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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: 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]

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]

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]

ABC transporters (ATP‐binding cassette transporter) traffic drugs and their metabolites across membranes, making ABC transporter expression levels a key factor regulating local drug concentrations in different tissues and individuals. Yet, quantification of ABC transporters remains challenging because they are large and low‐abundance transmembrane proteins. Here, we analysed 200 samples of crude and membrane‐enriched fractions from human liver, kidney, intestine, brain microvessels and skin, by label‐free quantitative mass spectrometry. We identified 32 (out of 48) ABC transporters: ABCD3 was the most abundant in liver, whereas ABCA8, ABCB2/TAP1 and ABCE1 were detected in all tissues. Interestingly, this atlas unveiled that ABCB2/TAP1 may have TAP2‐independent functions in the brain and that biliary atresia (BA) and control livers have quite different ABC transporter profiles. We propose that meaningful biological information can be derived from a direct comparison of these data sets. [ABSTRACT FROM AUTHOR]

Environmental food contaminants constitute a threat to human health. For instance, the globally spread mycotoxin Ochratoxin A (OTA) contributes to chronic kidney damage by affecting proximal tubule cells via unknown mechanisms. We applied a top-down approach to identify relevant toxicological mechanisms of OTA using RNA-sequencing followed by in-depth bioinformatics analysis and experimental validation. Differential expression analyses revealed that OTA led to the regulation of gene expression in kidney human cell lines, including for genes enriched in cell cycle-related pathways, and OTA-induced gap 1 and 2 (G1 and G2) cell-cycle arrests were observed. Weighted correlation network analysis highlighted cyclin dependent kinase 2 (CDK2) as a putative key regulator of this effect. CDK2 was downregulated by OTA exposure, and its overexpression partially blocked the OTA-induced G1 but not G2 cell-cycle arrest. We, therefore, propose CDK2 as one of the key regulators of the G1 cell-cycle arrest induced by low nanomolar concentrations of OTA. [ABSTRACT FROM AUTHOR]

Our current knowledge of the properties of renal ion channels responsible for electrolytes and cell energy homeostasis mainly relies on rodent studies. However, it has not been established yet to what extent their characteristics can be generalized to those of humans. The present study was designed to develop a standardized protocol for the isolation of well-preserved glomeruli and renal tubules from rodent and human kidneys and to assess the functional suitability of the obtained materials for physiological studies. Separation of nephron segments from human and rodent kidneys was achieved using a novel vibrodissociation technique. The integrity of isolated renal tubules and glomeruli was probed via electrophysiological analysis and fluorescence microscopy, and the purity of the collected fractions was confirmed using quantitative RT-PCR with gene markers for specific cell types. The developed approach allows rapid isolation of well-preserved renal tubules and glomeruli from human and rodent kidneys amenable for electrophysiological, Ca2+ imaging, and omics studies. Analysis of the basic electrophysiological parameters of major K+ and Na+ channels expressed in human cortical collecting ducts revealed that they exhibited similar biophysical properties as previously reported in rodent studies. Using vibrodissociation for nephron segment isolation has several advantages over existing techniques: it is less labor intensive, requires little to no enzymatic treatment, and produces large quantities of well-preserved experimental material in pure fractions. Applying this method for the separation of nephron segments from human and rodent kidneys may be a powerful tool for the indepth assessment of kidney function in health and disease. [ABSTRACT FROM AUTHOR]

Statistical shape analysis was conducted on 15 pairs (left and right) of human kidneys. It was shown that the left and right kidney were significantly different in size and shape. In addition, several common modes of kidney variation were identified using statistical shape analysis. Semi-automatic mesh morphing techniques have been developed to efficiently create subject specific meshes from a template mesh with a similar geometry. Subject specific meshes as well as probabilistic kidney meshes were created from a template mesh. Mesh quality remained about the same as the template mesh while only taking a fraction of the time to create the mesh from scratch or morph with manually identified landmarks. This technique can help enhance the quality of information gathered from experimental testing with subject specific meshes as well as help to more efficiently predict injury by creating models with the mean shape as well as models at the extremes for each principal component. [ABSTRACT FROM AUTHOR]

Podocytes play a key role in diabetic nephropathy pathogenesis, but alteration of their metabolism remains unknown in human kidney. By using a conditionally differentiating human podocyte cell line, we addressed the functional and molecular changes in podocyte energetics during in vitro development or under high glucose conditions. In 5 mM glucose medium, we observed a stepwise activation of oxidative metabolism during cell differentiation that was characterized by peroxisome proliferator‐activated receptor‐γ coactivator 1α (PGC‐1α)– dependent stimulation of mitochondrial biogenesis and function, with concomitant reduction of the glycolytic enzyme content. Conversely, when podocytes were cultured in high glucose (20 mM), stepwise oxidative phosphorylation biogenesiswas aborted, and a glycolytic switch occurred, with consecutive lactic acidosis. Expression of the master regulators of oxidative metabolism transcription factor A mitochondrial, PGC‐1α, AMPK, and serine– threonine liver kinase B1 was altered by high glucose, as well as their downstream signaling networks. Focused transcriptomics revealed that myocyte‐specific enhancer factor 2C (MEF2C) and myogenic factor 5(MYF5)expression was inhibited by high glucose levels, and endoribonuclease‐prepared small interfering RNA–mediated combined inhibition of those transcription factors phenocopied the glycolytic shift that was observed in high glucose conditions. Accordingly, a reduced expression of MEF2C, MYF5, and PGC‐1α was found in kidney tissue sections that were obtained from patients with diabetic nephropathy. These findings obtained in human samples demonstrate that MEF2C‐MYF5–dependent bioenergetic dedifferentiation occurs in podocytes that are confronted with a highglucose milieu.—Imasawa, T., Obre, E., Bellance, N., Lavie, J., Imasawa, T., Rigothier, C., Delmas, Y., Combe, C., Lacombe, D., Benard, G., Claverol, S., Bonneu, M., Rossignol, R. High glucose repatterns human podocyte energy metabolism during differentiation and diabetic nephropathy. FASEB J. 31, 294–307 (2017) www.fasebj.org [ABSTRACT FROM AUTHOR]

