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Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a rapidly advancing technology for biomedical research. As spatial resolution increases, however, so do acquisition time, file size, and experimental cost, which increases the need to perform precise sampling of targeted tissue regions to optimize the biological information gleaned from an experiment and minimize wasted resources. The ability to define instrument measurement regions based on key tissue features and automatically sample these specific regions of interest (ROIs) addresses this challenge. Herein, we demonstrate a workflow using standard software that allows for direct sampling of microscopy-defined regions by MALDI IMS. Three case studies are included, highlighting different methods for defining features from common sample typesmanual annotation of vasculature in human brain tissue, automated segmentation of renal functional tissue units across whole slide images using custom segmentation algorithms, and automated segmentation of dispersed HeLa cells using open-source software. Each case minimizes data acquisition from unnecessary sample regions and dramatically increases throughput while uncovering molecular heterogeneity within targeted ROIs. This workflow provides an approachable method for spatially targeted MALDI IMS driven by microscopy as part of multimodal molecular imaging studies. [ABSTRACT FROM AUTHOR]

Background: Telocytes are interstitial stromal cells identified in various human organs, including the kidney. Their presence and role in human diabetic kidney disease remain unknown. Methods: To identify and localize telocytes in glomerular and tubule-interstitial compartments, both normal and diabetic human renal tissues were examined using immunohistochemistry, immunofluorescence, and transmission electron microscopy. Results: Renal telocytes are elongated interstitial cells with long, thin telopodes, showing alternating thin and thick segments. They expressed CD34, Nestin, α-SMA, and Vimentin markers. Occasionally, c-Kit expression was observed in some rounded and spindle cells, while no positivity was detected for PDGFR-β and NG2. Telocytes were identified around Bowman's capsule, tubules, and peritubular capillaries in both normal and diabetic conditions. In diabetic renal samples, there was a significant increase in α-SMA expressing telocytes, leading to periglomerular fibrosis. These telocytes also exhibited a synthetic phenotype with proteoglycan deposition in the extracellular matrix and, in some cases, showed pre-adipocytic differentiation. Conclusions: Telocytes were identified in normal and diabetic human kidneys. These cells form an elastic mechanical scaffold in the interstitium and are present in all renal cortical compartments. In diabetic samples, their increased α-SMA expression and synthetic phenotype suggest their potential role in the pathogenesis of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Background and aimsThe immune system plays a crucial role in the development of kidney diseases. Chronic kidney disease (CKD) can lead to various complications, potentially affecting multiple systems throughout the body. Currently, the description of the immune system in human CKD is not comprehensive enough. Constructing a CKD kidney atlas using single-cell RNA sequencing (scRNA-seq) can provide deeper insights into the composition and functional changes of immune cells in CKD, facilitating the discovery of new therapeutic targets.MethodsWe processed and integrated scRNA-seq datasets from healthy and CKD kidneys from three independent cohorts using the same approach (including 42 normal samples and 23 chronic kidney disease samples). Subsequently, we conducted gene enrichment and intercellular communication analysis to construct an immune cell atlas of the kidneys in CKD patients.ResultsWe identified nine major kidney cell clusters. Further clustering analysis of different immune cell clusters revealed that, compared to normal kidneys, CKD patients’ kidneys had decreased CD16+ NK cells while CD4+ naive helper T cells and CCR7+ DC increased. Partial activation of the WNT signaling pathway was observed in T cells and NK cells of CKD patients, while some metabolism-related genes were inhibited. Myeloid cell subgroups also exhibited abnormal signaling pathway alterations. Additionally, we discovered a unique population of SPP1 macrophages in CKD, which are recruited by chemokines released from aPT and aTAL cell subpopulations. These SPP1 macrophages may promote cellular fibrosis through the signaling of SPP1, FN1, and various receptors.ConclusionWe established a human CKD kidney immune cell atlas and identified SPP1 macrophages as a unique cell type in CKD. The interaction between SPP1 macrophages and damaged cells may serve as a potential therapeutic target for treating CKD in the future.

