Optimized protocol for the multi-omics processing of cryopreserved human kidney tissue.

Biobanking of tissue from clinically obtained kidney biopsies for later use with multi-omic and imaging techniques is an inevitable step to overcome the need of disease model systems and towards translational medicine. Hence, collection protocols ensuring integration into daily clinical routines using preservation media not requiring liquid nitrogen but instantly preserving kidney tissue for clinical and scientific analyses are of paramount importance. Thus, we modified a robust single nucleus dissociation protocol for kidney tissue stored snap frozen or in the preservation media RNAlaterand CellCover. Using porcine kidney tissue as surrogate for human kidney tissue, we conducted single nucleus RNA sequencing with the Chromium 10X Genomics platform. The resulting data sets from each storage condition were analyzed to identify any potential variations in transcriptomic profiles. Furthermore, we assessed the suitability of the preservation media for additional analysis techniques (proteomics, metabolomics) and the preservation of tissue architecture for histopathological examination including immunofluorescence staining. In this study, we show that in daily clinical routines the RNAlater facilitates the collection of highly preserved human kidney biopsies and enables further analysis with cutting-edge techniques like single nucleus RNA sequencing, proteomics, and histopathological evaluation. Only metabolome analysis is currently restricted to snap frozen tissue. This work will contribute to build tissue biobanks with well-defined cohorts of the respective kidney disease that can be deeply molecularly characterized, opening new horizons for the identification of unique cells, pathways and biomarkers for the prevention, early identification, and targeted therapy of kidney diseases.

Recellularization of Acellular Xeno Kidney Scaffold: An In Vivo Method to Generate Bioartificial Kidney.

A significant hurdle for kidney tissue engineering is reproducing the complex three-dimensional structure of the kidney. In our study, a stepwise approach of generating a reproducible Xeno kidney scaffold from a goat kidney is described, which can be implanted and recellularized by host cells. We have proposed a combination of sodium dodecyl sulfate and Triton-X-100-based protocol to generate a reproducible Xeno kidney scaffold, which was then analyzed by histology, DNA quantification, SEM, and renal angiography. Further, a small portion from the cortico-medullar region of the acellular scaffold was implanted in the rat's kidney subcapsular pocket for a period of 1 month, to check the recruitment of host cells into the scaffold. Post implantation, the extracellular matrix of the scaffold was well preserved and it did not induce any damage or inflammation in the native kidney. Implantation of the Xeno scaffold resulted in apparent early vascularization which helped in the recruitment of the host cells, which was characterized by histology, immunohistochemistry, and scanning electron microscopy. Implanted Xeno scaffold showed AQP-1, Nephrin, α-SMA, and VEGF expression in proximal tubules and renal glomerulus. Importantly, Ki-67 and WTAP-expressing cells were also observed near proximal tubules suggesting a high level of proliferation in the scaffold. Thus, showing the potential of Xeno kidney development that can be recellularized by the host cell to engineer into a functional kidney.

The heterophilicic epitopes in conserved HA regions of human and avian influenza viruses can produce antibodies that bound to kidney tissue.

Influenza virus infection can cause kidney damage. However, the link between influenza infection and disease is still unclear. The purpose of this study was to analyze the relationship between heterophilic epitopes on H5N1 hemagglutinin (HA) and disease. The monoclonal antibody (mAb) against H5N1 was prepared, mAbs binding to human kidney tissue were screened, and the reactivities of mAbs with five different subtypes of influenza virus were detected. Design and synthesize the peptides according to the common amino acid sequence of these antigens, and analyze the distribution of the epitope on the crystal structure of HA. Immunological methods were used to detect whether the heterophilic epitopes could induce the production of antibodies that cross-react with kidney tissue. The results showed that H5-30 mA b binding to human kidney tissue recognized the heterophilic epitope 191-LVLWGIHHP-199 on the head of HA. The key amino acid were V192, L193, W194 and I196, which were highly conserved in human and avian influenza virus HA. The heterophilic epitope could induce mice to produce different mAbs binding to kidney tissue. Such heterophilic antibodies were also detected in the serum of the patients. It can provide materials for the mechanism of renal diseases caused by influenza virus infection.

