Tubule Cells Research Articles

ПАТОЛОГОАНАТОМИЧЕСКИЕ ИЗМЕНЕНИЯ У СВИНЕЙ ПРИ ФУМОНИЗИНОВОМ ТОКСИКОЗЕ

The material of the study was 16 weaned piglets that died at the age of 27–45 days, and samples of full-fledged granular mixed feed SK-3 – prestarter for piglets aged 10–42 days, fed in groups of the studied animals. Pathoanatomic examination and histological examination of lung, liver and kidney samples were performed. Conducted a study of the feed for the presence of fumonisins by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) by using Agilent 1290/AB SCIEX Triple Quad 5500 GOST 34140-2017. As a result of the study, it was established that about 4 mcg of fumonisins per 1 kg of weight were received into the body of the studied piglets per day with mixed feed at an acceptable rate of 2 mcg/kg, i.e. the daily dose of fumonisins exceeded the permissible dose by 2 times. At the autopsy, pulmonary edema, asphyxiated heart, protein liver dystrophy with foci of necrosis, catarrhal enteritis, cloudy swelling of the kidneys were found in piglets. Histological examination in the liver of piglets revealed granular dystrophy and necrosis of hepatocytes, and in some animals also leukocyte infiltration of parenchyma, in the kidneys, in the epithelial cells of the renal tubules – granular dystrophy and necrosis, in the lungs – accumulation of edematous fluid in the alveoli and bronchi.

Spatially resolved multi-omics unravels region-specific responses, microenvironment remodeling and metabolic reprogramming in aristolochic acid nephropathy

<p>Aristolochic acid nephropathy (AAN), primarily caused by overexposure to aristolochic acid I (AAI), is characterized by acute renal injury, interstitial nephritis, and metabolic dysfunction. Previous studies have revealed the cell-specific responses to AAI and its role in inducing metabolic dysfunction. However, the lack of structural information in these datasets hinders our full understanding of the spatially specific pathological mechanisms of AAN. To address this limitation, we propose an integration of spatial transcriptomics with spatial metabolomics methods to establish a spatial multi-omics analysis, which allows for deciphering region-specific responses, microenvironmental remodeling, and metabolic reprogramming <i>in situ</i> in AAN. The pathological differences between renal regions indicate that AAI-induced renal injury exhibits spatial heterogeneity. With prolonged AAI treatment, we observed an increased proportion and co-localization of the injured proximal tubule (PT-inj) and immune cells in the cortex region, accompanied by intercellular crosstalk involving the MHC-I and CCL pathways. In addition, we identified a divergent cellular response along nephron segments, with up-regulation of multiple renal stress markers and pathways after AAI treatment. Regional heterogeneity of metabolic activities was also observed, with PT-inj cells exhibiting dysregulation of carbohydrate, lipid, and amino acid metabolic pathways, as well as increased purine and pyrimidine metabolism after AAI treatment. These findings provide a more comprehensive understanding of the cellular and molecular mechanisms of AAN in a spatial context, and suggest potential intervention pathways to alleviate the global burden of AAN.</p>

Protein localization of aquaporins in the adult female disease vector mosquito, Aedes aegypti.

The female Aedes aegypti mosquito is a vector for several arboviral diseases, due to their blood feeding behavior and their association with urban communities. While ion transport in Ae. aegypti has been studied, much less is known about mechanisms of water transport. Rapid water and ion excretion occurs in the adult female mosquito post blood meal and involves a set of organs including the midgut, Malpighian tubules (MTs), and hindgut. The MTs are responsible for the formation of primary urine and are considered the most important site for active transport of ions. Within the cells of the MTs, along with various ion transporters, there are aquaporin water channels that aid in the transport of water across the tubule cell membrane. Six aquaporin genes have been molecularly identified in Ae. aegypti (AQP1-6) and found to be responsible for the transport of water and in some cases, small solutes such as glycerol. In this study, we used immunohistochemistry to localize AaAQP1, 2, 4, 5, and 6 in the adult female Ae. aegypti, in non-blood fed and post blood feeding (0.5 and 24hr) conditions. We further examined the main water transporting aquaporin, AaAQP1, using western blotting to determine protein abundance changes in isolated MTs pre- and post-blood feeding. Using fluorescence in situ hybridization, aqp1 mRNA was found exclusively in the principal cells of female MTs. Finally, we used immunogold staining with transmission electron microscopy to determine subcellular localization of AaAQP1 in the Malpighian tubules under non-blood fed conditions. Interestingly, AaAQP1 was found to be predominantly in the principal cells of the MTs, dispersed throughout the brush border; however, there was also evidence of some AaAQP1 localization in the stellate cells of the MTs.

