Periodic acid-Schiff Research Articles

Long-Term Follow-Up of Pediatric Excimer Laser-Assisted Penetrating Keratoplasty for Congenital Stromal Corneal Dystrophy.

The purpose of this study was to highlight characteristic clinical and microscopic findings and report the long-term follow-up of pediatric excimer laser-assisted penetrating keratoplasty (excimer-PKP) for congenital stromal corneal dystrophy (CSCD). A 2-year-old Greek child presented with CSCD at our department. Clinical examination showed bilateral flake-like whitish corneal opacities affecting the entire corneal stroma up to the limbus. Genetic testing identified a mutation of the decorin gene (c.962delA). The variant was not present in the parents and represented a de novo mutation. The uncorrected visual acuity was 20/100 in both eyes. Excimer-PKP (8.0/8.1 mm) was performed on the right eye at the age of 2.5 years and on the left eye at the age of 3 years. Postoperatively, alternating occlusion treatment was performed. The light microscopic examination demonstrated a disorganized extracellular matrix of the corneal stroma characterized by a prominent irregular arrangement of stromal collagen lamellae with large interlamellar clefts containing ground substance, highlighted by periodic acid-Schiff- and Alcian blue-positive reaction detecting acid mucopolysaccharides. Electron microscopy showed disorganization and caliber variation of collagen lamellae and thin filaments within an electron-lucent ground substance. The postoperative course was unremarkable. Both grafts remained completely clear 14 years postoperatively. Corneal tomography showed moderate regular astigmatism with normal corneal thickness. The corrected distance visual acuity was 20/25 in both eyes. Excimer-PKP for CSCD might be associated with excellent long-term results and a good prognosis, particularly when the primary surgery is performed at a very young age. However, this requires close postoperative follow-up examinations by an experienced pediatric ophthalmologist to avoid severe amblyopia.

Macroscopic, microscopic, and immunofluorescent characterization of the Greek tortoise (Testudo graeca graeca) oropharyngeal floor with concern to its feed adaptation as a herbivorous land reptile.

The current investigation focuses on gross anatomy, light, and scanning electron microscopy (SEM) of the Testudo graeca oropharyngeal floor, with particular reference to the immunofluorescence technique to examine its tongue. The T. graeca oropharyngeal floor showed many anatomical structures: the lower rhamphotheca, paralingual ridge, lower alveolar ridge, tongue, laryngeal mound, and glottis. The lower rhamphotheca appeared as a V-shaped jaw line with a highly serrated edge and a median tomium (beak). SEM observations of the lingual apex and the lingual body showed rectangular and conical filiform papillae with porous surfaces and taste pores. Meanwhile, the lingual root had two wings that carried papillae with different shapes: dagger-shaped, conical, bifurcated, and leaf-like papillae, and these papillae lacked taste pores. The laryngeal mound had openings for the laryngeal mucus gland and its secretions. Light microscopy findings showed mucous glands in the propria submucosa and near the mucosal surface of the lingual apex. The lingual root had lingual papillae and two hyaline cartilaginous skeletons between skeletal muscles, and the lingual papillae were elongated filiform, rectangular filiform papillae, and fungiform papillae. The lamina propria constituted the core of the lingual papillae and the mucous gland, they had a positive reaction with the periodic acid schiff (PAS) reagent. The apical surface of the fungiform papillae had taste pores. Under immunofluorescence, the vimentin was detected in taste bud cells, and synaptophysin reacted to the taste buds and nerve bundles. The current study of the Greek tortoise oropharyngeal floor investigated its herbivorous eating habits using its serrated lower rhamphotheca, a large tongue with differently shaped papillae, and numerous mucous glands. RESEARCH HIGHLIGHTS: The Greek tortoise (T. graeca graeca) oropharyngeal floor showed many anatomical structures: lower rhamphotheca, paralingual ridge, lower alveolar ridge, tongue, laryngeal mound, and glottis. SEM and light microscopy observations of the tongue revealed varied types and shapes of lingual papillae with a porous surface on the tongue apex (rectangular or conical filiform papillae), on the tongue body (filiform and fungiform papillae), and on the tongue root (dagger-shaped, conical, bifurcated, and leaf-like papillae). Light microscopy findings: the lamina propria constituted the core of the lingual papillae and had numerous mucous glands that had a slightly magenta-red color with PAS reagent. The apical surface of the fungiform papillae had taste pores. Vimentin and synaptophysin gave a reaction to the taste buds.

Aminophylline suppresses chronic renal failure progression by activating SIRT1/AMPK/mTOR-dependent autophagy.

Chronic renal failure (CRF) is a severe syndrome affecting the urinary system for which there are no effective therapeutics. In this study, we investigate the effects and mechanisms of aminophylline in preventing CRF development. A rat model of chronic renal failure is established by 5/6 nephrectomy. The levels of serum creatinine (SCR), urinary protein (UPR), and blood urea nitrogen (BUN) are detected by ELISA. Histological evaluations of renal tissues are performed by H&E, Masson staining, and PAS staining. Functional protein expression is detected by western blot analysis or immunofluorescence microscopy. Glomerular cell apoptosis is determined using the TUNEL method. Results show that Aminophylline significantly reduces the levels of SCR, UPR, and BUN in the CRF model rats. Histological analyses show that aminophylline effectively alleviates renal tissue injuries in CRF rats. The protein expression levels of nephrin, podocin, SIRT1, p-AMPK, and p-ULK1 are greatly increased, while p-mTOR protein expression is markedly decreased by aminophylline treatment. Additionally, the protein level of LC3B in CRF rats is significantly increased by aminophylline. Moreover, aminophylline alleviates apoptosis in the glomerular tissues of CRF rats. Furthermore, resveratrol promotes SIRT1, p-AMPK, and p-ULK1 protein expressions and reduces p-mTOR and LC3B protein expressions in CRF rats. Selisistat (a SIRT1 inhibitor) mitigates the changes in SIRT1, p-AMPK, p-ULK1, p-mTOR, and LC3B expressions induced by aminophylline. Finally, RAPA alleviates renal injury and apoptosis in CRF rats, and 3-MA eliminates the aminophylline-induced inhibition of renal injury and apoptosis in CRF rats. Aminophylline suppresses chronic renal failure progression by modulating the SIRT1/AMPK/mTOR-mediated autophagy process.