Establishment and characterization of a novel conditionally immortalized human parietal epithelial cell line.

Parietal epithelial cells (PECs) are epithelial cells in the kidney, surrounding Bowman's space. When activated, PECs increase in cell volume, proliferate, migrate to the glomerular tuft and excrete extracellular matrix. Activated PECs are crucially involved in the formation of sclerotic lesions, seen in focal segmental glomerulosclerosis (FSGS). In FSGS, a number of glomeruli show segmental sclerotic lesions. Further disease progression will lead to increasing number of involved glomeruli and gradual destruction of the affected glomeruli. Although the involvement of PECs in FSGS has been acknowledged, little is known about the molecular processes driving PEC activation. To get more insights in this process, accurate in vivo and in vitro models are needed. Here, we describe the development and characterization of a novel conditionally immortalized human PEC (ciPEC) line. We demonstrated that ciPECs are differentiated when grown under growth-restrictive conditions and express important PEC-specific markers, while lacking podocyte and endothelial markers. In addition, ciPECs showed PEC-like morphology and responded to IL-1ß treatment. We therefore conclude that we have successfully generated a novel PEC line, which can be used for future studies on the role of PECs in FSGS.

Renal reabsorption in 3D vascularized proximal tubule models.

Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular-vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium-endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.

MicroRNA Expression in Clear Cell Renal Cell Carcinoma Cell Lines and Tumor Biopsies: Potential Therapeutic Targets.

This study was carried out to quantitate the expression levels of microRNA-17, -19a, -34a, -155, and -210 (miRs) expressed in nine clear cell renal cell carcinoma (ccRCC) and one chromophobe renal cell carcinoma cell line with and without sarcomatoid differentiation, and in six primary kidney tumors with matching normal kidney tissues. The data in the five non-sarcomatoid ccRCC cell lines-RC2, CAKI-1, 786-0, RCC4, and RCC4/VHL-and in the four ccRCC with sarcomatoid differentiation-RCJ41T1, RCJ41T2, RCJ41M, and UOK-127-indicated that miR-17 and -19a were expressed at lower levels relative to miR-34a, -155, and -210. Compared with RPTEC normal epithelial cells, miR-34a, miR-155, and miR-210 were expressed at higher levels, independent of the sarcomatoid differentiation status and hypoxia-inducible factors 1α and 2α (HIFs) isoform expression. In the one chromophobe renal cell carcinoma cell line, namely, UOK-276 with sarcomatoid differentiation, and expressing tumor suppressor gene TP53, miR-34a, which is a tumor suppressor gene, was expressed at higher levels than miR-210, -155, -17, and -19a. The pilot results generated in six tumor biopsies with matching normal kidney tissues indicated that while the expression of miR-17 and -19a were similar to the normal tissue expression profile, miR-210, -155, -and 34a were expressed at a higher level. To confirm that differences in the expression levels of the five miRs in the six tumor biopsies were statistically significant, the acquisition of a larger sample size is required. Data previously generated in ccRCC cell lines demonstrating that miR-210, miR-155, and HIFs are druggable targets using a defined dose and schedule of selenium-containing molecules support the concept that simultaneous and concurrent downregulation of miR-210, miR-155, and HIFs, which regulate target genes associated with increased tumor angiogenesis and drug resistance, may offer the potential for the development of a novel mechanism-based strategy for the treatment of patients with advanced ccRCC.

Effect of the Lipid Fraction of Microalgae on Biochemical Parameters in Female C57BL/6 Mice.

We studied the effect of microalgae of various systematic groups added to the ration on the biochemical parameters of blood serum and liver and kidney tissue in rats. It was found that microalgae had different effects on the levels of proteins, lipids, and sex hormones, activity of aminotransaminases, and filtration capacity of the kidneys. Microalgae also affected the biochemical parameters of the liver and kidney tissues.

Metabolomic and elemental profiling of human tissue in kidney cancer.