In recent years, the renal interstitium has been identified as the site of multiple cell types, giving rise to multiple contiguous cellular networks with multiple fundamental structural and functional roles. Few studies have been carried out on the morphological and functional properties of the stromal/interstitial renal cells during the intrauterine life. This work was aimed at reviewing the peculiar features of renal interstitial stem/progenitor cells involved in kidney development. The origin of the renal interstitial progenitor cells remains unknown. During kidney development, besides the Six2 + cells of the cap mesenchyme, a self-renewing progenitor population, characterized by the expression of Foxd1, represents the first actor of the non-nephrogenic lineage. Foxd1 + interstitial progenitors originate the cortical and the renal medullary interstitial progenitors. Here, the most important stromal/interstitial compartments present in the developing human kidney will be analyzed: capsular stromal cells, cortical interstitial cells, medullary interstitial cells, the interstitium inside the renal stem cell niche, Hilar interstitial cells and Ureteric interstitial cells. Data reported here indicate that the different interstitial compartments of the developing kidney are formed by different cell types that characterize the different renal areas. Further studies are needed to better characterize the different pools of renal interstitial progenitors and their role in human nephrogenesis. [ABSTRACT FROM PUBLISHER]

The treatment of renal injury by autologous, patient-specific adult stem cells is still an unmet need. Unsolved issues remain the spatial integration of stem cells into damaged areas of the organ, the commitment in the required cell type and the development of improved bioengineered devices. In this respect, biomaterials and architectures have to be specialized to control stem cell differentiation. Here, we perform an extensive study on micropatterned extracellular matrix proteins, which constitute a simple and non-invasive approach to drive the differentiation of adult renal progenitor/stem cells (ARPCs) from human donors. ARPCs are interfaced with fibronectin (FN) micropatterns, in the absence of exogenous chemicals or cellular reprogramming. We obtain the differentiation towards tubular cells of ARPCs cultured in basal medium conditions, the tubular commitment thus being specifically induced by micropatterned substrates. We characterize the stability of the tubular differentiation as well as the induction of a polarized phenotype in micropatterned ARPCs. Thus, the developed cues, driving the functional commitment of ARPCs, offer a route to recreate the microenvironment of the stem cell niche in vitro , that may serve, in perspective, for the development of ARPC-based bioengineered devices. [ABSTRACT FROM AUTHOR]

The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na+-K+-ATPase (EC 3.6.3.9), secondary active Na+-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na+-K+-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1–OAT3- related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney. [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]

Objective and design: The aim of this study is to evaluate the expression of the histamine receptors, particularly focusing on the HR in human renal tubules. Material: The ex vivo evaluation was carried on specimens from human renal cortex. Primary and immortalized tubular epithelial cells (TECs) and the HK-2 cell line were used as in vitro models. Treatment: Cells were pretreated for 10 min with chlorpheniramine maleate 10 μM (HR antagonist), ranitidine 10 µM (HR antagonist), GSK189254 1 µM (HR antagonist) or JNJ7777120 10 µM (HR antagonist), and then exposed to histamine (3 pM-10 nM) for 30 min. Methods: The ex vivo evaluation on specimens from human renal cortex was performed by immunohistochemistry. The expression of histamine receptors on primary and immortalized TECs and the HK-2 cell line was evaluated at both gene (RT-PCR) and protein (immunocytofluorescence) levels. The pharmacological analysis was performed by TR-FRET measurements of second messenger (IP3 and cAMP) production induced by histamine with or without the selective antagonists. Results: Our data revealed the presence of all histamine receptors in human tubules; however, only TECs expressed all the receptors. Indeed, histamine elicited a sigmoid dose-response curve for IP production, shifted to the right by chlorpheniramine maleate, and elicited a double bell-shaped curve for cAMP production, partially suppressed by the selective HR, HR and HR antagonists when each added alone, and completely ablated when combined together. Conclusions: Herein, we report the identification of all four histamine receptors in human renal tubules. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes. [ABSTRACT FROM AUTHOR]

Nephron number at birth has relevant clinical importance with implications for long-term renal health. In recent years, the podocyte depletion hypothesis has emerged as an important concept in kidney pathology. This study was aimed at verifying whether human podocyte number changes significantly during intrauterine life. To this end, 62 subjects with gestational ages ranging from 20 to 41 wk were examined. Kidney sections were stained with hematoxylin and eosin and digitally scanned at ×400 magnification. Subjects were subdivided into fetuses (gestational age ×24 wk, n = 5), preterms (gestational age ≥25 and ≤36 wk, n = 39), and full-term newborns (gestational age ≥37 wk, n = 18). The average podocyte number of 1,908 ± 645, 1,394 ± 498, and 1,126 ± 256 was, respectively, observed in fetuses, preterms, and full-term newborns. A significant main effect (P = 0.0051) of gestational age on podocyte number was observed with a significantly lower number in full-term newborns than in fetuses (P < 0.01). Intragroup variability was also observed. We speculate that variations in podocyte number could be correlated with factors such as drugs and maternal diet occurring during intrauterine life. In conclusion, this study shows, for the first time, a decreasing trend in podocyte number during gestation. [ABSTRACT FROM AUTHOR]

Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1-P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the feasibility of in vivo Na imaging of the corticomedullary Na gradient and to measure Na transverse relaxation times (T2*) in human kidneys. Methods: In this prospective, IRB-approved study, eight healthy volunteers (4 female, 4 male; mean age 29.4 ± 3.6 years) were examined on a 7-T whole-body MR system using a Na-only spine-array coil. For morphological Na-MRI, a 3D gradient echo (GRE) sequence with a variable echo time scheme (vTE) was used. T2* times were calculated using a multiecho 3D vTE-GRE approach. Na signal-to-noise ratios (SNR) were given on a pixel-by-pixel basis for a 20-mm section from the cortex in the direction of the medulla. T2* maps were calculated by fitting the Na signal decay monoexponentially on a pixel-by-pixel basis, using least squares fit. Results: Mean corticomedullary Na-SNR increased from the cortex (32.2 ± 5.6) towards the medulla (85.7 ± 16.0). The SNR increase ranged interindividually from 57.2 % to 66.3 %. Mean Na-T2* relaxation times differed statistically significantly ( P < 0.001) between the cortex (17.9 ± 0.8 ms) and medulla (20.6 ± 1.0 ms). Conclusion: The aim of this study was to evaluate the feasibility of in vivo Na MRI of the corticomedullary Na gradient and to measure the Na T2* relaxation times of human kidneys at 7 T. Key Points: • High field MR offers new insights into renal anatomy and physiology. • Na MRI of healthy human kidneys is feasible at ultra- high field. • Renal Na concentration increases from the cortex in the medullary pyramid direction. • In vivo measurements of renal Na- T2* times are demonstrated at 7. 0 T. [ABSTRACT FROM AUTHOR]

Although knowledge of human renal cytochrome P450 ( CYP) and UDP-glucuronosyltransferase ( UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug-endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis. [ABSTRACT FROM AUTHOR]

Although knowledge of human renal cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug–endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20‐hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis. [ABSTRACT FROM AUTHOR]

Angiogenin ( ANG) is a potent angiogenic factor that is up-regulated by hypoxia. ANG expression is well documented in normal tissues and in common tumours, but its expression has not been reported in the normal human kidney or in Wilms' tumours ( WT). We examined ANG expression in WTs, human fetal kidney ( FK) and childhood kidney ( NK) samples and studied its relationship with microvascular density ( MVD) and with three other hypoxia-induced angiogenic factors: lactate dehydrogenase A ( LDHA), vascular endothelial growth factor (VEGFA) and BHLHE40 (basic helix-loop-helix transcription factor E40). Total ANG protein levels were significantly lower in WTs when compared with those in 15 matched-paired NKs. ANG immunoreactivity was observed in the glomeruli, proximal tubules and vessels in the FKs and NKs, indicating that ANG plays a physiological role in the human kidney. ANG cellular localization and distribution in 27 WTs reflected the pattern observed in the FKs. ANG colocalized with LDHA in the perinecrotic areas of untreated WTs suggesting up-regulation by hypoxia. There was a significant correlation between CD31- MVD and ANG- MVD. ANG, CD31, VEGFA and BHLHE40 mRNA levels were significantly lower in 15 WTs compared with matched-paired NKs. Univariable and multivariable statistical analyses showed significant correlations between ANG and CD31, ANG and BHLHE40 mRNAs and a weaker relationship between ANG and VEGFA mRNAs. ANG expression in WTs recapitulates that seen during nephrogenesis, and correlation with CD31- MVDs and mRNAs is consistent with a contribution to angiogenesis in WTs. Our study contributes to the understanding of angiogenesis during development and in WTs. [ABSTRACT FROM AUTHOR]

Abstract: Selective inhibitors of sodium-glucose cotransporter 2 (SGLT2)-mediated reabsorption of glucose in the proximal tubule of the kidney are being developed for the treatment of diabetes. SGLT2 shares high degree of homology with SGLT3; however, very little is known about the expression and functional role of SGLT3 in the human kidney. Indeed, the SGLT2 inhibitors that are currently in clinical trials might affect the expression and/or the activity of SGLT3. Therefore, the present study examined the expression of SGLT3 mRNA and protein in human kidney and in a human proximal tubule HK-2 cell line. The results indicated that human SGLT3 (hSGLT3) message and protein are expressed both in vivo and in vitro. We also studied the activity of hSGLT3 protein following its over-expression in mammalian kidney-derived COS-7 cells and in HK-2 cells treated with the imino sugar deoxynojirimycin (DNJ), a potent agonist of hSGLT3. Over-expression of hSGLT3 in COS-7 cells increased intracellular sodium concentration by 3-fold without affecting glucose transport. Activation of hSGLT3 with DNJ (50μM) increased sodium uptake in HK-2 cells by 5.5 fold and this effect could be completely blocked with SGLT inhibitor phlorizin (50μM). These results suggest that SGLT3 is expressed in human proximal tubular cells where it serves as a novel sodium transporter. Up-regulation of the expression of SGLT3 in the proximal tubule in diabetic patients may contribute to the elevated sodium transport in this segment of the nephron that has been postulated to promote hyperfiltration and renal injury. [Copyright &y& Elsevier]

Ciliogenesis in developing and post-natal human kidneys appears to influence cell proliferation and differentiation, apico-basal cell polarity, and tubular lumen formation. We have analyzed the appearance of primary cilia and differentiation of kidney cells in ten human conceptuses aged 6-22 weeks and in one 5-year-old kidney, using a double immunofluorescence labeling technique for α-tubulin, γ-tubulin, Oct-4, and Ki-67 and by electron microscopy. Immature forms of nephrons and ampullae were characterized by intense cell proliferation, which subsequently decreased during development. Primary cilia appeared on the surfaces of non-proliferating cells in developing nephrons, gradually increasing in length from 0.59 μm in renal vesicles to 0.81 μm in the S-forms of nephrons, ultimately reaching 3.04 μm in length in mature fetal and post-natal nephrons. Ciliary length increased from 0.59 μm in ampullae to 1.28 μm in post-natal collecting tubules. Mesenchymal to epithelial transformation of kidney cells coincided with the appearance of apico-basal polarity, both gap and tight junctions, and lumen formation. Up-regulation of Oct-4 expression correlated with the onset of kidney cell differentiation. Our results demonstrate the importance of proper primary cilia lengthening and Oct-4 expression for the normal development of fetal and post-natal kidneys and of apico-basal polarity for normal tubular lumen formation. Disturbances in these processes are associated with ciliopathies. [ABSTRACT FROM AUTHOR]