Fungi and bacteria occupy distinct spatial niches within carious dentin.

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal β-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.

Patient-derived parotid gland pleomorphic adenoma organoid model and its preliminary histological and functional characterization

Objective: To establish patient-derived organoid models of pleomorphic adenomas (PAs) of the parotid gland and preliminarily characterize their histology, related biomarkers and functions. Methods: Fresh tumor tissue specimens were collected from surgical procedures of Oral and Maxillofacial Department. The harvested tissues were processed and cultured in a head and neck tumor organoid culture system, resulting in the successful establishment of organoid models from four cases of parotid gland pleomorphic adenomas. The in vitro growth of PA organoids was recorded by light microscopy. The successfully established organoids were passaged and cryopreserved, and the cryopreserved PA organoids were revived and re-cultured to observe their viability and organoid regeneration ability. Histological characterization, as well as characterization and detection of related markers and functional proteins, were performed on the organoids, comparing them with the patient-derived tissues. Results: The constructed organoid model of pleomorphic adenoma exhibited a dense and compact three-dimensional spherical structure. Hematoxylin and eosin staining indicated morphological similarities between the organoid and its tissue of origin. Immunohistochemistry showed positive cytoplasmic staining for Calponin, CK7, and EMA in both the organoid and the source tumor tissue, suggesting consistent histopathological characteristics between the organoid and its tissue of origin. Periodic acid-Schiff staining of the organoid showed positive staining for glycogen, with positive staining located in the interior and periphery of the organoid, indicating that the organoid possessed secretory functions like the salivary gland. Conclusion: We successfully constructed organoids of pleomorphic adenoma derived from patient samples. This model faithfully replicates the tissue morphology and biomarkers of the source tissue and exhibits biological functions associated with mucus secretion. It serves as a valuable in vitro model for studying the development and progression of salivary gland tumors.

Shenfu injection alleviate gut ischemia/reperfusion injury after severe hemorrhagic shock through improving intestinal microcirculation in rats

BackgroundShenfu (SF) injection, a traditional Chinese medication, would improve microcirculation in cardiogenic shock and infectious shock. This study was aimed to explore the therapeutic potential of the SF injection in gut ischemia-reperfusion (I/R) injury after severe hemorrhagic shock (SHS) and resuscitation. Furthermore, we also investigated the optimal adm？ inistration timing. MethodsTwenty-four male SD rats were randomly divided into four groups: Sham group (sham, n = 6), Control group (n = 6), SF injection group (SF, n = 6), and Delayed Shenfu injection administration group (SF-delay, n = 6). In SHS and resuscitation model, rats were induced by blood draw to a mean arterial pressure (MAP) of 40 ± 5 mmHg within 1 h and then maintained for 40 min; HR, MAP ‘were recorded, microcirculation index [De Backer score, perfused small vessel density (PSVD), total vessel density (TVD), microcirculation flow index score (MFI), flow heterogeneity index (HI)] were analyzed. The blood gas index was detected, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), diamine oxidase (DAO), malondialdehyde (MDA) were measured by ELISA; ZO-1, and claudin-1 were measured by Western blotting. In addition, hematoxylin-eosin (HE) and periodic acid schiff (PAS) staining pathological sections of the intestinal mucosal tissues were also performed. ResultsSF injection increased the MAP, relieved the metabolic acidosis degree associated with the hypoperfusion, and improved the intestinal microcirculatory density and perfusion quality after I/R injury. The expression of DAO, MDA in intestinal tissue, and plasma IL-6, TNF-α significantly decreased in the SF injection group compared to the control group. The concentration of ZO-1 and claudin-1 is also higher in the SF injection group. In addition, the HE and PAS staining results also showed that SF injection could decrease mucosal damage and maintain the structure. In the SF-delay group, the degree of intestinal tissue damage was intermediate between that of the control group and SF injection group. ConclusionsSF injection protect the intestine from I/R injury induced by SHS and resuscitation, the mechanism of which might be through improving intestinal microcirculation, reducing the excessive release of inflammatory factors and increasing intestinal mucosal permeability. Furthermore, the protection effect is more pronounced if administration during the initial resuscitation phase.

#2281 SEM for the diagnosis of glomerular diseases (amyloidosis, minimal change, thin membrane, IgA, membranoproliferative and diabetic nephropathy)