INTRODUCTION: Kidney cancer is one of the most frequently diagnosed and the most lethal urinary cancer. Despite advances in treatment, no specific biomarker is currently in use to guide therapeutic interventions. OBJECTIVES: Major aim of this work was to perform metabolomic and elemental profiling of human kidney cancer and normal tissue and to evaluate cancer biomarkers. METHODS: Metabolic and elemental profiling of tumor and adjacent normal human kidney tissue from 50 patients with kidney cancer was undertaken using three different analytical methods. RESULTS: Five potential tissue biomarkers of kidney cancer were identified and quantified using with high-resolution nuclear magnetic resonance spectroscopy. The contents of selected chemical elements in tissues was analyzed using inductively coupled plasma optical emission spectrometry. Eleven mass spectral features differentiating between kidney cancer and normal tissues were detected using silver-109 nanoparticle enhanced steel target laser desorption/ionization mass spectrometry. CONCLUSIONS: Our results, derived from the combination of ICP-OES, LDI MS and 1H NMR methods, suggest that tissue biomarkers identified herein appeared to have great potential for use in clinical prognosis and/or diagnosis of kidney cancer.

Open microfluidic coculture reveals paracrine signaling from human kidney epithelial cells promotes kidney specificity of endothelial cells.

Endothelial cells (ECs) from different human organs possess organ-specific characteristics that support specific tissue regeneration and organ development. EC specificity is identified by both intrinsic and extrinsic cues, among which the parenchyma and organ-specific microenvironment are critical contributors. These extrinsic cues are, however, largely lost during ex vivo cultures. Outstanding challenges remain to understand and reestablish EC organ specificity for in vitro studies to recapitulate human organ-specific physiology. Here, we designed an open microfluidic platform to study the role of human kidney tubular epithelial cells in supporting EC specificity. The platform consists of two independent cell culture regions segregated with a half wall; culture media are added to connect the two culture regions at a desired time point, and signaling molecules can travel across the half wall (paracrine signaling). Specifically, we report that in the microscale coculture device, primary human kidney proximal tubule epithelial cells (HPTECs) rescued primary human kidney peritubular microvascular EC (HKMEC) monolayer integrity and fenestra formation and that HPTECs upregulated key HKMEC kidney-specific genes (hepatocyte nuclear factor 1 homeobox B, adherens junctions-associated protein 1, and potassium voltage-gated channel subfamily J member 16) and endothelial activation genes (vascular cell adhesion molecule-1, matrix metalloproteinase-7, and matrix metalloproteinase-10) in coculture. Coculturing with HPTECs also promoted kidney-specific genotype expression in human umbilical vein ECs and human pluripotent stem cell-derived ECs. Compared with culture in HPTEC conditioned media, coculture of ECs with HPTECs showed increased upregulation of kidney-specific genes, suggesting potential bidirectional paracrine signaling. Importantly, our device is compatible with standard pipettes, incubators, and imaging readouts and could also be easily adapted to study cell signaling between other rare or sensitive cells.

Renal tissue Po2 sensing during acute hemodilution is dependent on the diluent.