Background: A range of diseases can result in end-stage renal disease (ESRD), characterized by a gradual decline in kidney function and associated with significant morbidity and mortality. Currently, renal transplantation as the most effective treatment for managing ESRD. Tissue engineering presents a considerable opportunity to expand the available supply of donor organs for kidney transplants. The aim of this research was to develop a suitable technique for preparing decellularized kidney scaffolds from human tissues. Methods: The present study was carried out from April 2019 to August 2019 in Mashhad University of Medical Sciences. In this study, two decellularization protocols were compared using sections of human kidney tissue. Therefore, two human kidneys which collected from Ghaem and Imam Reza hospitals were used. In the first protocol, detergents such as 1% Triton X-100 (1A) and 1% SDS (Sodium Dodecyl Sulfate) (1B) were employed, followed by the application of DNase I. The second protocol utilized 0.5% SDS (2A) and 1% SDS (2B). The effectiveness of these techniques was evaluated using hematoxylin and eosin (H&E) staining, 4',6-diamidino-2-phenylindole (DAPI), DNA quantification, and immunohistochemistry (IHC). Results: Based on H&E staining results, comparison of the decellularized and native human kidney tissues showed a successful elimination of cell nuclei and the ameliorate extracellular matrix preservation in triton-treated scaffolds (1A) in comparison with the SDS-treated scaffolds (1B) at all times protocols. Furthermore, DNA quantification illustrated triton X-100 in removing DNA was more effective in eliminating DNA from kidney tissues compared to other protocols in renal tissues. In addition, IHC staining demonstrated that the expression of collagen IV and laminin was preserved throughout the decellularization process with Triton X-100 on day fifth. Also, IHC staining indicated human leukocyte antigen (HLA) was completely eliminated in the cortexmedulla of human scaffolds treated with Triton X-100 within day fifth. Conclusion: Our results demonstrated that triton X-100 outperformed SDS as a detergent for decellularizing human kidneys. Meanwhile these results indicate suitable method for decellularization of human kidneys to produce functional kidneys. [ABSTRACT FROM AUTHOR]

Background: A range of diseases can result in end-stage renal disease (ESRD), characterized by a gradual decline in kidney function and associated with significant morbidity and mortality. Currently, renal transplantation as the most effective treatment for managing ESRD. Tissue engineering presents a considerable opportunity to expand the available supply of donor organs for kidney transplants. The aim of this research was to develop a suitable technique for preparing decellularized kidney scaffolds from human tissues. Methods: The present study was carried out from April 2019 to August 2019 in Mashhad University of Medical Sciences. In this study, two decellularization protocols were compared using sections of human kidney tissue. Therefore, two human kidneys which collected from Ghaem and Imam Reza hospitals were used. In the first protocol, detergents such as 1% Triton X-100 (1A) and 1% SDS (Sodium Dodecyl Sulfate) (1B) were employed, followed by the application of DNase I. The second protocol utilized 0.5% SDS (2A) and 1% SDS (2B). The effectiveness of these techniques was evaluated using hematoxylin and eosin (H&E) staining, 4',6-diamidino-2-phenylindole (DAPI), DNA quantification, and immunohistochemistry (IHC). Results: Based on H&E staining results, comparison of the decellularized and native human kidney tissues showed a successful elimination of cell nuclei and the ameliorate extracellular matrix preservation in triton-treated scaffolds (1A) in comparison with the SDS-treated scaffolds (1B) at all times protocols. Furthermore, DNA quantification illustrated triton X-100 in removing DNA was more effective in eliminating DNA from kidney tissues compared to other protocols in renal tissues. In addition, IHC staining demonstrated that the expression of collagen IV and laminin was preserved throughout the decellularization process with Triton X-100 on day fifth. Also, IHC staining indicated human leukocyte antigen (HLA) was completely eliminated in the cortex-medulla of human scaffolds treated with Triton X-100 within day fifth. Conclusion: Our results demonstrated that triton X-100 outperformed SDS as a detergent for decellularizing human kidneys. Meanwhile these results indicate suitable method for decellularization of human kidneys to produce functional kidneys.

Health literacy plays a pivotal role in shaping an individual's adoption of a healthy lifestyle, particularly among students transitioning into adolescence and adulthood. Hence, integrating health literacy into educational curricula is imperative. For instance, the study of the human kidney excretory system, which necessitates the use of measuring instruments, can serve as a platform to enhance students' health literacy skills within the realm of biology education. This study was conducted to develop and determine the feasibility of human kidney health literacy instruments. The research method used in this study is ADDIE development research which consists of 5 stages, including (1) preliminary needs analysis (2) product design (3) Development (4) Implementation, and (5) evaluation. The trial results stated that the instrument was feasible to use with a linguist assessment of 9.8% very feasible category, and a material expert of 91% very feasible category. The empirical validity test was conducted with a limited trial to 47 students of Al-Azhar 9 Yogyakarta High School and the results showed that 92% of the items were declared valid with a high reliability value of 0.7. This study produced 16 statement items on the attitude aspect, 6 items on the knowledge aspect, and items on the skill aspect. The total question items produced in this study were 24 question items that were valid and feasible to use as a tool to improve the quality of education.