Abstract Background and Aims Treating renal diseases often requires ultrastructural details to establish or confirm diagnosis. Ultrastructural information that is not evidenced by optical microscopy includes, e.g., the podocyte foot processes effacement, thinning or alteration of basement membrane, presence of fibrils, Fabri's intracellular inclusions and subepithelial vs. subendothelial of deposits. Unfortunately, transmission electron microscopy (TEM), required to achieve the required resolution, has high costs, and is time-consuming. Here, we report our experience with scanning electron microscopy (SEM) on paraffin sections from standard pathology sections. Method We have retrospectively reanalyzed paraffin sections from renal biopsies of 50 patients with a diagnosis of IgAN, minimal change, diabetes, and amyloidosis. The paraffin sections were available and processed according to standard procedures (fixation, paraffin embedding), with a thickness of 3-4 µm. Sections were deparaffinized, stained with PASM (periodic acid Shiff + methenamine), mounted in aqueous mounting medium and imaged with 40x and 60x objectives. Sections were then unmounted, washed to remove glycerol, dehydrated in absolute alcohol, and air-dried. Finally, they were gold coated (“sputtering”) and analyzed in a scanning electron microscope (Zeiss). Results For the interpretation of SEM images, correlative analysis has been used, imaging precisely the same region of the section using standard optical microscopy and SEM. SEM can quickly identify the typical morphology of podocyte foot processes (Fig. 1A) and the podocyte effacement in minimal change disease (Fig. 1B). SEM can also easily allow the measurement of the thickness of the basement membrane of glomerular capillary walls (Fig. 1C-E) and its thinning in case of thin membrane disease (Fig. 1D) or its increased thickness in the case of diabetes (Fig. 1E). Amyloid fibrils (Fig. 1F) can also be identified in the capillary walls, confirming the diagnosis of amyloidosis Congo-red positive (Fig. 1G). The high resolution of the technique allows us to identify with high reliability the small ruptures of the capillary walls in IgA and membranoproliferative nephritis, with extravasation of erythrocytes and fibrin cloths (Fig. 1H). Mitochondria are well-demarcated, particularly in proximal tubule cells, and often, mitochondrial cristae are visible in samples with adequate fixation (Fig. 1I). Interstitial edema and fibrosis are evident in SEM microscopy (Fig. 1L). At very high magnification, in histological preparations with exceptional fixation, the filtration pores of endothelial cells could be observed (partially visible in Fig. 1E), as well as details such as the brush border of the tubular epithelial cells. The interpretation of the images is not always straightforward as it also requires knowledge of the artifacts induced by the fixation, processing, and inclusion of paraffin, such as, for example, the intracytoplasmic cellular structures containing lipids of the intratubular “foam cells” appear empty. For a correct interpretation of the SEM images, however, the recognition of the nuclei is essential because the chromatin is condensed into clods, especially around the periphery, and inside, some areas appear empty. Conclusion Scanning electron microscopy (SEM) is a powerful tool for the study of nephrological diseases, as it allows you to examine microscopic details at the nanometric scale (not detectable in optical microscopy), providing valuable information for diagnosis, understanding, and management of the disease, the possibility of carrying out Correlative Microscopy. Given the relatively simple and fast preparation of the samples to be examined, the low costs of execution, and the possibility of archiving the preparation in the histotheque for a possible revision of the case, this leads us to think that this method could be considered a valid alternative to transmission electron microscopy (TEM).

Unveiling the aluminum tolerance by Tithonia diversifolia grown in acid soil: Insights from morphological, anatomical, and nutritional analysis

AbstractAcidic soils cover two‐thirds of Brazil's land, significantly reducing pasture productivity and resilience. Tithonia diversifolia has emerged as a promising forage alternative for such soils. The objective of this study was to assess the effect of different soil acidity levels, determined by base‐cation saturation (BCS) levels, on the anatomical, and morphological analyses of the roots, the nutritional status of the plants, as well as the root and shoot biomass production. A 107‐days greenhouse experiment was carried out, employing a randomized block design with a 2 × 4 factorial arrangement. The factors included two propagation methods (seeds and stem cuttings) and four BCS levels (19%, 39%, 52%, and 63%). There was no observed interaction between propagation methods and BCS levels regarding root and shoot biomass production. This indicates that T. diversifolia's tolerance to acidic soils is not affected by the propagation method. However, stem‐propagated plants exhibited significantly greater root (31% increase) and shoot (26% increase) biomass production compared to seed‐propagated plants. Regardless of the propagation method, T. diversifolia plants grown in soil with a 19% BCS level showed a substantial reduction in root (45% decrease) and shoot (31% decrease) biomass compared to the 63% BCS level treatment. The root anatomy and morphology of T. diversifolia are affected when cultivated in acidic soils with a 19% BCS level. Nevertheless, the Periodic Acid Schiff (PAS) test detected some mucilage in T. diversifolia roots, suggesting a potential defense mechanism for Al3+. The shoot biomass of T. diversifolia plants cultivated in soil with a 19% BCS level exhibited greater nutritional values compared to the 63% BCS level. In conclusion, propagation methods have a limited impact on the tolerance of T. diversifolia to acidic soils. Despite the negative effects on root and shoot biomass production, T. diversifolia effectively maintains nutrient uptake even under acidic soil conditions with Al3+ availability of 5 mmolc dm−3.

#2339 All-trans retinoic acid enhances the efficacy of mesenchymal stem cells in acute kidney injury