Sensing changes in blood oxygen content ([Formula: see text]) is an important physiological role of the kidney; however, the mechanism(s) by which the kidneys sense and respond to changes in [Formula: see text] are incompletely understood. Accurate measurements of kidney tissue oxygen tension ([Formula: see text]) may increase our understanding of renal oxygen-sensing mechanisms and could inform decisions regarding the optimal fluid for intravascular volume resuscitation to maintain renal perfusion. In some clinical settings, starch solution may be nephrotoxic, possibly due to inadequacy of tissue oxygen delivery. We hypothesized that hemodilution with starch colloid solutions would reduce [Formula: see text] to a more severe degree than other diluents. Anesthetized Sprague-Dawley rats (n = 77) were randomized to undergo hemodilution with either colloid (6% hydroxyethyl starch or 5% albumin), crystalloid (0.9% saline), or a sham procedure (control) (n = 13-18 rats/group). Data were analyzed by ANOVA with significance assigned at P < 0.05. After hemodilution, mean arterial pressure (MAP) decreased marginally in all groups, while hemoglobin (Hb) and [Formula: see text] decreased in proportion to the degree of hemodilution. Cardiac output was maintained in all groups after hemodilution. [Formula: see text] decreased in proportion to the reduction in Hb in all treatment groups. At comparably reduced Hb, and maintained arterial oxygen values, hemodilution with starch resulted in larger decreases in [Formula: see text] relative to animals hemodiluted with albumin or saline (P < 0.008). Renal medullary erythropoietin (EPO) mRNA levels increased more prominently, relative to other hypoxia-regulated molecules (GLUT-1, GAPDH, and VEGF). Our data demonstrate that the kidney acts as a biosensor of reduced [Formula: see text] following hemodilution and that [Formula: see text] may provide a quantitative signal for renal cellular responsiveness to acute anemia. Evidence of a more severe reduction in [Formula: see text] following hemodilution with starch colloid solution suggests that tissue hypoxia may contribute to starch induced renal toxicity.

Pronounced kidney hypoxia precedes albuminuria in type 1 diabetic mice.

Intrarenal tissue hypoxia has been proposed as a unifying mechanism for the development of chronic kidney disease, including diabetic nephropathy. However, hypoxia has to be present before the onset of kidney disease to be the causal mechanism. To establish whether hypoxia precedes the onset of diabetic nephropathy, we implemented a minimally invasive electron paramagnetic resonance oximetry technique using implanted oxygen sensing probes for repetitive measurements of in vivo kidney tissue oxygen tensions in mice. Kidney cortex oxygen tensions were measured before and up to 15 days after the induction of insulinopenic diabetes in male mice and compared with normoglycemic controls. On day 16, urinary albumin excretions and conscious glomerular filtration rates were determined to define the temporal relationship between intrarenal hypoxia and disease development. Diabetic mice developed pronounced intrarenal hypoxia 3 days after the induction of diabetes, which persisted throughout the study period. On day 16, diabetic mice had glomerular hyperfiltration, but normal urinary albumin excretion. In conclusion, intrarenal tissue hypoxia in diabetes precedes albuminuria thereby being a plausible cause for the onset and progression of diabetic nephropathy.

Single adult kidney stem/progenitor cells reconstitute three-dimensional nephron structures in vitro.

The kidneys are formed during development from two distinct primordial tissues, the metanephric mesenchyme and the ureteric bud. The metanephric mesenchyme develops into the kidney nephron, the minimal functional unit of the kidney. A nephron consists of several segments and regulates water, electrolyte, and acid-base homeostasis in addition to secreting certain hormones. It has been predicted that the kidney will be among the last organs successfully regenerated in vitro due to its complex structure and multiple functions. Here, we show that adult kidney stem/progenitor cells (KS cells), derived from the S3 segment of adult rat kidney nephrons, can reconstitute a three-dimensional kidney-like structure in vitro. Kidney-like structures were formed when a cluster of KS cells was suspended in an extracellular matrix gel and cultured in the presence of several growth factors. Morphological analyses revealed that these kidney-like structures contained every substructure of the kidney, including glomeruli, proximal tubules, the loop of Henle, distal tubules, and collecting ducts, but no vasculature. Our results demonstrate that a cluster of tissue stem/progenitor cells has the ability to reconstitute the minimum unit of its organ of origin by differentiating into specialized cells in the correct location. This process differs from embryonic kidney development, which requires the mutual induction of two different populations of progenitors, metanephric mesenchymal cells and ureteric bud cells.

Assessment standards: comparing histopathology, digital image analysis, and stereology for early detection of experimental cisplatin-induced kidney injury in rats.

