of collagen fibers. These results not only indicate that the FOB is a common structure within mice, but also imply the physiological function of the LOB and its role in maintaining the microenvironment around the ovary, as well as regulating healthy reproduction.

Toll-like receptors (Tlrs) are sensors of danger signals which promote the activation of immune cells and intrinsic renal cells. Podocytes, the intrinsic cells of glomerulus, are continuously exposed to various plasma solutes and danger signals due to their unique location in the glomerulus. Herein, we show that Tlr9 is overexpressed in podocytes and the mechanisms which cause its injury and development of membranoproliferative glomerulonephritis (MPGN) in model BXSB/MpJ-Yaa (Yaa) mice. Yaa mice developed typical lesions of MPGN and showed strong expression of Tlr9 mRNA throughout the glomerulus particularly toward the periphery of the glomerulus. However, BXSB/MpJ (BXSB) mice showed no lesion for MPGN but a very weak expression of Tlr9 mRNA. Relative mRNA expression of Tlr9 and its downstream cytokines, including interleukin 1 beta (Il1b), Il6, interferon gamma (Ifng) and tumour necrosis factor alpha (Tnfa) was markedly increased in glomeruli isolated from Yaa mice. Tlr9 protein expression was almost absent in BXSB mice but intense expression was found in Yaa mice. Podocyte protein expression was normal in BXSB mice but decreased in Yaa mice and colocalized with Tlr9 protein. Furthermore, electron microscopy examination revealed podocyte injury and electron-dense materials in thickened glomerular basement membrane of Yaa mice. Glomerular Tlr9 mRNA expression was significantly correlated with anti-dsDNA antibody, proteinuria, renal function indices (sBUN and sCr), glomerular histopathology indices, downstream factors of Tlr family (Ilb and Tnfa), podocyte injury parameters (p .05 and p .01). In conclusion, overexpression of TLR9 correlates with podocyte injury and development of MPGN.

Ovulation and oocyte-pick-up are essential processes in fertilization. Herein, we found associations between autoimmune disease and the aforementioned processes in mice. At three and six months, along with the evaluation of autoimmune disease indices, the ovary, mesosalpinx, and oviducts were histologically examined in C57BL/6, MRL/MpJ, and MRL/MpJ-Fas lpr/lpr mice as healthy control, mild and severe models of autoimmune disease, respectively. In superovulated mice, the number of “oocyte cumulus complexes” found in the ampulla was macroscopically counted, and that of “ovulated oocytes” was histologically evaluated, as indicated by ruptured follicles or corpora hemorrhagica in ovaries. Finally, the oocyte-pick-up rate was calculated. In MRL/MpJ-Fas lpr/lpr mice, the oocyte-pick-up rate decreased with disease-related deterioration, unlike in other mouse strains. Further, more ovulated oocytes were found in MRL/MpJ mice than in C57BL/6 mice, and this number significantly decreased with aging in MRL/MpJ-Fas lpr/lpr mice. Numerous T-cells infiltrated into the infundibulum or a part of the mesosalpinx in aged MRL/MpJ-Fas lpr/lpr mice, and their infundibulum showed swelling and fewer ciliated epithelial cells compared to that of C57BL/6 mice. In conclusion, the progression of severe autoimmune disease affected the oocyte-pick-up process through histopathological changes in the infundibulum. These results provide important insights into female infertility associated with autoimmune disease.

According to our previous reports, impaired oocyte pickup was observed in the oviductal infundibulum of an autoimmune disease (AD) mouse model, suggesting a relationship between female infertility and AD. This study examines the relationship between AD and infundibulum morphofunction by focusing on the epithelial cilia. Healthy MRL/MpJ and AD-prone MRL/MpJ-Faslpr/lpr mice were examined at 3 and 6 months of age, representing early and late disease stages, respectively. Oocyte pickup indices decreased with AD progression indicated by splenomegaly, autoantibody production and increased T cell counts of infundibulum mucosa in MRL/MpJ-Faslpr/lpr mice. Ciliary beating frequency (CBF) and height in the infundibulum were faster and higher in MRL/MpJ-Faslpr/lpr mice than in MRL/MpJ mice at the early AD stages, although the absolute CBF values were lower at the late AD stage. At the late stage, ciliary height did not differ between mouse lines but the morphological index of cilia beating direction indicated randomized patterns in MRL/MpJ-Faslpr/lpr mice. The tracheal mucosa was also examined as a representative example of cilia morphology; its CBF decreased at the late AD stage in MRL/MpJ-Faslpr/lpr; however, there were no AD-related morphological changes. Our results demonstrate altered cilia motility in systemic and reproductive organs, with such morphological changes of the infundibulum likely impairing function, including oocyte pickup.