Development of mitochondrial dysfunction and oxidative stress in chronic kidney disease

Finding new pathogenic factors that affect the progression of chronic diseases is an important step in the fight against these diseases. In this review, the causes and role of mitochondrial dysfunction in the pathogenesis of chronic kidney disease (CKD) are considered. In this context, the importance of mitochondria for kidney cells in general and proximal tubule cells in particular, as well as the role of oxidative stress in the pathogenesis of CKD, are also discussed. The causes of mitochondrial dysfunction in CKD and the consequences of the development of pathological conditions as a result of impaired mitochondrial activity are analyzed in detail.

Online monitoring of epithelial barrier kinetics and cell detachment during cisplatin-induced toxicity of renal proximal tubule cells.

Real-time and non-invasive assessment of tissue health is crucial for maximizing the potential of microphysiological systems (MPS) for drug-induced nephrotoxicity screening. Although impedance has been widely considered as a measure of the barrier function, it has not been incorporated to detect cell detachment in MPS with top and bottom microfluidic channels separated by a porous membrane. During cell delamination from the porous membrane, the resistance between both channels decreases, while capacitance increases, allowing the detection of such detachment. Previously reported concepts have solely attributed the decrease in the resistance to the distortion of the barrier function, ignoring the resistance and capacitance changes due to cell detachment. Here, we report a two-channel MPS with integrated indium tin oxide (ITO) electrodes capable of measuring impedance in real time. The trans-epithelial electrical resistance (TEER) and tissue reactance (capacitance) were extracted from the impedance profiles. We attributed the anomalous initial increase observed in TEER, upon cisplatin administration, to the distortion of tight junctions. Cell detachment was captured by sudden jumps in capacitance. TEER profiles illuminated the effects of cisplatin and cimetidine treatments in a dose-dependent and polarity-dependent manner. The correspondence between TEER and barrier function was validated for a continuous tissue using the capacitance profiles. These results demonstrate that capacitance can be used as a real-time and non-invasive indicator of confluence and will support the accuracy of the drug-induced cytotoxicity assessed by TEER profiles in the two-channel MPS for the barrier function of a cell monolayer.

Radix Astragali and Its Representative Extracts for Diabetic Nephropathy: Efficacy and Molecular Mechanism.

Diabetic nephropathy (DN) is a common microvascular complication of diabetes mellitus (DM). Radix Astragali (RA), a frequently used Chinese herbal medicine in the Leguminosae family, Astragalus genus, with its extracts, has been proven to be effective in DN treatment both in clinical practice and experimental studies. RA and its extracts can reduce proteinuria and improve renal function. They can improve histopathology changes including thickening of the glomerular basement membrane, mesangial cell proliferation, and injury of endothelial cells, podocytes, and renal tubule cells. The mechanisms mainly benefited from antioxidative stress which involves Nrf2/ARE signaling and the PPARγ-Klotho-FoxO1 axis; antiendoplasmic reticulum stress which involves PERK-ATF4-CHOP, PERK/eIF2α, and IRE1/XBP1 pathways; regulating autophagy which involves SIRT1/NF-κB signaling and AMPK signaling; anti-inflammation which involves IL33/ST2 and NF-κB signaling; and antifibrosis which involves TGF-β1/Smads, MAPK (ERK), p38/MAPK, JNK/MAPK, Wnt/β-catenin, and PI3K/AKT/mTOR signaling pathways. This review focuses on the clinical efficacy and the pharmacological mechanism of RA and its representative extracts on DN, and we further document the traditional uses of RA and probe into the TCM theoretical basis for its application in DN.

Drug Screening Using Normal Cell and Cancer Cell Mixture in an Automated 3D Cell Culture System.

Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.

Fluid shear stress-induced changes in megalin trafficking enhance endocytic capacity in proximal tubule cells.

Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multi ligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells in vivo. To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is > 5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs in vivo compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.

Effect of confinement on the propagation patterns of lean hydrogen–air flames

The effect of confinement on the propagation of lean hydrogen–air flames in a Hele-Shaw cell, formed by two parallel plates separated by a small distance, is studied numerically. The influence of momentum loss on the flame structure and propagation rates is analyzed by examining two limits: (i) the limit of very small distance between the plates, for which a quasi-three-dimensional (quasi-3D) Darcy’s law-based approximation is used, and (ii) the limit of unconfined geometry, for which the full set of two-dimensional (2D) Navier-Stokes equations is considered. The influence of heat losses is also included. The chemistry is described by a one-step reduced kinetics with a simple model for transport properties. The results demonstrate that momentum loss primarily increases the flame surface area by elongating the cellular structures in a finger-like form, with a relatively small enhancement of the reactivity at the flame front, consistent with a mechanism of hydrodynamic nature. This elongation leads to approximately a 50% increase in flame speed compared to the absence of confinement. Conversely, heat losses are observed to flatten the large-scale continuous flame front and, if significant enough, can lead to the formation of isolated propagating flame cells of the two-headed or, ultimately, one-headed type. The present results are in good agreement with recent experiments.

SERCA2 dysfunction triggers hypertension by interrupting mitochondrial homeostasis and provoking oxidative stress

Background and aimSarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is critical in maintaining Ca2+ homeostasis. The cysteine 674 (C674) is the key redox regulatory cysteine in regulating SERCA2 activity, which is irreversibly oxidized in the renal cortex of hypertensive mice. We have reported that the substitution of C674 by serine causes SERCA2 dysfunction and increases blood pressure by induction of endoplasmic reticulum stress (ERS). This study is to explore whether the dysfunction of SERCA2 causes hypertension by interrupting mitochondrial homeostasis and inducing oxidative stress. Methods & resultsWe used heterozygous SERCA2 C674S gene mutation knock-in (SKI) mice, where one copy of C674 was substituted by serine to represent partial C674 oxidation. In renal proximal tubule (RPT) cells, the substitution of C674 by serine decreased mitochondrial Ca2+ content, increased mitochondrial membrane potential, ATP content, and reactive oxygen species (ROS), which could be reversed by ERS inhibitor 4-phenylbutyric acid or SERCA2 agonist CDN1163. In SKI RPT cells, the redox modulator Tempol alleviated oxidative stress, downregulated the protein expression of ERS markers and soluble epoxide hydrolase, upregulated the protein expression of dopamine D1 receptor, and reduced Na+/K+- ATPase activity. In SKI mice, SERCA2 agonists CDN1163 and [6]-Gingerol, or the redox modulator Tempol increased urine output and lowered blood pressure. ConclusionThe irreversible oxidation of C674 is not only an indicator of increased ROS, but also further inducing oxidative stress to cause hypertension. Activation of SERCA2 or inhibition of oxidative stress is beneficial to alleviate hypertension caused by SERCA2 dysfunction.

Silencing HE4 alleviates the renal fibrosis in lupus nephritis mice by regulating the C3/MMPs/prss axis

