Human Kidney Samples Research Articles

Obesity-related drug transporter expression alterations in human liver and kidneys.

Pathophysiological changes associated with obesity might impact various drug pharmacokinetics (PK) parameters. The liver and kidneys are the primary organs involved in drug clearance, and the function of hepatic and renal transporters is critical to efficient drug elimination (or reabsorption). Considering the impact of an increased BMI on the drug's PK is crucial in directing dosing decisions. Given the critical role of transporters in drug biodisposition, this study investigated how overweight and obesity affect the gene expression of renal and hepatic drug transporters. Human liver and kidney samples were collected post-mortem from 32 to 28 individuals, respectively, which were divided into the control group (lean subjects; 18.5 ≤ BMI < 25kg/m2) and the study group (overweight/obese subjects; BMI ≥ 25kg/m2). Real-time quantitative PCR was performed for the analysis of 84 drug transporters. Our results show significant changes in the expression of genes involved in human transporters, both renal and hepatic. In liver tissue, we found that ABCC4 was up-regulated in overweight/obese subjects. In kidney tissue, up-regulation was only observed for ABCC10, while the other differentially expressed genes were down-regulated: ABCA1, ABCC3, and SLC15A1. The observed alterations may be reflected by the differences in drug PK between lean and obese populations. However, these findings need further evaluation through the proteomic and functional study of these transporters in this patient population.

Dense Collagen I as a Biomimetic Material to Track Matrix Remodelling in Renal Carcinomas.

Aims: Renal tissue is a dynamic biophysical microenvironment, regulating healthy function and influencing tumor development. Matrix remodelling is an iterative process and aberrant tissue repair is prominent in kidney fibrosis and cancer. Biomimetic 3D models recapitulating the collagen composition and mechanical fidelity of native renal tissue were developed to investigate cell-matrix interactions in renal carcinomas. Methods: Collagen I and laminin hydrogels were engineered with renal cancer cells (ACHN and 786-O), which underwent plastic compression to generate dense matrices. Mechanical properties were determined using shear rheology and qPCR determined the gene expression of matrix markers. Results: The shear modulus and phase angle of acellular dense collagen I gels (474 Pa and 10.7) are similar to human kidney samples (1410 Pa and 10.5). After 21 days, 786-O cells softened the dense matrix (∼155 Pa), with collagen IV downregulation and upregulation of matrix metalloproteinases (MMP7 and MMP8). ACHN cells were found to be less invasive and stiffened the matrix to ∼1.25 kPa, with gene upregulation of collagen IV and the cross-linking enzyme LOX. Conclusions: Renal cancer cells remodel their biophysical environment, altering the material properties of tissue stroma in 3D models. These models can generate physiologically relevant stiffness to investigate the different matrix remodelling mechanisms utilized by cancer cells.

Automatic renal carcinoma biopsy guidance using forward-viewing endoscopic optical coherence tomography and deep learning

Percutaneous renal biopsy is commonly used for kidney cancer diagnosis. However, the biopsy procedure remains challenging in sampling accuracy. Here we introduce a forward-viewing optical coherence tomography probe for differentiating tumor and normal tissues, aiming at precise biopsy guidance. Totally, ten human kidney samples, nine of which had malignant renal carcinoma and one had benign oncocytoma, were used for system evaluation. Based on their distinct imaging features, carcinoma could be efficiently distinguished from normal renal tissues. Additionally, oncocytoma could be differentiated from carcinoma. We developed convolutional neural networks for tissue recognition. Compared to the conventional attenuation coefficient method, convolutional neural network models provided more accurate carcinoma predictions. These models reached a tissue recognition accuracy of 99.1% on a hold-out set of four kidney samples. Furthermore, they could efficiently distinguish oncocytoma from carcinoma. In conclusion, our convolutional neural network-aided endoscopic imaging platform could enhance carcinoma diagnosis during percutaneous renal biopsy procedures.