Abstract In humans, uric acid is an end-product of purine metabolism. Urate excretion from the human kidney is tightly regulated by reabsorption and secretion. At least eleven genes have been identified as human renal urate transporters. However, it remains unclear whether all renal tubular cells express the same set of urate transporters. Here, we show renal tubular cells are divided into three distinct cell populations for urate handling. Analysis of healthy human kidneys at single-cell resolution revealed that not all tubular cells expressed the same set of urate transporters. Only 32% of tubular cells were related to both reabsorption and secretion, while the remaining tubular cells were related to either reabsorption or secretion at 5% and 63%, respectively. These results provide physiological insight into the molecular function of the transporters and renal urate handling on single-cell units. Our findings suggest that three different cell populations cooperate to regulate urate excretion from the human kidney, and our proposed framework is a step forward in broadening the view from the molecular to the cellular level of transport capacity.

Background: Telocytes are interstitial stromal cells identified in various human organs, including the kidney. Their presence and role in human diabetic kidney disease remain unknown. Methods: To identify and localize telocytes in glomerular and tubule-interstitial compartments, both normal and diabetic human renal tissues were examined using immunohistochemistry, immunofluorescence, and transmission electron microscopy. Results: Renal telocytes are elongated interstitial cells with long, thin telopodes, showing alternating thin and thick segments. They expressed CD34, Nestin, α-SMA, and Vimentin markers. Occasionally, c-Kit expression was observed in some rounded and spindle cells, while no positivity was detected for PDGFR-β and NG2. Telocytes were identified around Bowman’s capsule, tubules, and peritubular capillaries in both normal and diabetic conditions. In diabetic renal samples, there was a significant increase in α-SMA expressing telocytes, leading to periglomerular fibrosis. These telocytes also exhibited a synthetic phenotype with proteoglycan deposition in the extracellular matrix and, in some cases, showed pre-adipocytic differentiation. Conclusions: Telocytes were identified in normal and diabetic human kidneys. These cells form an elastic mechanical scaffold in the interstitium and are present in all renal cortical compartments. In diabetic samples, their increased α-SMA expression and synthetic phenotype suggest their potential role in the pathogenesis of diabetic nephropathy.

The glomerulus is a multicellular functional tissue unit (FTU) of the nephron that is responsible for blood filtration. Each glomerulus contains multiple substructures and cell types that are crucial for their function. To understand normal aging and disease in kidneys, methods for high spatial resolution molecular imaging within these FTUs across whole slide images is required. Here we demonstrate a workflow using microscopy-driven selected sampling to enable 5 μm pixel size matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) of all glomeruli within whole slide human kidney tissues. Such high spatial resolution imaging entails large numbers of pixels, increasing the data acquisition times. Automating FTU-specific tissue sampling enables high-resolution analysis of critical tissue structures, while concurrently maintaining throughput. Glomeruli were automatically segmented using coregistered autofluorescence microscopy data, and these segmentations were translated into MALDI IMS measurement regions. This allowed high-throughput acquisition of 268 glomeruli from a single whole slide human kidney tissue section. Unsupervised machine learning methods were used to discover molecular profiles of glomerular subregions and differentiate between healthy and diseased glomeruli. Average spectra for each glomerulus were analyzed using Uniform Manifold Approximation and Projection (UMAP) and k-means clustering, yielding 7 distinct groups of differentiated healthy and diseased glomeruli. Pixel-wise k-means clustering was applied to all glomeruli, showing unique molecular profiles localized to subregions within each glomerulus. Automated microscopy-driven, FTU-targeted acquisition for high spatial resolution molecular imaging maintains high-throughput and enables rapid assessment of whole slide images at cellular resolution and identification of tissue features associated with normal aging and disease. [ABSTRACT FROM AUTHOR]