Abstract Background and Aims Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic strategy for acute kidney injury (AKI), but still suffer from the drawbacks of low survival rate, low homing rate, and uncertain differentiation. In search of better therapeutic strategies, we explored all-trans retinoic acid (ATRA) pretreatment of MSCs to observe whether it could improve the therapeutic efficacy on AKI. Method We established a renal ischemia/reperfusion (I/R) injury model induced by clamping of left renal artery for 30 minutes and contralateral nephrectomy. C57BL/6 mice were randomly divided into 4 groups: sham group, IRI group, MSCs group, and ATRA-MSCs group. MSCs or ATRA-pretreated MSCs were injected via tail vein. GFR was measured by transcutaneous monitoring the clearance of FITC-sinistrin. These mice were sacrificed 3 days after surgery, and the serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured. The renal pathological changes were examined by Periodic Acid-Schiff staining (PAS). The in vitro hypoxia/reoxygenation (H/R) model was established using human proximal tubular epithelial cells (HK-2 cells). Co-culturing HK-2 cells with MSCs or ATRA-pretreated MSCs. Western blot, quantitative real time-PCR (qRT-PCR), and immunohistochemistry were used to detect mRNA or protein expression. We performed RNA sequencing (RNA-seq) analysis in ATRA-pretreated MSCs. The content of hyaluronic acid (HA) in the supernatant of ATRA-pretreated MSCs culture was detected by ELISA. Hyaluronan synthase mRNA expression level in ATRA-pretreated MSCs assessed by qRT-PCR analysis. The HAS2 promoter-binding transcription factors were forecasted by the JASPAR Transcription Factor Binding Site database. Western blot was used to detect whether P13K/AKT pathway was activated by ATRA-pretreated MSCs. Whether this nephroprotective effect could be reversed were explored by silencing HAS2 or CD44 antibodies. Results The AKI mice showed a significant decrease in GFR and even complete loss of renal function. Administration of MSCs significantly increased GFR, and MSCs pretreated with ATRA further increased GFR. Mice injected with ATRA-pretreated MSCs had significantly lower levels of SCr, BUN and ATN scores compared with untreated MSCs. Western blot, quantitative real-time PCR (qRT-PCR), and immunohistochemistry showed that ATRA pretreated MSCs were more effective in reducing AKI inflammation and apoptosis and promoting proliferative repair than untreated MSCs. GO enrichment analysis demonstrated that the hyaluronan biosynthetic process may be a significantly expressed. HAS2 expression and HA content of MSCS were significantly increased after the addition of ATRA. Analysis for potential transcription factor binding sites revealed that the HAS2 promoter region has multiple potential binding sites for the retinoic acid receptor. Based on the predictions from the JASPAR database, the RXR-RAR heterodimer had the highest prediction score. We then performed ChIP analysis to experimentally verify the binding of RXR-RAR heterodimer transcription factor to HAS2 promoters. We further found that ATRA-pretreated MSCs activated the PI3K/AKT signaling pathway in AKI. As expected, we found that ATRA-pretreated MSCs anti-inflammatory, anti-apoptosis, and pro-proliferation effects were substantially reversed by silencing HAS2 or blocking CD44. Conclusion ATRA promotes HA secretion from MSCs and activates the PI3K/AKT pathway, which in turn enhances the therapeutic efficacy of MSCs on AKI.

#1902 Remdesivir ameliorates ferroptotic renal injury in hamster after SARS-CoV-2 infection

Abstract Background and Aims The purpose of this study was to see how remdesivir affected renal ferroptotic damage in hamsters following a human Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. We evaluated the inhibitory effects of remdesivir on the ferroptosis of SARS-CoV-2 infection in a hamster infection model and in in vivo assays. Method To investigate the effect of SARS-CoV-2 on renal ferroptosis, we administered 2.5 × 104 p.f.u. SARS-CoV-2 via intranasal to hamster. In the initial study, animals were treated with remdesivir subcutaneously (s.c.) for 7 days beginning at various times in relation to virus challenge. After 5 days, kidney tissues were harvested. The effect of REMDESIVIR on SARS-CoV-2 infection and ferroptosis were evaluated by immunohistochemistry. Next, ferroptosis-related markers, glutathione peroxidase 4 (Gpx4), 4-hydroxynonenal (4-HNE), Acyl-CoA synthetase long chain family member 4 (Acsl4), nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (Ho-1) were evaluated by quantitative PCR. Results In hamsters, SARS-CoV-2 resulted in high viral protein expression in the renal tubules and decreased renal tubular luminal diameter in periodic acid-Schiff (PAS) stained kidney sections. Remdesivir treatment decreased SARS-CoV-2-induced viral spike protein, HO-1, ferritin heavy chain1 (FTH1), ferritin light chain (FTL), and 4-HNE expression in renal tubular epithelial cells of hamsters in a dose-dependent manner. GPX4 expression suppression after infection was ameliorated by remdesivir administration. Increased ferroptosis-related mRNA marker (Gpx4, 4-HNE, Acsl4, Nrf2, Ho-1) expression in hamster kidneys was suppressed by remdesivir treatment. Conclusion All of our data suggest that remdesivir treatment ameliorated SARS-CoV-2 infection-induced ferroptosis in the kidney in a hamster model.

#1957 Microbial and metabolomic remodeling by Sichuan dark tea-based formula improves obesity-induced renal lipid metabolism disorder in mice

Abstract Background and Aims The theory of medicine-food homology is long-standing and has been widely applied in traditional Chinese medicine. The Sichuan dark tea-based medicated dietary formula (alternatively referred to as Qing, or clarity in Chinese) is previously used for its lipid-lowering properties in apoE mice. This study further investigated the renoprotective effects of Qing on diet-induced obesity (DIO) mice and explored the specific mechanisms involved. Method Male C57BL/6 mice were randomly assigned to three groups, a control group, a DIO group, and a Qing treatment group, or the Qing group, with 8 mice in each group. The mice in the control group were given normal maintenance feed and purified water, and the other two groups were fed a high-fat diet for 12 weeks to establish the DIO model. After that, high-fat diet continued in the DIO group, while the Qing group was given Qing at the same time for 12 weeks, during which period the weight of the mice was monitored and recorded every week. The mice were sacrificed after 12 weeks. Serum samples were collected and the levels of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin were measured to evaluate liver function. In addition, renal lipids were extracted to determine the levels of TG and TC in the kidney and periodic acid-Schiff (PAS) and oil red O stainings were performed to evaluate kidney pathological injury. Western blot was performed to determine the phosphorylated AMPK (pAMPK)/AMPK ratio in the kidney tissue. RT-qPCR and Western blot were used to determine the expression of proteins related to fatty acid oxidation and key proteins related to lipid synthesis. 16SrRNA and metabolomics were applied to analyze the gut microbiota in the intestinal contents and its metabolites. Results Compared with those of the control group, the levels of liver mass (P=0.0003), serum ALT(P < 0.0001), AST (P=0.0001), TC (P=0.0191) and TG (P=0.0101) in the model group were significantly increased. Qing treatment can effectively reduce liver mass (P=0.0316), and improve AST (P=0.0012), ALT (P=0.0027), TC (P=0.0200), TG (P=0.0499) levels of mice compared to those of the DIO group. Qing treatment inhibited the expression of lipid synthesis related genes (SREBP-1, FASN and SCD1) and promoted the expression of fatty acid oxidation related genes (CPT1A, PGC1α and PPARγ) in kidneys (Fig. 1). Obesity-triggered dysfunction of structure and composition of gut microbiota were improved by Qing (Fig. 2), where the content of short-chain fatty acids, especially isovaleric acid and propionic acid, were also restored (Fig. 3). Conclusion Our data suggested that Sichuan dark tea-based medicated dietary formula may improve renal lipid metabolism by regulating gut microbiota and the levels of intestinal short-chain fatty acids, thereby protecting obesity-related kidney injury. Isovaleric acid and propionic acid may be the metabolites key to its regulation of gut microbiota.