#71 Dysregulation of ITGAM and ITGB2 in the pathogenesis of diabetic kidney disease—results from bioinformatics analysis and in vitro studies

Abstract Background and Aims Activated neutrophils can release web-like chromatin structures known as neutrophil extracellular traps (NETs), but their roles in the immunopathogenesis of diabetic kidney disease (DKD) remains elusive. Method The characteristic NETs associated targets of differentially expressed genes (DEGs) from human kidney biopsy datasets of patients with DKD (GSE30528 and GSE30529) were identified by machine learning method (LASSO and SVM-RFE). The genes expression was further validated using kidney biopsy dataset of GSE142025. The most abundant single cell of the targets in DKD was further selected by single-nucleus RNA sequencing datasets (GSE131882). Predictive pathway network of targeted genes was constructed using GENEMANIA database. The expressions and potential signaling pathway of these candidate genes were validated using flow cytometry and ELISA. Results Three predicting NETs related genes (ITGAM, ITGB2 and TLR7), identified by machine learning algorithms, which all showed significant upregulation in both glomerular and tubulointerstitial compartments in human DKD kidney samples. Single-cell analysis suggested that ITGAM, ITGB2 and TLR7 were most enriched in leucocytes. DKD patients (n = 18) showed significantly higher activated neutrophils as well as increased expression of ITGAM and ITGB2 compared to healthy controls (HCs, n = 9). DKD patients also exhibited higher serum levels of IL6 but lower IL-10 than HCs (all p &amp;lt; 0.01). The significant network of IL10, IL6, ITGAM and ITGB2 was generated. This network might be associated with leukocyte activation, macrophage activation, JAK-STAT pathway, acute inflammatory response and lymphocyte immune response (all p &amp;lt; 0.05). Conclusion Aberrant ITGAM and ITGB2 expressions in activated neutrophils contribute to DKD pathogenesis and may serve as novel therapeutic targets for DKD.

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures.

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and embedding conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

Piezo2 sensory innervation during kidney development

Piezo2 is a mechanically activated ion channel expressed in a subset of sensory neurons, including in the bladder. Our lab has recently mapped innervation of the developing kidney and found that renal nerve development begins at embryonic day 13.5 (E13.5) and is tightly aligned with smooth muscle actin (SMA+) arterial development. However, the presence and spatiotemporal innervation of Piezo2+ sensory nerves in the kidney remains unknown. In the current study, we hypothesized that Piezo2+ sensory nerves would be present early in kidney development adjacent developing nephrons and that these axons innervating the kidney could be traced back to Piezo2+ cell bodies in the dorsal root ganglia (DRGS). To test this hypothesis, we performed immunofluorescent staining to label all nerves (Tubb3+, pan-neuronal marker) and Piezo2 in kidneys from wildtype mice at early (E13.5), mid (E15.5) and late (post-natal day 0, P0) nephrogenesis. To determine the signaling pathways of Piezo2+ nerves in the kidney to the peripheral nervous system (PNS), we utilized retrograde tracing with a Cre-driven fluorescently (pAAV-Flex-tdTomato) labeled virus injected into the kidney of heterozygous Piezo2Cre/+ adult mice (n=4, 10 weeks). The injected kidney, the contralateral control, and cervical to sacral DRGs were collected and fluorescent imaging was performed with confocal microscopy. We found that by E15.5, Piezo2+ sensory nerves were present in the developing kidney which constitute a small subset of all nerves (Tubb3+). These Tubb3+Piezo2+ sensory nerves persist through nephrogenesis (P0) and in the adult mouse kidney. Piezo2+ sensory nerves were found in nerve bundles running along SMA+ vasculature as well as observed adjacent glomeruli (Nphs2+) and proximal tubules (Lrp2+). Importantly, we also identified Piezo2+ sensory nerves in the renal cortex from human kidney samples. Retrograde tracing experiments revealed Piezo2+ sensory nerves in the kidney originate from lower thoracic and lumbar DRGs. Taken together, these data suggest that Piezo2+ sensory nerves are present in the mouse and human kidney and are capable of transmitting signals back to the PNS. As Piezo2 is known to be expressed in juxtaglomerular cells, Piezo2+ sensory nerves observed in close proximity suggests a previously unrecognized kidney cell-nerve crosstalk. Future studies will investigate Piezo2+ sensory activity and feedback in the kidney to understand the role of Piezo2 in renal mechanosenstion. NIH R01DK121014 to LO. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of HDAC6 with CAY10603 alleviates acute and chronic kidney injury by suppressing the ATF6 branch of UPR