The macula densa (MD) is an anatomical structure having a plaque shape, placed in the distal end of thick ascending limb of each nephron and belonging to juxtaglomerular apparatus (JGA). The aim of the present investigation is to investigate the presence of ZO‐1, a specific marker of tight juncions (TJs), in MD cells. Six samples of normal human renal tissue were embedded in paraffin for ZO‐1 expression analysis by immunohistochemical and immunofluorescence techniques. We detected ZO‐1 expression in the apical part of cell membrane in MD cells by immunohistochemistry. In addition, ZO‐1 and nNOS expressions (a specific marker of MD) were colocalized in MD cells providing clear evidence of TJs presence in normal human MD. Since ZO‐1 is responsible for diffusion barrier formation, its presence in the MD supports the existence of a tubulomesangial barrier that ensures a regulated exchange between MD and JGA effectors in renal and glomerular haemodynamic homeostasis. [ABSTRACT FROM AUTHOR]

Summary: Background: Estimation of glomerular function is necessary to diagnose kidney diseases. However, the study of glomeruli in the clinic is currently done indirectly through urine and blood tests. A recent imaging technique called Ultrasound Localization Microscopy (ULM) has appeared. It is based on the ability to record continuous movements of individual microbubbles in the bloodstream. Although ULM improved the resolution of vascular imaging up to tenfold, the imaging of the smallest vessels had yet to be reported. Methods: We acquired ultrasound sequences from living humans and rats and then applied filters to divide the data set into slow-moving and fast-moving microbubbles. We performed a double tracking to highlight and characterize populations of microbubbles with singular behaviors. We decided to call this technique “sensing ULM” (sULM). We used post-mortem micro-CT for side-by-side confirmation in rats. Findings: In this study, we report the observation of microbubbles flowing in the glomeruli in living humans and rats. We present a set of analysis tools to extract quantitative information from individual microbubbles, such as remanence time or normalized distance. Interpretation: As glomeruli play a key role in kidney function, it would be possible that their observation yields a deeper understanding of the kidney. It could also be a tool to diagnose kidney diseases in patients. More generally, it will bring imaging capabilities closer to the functional units of organs, which is a key to understand most diseases, such as cancer, diabetes, or kidney failures. Funding: This study was funded by the European Research Council under the European Union Horizon H2020 program (ERC Consolidator grant agreement No 772786-ResolveStroke).

Healthcare is the most important need of today's era. Healthcare refers to the improvement of the human health by preventing, curing, diagnosing, recovering from a health hazard caused. Thus, to improve the health condition of a human system technology, such as machine learning, deep learning and artificial intelligence has come into play. The combination of artificial technology with the health sector has made a huge impact and success on the world. Curing millions of diseases, analysis of various infections, providing accurate test results and high-level maintenance check are now all possible with the evolution of technology. Every part of human body can now be diagnosed and analyze to study all kinds of tissues, blood vessels, organs, cells for improvement of health and curing of diseases. Research sector has been working with a continuous pace to accomplish various studies to identify different body organs and have a descriptive study for the identification of proper working mechanism of the human body. One such study has also shown a huge progress in the recent times, the identification of glomeruli in human kidney tissue. The tiny ball like structured which is composed of blood vessels that has an actively participation in the filtration of the blood to form urine. Thus, the paper presents a deep learning-based model formed for the identification of these glomeruli present in the human kidney. After implementing, the proposed model obtained an accuracy of 99.68% with a dice coefficient of 0.9060. [ABSTRACT FROM AUTHOR]

In the context of renal proteinuric diseases, TRPC6 has been shown to play an important role in ultrafiltration associated with the slit diaphragm through the control of the intracellular Ca2+ concentration in the podocytes of glomeruli. However, to date, the properties of TRPC6 have been studied mainly in cell lines or in animal models. Therefore, the aim of the study presented here was to investigate the presence and distribution of TRPC6 in human kidneys in order to possibly verify the applicability of the results previously obtained in nonhuman experiments. For this purpose, kidneys from nine cadavers were prepared for immunohistochemical staining and were supplemented with a fresh human kidney obtained by nephrectomy. TRPC6 was detected in glomeruli and in the parietal epithelial cells of Bowman’s capsule. Larger amounts were detected in the tubular system and collecting ducts. In contrast to the peritubular capillary bed, which showed no immune reaction, the cortical resistance vessels showed mild TRPC6 staining. In conclusion, our studies on the expression of TRPC6 in human kidney tissue support the translational concept of the involvement of TRPC6 in various renal diseases and reveal new aspects of the distribution of TRPC6 in the human kidney.