#2599 Adam17 inhibition moderates interstitial fibrosis and macrophage infiltration in TD1 mouse model

Abstract Background and Aims ADAM17 participates in the release into circulation of inflammatory and fibrotic molecules, such as TNF-α actively involved in the progression of Diabetic Nephropathy. We studied the effect of specific deletion of ADAM17 in the endothelium and renal tubule in a T1 diabetic mouse model demonstrating its participation in the progression of kidney damage. As ADAM17 is also expressed in other cell types we wanted to study the effect of the complete deletion of Adam17 in type 1 diabetic mouse. Method We studied total inducible (by TAM) Adam17 knockout male mice (ADAM17KO) and their control littermates. Animals were rendered diabetic by STZ injection (DB group). Blood glucose, mesangial index (PAS staining), number of podocytes and positive area of α-SMA (by immunohistochemistry) were determined. We also studied the protein expression of the cytokine MCP-1 in renal cortex by Western Blot. Results The mesangial index value observed in the 20wk-DB group was compared with the diabetic ADAM17KO group. In the latter, the value significantly decreased. Podocyte loss detected in DB group was not observed in the KO-DB group. For interstitial fibrosis, the intensity and location of α-SMA was significantly lower in the ADAM17KO group. In addition, complete deletion of Adam17 partly prevented the infiltration of macrophages assessed by the expression of MCP-1 in the protein extract of the renal cortex. Conclusion Complete inhibition of Adam17 expression in T1DM mice prevents fibrosis progression and protects the glomerulus from hypertrophy. At the same time, it reduces macrophage infiltration, limiting the inflammatory response induced by diabetes. We demonstrate the influence of Adam17 on macrophages to reduce the expression of TNF-a partly responsible for the progression of diabetic nephropathy.

#2592 Tubular cell polyploidy is involved in kidney compensatory hypertrophy

Abstract Background and Aims Chronic kidney disease (CKD) affects more than 10% of the population and it is associated with high risk of death for cardiovascular causes or progression towards end stage kidney disease (ESKD) requiring dialysis or kidney transplantation. CKD is characterized by progressive loss of nephrons, with consequent structural degeneration and fibrosis. Nephron number is established during prenatal kidney development and cannot increase. Consequently, following a reduction in kidney mass or in condition of altered metabolic demands, the remaining nephrons undergo compensatory hypertrophy. Compensatory hypertrophy results in increased functional output and has been observed in the absence of direct tissue injury after nephrectomy. Recently, we have identified a previously unknown mechanism of cellular adaptation: polyploidization of tubular epithelial cells (TC), and demonstrated that this is the essential mediator of kidney hypertrophy after acute kidney injury (AKI). Here, we hypothesize that TC polyploidy is involved in compensatory hypertrophy following nephrectomy. Method Polyploid TC are characterized by an increased DNA content in the absence of cell division. To discriminate polyploid cells from actively proliferating cells, we employed a series of in vitro and in vivo transgenic models based on the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI2aR) technology. Nephrectomy was performed by removing the left kidney and mice were sacrificed at different days after uni- Nephrectomy (Nfx). Cytofluorimetric techniques were employed to characterize a subpopulation of polyploid TC and histology investigation was employed analyze fibrosis development. These results were further corroborated by single cell RNA-sequencing (scRNA-seq) analyses in vitro and in vivo. Results Employing mice that express the FUCCI2aR reported under the Pax8 promoter (which marks all TC), we found that TC polyploidy increases immediately after uni-Nfx. This was accompanied by an increased kidney to body mass ratio with a maximum peaks 3 days afterwards. Importantly, after uni-Nfx, YAP1 expression and nuclei localization (i.e., implying pathway activation) was exponentially increased, suggesting that the same mechanism that drives TC polyploidy after AKI controls TC polyploidization response after uni-Nfx. However, unlike in AKI, polyploidy after nephrectomy was not accompanied by structural alterations as shown by PAS and Masson's trichrome staining. Conversely, after uni-Nfx YAP1 knock-out mice, that are unable to undergo rapid polyploidization, displayed an impaired kidney function in the long term, characterized by extensive tubular dilations. Conclusion Polyploidization of TC mediates compensatory hypertrophy following nephrectomy via YAP1 activation

#410 Novel renal pathology and treatment of TIN with monoclonal plasma cells invasion in a patient with Sjögren's syndrome and IgA monoclonal gammopathy

