Uptake of Fluorescein via a pH-Dependent Monocarboxylate Transporter by Human Kidney 2 (HK-2) Cells.

Herein, we investigated whether a fluorescent probe for an organic anion transporter (OAT), fluorescein (FLS), could be accumulated by human kidney 2 (HK-2) cells derived from human kidney proximal tubular epithelia. HK-2 cells took up FLS in a pH-dependent and concentration-dependent manner. FLS accumulation by HK-2 cells was inhibited by monocarboxylic acids, ibuprofen, rosuvastatin, and indoleacetic acid but not by typical substrates for OATs. A typical protonophore, carbonyl cyanide p-trichloromethoxyphenylhydrazone completely abolished FLS accumulation by HK-2 cells. The FLS efflux process from the preloaded HK-2 cells exhibited substantial trans-stimulation by the excess amount of extracellular FLS transport inhibitable monocarboxylate compounds such as 2,4-dichloro phenoxyacetic acid, fluvastatin, ibuprofen, indoleacetic acid, salicylic acid and rosuvastatin, indicating that the FLS transporter can recognize and accumulate them into the cells in a pH-dependent manner. The involvement of the FLS transporter in the reabsorption of monocarboxylic compounds was indicated by demonstrating that the pH-dependent FLS uptake is inhibited by various monocarboxylates in rabbit renal brush border membrane vesicles. pH-dependent FLS uptake was trans-stimulated by the inhibitable monocarboxylates. Collectively, the present data indicate that the pH-dependent transporters expressed in HK-2 cells are involved in the reabsorption of monocarboxylates from the urinary fluid into the tubular epithelia.

Molecular architecture of proliferative lupus nephritis as elucidated using 50-plex imaging mass cytometry proteomics.

Due to unique advantages that allow high-dimensional tissue profiling, we postulated imaging mass cytometry (IMC) may shed novel insights on the molecular makeup of proliferative lupus nephritis (LN). This study interrogates the spatial expression profiles of 50 target proteins in LN and control kidneys. Proliferative LN glomeruli are marked by podocyte loss with immune infiltration dominated by CD45RO+, HLA-DR+ memory CD4 and CD8 T-cells, and CD163+ macrophages, with similar changes in tubulointerstitial regions. Macrophages are the predominant HLA-DR expressing antigen presenting cells with little expression elsewhere, while macrophages and T-cells predominate cellular crescents. End-stage sclerotic glomeruli are encircled by an acellular fibro-epithelial Bowman's space surrounded by immune infiltrates, all enmeshed in fibronectin. Proliferative LN also shows signs indicative of epithelial to mesenchymal plasticity of tubular cells and parietal epithelial cells. IMC enabled proteomics is a powerful tool to delineate the spatial architecture of LN at the protein level.

ZO-1 expression in normal human macula densa: Immunohistochemical and immunofluorescence investigations.

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.

A murine monoclonal antibody against H5N1 avian influenza virus cross-reacts with human kidney cortex cells.

To investigate the biological characteristics of monoclonal antibodies (mAbs) against avian influenza virus (AIV) and the possible mechanism of AIV-related kidney injury. BALB/c mice were immunized with inactivated H5N1 AIV to prepare monoclonal antibody H5-32, and its subtype, titer and cross-reactivity with other influenza viruses were identified. The reactivity of monoclonal antibody with normal human tissue was analyzed by immunohistochemistry. Immunofluorescence and confocal laser scanning technique were used to detect the binding sites between mAb and human renal cortical cells, and Western blotting was used to detect the size of binding fragments. Immunohistochemical analysis confirmed that monoclonal antibody H5-32 cross-reacted with normal human kidney tissue. In human kidney, mAb H5-32 was localized in the cytoplasm of human renal tubular epithelial cells, and its binding fragment size was about 43 kDa. H5N1 AIV appears to bind to human renal tubular epithelial cells, which may be one of the mechanisms of kidney injury caused by AIV infection.

3-tert-Butyl-4-hydroxyanisole perturbs renal lipid metabolism in vitro by targeting androgen receptor-regulated de novo lipogenesis.