To explore the regulatory effect of human epididymis protein 4 (HE4) on renal fibrosis in mice with lupus nephritis (LN) and the underlying mechanism. Ten-week old MRL/LPR mice were injected with HE4 shRNA adenovirus vector through the renal pelvis for 5 days. Renal tissues were extracted for HE and Masson staining to evaluate pathological changes and fibrosis in lupus nephritis mice. The level of urine protein was measured using a biochemical analyzer, while the expression level of HE4 and p-NF-κB p65 in renal tissues was visualized using an immunofluorescence assay. The level of β2-microglobulin (β2-MG), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (Kim-1) was determined by the immunohistochemical assay. Western blotting was used to determine the levels of C3, HE4, matrix metalloproteinase-2 (MMP2), MMP9, p-p65, prss23, and prss35 in renal tissues. Compared to wild-type C57BL/6 mice, MRL/LPR mice showed a marked increase in the number of glomeruli, hyperplasic basement membrane, severe infiltration of inflammatory cells in renal tubules and glomeruli, obvious necrosis in glomeruli, elevated fibrosis levels, and increased levels of urine protein, β2-MG, NGAL, Kim-1, C3, HE4, MMP2, MMP9, and p-p65; and decreased levels of prss23 and prss35 were observed in MRL/LPR mice. After the administration of the HE4 shRNA adenovirus vector, the repaired structure of renal tubules and glomeruli improved infiltration of inflammatory cells, reduced collagen fiber and urine protein, suppressed levels of C3, HE4, MMP2, MMP9, and p-P65, and facilitated the expression of prss23 and prss35 which were observed. Silencing HE4 improved renal fibrosis and inhibited inflammation in mice with lupus nephritis, which may play a role in inhibiting C3/MMPs and promoting prss-related protein expression.

Peculiarities of the microscopic structure of rat testis under the influence of Vipera berus berus venom

Morpho-functional changes in the organs of the male reproductive system are usually associated with injuries, infectious diseases, age, lifestyle, the presence of bad habits (abuse of alcohol, drugs), the influence of environmental factors, etc. However, the action of natural toxins, in particular those that are components of animal venoms, including the venoms of snakes and vipers, is no less important. The aim of the research is to study the features of the microscopic structure of the testis of rats under the influence of Vipera berus berus venom. Experimental studies were carried out on white non-linear male rats. Animals were conditionally divided into two groups – control and experimental, 10 individuals in each. Experimental rats were injected intraperitoneally with a semi-lethal dose (LD50) (1.576 mg/g-1) of Vipera berus berus venom in physiological solution. Animals of the control group were injected intraperitoneally with only saline solution. Rats were removed from the experiment 24 hours after exposure of the venom, anesthetized by cervical dislocation. Testis samples were taken for microscopic examination. Fixation of the material and preparation of paraffin blocks were carried out according to generally accepted methods. Staining of histological preparations of the testis was carried out with hematoxylin and eosin. Histological preparations were studied using a SEO SСAN light microscope. Administration of Vipera berus berus venom to rats leads to the development of pathogistological changes in the parenchymal elements of the testis of animals, among which desquamation and disorganization of all stages of spermatogenic cells development, disruption of spermatogenesis processes were the most pronounced. Spermatogenic cells of the seminiferous tubules of the testis were distinguished by changes in morphology and location, lost the regularity of their placement, and their number decreased. The cells detached from the basement membrane and did not form a continuous layer. A characteristic feature was the presence of clusters of erythrocytes in the lumen of the seminiferous tubules, which may indicate the development of hemorrhagic complications. In addition, an increase in the number of Leydig cells was detected, which is probably the cause of changes in the hormonal regulation of the organ's function.

Cellularization and intrinsic instability characteristics of 2-methylfuran spherical expanding flame

ABSTRACT The flame morphology, flame instability and thermodynamic parameters of 2-methylfuran (2-MF) spherically expanding flame were studied at different initial temperatures, initial pressures, and equivalence ratios. Quantitative analysis of 2-MF flame surface cracks and cell structures was performed. The radius for the crack to enter the accelerated growth stage is reduced by 52.9% as the initial pressure increases from 2 bar to 4 bar. Also, the radius at which the crack enters the accelerated growth stage is reduced by 16.4% when the equivalence ratio is increased by 0.1. The radius required for the average cell area to stabilize is reduced by 31.5% when the initial pressure increases from 2 bar to 4 bar. Additionally, instability parameters were studied, and the results revealed that hydrodynamic instability and thermal-diffusion stability increase with increasing Peclet number. The relationship between the local instability and stability curves was discussed. The average wavenumber fitting line is closer to the upper limit of the instability curve when the equivalence ratio is 1.4, which is 8.5% higher than that of the equivalence ratio of 1.3. Next, the flame self-acceleration was studied, the initial pressure and equivalence ratio greatly promote the self-acceleration of the flame, but the temperature has no obvious effect, and the self-acceleration exponent of 2-MF flame is less than 1.5. Finally, the flame image information was reconstructed by a reconstruction algorithm and the cellularity factor was formulated to measure the degree of flame cellular. The cellularity factor is affected by initial pressure and equivalence ratio. Comparing and analyzing the evolution of flame cracks, flame self-acceleration, and cellularity factor, it was found that flame cracks promote flame self-acceleration and exacerbate flame cellularization and instability.