Abstract Background and Aims Patients with Sjögren's syndrome (SS) may occasionally present with monoclonal gammopathy of undetermined significance (MGUS), of the rare IgA type. Additionally, they may present with tubulointerstitial nephritis (TIN) infiltrated by polyclonal plasmacytes and lymphocytes. Previous reports indicate the development of IgA-type MGUS and TIN with monoclonal plasma cells in some SS patients, which are considered distinct from monoclonal gammopathy of renal significance (MGRS) because there is no evidence that monoclonal gammopathy affects nephropathy. Although treatment with prednisolone (PSL) has shown beneficial effects, the influence of monoclonal plasma cells on nephropathy remains unclear and a standardized treatment approach is lacking. Here we present a new renal pathological insight through a case of such a patient as described above, who underwent renal biopsy before and after PSL treatment. The aim is to propose a treatment strategy based on the findings of the second renal biopsy. Method and Results (Case Presentation) A 75-year-old Japanese man with primary complaint of dry mouth was diagnosed with SS by lip biopsy and anti-Ro/SSA and La/SSB antibody tests. Monoclonal proteins were found in both blood and urine. After immunoelectrophoresis, bone marrow puncture, and 18F-FDG PET/CT, we identified it as IgA kappa-type MGUS. At the same time, he developed renal dysfunction and was admitted to our hospital (week 1, Fig. 1) for a renal biopsy. The biopsy revealed severe and diffuse TIN infiltrated by lymphocytes with abundant interstitial plasma cell invasion (Fig. 2a). Minimal changes were observed in the glomeruli. Periodic acid–Schiff staining revealed Dutcher bodies in the nucleus of plasma cells. Immunohistochemical staining for immunoglobulins and light chains showed predominantly positive results for IgA and kappa (Fig. 2b). Monoclonality was confirmed by in situ hybridization. The infiltrates were positive for CD 45, CD 79a, and CD 138, partially positive for CD 19 and CD 20, and negative for CD 5, CD 10, and CD 23. Congo-red staining showed no evidence of amyloid. Immunofluorescence and electron microscopy revealed no detectable immunological deposits in the glomeruli and tubules. Mutations in the myeloid differentiation primary response gene 88 were not detected in the kidney. We diagnosed the condition as acute TIN by lymphocytes with IgA kappa-positive monoclonal plasma cells, distinct from MGRS. Considering the lack of evidence from previous reports on the impact of monoclonal plasma cells on nephropathy, we decided to treat the patient primarily for TIN with SS, initiating PSL at 30 mg/day (0.5 mg/kg/day). Renal function improved rapidly with the treatment, allowing for a gradual reduction in the dose of PSL; however, a low dose of PSL (10 mg/day) was required to prevent relapse. As a complication of PSL, the patient eventually developed a lumbar compression fracture, prompting us to evaluate the effect of PSL on renal function by a renal re-biopsy. The re-biopsy revealed significant recovery of TIN by lymphocytes (Fig. 2c). Other findings, including abundant interstitial plasma cells invasion were comparable to the initial biopsy. We diagnosed the condition as recovered TIN by lymphocytes with IgA kappa-positive monoclonal plasma cells. We concluded that TIN by lymphocytes, rather than monoclonal plasma cell invasion, primarily affected the nephropathy, and PSL mainly improved the former. The Nudix hydrolase 15 codon139 in blood was identified as Cys/Cys, indicating a high risk of severe adverse reactions to azathioprine in Asians. Therefore, we decided to concurrently administer mycophenolate mofetil, proven effective in the treatment of TIN with SS. Conclusion In the management patients with SS and IgA monoclonal gammopathy, the focus should be on TIN by lymphocytes rather than monoclonal plasma cell invasion. This case, considering similar cases, suggests that it should be defined as a new disease category. We demonstrate new renal pathology and treatment insights based on renal re-biopsy in a patient treated with PSL.

#1918 L-NAME accelerates the progression of diabetic kidney disease in mice

Abstract Background and Aims Diabetic kidney disease (DKD) represents one of the major causes of end stage kidney disease worldwide. Despite DN having such a high incidence, mouse models of DN rarely mimic all aspects of the human disease, besides showing very slow progression and sometimes maintenance issues. For this reason, in this project we aimed to develop an accelerated, reproducible and accessible mouse model of DN. Method For our experiments we chose 9-week-old female and male of the leptin receptor knock-out (BKS.Cg-Dock7m +/+ Leprdb/J) mice as type 2 diabetes model. As these mice are still not prone to develop severe kidney injury despite their diabetic condition, we administered the eNOS inhibitor L-NAME for 6 weeks, in order to suppress nitric oxide production and therefore to aggravate kidney damage. Mice were divided in three groups: one vehicle control group, one receiving 40/mg/kg/day of L-NAME and one receiving 80/mg/kg/day of L-NAME dissolved in drinking water. Blood pressure and glomerular filtration rate (GFR) were measured at different time points, together with glycemia and albuminuria. Weight and water intake were also monitored throughout the study. At the end of the experiment, animals were killed and blood and organs collected for further analysis. Results Mice treated with 80 mg/kg/day of L-NAME showed an average increase of 10 mmHg in systolic blood pressure, with a peak of 30 mmHg increase at week 4 of treatment. On the other hand, 40 mg/kg/day of L-NAME did show any effect on blood pressure. Despite the different effect on blood pressure, three weeks of L-NAME administration caused a significant reduction in GFR at both doses, with 5% reduction for the 40 mg-group and 23% for the 80 mg-group. For the 80 mg-group we observed an even further GFR decline after 5 weeks of treatment, with a 28% reduction. No change in GFR was observed in the controls. This decrease in GFR was accompanied by a 10-fold increase in urinary albumin for both experimental groups already after 3 weeks of treatment, which after 4 weeks slightly declined but remained significantly higher than the controls (p < 0.0001). At a histological level, PAS staining of kidney sections revealed that in particular mice treated with 80 mg of L-NAME have enlarged Bowman capsule and glomerular tuft area, besides a reduced glomerular capillary density, coinciding with mesangial expansion. We also stained for overall collagen deposition with picrosirius red staining in order to detect possible fibrosis formation. We then observed that 80 mg of L-NAME accelerates interstitial fibrosis in the cortical area of the kidney, which also overlapped with an increased deposition of collagen I. This increase in collage I was also detected in kidney cortex of mice treated with 40 mg of L-NAME, although their overall collagen deposition and therefore fibrotic tissue did not differ from the control group. The medullar region of kidneys was also analyzed, but none of the two experimental groups revealed significant differences in terms overall collagen or collagen I deposition. Unlike mesangial expansion and fibrosis, L-NAME did not influence microphages infiltration, as revealed by CD68 staining in kidney sections. This suggests that L-NAME treatment does not accelerate kidney damage by triggering or accelerating inflammatory processes. This was also confirmed by qPCR analysis of kidney tissue, where the expression pro-inflammatory markers, such as IL1β, TNFα and MCP-1, did not change in the experimental groups. Conclusion Our results show that L-NAME considerably accelerates the progression of DKD in diabetic mice, with GFR declining and progressive albuminuria already after 3 weeks. Such disfunction is confirmed at 6 weeks by histological analysis, where mesangial expansion and fibrosis were detected. This model can be used to assess the therapeutic potential of novel interventions aimed to slow down DKD.