The widespread usage of 3-tert-butyl-4-hydroxyanisole (3-BHA) as an anthropogenic antioxidant has caused considerable environmental contamination and frequent detection in diverse human-derived samples. 3-BHA can promote adipogenesis and impair hepatic lipid metabolism, while its effects on renal lipid homeostasis remain to be uncertain. Herein, using the human kidney 2 (HK-2) cell experiments, 3-BHA was found to cause a significant reduction in lipid accumulation of the HK-2 cells in both exposure concentration- and duration-dependent manners. Exposure to 3-BHA lowered the transcriptional expressions of sterol regulatory element-binding protein 1 (SREBP1) and acetyl-CoA carboxylase (ACC), as well as ACC activity, indicating the inhibition in the process of de novo lipogenesis in HK-2 cells. On this basis, the mechanism study suggested that the reduced glucose absorption and accelerated glycolysis were concomitantly involved. The antagonism of 3-BHA on the transactivation of androgen receptor (AR) contributed to the lowered de novo lipogenesis and the consequent intracellular lipid reduction. The metabolomics data further confirmed the imbalance of lipid homeostasis and dysregulation of de novo lipogenesis. The new findings on the impaired renal lipid metabolism induced by 3-BHA warranted proper care about the usage of this chemical as a food additive.

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

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

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

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

Scattered Tubular Cells Markers in Macula Densa of Normal Human Adult Kidney.

BACKGROUND: The scattered tubular cells (STCs) are a population of resident progenitor tubular cells with expansion, self-renewal and epithelial differentiation abilities. Although these cells are localized within the proximal (PTs) and distal (DTs) tubules in a normal adult kidney, their presence has never been demonstrated in human macula densa (MD). The purpose of the present study is to describe the presence of STCs in MD using specific markers such as prominin-1 (CD133), cytokeratin 7 (KRT7) and vimentin (VIM). METHODS: We analyzed two sets of three consecutive serial sections for each sample. The first sections of each set were immunostained for nNOS to identify MD, the second sections were immune-stained for CD133 (specific STCs marker) while the third sections were analyzed for KRT7 (another STCs specific marker) and VIM (that stains the basal pole of the STCs) in the first and second sets, respectively, in order to study the co-expression of KRT7 and VIM with the CD133 marker. RESULTS: CD133 was localized in some MD cells and in the adjacent DT cells. Moreover, CD133 was detected in the parietal epithelial cells of Bowman's capsule and in some proximal tubules (PT). KRT7-positive cells were identified in MD and adjacent DT cells, while KRT7 positivity was mostly confined in both DT and collecting ducts (CD) in the other areas of the renal parenchyma. CD133 and KRT7 were co-expressed in some MD and adjacent DT cells. Some of the latter cells were positive both for CD133 and VIM. CD133 was always localized in the apical part of the cells, whereas the VIM expression was evident only in the cellular basal pole. Although some cells of MD expressed VIM or CD133, none of them co-expressed VIM and CD133. CONCLUSIONS: The presence of STCs was demonstrated in human adult MD, suggesting that this structure has expansion, self-renewal and epithelial differentiation abilities, similar to all other parts of renal tubules.

Impact of COVID-19 on Kidney of Diabetic Patients.

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.

In Situ Classification of Cell Types in Human Kidney Tissue Using 3D Nuclear Staining.

To understand the physiology and pathology of disease, capturing the heterogeneity of cell types within their tissue environment is fundamental. In such an endeavor, the human kidney presents a formidable challenge because its complex organizational structure is tightly linked to key physiological functions. Advances in imaging-based cell classification may be limited by the need to incorporate specific markers that can link classification to function. Multiplex imaging can mitigate these limitations, but requires cumulative incorporation of markers, which may lead to tissue exhaustion. Furthermore, the application of such strategies in large scale 3-dimensional (3D) imaging is challenging. Here, we propose that 3D nuclear signatures from a DNA stain, DAPI, which could be incorporated in most experimental imaging, can be used for classifying cells in intact human kidney tissue. We developed an unsupervised approach that uses 3D tissue cytometry to generate a large training dataset of nuclei images (NephNuc), where each nucleus is associated with a cell type label. We then devised various supervised machine learning approaches for kidney cell classification and demonstrated that a deep learning approach outperforms classical machine learning or shape-based classifiers. Specifically, a custom 3D convolutional neural network (NephNet3D) trained on nuclei image volumes achieved a balanced accuracy of 80.26%. Importantly, integrating NephNet3D classification with tissue cytometry allowed in situ visualization of cell type classifications in kidney tissue. In conclusion, we present a tissue cytometry and deep learning approach for in situ classification of cell types in human kidney tissue using only a DNA stain. This methodology is generalizable to other tissues and has potential advantages on tissue economy and non-exhaustive classification of different cell types.