The kidney is characterised by the intensive processes of maturation, but also changes of involution. These processes begin during the foetal life when predominate the process of maturation and some changes of involution and continue after birth. After birth there is a process of the structural maturation of the kidney; it reaches its full anatomical maturity by the third-fourth decade of life. From then on the human kidney is characterised by the changes of involution. These changes of involution are found in all anatomical segments of the kidney and initially they are slow but after the end of sixth decade they are more rapid in progression. The changes of involution can be separate, but they can coincide with renal diseases. Understanding the age renal changes may help to prevent life-threatening kidney disease, and these are important for the kidney transplant. This work will focus on anatomical changes of the kidney during the life, using the qualitative and quantitative methods and we examined some of the current literature for understanding of the complex aging of the kidney. [ABSTRACT FROM AUTHOR]

Abstract: 11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) regulates active glucocorticoid access to glucocorticoid and mineralocorticoid receptors by metabolizing it to an inactive form. Perfluoroalkylated substances (PFASs) are man-made polyfluorinated compounds that are widely used and persistent in the environment. We tested the inhibitory potencies of four PFASs including perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexanesulfonate (PFHxS) and perfluorobutane sulfonate (PFBS) on human and rat 11β-HSD2. PFOS was a potent inhibitor of both human (IC50 =48nM) and rat (IC50 =293nM) 11β-HSD2 activities. The potencies for the inhibition of human and rat 11β-HSD2 activities were PFOS>PFOA>PFHxS>PFBS. PFASs showed competitive inhibition of both human and rat 11β-HSD2 activities. This observation indicates that PFOS is a potent endocrine disruptor for glucocorticoid metabolism. Article from the Special issue on Targeted Inhibitors. [Copyright &y& Elsevier]

The role of the kidney in drug and chemical disposition has traditionally focused on the excretion of polar xenobiotics and metabolites. However, there is increasing evidence demonstrating that renal UGTs are integral to the “local” intrarenal, and, possibly, systemic, metabolic clearance of numerous drugs and nondrug xenobiotics, as well as to the maintenance of renal homeostasis through limiting the biological activity of endogenous renal mediators that control electrolyte balance and renal blood flow. The common involvement of UGT1A9 and UGT2B7 in the metabolism of both endogenous and exogenous compounds in kidney predicates significant renal drug-endobiotic interactions that may explain, in part, the adverse renal effects of some drugs. [ABSTRACT FROM AUTHOR]

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]

Postdiarrhea hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children in Argentina. It is well established that Shiga toxin type 2 (Stx2) causes direct damage to glomerular endothelial cells and tubular epithelial cells, leading to a reduction in the water handling capacity of the kidney. In this study, we demonstrate that Stx2 and its B subunit (Stx2B) were able to inhibit water absorption across human renal tubular epithelial cell (HRTEC) monolayers without altering the short circuit current and the 3H-mannitol permeability. Quantitative evaluation of 14C-inulin transport across HRTEC monolayers showed a similar transport rate both before and after HRTEC treatment with Stx2 that confirmed the integrity of the paracellular pathway. Furthermore, Stx2 produced significant protein synthesis inhibition of HRTEC at concentrations as low as 0.001 ng/ml and 1 h of incubation, whereas Stx2B did not modify it at concentrations as high as 10,000 ng/ml and 6 h of incubation. Our findings suggest that whereas the action of Stx2 appears to be caused mainly by the inhibition of protein synthesis mediated by the A subunit, the binding of Stx2B subunit to the Gb3 receptor may affect the membrane mechanisms related to water absorption. We speculate that inhibition of water absorption may occur in proximal tubular cells in vivo in response to Stx2 and may contribute to the early event of HUS pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: In order to gain insights into the renal and hepatic glucuronidation of frusemide (FSM), this study: (i) characterised the kinetics of FSM glucuronidation by human liver microsomes (HLM) and human kidney cortical- (HKCM) and medullary- (HKMM) microsomes, and (ii) identified the human UDP-glucuronosyltransferase enzyme(s) involved in this pathway. HLM, HKCM and HLMM efficiently glucuronidated FSM. FSM glucuronide (FSMG) formation followed Michaelis–Menten kinetics in all tissues. While the mean K m for FSMG formation by HKMM (386±68μM) was lower than the K m values for HLM (988±271μM) and HKCM (704±278μM), mean V max/K m values were comparable for the three tissues. A panel of recombinant UGT enzymes was screened for the capacity to glucuronidate FSM. UGT 1A1, 1A3, 1A6, 1A7, 1A9, 1A10 and 2B7 metabolised FSM. Of the renally and hepatically expressed enzymes, comparison of kinetic parameters suggests a predominant role of UGT1A9 in FSM glucuronidation, although UGT1A1 may also contribute to FSMG formation by HLM. Consistent with these observations, the UGT1A selective inhibitors phenylbutazone and sulfinpyrazone decreased FSMG formation by HLM, HKCM and HKMM by 60–80%, whereas the UGT2B7 selective inhibitor fluconazole reduced FSM glucuronidation by ≤20%. The ability of HKCM and HKMM to form FSMG supports the proposition that the kidney is the main organ involved in FSM glucuronidation in vivo, although a role for hepatic metabolism remains a possibility in renal dysfunction. The data further demonstrate the potential importance of both the medulla and cortex in renal drug metabolism and detoxification. [Copyright &y& Elsevier]