Biological effects of Viscum album homeopathic preparations in kidney proximal tubule cells: an in vitro study

Viscum album (VA) preparations of different host trees have been used for the treatment of different diseases with mainly on cancer1 and cardiovascular diseases2. However, the mechanisms related to the mistletoe hypotensive potential are poorly investigated. Therefore, the objective of this work was to evaluate the cytotoxicity, the Na+/K+ ATPase activity and the Reactive Oxygen Species (ROS) generation induced by winter and summer Viscum album mother tinctures (TMVA), as well as by their potentized samples (6cH, 12cH and 30cH), in porcine proximal tubule renal cells (LLC-PK1). For the cytotoxicity evaluation, after 24h of treatment with TMVA (from Quercus robur, Malus domestica, Ulmus carpinifolia, Pinus sylvestris and Abies alba), at concentrations ranging from 0.5% to 2.5% (V/V), the 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was added. The same procedure was done with the potentized samples, after systematic control evaluation, in which the dynamized water was blindly tested. For the Na+/K+ ATPase and ROS assays, cells were treated only for 30 min, using 1.5% (V/V) of TMVA of Malus domestica, Pinus sylvestris, and Abies alba and their respective homeopathic potencies. The MTT assay showed a dose-dependent cytotoxicity related only to the summer Quercus Robur TMVA (p<0.05). Since for the enzymatic and ROS assays cells need to be metabolically active, this TMVA was excluded from these experimental evaluations. Regarding the ROS generation (n=5), no significant effects were triggered neither by TMVA nor by the potentized samples (p>0.05). The Na+/K+ ATPase evaluation (n=9) indicated no enzymatic activity alterations at 30min of incubation time. Further experiments are under development with higher incubation cellular times (24 and 48 h) to confirm the Na+/K+ ATPase effects induced by potentized and non-potentized V. album samples. Additionally, the gene expression of Na+/K+ ATPase, by western blot, and the ultrastructural cellular aspects, using transmission electron microscopy, will be performed.

APOBEC-1 deletion enhances cisplatin-induced acute kidney injury

Cisplatin (CP) induces acute kidney injury (AKI) whereby proximal tubules undergo regulated necrosis. Repair is almost complete after a single dose. We now demonstrate a role for Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (Apobec-1) that is prominently expressed at the interface between acute and chronic kidney injury (CKD), in the recovery from AKI. Apobec-1 knockout (KO) mice exhibited greater mortality than in wild type (WT) and more severe AKI in both CP- and unilateral ischemia reperfusion (IR) with nephrectomy. Specifically, plasma creatinine (pCr) 2.6 ± 0.70 mg/dL for KO, n = 10 and 0.16 ± 0.02 for WT, n = 6, p < 0.0001 in CP model and 1.34 ± 0.22 mg/dL vs 0.75 ± 0.06, n = 5, p < 0.05 in IR model. The kidneys of Apobec-1 KO mice showed increased necrosis, increased expression of KIM-1, NGAL, RIPK1, ASCL4 and increased lipid accumulation compared to WT kidneys (p < 0.01). Neutrophils and activated T cells were both increased, while macrophages were reduced in kidneys of Apobec-1 KO animals. Overexpression of Apobec-1 in mouse proximal tubule cells protected against CP-induced cytotoxicity. These findings suggest that Apobec-1 mediates critical pro-survival responses to renal injury and increasing Apobec-1 expression could be an effective strategy to mitigate AKI.