BackgroundHistone deacetylase 6 (HDAC6) inhibitor CAY10603 has been identified as a potential therapeutic agent for the treatment of diabetic kidney disease (DKD). The objective of this study was to investigate the therapeutic effects of CAY10603 in mice with acute kidney injury (AKI) and chronic kidney diseases (CKD). MethodsRenal immunohistology was performed to assess the expression levels of HDAC6 in both human and mouse kidney samples. C57BL/6J mice were intraperitoneal injected with lipopolysaccharide (LPS) to induce AKI; CD-1 mice were fed with adenine diet to induce adenine-nephropathy as CKD model. Serum creatinine, blood urea nitrogen and uric acid were measured to reflect renal function; renal histology was applied to assess kidney damage. Western blot and immunohistology were used to analyze the unfolded protein response (UPR) level. ResultsHDAC6 was significantly upregulated in renal tubular epithelial cells (RTECs) of both AKI and CKD patients as well as mice. In the murine models of AKI induced by LPS and adenine-induced nephropathy, CAY10603 exhibited notable protective effects, including improvement in biochemical indices and pathological changes. In vivo and in vitro studies revealed that CAY10603 effectively suppressed the activation of activating transcription factor 6 (ATF6) branch of UPR triggered by thapsigargin (Tg), a commonly employed endoplasmic reticulum (ER) stressor. Consistent with these findings, CAY10603 also displayed substantial inhibition of ATF6 activation in RTECs from both murine models of LPS-induced AKI and adenine-induced nephropathy. ConclusionsCollectively, these results suggest that CAY10603 holds promise as a potential therapeutic agent for both acute and chronic kidney injury.

Abstract 6169: High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue

Abstract We have developed an high throughput high resolution in situ multi-omic platform to enable studies in the rapidly evolving landscape of oncology and immunology research. Despite the potential of existing tools, the speed and throughput of current methodologies pose significant limitations. Here, we introduce a novel spatial sequencing platform employing a rapid 4-color SBS chemistry with sub-micron resolution imaging and an ultra-high throughput capacity. The platform simultaneously profiles RNA transcripts and proteins within formalin-fixed paraffin-embedded (FFPE) tissues. With up to 40 cm^2 of tissue area imaging within a 24-hour timeframe, the platform can power extensive, multi-omic studies in cancer research. To evaluate the performance of the platform, we prepared 5 um serial sections from healthy and tumor kidney FFPE block and used a novel tool to precisely position multiple tissue sections on a slide. Following probe binding and amplification, we used SBS readout to profile 105 transcripts and 6 proteins (via DNA-conjugated antibodies). Images were analyzed using a custom pipeline for feature detection and decoding, and cell segmentation using Cellpose. Results showed strong correlation to single-cell transcription profiles and cell type signatures defined by scRNA-Seq. Additionally, we see good agreement between single cell transcript and protein profiles. Existing spatial biology platforms are known to struggle with degraded and challenging clinical sample types (e.g. decalcified bone marrow), due to RNA fragmentation. We successfully profiled immune cells in FFPE-embedded bone marrow biopsies, measuring 143 transcript targets and 8 proteins. We believe this represents a significant step forward for in-situ analysis of bone marrow samples, and could be valuable for studying the pathology and response to treatment in blood cancers such as AML and CML. In-situ sequencing of antibody genes and T-cell receptors provides a deeper view of cellular immune response. As a step towards this goal, we demonstrated in-situ sequencing of antibody heavy and light chains within a B cell line, resulting in accurate readout of 28 bases within the CDR3 regions. In conclusion, we present an SBS-based approach for in-situ detection of targeted gene transcripts and proteins at an unprecedented throughput, and its application to the analysis of human kidney and bone marrow FFPE tissue samples. The capability to deeply profile tissues at high throughput could enable larger translational studies, and assembly of 3D maps of gene and protein expression at sub-cellular resolution. Citation Format: Michael Lawson, Yuji Ishitsuka, Andrew Pawlowski, Zhenmin Hong, Jake Koh, Kenneth Gouin, Nathan Ing, Richard Que, Yeoan Youn, Sara Ding, Tony Facchini, Ryan Costello, Katelyn Nelson, Jamie Stover, Howon Lee, Sarthak Duggal, Taylor Plaziak, Kaitlin Cameron, Mimi Abdu, Daan Witters, Martin Fabani, Eli Glezer. High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6169.