Abstract: The local renal metabolism of glucocorticoids (GCs) by isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) determines their biological effects. 11β-HSD2, located in collecting duct epithelial cells of the mammalian and human kidney, serves as a putative “guardian” preventing GCs from binding to mineralocorticoid receptors. Various investigators have shown that both isoforms are present in kidney tissue from the rat, dog and other mammals. There is controversy as to whether 11β-HSD1 exists and functions in human kidney. The current studies examine the locale and function of both isoforms in human kidney. The expression of 11β-HSD1 was similar to that of 11β-HSD2 by Western blot. Two distinct Lineweaver Burke plots could be drawn providing enzyme kinetics for both isoforms. The apparent Km for the NADP dependent 11β-HSD1 enzyme was 0.42 μM while the apparent Km for the NAD dependent 11β-HSD2 enzyme was 10.2 nM. Human renal 11β-HSD1 appears to function as a dehydrogenase with no significant “reverse” reductase activity. Using immuno-histochemistry and Western blot analysis, 11β-HSD1 was found to co-localize with COX-2 in proximal tubule cells; COX-2 was not seen with 11β-HSD2 in cortical collecting duct. Thus, normal human kidney contains active 11β-HSD1 and 11β-HSD2. 11β-HSD1 co-localizes with COX-2 in proximal tubule cells. [Copyright &y& Elsevier]

Abstract: We previously catalogued expression and activity of organic anion and cation, amino acid, and peptide transporters in primary cultures of human proximal tubular (hPT) cells to establish them as a cellular model to study drug transport in the human kidney [Lash, L.H., Putt, D.A., Cai, H., 2006. Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228, 200–218]. Here, we extend our analysis to drug metabolism enzymes. Expression of 11 cytochrome P450 (CYP) enzymes was determined with specific antibodies. CYP1B1, CYP3A4, and CYP4A11 were the only CYP enzymes readily detected in total cell extracts. These same CYP enzymes, as well as CYP3A5 and possibly CYP2D6, were detected in microsomes from confluent hPT cells, although expression levels varied among kidney samples. In agreement with Western blot data, only activity of CYP3A4/5 was detected among the enzyme activities measured. Expression of all three glutathione S-transferases (GSTs) known to be found in hPT cells, GSTA, GSTP, and GSTT, was readily detected. Variable expression of three sulfotransferases (SULTs), SULT1A3, SULT1E, and SULT2A1, and three UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A6, and UGT2B7, was also detected. When examined over the course of cell growth to confluence, expression of all enzymes was generally maintained at readily measurable levels, although they were often lower than in fresh tissue. These results indicate that primary cultures of hPT cells possess significant capacity to metabolize many classes of drugs, and can be used as an effective model to study drug metabolism. [Copyright &y& Elsevier]

The toxicology of hydroquinone has been reviewed on a number of previous occasions. This review targets its potential for carcinogenicity and possible modes of carcinogenic action. The evaluation made by IARC (1999) of its carcinogenic risk to humans was that hydroquinone is not classifiable as to its carcinogenicity to humans (Group 3). This evaluation was based on inadequate evidence in humans and limited evidence in experimental animals. The epidemiological information comes from four cohort studies involving occupational exposures. A cohort of lithographers, some of whom had worked with hydroquinone, had an excess of malignant melanoma based on five cases, but only two of the cases had reported exposure to hydroquinone. In a study of photographic processors the number of exposed individuals was uncertain and the numbers of cases of individual cancer sites were small. In view of the statistical power limitations of these studies for individual diagnostic categories of cancers, they are not considered to be informative with regard to the carcinogenicity of hydroquinone. A cohort of workers with definite and lengthy exposure to hydroquinone, during either its manufacture or its use, had low cancer rates compared with two comparison populations; the reason for the lower than expected rates is unclear... [ABSTRACT FROM AUTHOR]

Abstract: Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), is used commonly to treat menorrhagia. This study investigated the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7. Using HKCM Michaelis–Menten (MM) kinetics were observed for mefenamic ( 23μM) and niflumic acid ( 123μM) glucuronidation, while flufenamic acid exhibited non-hyperbolic (atypical) glucuronidation kinetics. Notably, the intrinsic renal clearance of mefenamic acid (CLint 17±5.5μL/minmg protein) was fifteen fold higher than that of niflumic acid (CLint 1.1±0.8μL/minmg protein). These data suggest that renal glucuronidation of mefenamic acid may result in high intrarenal exposure to mefenamic acyl-glucuronide and subsequent binding to renal proteins. Diverse kinetics were observed for fenamate glucuronidation by UGT2B7 and UGT1A9. Using UGT2B7 MM kinetics were observed for flufenamic ( 48μM) and niflumic acid ( 135μM) glucuronidation and atypical kinetics with mefenamic acid. Similarity in between HKCM and UGT2B7 suggests that UGT2B7 may be the predominant renal UGT isoform catalysing niflumic acid glucuronidation. In contrast, UGT1A9 glucuronidation kinetics were characterised by negative cooperativity with mefenamic (S 50 449μM, h 0.4) and niflumic acid (S 50 7344μM, h 0.4) while atypical kinetics were observed with flufenamic acid. Additionally, potent inhibition of the renal glucuronidation of the UGT substrate ‘probe’ 4-methylumbelliferone by flufenamic, mefenamic and niflumic acid was observed. These data suggest that inhibitory metabolic interactions may occur between fenamates and other substrates metabolised by UGT2B7 and UGT1A9 in human kidney. [Copyright &y& Elsevier]

Changes in glomerular volume ( Vglom) play an important role in the initiation and progression of various glomerulopathies. Estimation of Vglom in the normal kidney provides baseline values for studies of glomerular hypertrophy in disease. The traditional model-based method of Weibel and Gomez is widely applied to estimate Vglom in clinical biopsy specimens. Assumptions of glomerular size distribution and shape required by this method are potential sources of bias that have not been verified. We evaluated the applicability of the glomerular size distribution coefficient in estimating Vglom in human kidneys. Vglom of 720 non-sclerotic glomeruli in histologically normal kidneys of 24 males (20–69 years) was estimated by the unbiased disector/Cavalieri approach. Accurate glomerular diameters were calculated from Cavalieri estimates of Vglom assuming glomerular sphericity. The coefficients of variation (CV) of glomerular diameters were compared with the corresponding values of the size distribution coefficient predicted by the Weibel and Gomez method. Mean (SD) glomerular diameter was 201 (28) mm (range 110–276 mm). The CV of glomerular diameter within each kidney ranged from 4.9 to 14.6%. Corresponding glomerular size distribution coefficients predicted by the formula of Weibel and Gomez ranged from 1.00 to just 1.03. The value of the size distribution coefficient required by the Weibel and Gomez technique when estimating Vglom in normal human kidneys is remarkably constant. This is despite large variations in Vglom. Future studies should examine the extent of bias introduced by the glomerular shape assumptions of this method. [ABSTRACT FROM AUTHOR]