Comparative effects of chemical and green zinc oxide nanoparticles in caprine testis: ultrastructural and steroidogenic enzyme analysis

ABSTRACT Recent advancements in nanotechnology has opened up enormous possibilities in diverse sectors such as industries, agriculture, environmental remediation, electronics, medicine and varied industries. Among metal oxide nanoparticles zinc oxide nanoparticles has gained considerable attention due to their fascinating physiochemical properties. Rapid growth in the use of zinc oxide nanoparticles (ZnONPs) in daily household products, food and feed additives, biological products, medicine, as antimicrobial agents, electronics and agriculture, creates serious toxic potential risks of these engineered nanoparticles on living organisms. The aim of present study was to assess the effects of synthesized chemical ZnONPs and green ZnONPs on testicular tissue of Capra hircus (goat) in vitro. The reproductive stress was analyzed by ultrastructural damage, change in frequency of apoptotic cells and alteration in steroidogenic enzyme activity. The transmission electron micrographs of testicular cells after treatment with chemical and green ZnONPs at three doses (10 µg/ml, 20 µg/ml and 30 µg/ml) for exposure duration 4 h and 8 h illustrated that chemical nanoparticles induced more alterations, identified as ruptured nuclear membrane, condensation and margination of chromatin material in somatic cells and germ cells in the seminiferous tubules, presence of apoptotic bodies in nucleus of spermatocytes and spermatids, reduction in number of cell organelles, vacuolization and hyalinization of cytoplasm. Maximum damage was observed after treatment of testicular tissues with 30 µg/ml of chemical ZnONPs for 8 h exposure duration. However, the green ZnONPs were found to be less toxic as evidenced by few apoptotic characteristics in testicular cells. The results of fluorescence assay by acridine orange staining showed significant increase in the percentage of apoptotic cells in chemical treated groups as compared to green and control groups. Decreased enzyme activity of 3β-Hydroxysteroid dehydrogenase and 17β-Hydroxysteroid dehydrogenase was assayed in chemical ZnONPs than green ZnONPs treated groups. Our results confirm that chemical ZnONPs are significantly more toxic in comparison to green ZnONPs and adversely affects the male fertility.

Salvianolic acid B attenuates diabetic nephropathy through alleviating ADORA2B, NALP3 in flammasome, and NFκB activity.

Diabetic nephropathy is one of the microvascular complications of diabetes. This study is aimed at investigating the role and mechanisms of salvianolic acid B (Sal B) in diabetic nephropathy. High glucose (HG)-induced human renal tubular epithelial HK-2 cells were treated with Sal B, BAY-60-6583 (agonist of adenosine 2B receptor), or PSB-603 (antagonist of adenosine 2B receptor) for 24 h. Adenosine A2b receptor (ADORA2B), NACHT, leucine-rich repeat (LRR), and pyrin (PYD) domains-containing protein 3 (NALP3), and nuclear factor Kappa B (NFκB) expressions, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) levels were examined. Following 6 weeks of Sal B treatment, db/db mice blood and kidney tissue were harvested for biochemical detection with hematoxylin-eosin (H&E), Masson's, periodic acid schiff (PAS), and Sirius red staining and detection of ADORA2B, NALP3, NFκB, interleukin 1β (IL-1β), and toll-like receptor 4 (TLR4) activity. NFκB, NALP3, and ADORA2B were found to be downregulated in Sal B treated HK-2 cells exposed to high glucose (HG), accompanied by elevated levels of MMPs and reduced intracellular ROS production. Sal B-treated diabetic mice had the improvement in body weight, water intake, hyperglycemia, hyperlipidemia, and liver and kidney function. Altogether, Sal B attenuates HG-induced kidney tubule cell injury and diabetic nephropathy in diabetic mice, providing clues to other novel mechanisms by which Sal B is beneficial in diabetic nephropathy.

Changes in adhesion molecules: β-catenin, E-cadherin and Galectin-3 in cells of testicular seminoma.