Antagonistic Effect of Jiawei Shengjiang San on a Rat Model of Diabetic Nephropathy: Related to EGFR/MAPK3/1 Signaling Pathway.

We aimed to delve into the mechanisms underpinning Jiawei Shengjiang San's (JWSJS) action in treating diabetic nephropathy and deploying network pharmacology. Employing network pharmacology and molecular docking techniques, we predicted the active components and targets of JWSJS and constructed a meticulous "drug-component-target" network. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were utilized to discern the therapeutic pathways and targets of JWSJS. Autodock Vina 1.2.0 was deployed for molecular docking verification, and a 100-ns molecular dynamics simulation was conducted to affirm the docking results, followed by in vivo animal verification. The findings revealed that JWSJS shared 227 intersecting targets with diabetic nephropathy, constructing a protein-protein interaction network topology. KEGG enrichment analysis denoted that JWSJS mitigates diabetic nephropathy by modulating lipids and atherosclerosis, the PI3K-Akt signaling pathway, apoptosis, and the HIF-1 signaling pathway, with mitogen-activated protein kinase 1 (MAPK1), MAPK3, epidermal growth factor receptor (EGFR), and serine/threonine-protein kinase 1 (AKT1) identified as collective targets of multiple pathways. Molecular docking asserted that the core components of JWSJS (quercetin, palmitoleic acid, and luteolin) could stabilize conformation with three pivotal targets (MAPK1, MAPK3, and EGFR) through hydrogen bonding. In vivo examinations indicated notable augmentation in body weight and reductions in glycated serum protein (GSP), low-density lipoprotein cholesterol (LDL-C), uridine triphosphate (UTP), and fasting blood glucose (FBG) levels due to JWSJS. Electron microscopy coupled with hematoxylin and eosin (HE) and Periodic acid-Schiff (PAS) staining highlighted the potential of each treatment group in alleviating kidney damage to diverse extents, exhibiting varied declines in p-EGFR, p-MAPK3/1, and BAX, and increments in BCL-2 expression in the kidney tissues of the treated rats. Conclusively, these insights suggest that the protective efficacy of JWSJS on diabetic nephropathy might be associated with suppressing the activation of the EGFR/MAPK3/1 signaling pathway and alleviating renal cell apoptosis.

Coptidis rhizoma extract alleviates oropharyngeal candidiasis by gC1qR-EGFR/ERK/c-fos axis-induced endocytosis of oral epithelial cells

Ethnopharmacological relevanceCoptidis rhizoma, first recorded in the “Shen Nong's Herbal Classic”, is one of the traditional Chinese medicine (TCM) used to treat infectious diseases, with reputed effectiveness against oropharyngeal candidiasis (OPC). Studies have demonstrated the inhibitory properties of C. rhizoma (CRE) against Candida albicans, yet there is limited information available regarding its treatment mechanism for OPC. Aim of the studyOur previous research has suggested that CRE can prevent the formation of C. albicans hyphae and their invasion of the oral mucosa, thereby exerting a therapeutic effect on OPC. Nevertheless, the precise therapeutic mechanisms remain incompletely understood. Previous studies have revealed that a receptor for globular heads of C1q (gC1qR), a crucial co-receptor of the epidermal growth factor receptor (EGFR), facilitates the EGFR-mediated internalization of C. albicans. Therefore, this study aims to investigate the potential mechanism of action of CRE and its primary component, berberine (BBR), in treating OPC by exploring their effects on the gC1qR-EGFR co-receptor. Materials and methodsTo identify the chemical components of CRE, we utilized Ultra-high performance liquid chromatography in conjunction with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MSE), revealing the presence of at least 18 distinct components. To observe the therapeutic effects of CRE on OPC at the animal level, we employed hematoxylin and eosin staining, periodic acid-Schiff staining, scanning electron microscopy, and fungal load detection. Subsequently, we evaluated the anti-inflammatory properties of CRE and its main component, BBR, in treating OPC. This was achieved through enzyme-linked immunosorbent assay (ELISA) both at the animal and cellular levels. Additionally, we assessed the ability of C. albicans to disrupt the epithelial barrier of FaDu cells by studying the protective effects of BBR on the fusion barrier using the transwell assay. To further explore the underlying mechanisms, we analyzed the effects of BBR on the gC1qR-EGFR/extracellular signal-regulated kinase/c-Fos signaling pathway at the cellular level using qRT-PCR, western blotting, and immunofluorescence. Furthermore, we validated the effects of BBR on the gC1qR-EGFR co-receptor through ELISA, qRT-PCR, and western blotting. Finally, to confirm the outcomes observed at the cellular level, we validated the impact of CRE on the gC1qR-EGFR co-receptor in vivo using qRT-PCR, western blotting, and immunofluorescence. These comprehensive methods allowed us to gain a deeper understanding of the therapeutic mechanisms of CRE and BBR in treating OPC. ResultsOur findings indicate that CRE and its primary component, BBR, effectively alleviated the symptoms of OPC by modulating the gC1qR-EGFR co-receptor. The chemical composition of CRE and BBR was accurately identified using UPLC-Q/TOF-MSE. The gC1qR-EGFR co-receptor plays a crucial role in regulating downstream signaling pathways, emerging as a potential therapeutic target for OPC treatment. Through both in vitro and in vivo experiments, we explored the therapeutic potential of CRE and BBR in OPC. Additionally, we employed overexpression and silencing techniques to confirm that BBR can indeed influence the gC1qR-EGFR co-receptor and regulate the gC1qR-EGFR/extracellular signal-regulated kinase (ERK)/c-Fos signaling pathway, leading to improved OPC outcomes. Furthermore, the significance of CRE's effect on the gC1qR-EGFR co-receptor was validated in vivo. ConclusionOur study demonstrates that CRE and its main component, BBR, can effectively alleviate OPC symptoms by targeting the gC1qR-EGFR heterodimer receptor. This discovery offers a promising new therapeutic approach for the treatment of OPC.