Given the current state of COVID-19, it is crucial to reveal its evolving relationship with and effect on different body organ systems and their diseases. The severity and outcome of COVID-19 have a very complex relationship, especially to the vital organs including the kidney, either in their state of health or disease. Additionally, it is well known that diabetes affects the kidney, leading to diabetic nephropathy. The kidney is also affected by different pathological and immunopathological reactions with COVID-19 infection, leading to acute kidney injury. Therefore, this review intended to extract the recent advances, updates, and discoveries about the effects of COVID-19 on diabetic patients and the relationship between COVID-19 invasion and the diabetic kidney and to discuss the current state of knowledge that has not yet been proved or disproved, leading to numerous controversial issues in looking for the effect of COVID-19 associated with diabetes mellitus on the human kidney. [ABSTRACT FROM AUTHOR]

Pannexins are transmembrane glycoproteins that constitute channels involved in purinergic signaling through ATP release from cells in various physiological and pathological processes. In this study, the distribution of Panx1 expression in different cell populations of healthy postnatal human kidneys and during human embryonic and early fetal development was investigated by double immunohistochemistry. In addition, the glomerular and tubular expression of Panx1 was examined in patients with type 2 diabetes mellitus (DM2) and the control group, and renal Panx1 expression was correlated with serum creatinine. In the 6th week of embryonic development (DW), Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from 6th to 7th DW, Panx1 immunoreactivity was found in the mesonephric tubules and mesonephric duct, as well as in the metanephric ureteric bud and ampullae. In the 7th DW, strong Panx1 immunoreactivity was observed in the developing ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. In the 8th DW, Panx1 expression was also found in the ureteric bud of the metanephros, the renal vesicle and comma-shaped nephron, and the epithelial cells of Bowman's capsule. Expression of Panx1 was found at an early stage in both the paramesonephric duct and the mesonephric duct and diminished toward the 8th DW. During the 6th–10th DW, colocalization of Panx1 with alpha smooth actin (aSMA) was found in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and the thin descending limbs of the loop of Henle. Panx1 immunoreactivity was also found in nephrin-immunoreactive podocytes. Panx1 was not colocalized with aSMA immunoreactivity in the vessels of the postnatal human kidney, but it was present in the endothelium. A significant positive correlation was found between Panx1 expression in glomeruli and serum creatinine only in diabetic patients and was not found in the nondiabetic group. The spatiotemporal expression of Panx1 during the early stages of human kidney development supports its possible role in cellular differentiation, migration, and positioning in the developing human kidney. In addition, our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

Graphical Abstract

Given the current state of COVID-19, it is crucial to reveal its evolving relationship with and effect on different body organ systems and their diseases. The severity and outcome of COVID-19 have a very complex relationship, especially to the vital organs including the kidney, either in their state of health or disease. Additionally, it is well known that diabetes affects the kidney, leading to diabetic nephropathy. The kidney is also affected by different pathological and immunopathological reactions with COVID-19 infection, leading to acute kidney injury. Therefore, this review intended to extract the recent advances, updates, and discoveries about the effects of COVID-19 on diabetic patients and the relationship between COVID-19 invasion and the diabetic kidney and to discuss the current state of knowledge that has not yet been proved or disproved, leading to numerous controversial issues in looking for the effect of COVID-19 associated with diabetes mellitus on the human kidney.