Abstract: To further develop primary cultures of human proximal tubular (hPT) cells for study of drug disposition, we determined kinetics and protein expression of several key transporters for organic anions and cations, peptides, and neutral amino acids. p-Aminohippurate uptake exhibited similar kinetics as published values, was inhibited by cephaloridine, cimetidine, methotrexate, and urate, consistent with function of both organic anion transporter 1 (OAT1) and OAT3. Transport rates by organic cation transporters (OCTs) were up to three-fold higher than those of OATs. Of the OCT substrates tested, triethanolamine exhibited the highest transport rates across the basolateral membrane (BLM). OCTN1 exhibited high-affinity, low-capacity BLM transport of l-carnitine. Glycylsarcosine transport by PepT2 was rapid and comparable to that of OCTs. Amino acid System L on the BLM exhibited comparable kinetic parameters for transport of l-leucine as the OATs. Efflux of verapamil across the brush-border membrane by P-glycoprotein was very rapid. Expression of carriers was generally maintained throughout 5 days of culture. Of the four OAT proteins studied (OAT1-4), expression of OAT1 and OAT3 was the most readily detected and exhibited interindividual variation. OCTN2 was the major OCT in hPT cells. Expression was also quantified for multidrug resistance-associated proteins 2 and 5 and P-glycoprotein. These results show that primary cultures of hPT cells express a diverse array of transporters for major classes of important drugs and are suitable for study of drug transport and disposition and assessment of potential drug–drug interactions in human kidney. [Copyright &y& Elsevier]

Abstract: The present study examined accumulation of the metal toxins cadmium (Cd) and lead (Pb) in relation to the abundance of cytochrome P450 4F2 (CYP4F2), CYP2E1 and concentrations of zinc and copper in liver and kidney samples using immunoblotting coupled with metal analysis. The post mortem liver and kidney cortex samples were from 23 males and 8 females aged 3–89 years. All were Caucasians who had not been exposed to metals in the workplace. Average kidney cortex Cd load of 17.4μg/g w.w. was 17 times greater than average liver Cd load (1.1μg/g w.w.). In contrast, average kidney cortex Pb load of 0.09μg/g w.w. was two times lower than liver Pb load of 0.19μg/g w.w. Average Zn and Cu concentrations in the kidney cortex samples were 67% and 33% lower than those in the liver. Liver and kidney Cd loads, but not liver or kidney Pb loads, correlated positively with donors’ age. After controlling for liver Cd load, an inverse correlation was seen between Zn and age (partial r =−0.39, P =0.02), suggesting reduction in liver Zn levels in old age. Liver CYP2E1 protein abundance correlated with age-adjusted Cd load (partial r =0.37, P =0.02) whereas kidney CYP4F2 protein abundance showed a positive correlation with age-adjusted Cd loads (partial r =0.40, P =0.02). These findings suggest that Cd may be an inducer of renal CYP4F2 and hepatic CYP2E1 and that increased renal CYP4F2 expression may implicate in Cd-linked renal tubular dysfunction and high blood pressure, involving CYP4F2-dependent arachidonic acid metabolism. [Copyright &y& Elsevier]

Abstract: Difficulties in evaluation of trichloroethylene (TRI)-induced toxicity in humans and extrapolation of data from laboratory animals to humans are due to the existence of multiple target organs, multiple metabolic pathways, sex-, species-, and strain-dependent differences in both metabolism and susceptibility to toxicity, and the lack or minimal amount of human data for many target organs. The use of human tissue for mechanistic studies is thus distinctly advantageous. The kidneys are one target organ for TRI and metabolism by the glutathione (GSH) conjugation pathway is responsible for nephrotoxicity. The GSH conjugate is processed further to produce the cysteine conjugate, S-(1,2-dichlorovinyl)-l-cysteine (DCVC), which is the penultimate nephrotoxic species. Confluent, primary cultures of human proximal tubular (hPT) cells were used as the model system. Although cells in log-phase growth, which are undergoing more rapid DNA synthesis, would give lower LD50 values, confluent cells more closely mimic the in vivo proximal tubule. DCVC caused cellular necrosis only at relatively high doses (>100 μM) and long incubation times (>24 h). In contrast, both apoptosis and enhanced cellular proliferation occurred at relatively low doses (10–100 μM) and early incubation times (2–8 h). These responses were associated with prominent changes in expression of several proteins that regulate apoptosis (Bcl-2, Bax, Apaf-1, Caspase-9 cleavage, PARP cleavage) and cellular growth, differentiation and stress response (p53, Hsp27, NF-κB). Effects on p53 and Hsp27 implicate function of protein kinase C, the mitogen activated protein kinase pathway, and the cytoskeleton. The precise pattern of expression of these and other proteins can thus serve as molecular markers for TRI exposure and effect in human kidney. [Copyright &y& Elsevier]