Metabolomic and elemental profiling of human tissue in kidney cancer.

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

Kidney organoids: Research in developmental biology and emerging applications.

Kidney organoids generated from human pluripotent stem cells (hPSCs) have drastically changed the field of stem cell research on human kidneys within a few years. They are self-organizing multicellular structures that contain nephron components such as glomeruli and renal tubules in most cases, but hPSC-derived ureteric buds, the progenitors of collecting ducts and ureters, can also form three-dimensional organoids. Today's challenges facing human kidney organoids are further maturation and anatomical integrity in order to achieve a complete model of the developing kidneys and ultimately a complete adult organ. Since chronic kidney disease (CKD) and impaired kidney function are an increasing burden on public health worldwide, there is an urgent need to develop effective treatments for various renal conditions. In this regard, hPSC-derived kidney organoids may impact medicine by providing new translational approaches. The unique ability of kidney organoids derived from disease-specific hPSCs to reproduce human diseases caused by genetic alterations may help provide the next generation of kidney disease models. Recent advances in the field of kidney organoid research have been generally accompanied by progress in developmental biology and other technological breakthroughs. In this review, we consider the current trends in kidney organoid technology, especially focusing on the relationship to the study of human kidney development, and discuss the remaining hurdles and prospects in regenerating human kidney structures beyond organoids.

Downregulation of miR-574-5p inhibits HK-2 cell viability and predicts the onset of acute kidney injury in sepsis patients.

BACKGROUND: Increased levels of microRNA-574-5p (miR-574-5p) have been found to be associated with increased survival of septic patients, indicating the potential role of miR-574-5p in protecting against septic progression and complications. Acute kidney injury (AKI) is one of the most common and serious complications of sepsis. Therefore, the aim of this study was to test these hypotheses: (1) in a renal cell culture line (HK-2), upregulated expression of miR-574-5p increases, and downregulated expression of miR-574-5p decreases cell viability, and (2) serum levels of miR-574-5p from patients with sepsis and AKI are lower than those of patients with sepsis but no AKI. METHODS: The expression of miR-574-5p was regulated by cell transfection in HK-2 cells, and HK-2 cell viability was measured using the Cell Counting Kit-8. Serum miR-574-5p expression was analyzed using qRT-PCR. The predictive value of miR-574-5p for AKI onset was evaluated using the receiver operating characteristic curve and logistic regression analysis. RESULTS: The overexpression of miR-574-5p promoted HK-2 cell viability. Fifty-eight sepsis patients developed AKI, who had significantly lower miR-574-5p expression. miR-574-5p expression was decreased with AKI stage increase and correlated with kidney injury biomarker and had relatively high accuracy to predict AKI occurrence from sepsis patients. CONCLUSION: Overexpression of miR-574-5p in cultured HK-2 cells increases cell viability and knocked-down expression of miR-574-5p decreases cell viability. Consistently, septic patients with AKI were found to have less upregulation of miR-574-5p expression compared to septic patients without AKI. Thus, serum miR-574-5p may provide a novel biomarker for septic AKI.

Expression Pattern of α-Tubulin, Inversin and Its Target Dishevelled-1 and Morphology of Primary Cilia in Normal Human Kidney Development and Diseases.

The spatiotemporal expression of α-tubulin, inversin and dishevelled-1 (DVL-1) proteins associated with the Wnt-signaling pathway, and primary cilia morphology were analyzed in developing kidneys (14th-38th developmental weeks), healthy postnatal (1.5- and 7-years old) and pathologically changed human kidneys, including multicystic dysplastic kidneys (MCDK), focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome of the Finnish type (CNF). The analysis was performed by double immunofluorescence, electron microscopy, semiquantitative and statistical methods. Cytoplasmic co-expression of α-tubulin, inversin and DVL-1 was observed in the proximal convoluted tubules (pct), distal convoluted tubules (dct) and glomeruli (g) of analyzed tissues. During kidney development, the overall expression of α-tubulin, inversin and DVL-1 decreased, while in the postnatal period slightly increased. The highest expressions of α-tubulin and inversin characterized dct and g, while high DVL-1 characterized pct. α-tubulin, inversin and DVL-1 expression pattern in MCDK, FSGS and CNF kidneys significantly differed from the healthy control. Compared to healthy kidneys, pathologically changed kidneys had dysmorphic primary cilia. Different expression dynamics of α-tubulin, inversin and DVL-1 during kidney development could indicate that switch between the canonical and noncanonical Wnt-signaling is essential for normal kidney morphogenesis. In contrast, their disturbed expression in pathological kidneys might be associated with abnormal primary cilia, leading to chronic kidney diseases.