Pannexins are transmembrane glycoproteins that constitute channels involved in purinergic signaling through ATP release from cells in various physiological and pathological processes. In this study, the distribution of Panx1 expression in different cell populations of healthy postnatal human kidneys and during human embryonic and early fetal development was investigated by double immunohistochemistry. In addition, the glomerular and tubular expression of Panx1 was examined in patients with type 2 diabetes mellitus (DM2) and the control group, and renal Panx1 expression was correlated with serum creatinine. In the 6th week of embryonic development (DW), Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from 6th to 7th DW, Panx1 immunoreactivity was found in the mesonephric tubules and mesonephric duct, as well as in the metanephric ureteric bud and ampullae. In the 7th DW, strong Panx1 immunoreactivity was observed in the developing ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. In the 8th DW, Panx1 expression was also found in the ureteric bud of the metanephros, the renal vesicle and comma-shaped nephron, and the epithelial cells of Bowman’s capsule. Expression of Panx1 was found at an early stage in both the paramesonephric duct and the mesonephric duct and diminished toward the 8th DW. During the 6th–10th DW, colocalization of Panx1 with alpha smooth actin (aSMA) was found in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and the thin descending limbs of the loop of Henle. Panx1 immunoreactivity was also found in nephrin-immunoreactive podocytes. Panx1 was not colocalized with aSMA immunoreactivity in the vessels of the postnatal human kidney, but it was present in the endothelium. A significant positive correlation was found between Panx1 expression in glomeruli and serum creatinine only in diabetic patients and was not found in the nondiabetic group. The spatiotemporal expression of Panx1 during the early stages of human kidney development supports its possible role in cellular differentiation, migration, and positioning in the developing human kidney. In addition, our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes.

Introduction: Renal transplantation is currently the most effective treatment for ESRD. However, the number of available donor kidneys is drastically insufficient to meet the demand. For this reason, tissue engineering by way of seeding cells on scaffold, offers a great potential to increase the pool of donated organs for kidney transplantation. Methods and Materials: In the current work, adipose tissue derived MSCs were seeded onto the triton-treated (as selected method) cortex and medulla scaffolds for 3 weeks. Proliferation and differentiation of MSCs into kidneyspecific cell types on these scaffolds were then analyzed by H&E, Scanning electron microscopy (SEM), IHC staining and qRT-PCR. Results: After recellularization of the renal human scaffolds with MSCs, the cell attachment and migration into the triton-treated scaffold, were confirmed by histological studies and SEM results, as compared with non-seeded scaffolds at same days. Furthermore, the results of IHC staining showed that Na-K ATPase, Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) and Aquaporin 2 (AQP2) expressed in seeded human scaffold compared with non-seeded scaffolds after 7, 14, and 21 days from cell seeding. Also, expressions of these markers increased with time. Differentiation of MSCs into epithelial and endothelial cells was confirmed by qRT-PCR that analyzed expression of E-cadherin (E-CDH), Paired box 2 (PAX2) and platelet and endothelial cell adhesion molecule 1 (PECAM1) in human scaffolds compared with MSCs which cultured without scaffolds at the same time. Conclusions: These results indicate optimized method for decellularization and recellularization of human kidneys to produce functional kidneys. In this regard, these natural scaffolds supported the growth of MSCs and could also induce their differentiation into epithelial and endothelial cells. [ABSTRACT FROM AUTHOR]

Introduction: Various diseases can lead to end stage renal disease (ESRD) that is a progressive loss of kidney function with a high rate of morbidity and mortality. Natural biological scaffolds that are prepared by removing cellular components from the donor organs using a decellularization process have many advantages compared with synthetic scaffold. Applying natural kidney scaffolds beside regenerative medicine and tissue engineering techniques are promising to achieve functional kidney. Methods and Materials: In the current work two decellularization protocols were compared on human kidney sections (cortex, medulla and cortex-medulla). In protocol 1 detergents including 1% Triton X-100 (1A) and 1% SDS (1B) followed by DNase I were used. In the second protocol 0.5% SDS (2A) and 1% SDS (2B) were used. The efficiency of these methods was assessed by using hematoxylin and eosin (H&E), 4', 6 diamidino-2-phenylindole (DAPI), DNA quantification and immunohistochemistry (IHC) staining. Result: H&E and DAPI staining results revealed a successful elimination of cell nuclei in all sections of protocols 1 and 2. The histological examinations revealed a better extracellular matrix preservation in triton-treated scaffolds (1A) as compared with the SDS-treated scaffolds (1B) at all times protocols. In addition, DNA quantification showed triton X-100 was more efficient as compared with other protocols for removing DNA in kidney sections. Furthermore, IHC staining showed the expression of collagen IV and laminin was preserved during decellularization with triton X-100 on days 5 and 10. In addition, IHC staining showed human leukocyte antigen (HLA) was completely removed in the cortex and medulla of human triton-treated scaffolds on days 5 and 10. Conclusion: the results of this study showed that triton X-100 was a more effective detergent for decellularization of human kidney compared with SDS. [ABSTRACT FROM AUTHOR]