We investigated the effects of nitric oxide (NO) on activity of the inwardly rectifying K+ channel in cultured human proximal tubule cells, using the cell attached mode of the patch-clamp technique. An inhibitor of NO synthases, N ω-nitro-L-arginine methyl ester (L-NAME; 100 µM), reduced channel activity, which was restored by an NO donor, sodium nitroprusside (SNP; 10 µM) or 8-bromo-cGMP (8BrcGMP; 100 µM). However, SNP failed to activate the channel in the presence of an inhibitor of soluble guanylate cyclase, 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 µM). Similarly, the SNP effect was abolished by a protein kinase G (PKG)-specific inhibitor, KT-5823 (1 µM), but not by a protein kinase A-specific inhibitor, KT-5720 (500 nM). Another NO donor, S-nitroso-N-acetyl-D,L-penicillamine (10 µM), mimicked the SNP-induced channel activation. In contrast to the stimulatory effect of SNP at a low dose (10 µM), a higher dose of SNP (1 mM) reduced channel activity, which was not restored by 8-BrcGMP. Recordings of membrane potential with the slow whole cell configuration demonstrated that L-NAME (100 µM) and the high dose of SNP (1 mM) depolarized the cell by 10.1 ± 2.6 and 9.2 ± 1.0 mV, respectively, whereas the low dose of SNP (10 µM) hyperpolarized it by 7.1 ± 0.7 mV. These results suggested that the endogenous NO would contribute to the maintenance of basal activity of this K + channel and hence the potential formation via a cGMP/PKG dependent mechanism, whereas a high dose of NO impaired channel activity independent of cGMP/PKG-mediated processes. [ABSTRACT FROM AUTHOR]

In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT-PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO•. Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.British Journal of Cancer (2004) 90, 2364-2369. doi:10.1038/sj.bjc.6601809 www.bjcancer.com Published online 11 May 2004 [ABSTRACT FROM AUTHOR]

We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca2+ removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular. [ABSTRACT FROM AUTHOR]

To study the role of insulin-like growth factors (IGF) in podocyte maturation, we isolated and characterized fetal visceral glomerular epithelial cells from human kidneys obtained at 818 weeks gestation. Cells were identified as podocyte lineage by their cobblestone morphology and immunoreactivity with synaptopodin, Wilms tumor-1 suppressor gene product (WT-1), complement receptor CR1, and cytoskeletal proteins smooth muscle actin and vimentin. Stimulation of the podocyte cell monolayers with IGF-II resulted in a slight increase in mitogenesis, an effect that was concentration and time dependent and abrogated by co-incubation with exogenous IGF binding protein 2 (IGFBP-2). Western blot analysis of conditioned media revealed that cultured podocytes expressed endogenous IGFBP-2 exclusively. IGF-II stimulation enhanced IGFBP-2 production in a dose- and time-dependent fashion and was associated with an increase in IGFBP-2 mRNA production. These data demonstrate that IGF-II-stimulated IGFBP-2 production appears to inhibit the mitogenic effect of IGF-II, and may have an autocrine effect on the maturation, differentiation, and survival of fetal podocytes. [ABSTRACT FROM AUTHOR]

The Na+–HCO3− cotransporter (NBC-1) plays a major role in bicarbonate absorption from proximal tubules. However, which NBC-1 variant mediates proximal bicarbonate absorption has not been definitely determined. Moreover, the localization of this cotransporter in human kidney and renal cell carcinoma (RCC) tissues has not been clarified. To clarify these issues, immunohistochemical analysis was performed using the specific antibodies against kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters. In Western blot analysis the expression of kNBC-1 but not of pNBC-1 was detected in both normal human kidney and RCC tissues. In immunofluorescence analysis on normal renal tissues the anti-kNBC-1 antibody strongly and exclusively labeled the basolateral membranes of proximal tubules, which was confirmed by electron microscopic observation. In RCC cells, the anti-kNBC-1 antibody labeled both plasma membranes and intracellular organelles. The labeling by anti-pNBC-1 antibody was not detected in both normal kidney and RCC tissues. These results indicate that kNBC-1 is the dominant variant that mediates bicarbonate absorption from human renal proximal tubules. They also suggest that NBC-1 may have distinct roles in cancer cells. [Copyright &y& Elsevier]

Abstract We review our evidence in favour of the hypothesis that gap-junctional hemichannels (GJH) are activated by depletion of adenosine triphosphate (ATP) in human renal proximal tubule cells in primary culture (hPT cells). Undocked GJH permit fluxes of ions and hydrophilic molecules up to 1 kDa, and thus their opening can cause alterations of cell composition conducive to cell damage. We show that hPT cells express connexin 43 (Cx43) (at the mRNA and protein levels) and that the protein is expressed on the plasma membrane. Moderate levels of pharmacological depletion of ATP increased plasma-membrane permeability, as shown by loading with the hydrophilic dye 5/6 carboxyfluorescein (CF, 376 Da) and other low-molecular weight dyes, but not with fluorescein-labelled dextran (>1500 Da). Roles for endocytosis and activation of purinergic-receptor channels were experimentally ruled out. Moderate ATP depletion also caused necrosis, assessed by cell permeabilization to propidium iodide. Prolonged exposure to gadolinium reduced both the dye loading and the necrosis induced by ATP depletion, i.e. it protected the cells. Cx43 overexpressed in insect cells, purified to homogeneity and reconstituted in proteoliposomes formed hemichannels that are activated by dephosphorylation of Ser368, a residue in a protein-kinase-C consensus phosphorylation sequence near the end of the C-terminal domain. Conclusions: (1) ATP depletion of hPT cells induces a Gd[sup 3+] -sensitive permeability of the plasma membrane to hydrophilic dyes with a cut-off size consistent with Cx43 GJH. (2) ATP depletion also increases the percentage of necrotic cells, an effect also reduced by Gd[sup 3+] . (3) The experiments with purified Cx43 reconstituted in liposomes suggest that dephosphorylation of Ser368 is sufficient to activate GJH, although other mechanisms may be involved in some cells. [ABSTRACT FROM AUTHOR]