Histopathology generally represents the reference standard for performance evaluation of nonclinical biomarkers used to inform regulatory decision making. This study uses drug-induced nephrotoxicity in rats to evaluate histopathology methods utilized in biomarker performance assessments. Male Sprague-Dawley rats received a single dose of cisplatin (0.5-5.0 mg/kg, intraperitoneally) to produce mild renal injury. Animals were euthanized 72 hr postdose and perfusion fixed. Kidneys were processed for histology and stereology procedures. Kidney injury molecule-1 (Kim-1) was measured in urine and in kidney tissue. Digital slide images were generated and analyzed by pathologists after collaborating on a training set of glass slides and digital images. Image analysis identified immunohistochemistry (IHC)-defined tubular injury. Stereology methods yielded estimations of proximal tubular cell number and volume. Statistical relationships among data sets were determined using correlation coefficients. Receiver operator characteristic (ROC) analyses determined the effect of method on biomarker assessment. Urinary Kim-1 was strongly correlated with digital image analysis and secondarily to histopathology evaluations. Stereology demonstrated weak or no correlation to pathology and urinary Kim-1. In ROC analyses, semiquantitative evaluations determined higher values for urinary Kim-1 performance than did IHC-based qualitative digital analyses. Semiquantitative evaluation as used in this study was most predictive of urinary Kim-1 values.

Kidney tissue targeted metabolic profiling of glucocorticoid-induced osteoporosis and the proposed therapeutic effects of Rhizoma Drynariae studied using UHPLC/MS/MS.

Traditional Chinese medicine and modern science have indicated that there is a close relationship between bone and kidney. In light of this, this project was designed to study the metabolic profiling by UHPLC/MS/MS of glucocorticoid-induced osteoporosis in kidney tissue and the possible therapeutic effects of Rhizoma Drynariae (RD), a classic traditional Chinese medicine, in improving the kidney function and strengthening bone. Twenty-one Wistar rats were divided into three groups: control group (rats before prednisolone inducing), a model group (prednisolone-induced group) and a treatment group (prednisolone-induced rats that were then administered RD ethanol extracts). By using pattern recognition analysis, a significant change in the metabolic profile of kidney tissue samples was observed in the model group and restoration of the profile was observed after the administration of RD ethanol extracts. Some significantly changed biomarkers related to osteoporosis such as sphingolipids (C16 dihydrosphingosine, C18 dihydrosphingosine, C18 phytosphingosine, C20 phytosphingosine), lysophosphatidycholines (C16:0 LPC, C18:0 LPC) and phenylalanine were identified. As a complement to the metabolic profiling of RD in plasma, these biomarkers suggest that kidney damage, cell cytotoxicity and apoptosis exist in osteoporosis rats, which is helpful in further understanding the underlying process of glucocorticoid-induced osetoporosis and the suggested therapeutic effects of RD. The method shows that tissue target metabonomics might provide a powerful tool to further understand the process of disease and the mechanism of therapeutic effect of Chinese medicines.

n-Acetylcysteine protects against diazinon-induced histopathological damage and apoptosis in renal tissue of rats.

Diazinon (DZN) is a member of organophosphorus insecticides that has cytotoxic effects on different organs. n-Acetyl cysteine (NAC) is a widely used antioxidant in clinical, in vivo and in vitro studies. We evaluated the protective role of NAC against DZN-induced toxicity in kidney tissue of Wistar rats. 30 male Wistar rats were divided into 5 groups of control, single dose of DZN, continuous dose of DZN, single doses of DZN + NAC and continuous doses of DZN + NAC. Kidney function test (blood urea nitrogen, creatinine and uric acid) was provided. Levels of malondialdehyde (MDA), total antioxidant capacity (TAC) and total sulfhydryl (T-SH) were determined in renal tissues. Renal cells apoptosis was detected using TUNEL assay. The mRNA expressions of apoptosis, oxidative stress and inflammatory mediators, including B-cell lymphoma-2 (Bcl2), Bcl-2-associated X protein (Bax), superoxide dismutase (SOD), catalase (CAT), Interleukin 10 (IL-10), Tumor necrosis factor-α (TNF-α), Caspase-3 and Caspase-8 were analyzed in kidney tissues using Real Time PCR method. Chronic exposure to DZN was associated with severe morphological changes in the kidney, as well as impairment of its function and decreased kidney weights. Continues treatment with DZN significantly decreased the percentage of renal apoptotic cells as compared to rats treated with continuous dose of DZN alone (17.69 ± 3.67% vs. 39.46% ± 2.44%; p < 0.001). Continuous exposure to DZN significantly decreased TAC and T-SH contents, as well as SOD and CAT expression, but increased MDA contents in the kidney tissues (p < 0.001). A significant increase was observed in mRNA expression of Bax, Caspase-3, Caspase-8, as well as TNF-α following exposure to DZN, but the expression of IL-10 and Bcl2 was significantly decreased. NAC can protect kidney tissue against DZN-induced toxicity by elevating antioxidants capacity, mitigating oxidative stress, inflammation and apoptosis.