[Display omitted] • Simple and fast calculation of the broadband spectral optical properties of biological tissues can be made using experimental spectra acquired from ex vivo samples. • The spectral optical properties provide valuable information to develop light propagation models to be used in diagnostic and treatment procedures. • The spectral absorption coefficient contains information about the tissue components, namely hemoglobin (blood), DNA, lipids, water and melanin-like pigments. • Differentiated hemoglobin (blood) and melanin-like pigment contents can be retrieved from the analysis of the spectral absorption coefficient of normal and pathological tissues. • Estimation of differentiated absorption coefficient spectra for normal and pathological tissues can be made using noninvasive reflectance spectra in deep-learning algorithms to obtain a diagnosis in vivo. A fast calculation method was used to obtain the spectral optical properties of human normal and pathological (chromophobe renal cell carcinoma) kidney tissues. Using total transmittance, total reflectance and collimated transmittance spectra acquired from ex vivo kidney samples, the spectral optical properties of both tissues, namely the absorption, the scattering and the reduced scattering coefficients, as well as the scattering anisotropy, dispersion and light penetration depth, were calculated between 200 and 1000 nm. Analysis of the mean absorption coefficient spectra of the kidney tissues showed that both contain melanin and lipofuscin, and that 83 % of the melanin in the normal kidney converts into lipofuscin in the pathological kidney. [ABSTRACT FROM AUTHOR]

Background: Estimation of glomerular function is necessary to diagnose kidney diseases. However, the study of glomeruli in the clinic is currently done indirectly through urine and blood tests. A recent imaging technique called Ultrasound Localization Microscopy (ULM) has appeared. It is based on the ability to record continuous movements of individual microbubbles in the bloodstream. Although ULM improved the resolution of vascular imaging up to tenfold, the imaging of the smallest vessels had yet to be reported., Methods: We acquired ultrasound sequences from living humans and rats and then applied filters to divide the data set into slow-moving and fast-moving microbubbles. We performed a double tracking to highlight and characterize populations of microbubbles with singular behaviors. We decided to call this technique "sensing ULM" (sULM). We used post-mortem micro-CT for side-by-side confirmation in rats., Findings: In this study, we report the observation of microbubbles flowing in the glomeruli in living humans and rats. We present a set of analysis tools to extract quantitative information from individual microbubbles, such as remanence time or normalized distance., Interpretation: As glomeruli play a key role in kidney function, it would be possible that their observation yields a deeper understanding of the kidney. It could also be a tool to diagnose kidney diseases in patients. More generally, it will bring imaging capabilities closer to the functional units of organs, which is a key to understand most diseases, such as cancer, diabetes, or kidney failures., Funding: This study was funded by the European Research Council under the European Union Horizon H2020 program (ERC Consolidator grant agreement No 772786-ResolveStroke)., Competing Interests: Declaration of interests O.C. and V.H., hold patents in the field of ultrasound localization microscopy (EP4011299A1). O.C. and V.H. are founders and shareholders of the ResolveStroke startup. O.C. has received several grants: ERC Consolidator grant agreement no 772786-Resolve Stroke, ERC POC (2022–2023), Maturation SATT Lutech for ResolveStroke Startup, ANR FightClot, CNRS Prematuration for ResolveStroke Startup, RHU Quid NASH. O.C. gave a lecture at Bracco SAS and was an invited speaker at several conferences such as BMT Innsbruck. Other authors have no conflict of interest to disclose., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Despite recent advances made in short-term outcomes; minimal improvements have been observed in long-term kidney transplantation outcomes. Due to an imbalance between organ transplant availability and patient waiting list, expanding kidney allograft longevity is a critical need in the field. Prior studies have either focused on early ischemic and immunological conditions affecting kidney allografts (e.g., delayed graft function, acute rejection) or late stage chronic injury when interventions are no longer feasible. However, studies characterizing kidney allografts with normal function by its cellular distribution, cell-cell interactions, and associated molecular pathways are lacking. Herein, we used single nuclei RNA-sequencing to uncover the cellular landscape and transcriptome of the normal kidney allograft. We profiled 40,950 nuclei from seven human kidney biopsies (normal native, N = 3; normal allograft, N = 4); normal allograft protocol biopsies were collected ≥15-months post-transplant. A total of 17 distinct cell clusters were identified with proximal tubules (25.70 and 21.01%), distal tubules (15.22 and 18.20%), and endothelial cells (EC) (4.26 and 9.94%) constituting the major cell populations of normal native and normal allograft kidneys, respectively. A large proportion of cycling cells from normal native kidneys were in G1-phase (43.96%) whereas cells from normal allograft were predominantly in S-phase (32.69%). This result suggests that transcriptional differences between normal native and normal allograft biopsies are dependent on the new host environment, immunosuppression, and injury-affliction. In the normal allograft, EC-specific genes upregulated metabolism, the immune response, and cellular growth, emphasizing their role in maintaining homeostasis during the ongoing alloreactive stress response. Immune cells, including B (2.81%), macrophages (24.96%), monocytes (15.29%), natural killer (NK) (12.83%), neutrophils (8.44%), and T cells (14.41%, were increased in normal allografts despite lack of histological or clinical evidence of acute rejection. Phenotypic characterization of immune cell markers supported lymphocyte activation and proinflammatory cytokines signaling pathways (i.e., IL-15, IL-32 ). The activation of B, NK, and T cells reveals potential immune cells underlying subclinical inflammation and repair. These single nuclei analyses provide novel insights into kidney and immune cell associated signaling pathways that portray kidney grafts with normal allograft function beyond 2-years post-transplant, revealing a novel perspective in understanding long-term allograft graft survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McDaniels, Shetty, Rousselle, Bardhi, Maluf and Mas.)