Increased incidence of kidney disease (KD) is a common concern in human and companion animals. Cats, in particular, are highly susceptible to KD. Novel KD biomarkers would help to address these problems. Therefore, we are focusing on microRNA, a highly conserved nucleic acid, as a KD biomarker for various animals. We previously reported that altered levels of urinary exosome (UExo)-derived microRNAs indicate renal pathologies in dogs. This study comprehensively examined UExo-derived microRNAs, which reflected the KD status in cats. The examined cats were divided into two groups: normal renal function (NR) and KD. Based on our previous data in clogs and cats, as well as the present data on UExo-derived microRNAs in cats by next-generation sequencing, let-7b, let-7f, miR-10a, miR-10b, miR-21a, miR-22, miR-26a, miR-27b, miR-146a, miR-181a, miR-191, and miR-486a were identified as biomarker candidates. In summary, the levels of UExo-derived let-7b, miR-22, and miR-26a significantly decreased in cats with KD from the early stages of the disease. UExo-derived miRNA levels normalized to urinary creatinine or total RNA of miR-21a was significantly higher in the KD group. Importantly, the ratio of UExo-derived miR-21a to let-7b showed a significant and strongest correlation with serum creatinine (rho = 0.751), blood urea nitrogen (rho = 0.754), and urinary creatinine (rho = -0.421) among all examined indices. Further, the ratio of miR-181a to let-7b or miR-10b significantly correlated with the progression of renal dysfunction in the KD group. Thus, we identified that UExo-derived microRNAs in cats, and their raw and normalized levels could indicate altered renal function.

This study evaluated endothelial cells and podocytes, both being primary components of the glomerular filtration barrier, in the progression of membranoproliferative glomerulonephritis (MPGN) using modified scanning electron microscopy (mSEM) analysis. BXSB/MpJ-Yaa model mice exhibited autoimmune-mediated MPGN characterised by elevated serum autoantibody levels, albuminuria, renal dysfunctional parameters, and decreased glomerular endothelial fenestrations (EF) and podocyte foot process (PFP) effacement with immune cell infiltration. Similar to transmission electron microscopy, mSEM revealed a series of pathological changes in basement membrane and densities of EF and PFP in BXSB/MpJ-Yaa compared with control BXSB/MpJ at different stages. Further, immunopositive area of endothelial marker (CD34), podocyte functional molecules (Nephrin, Podocin, Synaptopodin, and Wilms’ tumour 1 (WT1)), and vascular endothelial growth factor A (VEGF A) significantly decreased in the glomerulus of BXSB/MpJ-Yaa compared with BXSB at final stage. The indices of glomerular endothelial injuries (EF density and immunopositive area of CD34 and VEGF A) and podocyte injuries (PEP density and immunopositive area of podocyte functional molecules) were also significantly correlated with each other and with indices of autoimmune disease and renal dysfunction. Thus, our results elucidated the pathological crosstalk between endothelial cells and podocytes in MPGN progression and the usefulness of mSEM for glomerular pathological analysis.

MicroRNAs act as post-transcriptional regulators, and urinary exosome (UExo)-derived microRNAs may be used as biomarkers. Herein, we screened for UExo-derived microRNAs reflecting kidney disease (KD) status in dogs. Examined dogs were divided into healthy kidney control (HC) and KD groups according to renal dysfunction. We confirmed the appearance of UExo having irregular globe-shapes in a dog by immunoblot detection of the exosome markers, TSG101 and CD9. Based on our previous data using KD model mice and the data obtained herein by next generation sequencing of UExo-derived microRNAs in dogs, miR-26a, miR-146a, miR-486, miR-21a, and miR-10a/b were selected as candidate microRNAs. In particular, UExo-derived miR-26a and miR-10a/b were significantly decreased in KD dogs, and miR-26a levels negatively correlated with deteriorated renal function compared to the other miRNAs. UExo-derived miR-21a levels corrected or not to that of internal control microRNAs in UExo, miR-26a and miR-191, significantly increased with renal dysfunction. In kidney tissues, the decrease of miR-26a and miR-10a/b in the glomerulus and miR-10b in the tubulointerstitium negatively correlated with deteriorated renal function and histopathology. Increased miR-21a in the tubulointerstitium rather than in the glomerulus correlated with deteriorated renal histopathology. In conclusion, microRNAs reflecting the changes in renal function and histopathology in dogs were identified in this study.