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

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

HLA-G dimer targets Granzyme B pathway to prolong human renal allograft survival.

Human leukocyte antigen G (HLA-G), a nonclassic HLA class Ib molecule involved in the maintenance of maternal tolerance to semiallogeneic fetal tissues during pregnancy, has emerged as a potential therapeutic target to control allograft rejection. We demonstrate here that the level of soluble HLA-G dimer was higher in a group of 90 patients with a functioning renal allograft compared with 40 patients who rejected (RJ) their transplants. The HLA-G dimer level was not affected by demographic status. One of the potential mechanisms in tissue-organ allograft rejection involves the induction of granzymes and perforin, which are the main effector molecules expressed by CD8+ cytotoxic T lymphocytes and function to destroy allogeneic transplants. Using genomics and molecular and cellular analyses of cells from T-cell-mediated RJ and nonrejected kidney transplant patients, cells from leukocyte Ig-like receptor B1 (LILRB1) transgenic mice, humanized mice, and genetically engineered HLA-G dimer, we demonstrated a novel mechanism by which HLA-G dimer inhibits activation and cytotoxic capabilities of human CD8+ T cells. This mechanism implicated the down-regulation of Granzyme B expression and the essential involvement of LILRB1. Thus, HLA-G dimer has the potential to be a specific and effective therapy for prevention of allograft rejection and prolongation of graft survival.-Ajith, A., Portik-Dobos, V., Nguyen-Lefebvre, A. T., Callaway, C., Horuzsko, D. D., Kapoor, R., Zayas, C., Maenaka, K., Mulloy, L. L., Horuzsko, A. HLA-G dimer targets Granzyme B pathway to prolong human renal allograft survival.

α1-Microglobulin (A1M) Protects Human Proximal Tubule Epithelial Cells from Heme-Induced Damage In Vitro.

Oxidative stress is associated with many renal disorders, both acute and chronic, and has also been described to contribute to the disease progression. Therefore, oxidative stress is a potential therapeutic target. The human antioxidant α1-microglobulin (A1M) is a plasma and tissue protein with heme-binding, radical-scavenging and reductase activities. A1M can be internalized by cells, localized to the mitochondria and protect mitochondrial function. Due to its small size, A1M is filtered from the blood into the glomeruli, and taken up by the renal tubular epithelial cells. A1M has previously been described to reduce renal damage in animal models of preeclampsia, radiotherapy and rhabdomyolysis, and is proposed as a pharmacological agent for the treatment of kidney damage. In this paper, we examined the in vitro protective effects of recombinant human A1M (rA1M) in human proximal tubule epithelial cells. Moreover, rA1M was found to protect against heme-induced cell-death both in primary cells (RPTEC) and in a cell-line (HK-2). Expression of stress-related genes was upregulated in both cell cultures in response to heme exposure, as measured by qPCR and confirmed with in situ hybridization in HK-2 cells, whereas co-treatment with rA1M counteracted the upregulation. Mitochondrial respiration, analyzed with the Seahorse extracellular flux analyzer, was compromised following exposure to heme, but preserved by co-treatment with rA1M. Finally, heme addition to RPTE cells induced an upregulation of the endogenous cellular expression of A1M, via activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-pathway. Overall, data suggest that A1M/rA1M protects against stress-induced damage to tubule epithelial cells that, at least partly, can be attributed to maintaining mitochondrial function.

PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells.

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1-4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1-4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer), globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) and Forssman GSL (GalNAcα1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) revealed balanced adhesion toward the three GSLs for PapG I-mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III-harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface-exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.

Vibrodissociation method for isolation of defined nephron segments from human and rodent kidneys.

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.