Platelet-activating factor (PAF) is one of the most potent inflammatory mediators. It is biosynthesized by either the de novo biosynthesis of glyceryl ether lipids or by remodeling of membrane phospholipids. PAF is synthesized and catabolized by various renal cells and tissues and exerts a wide range of biological activities on renal tissue suggesting a potential role during renal injury. The aim of this study was to identify whether cortex and medulla of human kidney contain the acetyl-CoA:1-O -alkyl-sn -glycero-3-phosphocholine acetyltransferase (lyso-PAF AT) activity which catalyses the last step of the remodeling biosynthetic route of PAF and is activated in inflammatory conditions. Cortex and medulla were obtained from nephrectomized patients with adenocarcinoma and the enzymatic activity was determined by a trichloroacetic acid precipitation method. Lyso-PAF AT activity was detected in both cortex and medulla and distributed among the membrane subcellular fractions. No statistical differences between the specific activity of cortical and medullary lyso-PAF AT was found. Both cortical and medullary microsomal lyso-PAF ATs share similar biochemical properties indicating common cellular sources. [ABSTRACT FROM AUTHOR]

Expression of NOS1 and soluble guanylyl cyclase by human kidney epithelial cells: Morphological evidence for an autocrine/paracrine action of nitric oxide. Background. Nitric oxide plays an important role in the kidney through effects on both renal hemodynamics and tubular functions. Tubular epithelial cells are thus a target for nitric oxide. However, as to whether tubular epithelial cells endogeneously produce nitric oxide under physiologic conditions in human kidney is currently unknown. The aim of the present study was to characterize and localize in situ the nitric oxide synthase (NOS) isoforms (NOS1, NOS2, and NOS3) expressed in human normal kidney, and soluble guanylyl cyclase, the well-known target for nitric oxide. Methods. Five complementary experimental approaches were used: (1 ) detection of NOS reductase activity by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, (2 ) immunolocalization of the NOS isoforms (NOS1, NOS2, NOS3), (3 ) immunoblot analysis, (4 ) quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of NOS mRNA, and (5 ) measurement of NOS activity as the conversion rate of l-[14 C]-arginine to l-[14 C]-citrulline. In addition, in situ detection of soluble guanylyl cyclase was assessed by immunohistochemistry. Results. All these techniques led to consistent results showing that epithelial cells of most tubules along the human nephron exhibit functional NOS1, with a corticomedullary gradient observed both at the protein and mRNA levels. Moreover, epithelial cells expressing NOS1 also express soluble guanylyl cyclase, indicating that these cells possess the machinery for autocrine/paracrine effect of nitric oxide. Conclusion. The present study demonstrates that NOS1 is strongly expressed in most tubules of the human nephron and therefore invites to consider epithelial cells as one of the major source of nitric oxide in the human kidney under physiologic conditions. [ABSTRACT FROM AUTHOR]

Man9-mannosidase, an α1,2-specific exo-enzyme involved in N-linked oligosaccharide processing, has been cloned recently from a human kidney cDNA library [Bause, E., Bieberich, E., Rolfs, A., Völker, C. & Schmidt, B, (1993) Eur. J. Biochem. 217, 533–540]. Transient expression in COS 1 cells of the enzyme resulted in a more than 20-fold increase of a catalytic activity cleaving specifically α1,2-mannosidic linkages in [14C]Man9-GlcNAc2 or [14C]Man5-GlcNAc2. Man9-mannosidase is expressed as a N-glycoprotein with a molecular mass of 73 kDa. Its enzymic activity is metal ion dependent and inhibited strongly by 1-deoxymannojirimycin (50% at 100 μM). Proteolytic studies with the membrane-associated form of Man9-mannosidase support the view that the enzyme is a type II transmembrane protein as predicted from its cDNA sequence. Several lines of evidence suggest that Man9-mannosidase, as expressed, is N-glycosylated at one of three potential Asn-Xaa-Thr/Ser/Cys acceptor sites. Approximately 50% of the N-linked oligosaccharide chains are removed by endoglycosidase H treatment, whereas complete deglycosylation of the enzyme is observed, when transfected cells were cultured in the presence of the Golgi mannosidase II inhibitor swainsonine, indicating that the sugar moiety of Man9-mannosidase is processed partially by Golgi-resident enzymes. This observation is consistent with the results of indirect immunofluorescence studies, pointing to a localization of the Man9-mannosidase predominantly in the juxtanuclear Golgi region. This localization clearly differs from that of pig liver Man9-mannosidase which appears to be located in the endoplasmic reticulum and transient vesicles. [ABSTRACT FROM AUTHOR]

The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions. • scRNA-seq identifies distinct transcriptional profiles of human podocyte development • Comparative analyses show similarities and differences of in vitro -derived podocytes • Implantation study suggests vascular interactions improve in vitro podocyte maturation Tran et al. performed single-cell RNA sequencing to provide an understanding of human podocyte development. Insights from the in vivo analysis was applied to extensively evaluate the formation of podocytes in vitro , highlighting autonomous programs of development and those requiring an interplay with adjacent cell types. [ABSTRACT FROM AUTHOR]

Background/Aims: The human proximal tubule (PT) epithelium is distinguished from other nephron segments via several unique characteristics. Studies assessing PT epithelium increasingly employ cell lines, bypassing the complexity of primary cell cultures. However, few human model systems exist for studying PT cells in vitro. The current work involves an intensive characterization of Caki-1 cells, a commercially available human renal cell line. Methods: Caki-1 cells were validated as a representative model system for PT cell research via morphological, physiological and biochemical investigations including light and transmission electron microscopy, transepithelial electrical resistance (TER) measurements and the detection of PT markers. Results: Morphologically, these cells form a polarized monolayer with apical located microvilli and multiple mitochondria per cell. Low TER ranging from 2 to 28 Ω cm2 was determined for Caki-1 cells, characteristic of the ‘leaky’ PT epithelium in vivo. Expression of the PT markers: NHE3, GGT, DPP IV, APM and AP were present in Caki-1 cells. Two epithelial markers, E-cadherin and Na+/K+-ATPase, were additionally observed. Conclusion: The current work is a concise summary which confirms that Caki-1 cells represent well-differentiated polarized PT cells in vitro, regardless of its cancerous origin and multiple passaging. They prove to be a significant contribution to the field of PT research. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]