Near-infrared microscopy reveals diabetic nephropathy in ob/ob mice.

Diabetic nephropathy (DN) is a major cause of global kidney failure. While histological kidney biopsy is the gold standard for diagnosis, it primarily reveals tissue morphology. In contrast, near-infrared (NIR) microscopy offers a label-free method for detailed molecular characterization of kidney tissue. Hematoxylin and eosin-stained kidney tissue samples from 17 ob/ob mice with DN and 14 healthy mice were examined using Fourier transform-NIR microscopy. Four different spectra were obtained from both the mesangium and tubulus. NIR spectral analysis unveiled distinct differences in wavenumbers between DN-affected and healthy kidneys, notably in the carbohydrate and protein-associated region (5500-4200 cm-1). In the mesangium, DN tissue samples exhibited higher median values at 4235 cm-1, 4659 cm-1, 4844 cm-1, 4906 cm-1, and 5222 cm-1 compared to controls (P < 0.05, P < 0.01, P < 0.05, P < 0.05 and P < 0.001, respectively). In tubular spectra, higher median values were found at 4258 cm-1, 4659 cm-1, 5222 cm-1, and 5346 cm-1 in the DN group (P < 0.01, P < 0.05, P < 0.05 and P < 0.01, respectively). These spectral differences strongly correlated with metabolic, histologic, and urinary parameters, providing valuable DN progression insights. The classification model achieved a visible clustering between the control and DN group for both the mesangial and tubular measurements. NIR microscopy demonstrated significant spectral differences between DN and healthy kidney tissues in mice, hinting at its potential for providing chemical insights, aiding in more accurate diagnoses, and offering a foundation for further clinical exploration and potential therapeutic advancements in DN.

MicroRNA-218 inhibits cell migration and invasion in renal cell carcinoma through targeting caveolin-2 involved in focal adhesion pathway.

PURPOSE: Our microRNA expression signature of renal cell carcinoma revealed that miR-218 expression was significantly decreased in cancer tissues, suggesting that miR-218 is a candidate tumor suppressor. We investigated the functional significance of miR-218 in cancer cells and identified what are to our knowledge novel miR-218 mediated cancer pathways in renal cell carcinoma. MATERIALS AND METHODS: Gain of function studies using mature miR-218 were performed to investigate cell proliferation, migration and invasion in the A498 and 786-O renal cell carcinoma cell lines. To identify miR-218 mediated molecular pathways and responsible genes in renal cell carcinoma, we used gene expression and in silico database analyses. Loss of function assays were performed to investigate the functional significance of miR-218 target genes. RESULTS: Restoration of mature miR-218 significantly inhibited RCC cell proliferation, migration and invasion. Gene expression studies and luciferase reporter assays showed that CAV2 involved in the focal adhesion pathway was directly regulated by miR-218. A silencing study of CAV2 revealed significant inhibition of cell proliferation, migration and invasion. CAV2 mRNA and protein expression was significantly up-regulated in renal cell carcinoma clinical specimens. CONCLUSIONS: Loss of tumor suppressive miR-218 enhances cancer cell migration and invasion through dysregulation of the focal adhesion pathway, especially CAV2 as an oncogenic function in renal cell carcinoma. Tumor suppressive microRNA mediated cancer pathways and responsible genes provide new insights into the potential mechanisms of renal cell carcinoma oncogenesis and metastasis.