The most common testicular tumors are seminomas. They are characterized by rapid growth and a very high potential for metastasis to other organs. Mutual interactions of tumor cells play an important role in the invasiveness and metastatic capacity, in which complexes of adhesion proteins play a special role. There is a lack of studies on changes in these molecules and their behaviour in testicular cancer. The aim of the study was immunohistochemical identification and evalutaion of adhesive molecules β-catenin, E-cadherin, galectin-3 in testicular cancer - seminoma. Tests were performed on sections of testicular cancer - seminoma in comparison with unchanged tissue samples as a control. Material was taken from 30 patients who underwent orchiectomy. Immunohistochemistry and PCR were used to identify β-catenin, E-cadherin and galectin-3 and gene expression. Immunoreactivity and expression of β-catenin and E-cadherin in seminomas were markedly decreased compared to non-cancerous testicular tissue. Galectin-3 immunoreactivity was found in both control and cancerous tissue, but in different location. In non-cancerous tissue, it was localized in the cytoplasm of the cells of the seminiferous tubules, in seminomas it was localized mainly in the endothelium. The expression of the Lgals3 gene encoding galectin-3 in seminomas was slightl higher in relation to the tissue unchanged by the carcinogenetic process. The results of the study suggest a significant role of β-catenin, E-cadherin and galectin-3 in the carcinogenesis of seminomas and may indicate new aspects of the patomechanism of seminomas formation, and thus time lead to better understand the biology of these tumors.

Zoledronic acid and ibandronate-induced nephrotoxicity in 2D and 3D proximal tubule cells derived from human and rat.

Drug-induced proximal tubule (PT) injury remains a serious safety concern throughout drug development. Traditional in vitro 2-dimensional (2D) and preclinical in vivo models often fail to predict drug-related injuries presented in clinical trials. Various 3-dimensional (3D) microphysiological systems (MPSs) have been developed to mimic physiologically relevant properties, enabling them to be more predictive toward nephrotoxicity. To explore the capabilities of an MPS across species, we compared cytotoxicity in hRPTEC/TERT1s and rat primary proximal tubular epithelial cells (rPPTECs) following exposure to zoledronic acid and ibandronate (62.5-500 µM), and antibiotic polymyxin B (PMB) (50 and 250 µM, respectively). For comparison, we investigated cytotoxicity using 2D cultured hRPTEC/TERT1s and rPPTECs following exposure to the same drugs, including overlapping concentrations, as their 3D counterparts. Regardless of the in vitro model, bisphosphonate-exposed rPPTECs exhibited cytotoxicity quicker than hRPTEC/TERT1s. PMB was less sensitive toward nephrotoxicity in rPPTECs than hRPTEC/TERT1s, demonstrating differences in species sensitivity within both 3D and 2D models. Generally, 2D cultured cells experienced faster drug-induced cytotoxicity compared to the MPSs, suggesting that MPSs can be advantageous for longer-term drug-exposure studies, if warranted. Furthermore, ibandronate-exposed hRPTEC/TERT1s and rPPTECs produced higher levels of inflammatory and kidney injury biomarkers compared to zoledronic acid, indicating that ibandronate induces acute kidney injury, but also a potential protective response since ibandronate is less toxic than zoledronic acid. Our study suggests that the MPS model can be used for preclinical screening of compounds prior to animal studies and human clinical trials.

Proximal tubule cells in blood and urine as potential biomarkers for kidney disease biopsy

Early diagnosis and treatment are crucial for managing kidney disease, yet there remains a need to further explore pathological mechanisms and develop minimally invasive diagnostic methods. In this study, we employed single-cell RNA sequencing (scRNA-seq) to assess the cellular heterogeneity of kidney diseases. We analyzed gene expression profiles from renal tissue, peripheral blood mononuclear cells (PBMCs), and urine of four patients with nephritis. Our findings identified 12 distinct cell subsets in renal tissues and leukocytes. These subsets encompassed fibroblast cells, mesangial cells, epithelial cells, proximal tubule cells (PTCs), and six immune cell types: CD8+ T cells, macrophages, natural killer cells, dendritic cells, B cells, and neutrophils. Interestingly, PTCs were present in both PBMCs and urine samples but absent in healthy blood samples. Furthermore, several populations of fibroblast cells, mesangial cells, and PTCs exhibited pro-inflammatory or pro-apoptotic behaviors. Our gene expression analysis highlighted the critical role of inflammatory PTCs and fibroblasts in nephritis development and progression. These cells showed high expression of pro-inflammatory genes, which could have chemotactic and activating effect on neutrophils. This was substantiated by the widespread in these cells. Notably, the gene expression profiles of inflammatory PTCs in PBMCs, urine, and kidney tissues had high similarity. This suggests that PTCs in urine and PBMCs hold significant potential as alternative markers to invasive kidney biopsies.