Regeneration of discarded human kidneys has been considered as an ideal approach to overcome organ shortage for the end-stage renal diseases (ESRDs). The aim of this study was to develop an effective method for preparation of kidney scaffolds that retain the matrix structure required for proliferation and importantly, differentiation of human adipose-derived mesenchymal stem cells (hAd-MSCs) into renal cells. We first compared two different methods using triton X-100 and sodium dodecyl sulfate (SDS) for human kidney decellularization; followed by characterization of the prepared human renal extracellular matrix (ECM) scaffolds. Then, hAd-MSCs were seeded on the scaffolds and cultured for up to 3 weeks. Next, viability, proliferation, and migration of seeded hAd-MSCs underwent histological and scanning electron microscopy (SEM) assessments. Moreover, differentiation of hAd-MSCs into kidney-specific cell types was examined using immunohistochemistry (IHC) staining and qRT-PCR. Our results indicated that triton X-100 was a more effective detergent for decellularization of human kidneys compared with SDS. Moreover, attachment and proliferation of hAd-MSCs within the recellularized human kidney scaffolds, were confirmed. Seeded cells expressed epithelial and endothelial differentiation markers, and qRT-PCR results indicated increased expression of platelet and endothelial cell adhesion molecule 1 (PECAM-1), paired box 2 (PAX2), and E -cadherine (E-CDH) as markers of differentiation into epithelial and endothelial cells. These observations indicate the effectiveness of decellularization with triton X-100 to generate suitable human ECM renal scaffolds, which supported adhesion and proliferation of hAd-MSCs and could induce their differentiation towards a renal lineage. [Display omitted] [ABSTRACT FROM AUTHOR]

Objective: Establishment of an efficient method of preparing human kidney single cell suspension, using a very small amount of tissue puncture. Methods: Samples of human kidney tissue puncture were cut into pieces, and then 80 μL of the digestive enzyme were added to each punctured tissue to induce enzymatic digestion. The enzyme combination is composed of collagenases, DNase and hyaluronidase and the sample was incubated 20 min at 37°C. The obtained cell suspension was filtered through a 70 μm cell strainer, centrifuged at 300 g for 5 min and the supernatant was removed, then the pellet was resuspended in 3 ml of DMEM (Dulbecco's Modified Eagle's Medium). Cell suspension was sorted and purified by flow sorting to remove dead cells and obtain a cell suspension with higher viability rate. Results: We found that 1) diverse single cells of human kidney can be obtained by the digestive enzyme, as observed under the light microscope, with different sizes, normal cell morphology and good dispersion. 2) (2-3) × 10 6 single cells can be extracted from one fresh punctured kidney tissue of about 10 mg, with a cell viability rate of more than 80%. Conclusion: In this work we generated a comprehensive and high-resolution single-cell method, which is simple and efficient for preparing single cell suspension from a minimal amount of human kidney tissue. This method can facilitate the study of renal cell biology and the pathogenesis of kidney diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yaigoub, Tirichen, Xin, Shi, Wu, Li and Li.)