Effects of Chronic Heat Stress on Kidney Damage, Apoptosis, Inflammation, and Heat Shock Proteins of Siberian Sturgeon (Acipenser baerii).

Chronic heat stress caused by global warming can have serious implications for fish survival. The kidney plays a central role in many homeostatic functions, including water and electrolyte regulation. However, there is limited knowledge about the effect of heat stress on fish kidneys. In this study, water temperatures were increased from 20 °C to 24 °C and 28 °C in 8 days at a warming rate of 1 °C/d, and then maintained for 12 days. We investigated the effects of mild heat stress (24 °C) and high heat stress (28 °C) on Siberian Sturgeon (Acipenser baerii) kidneys using histological observation, flow cytometry detection, and RT-qPCR. Our histological observations revealed that heat stress caused significant infiltration of inflammatory cells in the kidney, especially at 28 °C. The flow cytometry assay demonstrated a significant increase in the number of apoptotic cells after heat stress at 28 °C compared to a control group at 20 °C (p = 0.033). The level of plasma creatinine was significantly increased in the 28 °C group compared to the control group (p = 0.001). In addition, the mRNA expression levels of heat shock protein GRP75 increased (p = 0.009). The results indicate that heat stress at 28 °C caused damage to the kidneys of A. baerii and triggered the protective response of heat shock proteins. In conclusion, this study contributes to the understanding of the coping strategies of the kidney of A. baerii for chronic heat stress.

Automatic Identification of Glomerular in Whole-Slide Images Using a Modified UNet Model.

Glomeruli are interconnected capillaries in the renal cortex that are responsible for blood filtration. Damage to these glomeruli often signifies the presence of kidney disorders like glomerulonephritis and glomerulosclerosis, which can ultimately lead to chronic kidney disease and kidney failure. The timely detection of such conditions is essential for effective treatment. This paper proposes a modified UNet model to accurately detect glomeruli in whole-slide images of kidney tissue. The UNet model was modified by changing the number of filters and feature map dimensions from the first to the last layer to enhance the model's capacity for feature extraction. Moreover, the depth of the UNet model was also improved by adding one more convolution block to both the encoder and decoder sections. The dataset used in the study comprised 20 large whole-side images. Due to their large size, the images were cropped into 512 × 512-pixel patches, resulting in a dataset comprising 50,486 images. The proposed model performed well, with 95.7% accuracy, 97.2% precision, 96.4% recall, and 96.7% F1-score. These results demonstrate the proposed model's superior performance compared to the original UNet model, the UNet model with EfficientNetb3, and the current state-of-the-art. Based on these experimental findings, it has been determined that the proposed model accurately identifies glomeruli in extracted kidney patches.

Non-Perturbative Identification and Subtyping of Amyloidosis in Human Kidney Tissue with Raman Spectroscopy and Machine Learning.

Amyloids are proteins with characteristic beta-sheet secondary structures that display fibrillary ultrastructural configurations. They can result in pathologic lesions when deposited in human organs. Various types of amyloid protein can be routinely identified in human tissue specimens by special stains, immunolabeling, and electron microscopy, and, for certain forms of amyloidosis, mass spectrometry is required. In this study, we applied Raman spectroscopy to identify immunoglobulin light chain and amyloid A amyloidosis in human renal tissue biopsies and compared the results with a normal kidney biopsy as a control case. Raman spectra of amyloid fibrils within unstained, frozen, human kidney tissue demonstrated changes in conformation of protein secondary structures. By using t-distributed stochastic neighbor embedding (t-SNE) and density-based spatial clustering of applications with noise (DBSCAN), Raman spectroscopic data were accurately classified with respect to each amyloid type and deposition site. To the best of our knowledge, this is the first time Raman spectroscopy has been used for amyloid characterization of ex vivo human kidney tissue samples. Our approach, using Raman spectroscopy with machine learning algorithms, shows the potential for the identification of amyloid in pathologic lesions.