Comparative study of the reptilian cornea's microstructure

BackgroundThe cornea of various vertebrate is considered a major part in the glop, which acts as a powerful lens, providing a sharp retinal image, and meantime acts as an excellent defensor for other corneal layers.ResultsThe four reptilian families [Scincidae (Chalcides ocellatus); Chamaeleontidae (Chameleon chameleon); Cheloniidae (Chelonia mydas) and Testudiniae (Testudo kleinmanni)] investigated in the current study were gathered across Egypt. After being taken out of the orbit, the cornea under inquiry was ready for light, specific stain and scanning electron microscopy. The epithelium, stroma, and endothelium are the three corneal layers that are common to all four species of reptiles. All other species lack Bowmen's and Descemet's membranes, with the exception of C. ocellatus and T. kleinmanni. The latter layers of Chalcides ocellatus display a strong affinity for Periodic Acid Schiff stain. Epithelial cells with a variety of forms, from hexagonal to atypical polygonal cells, cover the outer corneal surface. These epithelial cells are coated in short microplicae, microvilli, and microholes of varied diameters. There are a few blebs scattered around their surface.ConclusionThe current study concluded that various habitats have various significant effects on the cornea's microstructure characteristics and its physiological trends to accommodate different environmental surroundings.

Histological Changes in Hepatocellular Carcinoma Post-locoregional Therapy

Locoregional therapies are widely practiced in treatment of primary hepatocellular carcinoma and metastatic carcinoma to the liver as they provide a more targeted treatment with lesser side effects. Transarterial chemo-embolisation and selective internal radiotherapy (SIRT) are a few examples. However, histopathological findings after locoregional therapy of hepatocellular carcinoma are rarely reported. We reported a case of a 61-year-old man with underlying Hepatitis B and subsequent hepatocellular carcinoma who was treated with SIRT followed by wedge liver resection. Macroscopically, the specimen showed a well-defined mass with areas of fibrosis and haemorrhage. Microscopic examination showed 70% tumour necrosis with numerous microspheres surrounded by multinucleated giant cells with histiocytes and chronic inflammatory cells. Currently, the patient is well with regular follow-up. The latest ultrasound findings showed no residual liver lesion in the background of liver cirrhosis. Although assessment of treatment response following locoregional therapy based on histopathological findings are not widely practiced, recognising histopathological changes such as tumour necrosis, provide valuable insight to a patient’s prognosis. Minimal to absent tumour necrosis implies poor response, limiting further locoregional therapy option. Findings of numerous microspheres might pose a diagnostic challenge as they mimic fungal organisms. Hence, histological examination with aid of special stains like periodic acid Schiff (PAS) and Grocott’s methenamine silver stain may be useful.

Quantifying Renal Changes: A Study of Area of Bowman's Space Following Prolonged Itopride Hydrochloride Exposure in Albino Rats

Objective: To see the effects of long-term administration of itopride hydrochloride on the histomorphology of kidney of the male Wistar albino rats.Study Design: A Laboratory based experimental study.Place and Duration of Study: The study was conducted at the Department of Anatomy, CMH Multan Institute of Medical Sciences Multan, Pakistan from September 2020 to March 2022.Methods: The study involved two groups: Group A served as the control, and Group B functioned as the experimental group. Each group consisted of 30 adult male albino rats. The animals of group A were administered 2ml/100g of distilled water by oral gavage along with a diet for laboratory rodents daily for 15 days. The animals of group B were given 4.4mg/kg of itopride hydrochloride dissolved in 2ml/100g body weight of distilled water by oral gavage three times a day for 15 days. The kidney was processed for paraffin embedding and stained with Periodic Acid Schiff (PAS). The area of Bowman's space was measured by linear micrometer after calibration with a stage micrometer and image J software. The student's t-test was utilized to assess potential differences between the two groups, with significance set at a p-value of ≤ 0.05.Result: The area of Bowman's space was significantly increased in the experimental group as compared to the control group (p= 0.045).Conclusion: Long-term Itopride Hydrochloride administration appears to influence the area of Bowman's Space in Wistar albino rats. How to cite this: Hashmi A, Syed H, Irfan A, Rabbani A, Mir A, Razzaq M. Quantifying Renal Changes: A Study of Area of Bowman's Space Following Prolonged Itopride Hydrochloride Exposure in Albino Rats. Life and Science. 2024; 5(2): 138-143. doi: http://doi.org/10.37185/LnS.1.1.539