Predicting proximal tubule failed repair drivers through regularized regression analysis of single cell multiomic sequencing

Renal proximal tubule epithelial cells have considerable intrinsic repair capacity following injury. However, a fraction of injured proximal tubule cells fails to undergo normal repair and assumes a proinflammatory and profibrotic phenotype that may promote fibrosis and chronic kidney disease. The healthy to failed repair change is marked by cell state-specific transcriptomic and epigenomic changes. Single nucleus joint RNA- and ATAC-seq sequencing offers an opportunity to study the gene regulatory networks underpinning these changes in order to identify key regulatory drivers. We develop a regularized regression approach to construct genome-wide parametric gene regulatory networks using multiomic datasets. We generate a single nucleus multiomic dataset from seven adult human kidney samples and apply our method to study drivers of a failed injury response associated with kidney disease. We demonstrate that our approach is a highly effective tool for predicting key cis- and trans-regulatory elements underpinning the healthy to failed repair transition and use it to identify NFAT5 as a driver of the maladaptive proximal tubule state.

Unraveling the epigenetic code: human kidney DNA methylation and chromatin dynamics in renal disease development

Epigenetic changes may fill a critical gap in our understanding of kidney disease development, as they not only reflect metabolic changes but are also preserved and transmitted during cell division. We conducted a genome-wide cytosine methylation analysis of 399 human kidney samples, along with single-nuclear open chromatin analysis on over 60,000 cells from 14 subjects, including controls, and diabetes and hypertension attributed chronic kidney disease (CKD) patients. We identified and validated differentially methylated positions associated with disease states, and discovered that nearly 30% of these alterations were influenced by underlying genetic variations, including variants known to be associated with kidney disease in genome-wide association studies. We also identified regions showing both methylation and open chromatin changes. These changes in methylation and open chromatin significantly associated gene expression changes, most notably those playing role in metabolism and expressed in proximal tubules. Our study further demonstrated that methylation risk scores (MRS) can improve disease state annotation and prediction of kidney disease development. Collectively, our results suggest a causal relationship between epigenetic changes and kidney disease pathogenesis, thereby providing potential pathways for the development of novel risk stratification methods.

Single-cell transcriptomics and chromatin accessibility profiling elucidate the kidney-protective mechanism of mineralocorticoid receptor antagonists.

Mineralocorticoid excess commonly leads to hypertension (HTN) and kidney disease. In our study, we used single-cell expression and chromatin accessibility tools to characterize the mineralocorticoid target genes and cell types. We demonstrated that mineralocorticoid effects were established through open chromatin and target gene expression, primarily in principal and connecting tubule cells and, to a lesser extent, in segments of the distal convoluted tubule cells. We examined the kidney-protective effects of steroidal and nonsteroidal mineralocorticoid antagonists (MRAs), as well as of amiloride, an epithelial sodium channel inhibitor, in a rat model of deoxycorticosterone acetate, unilateral nephrectomy, and high-salt consumption-induced HTN and cardiorenal damage. All antihypertensive therapies protected against cardiorenal damage. However, finerenone was particularly effective in reducing albuminuria and improving gene expression changes in podocytes and proximal tubule cells, even with an equivalent reduction in blood pressure. We noted a strong correlation between the accumulation of injured/profibrotic tubule cells expressing secreted posphoprotein 1 (Spp1), Il34, and platelet-derived growth factor subunit b (Pdgfb) and the degree of fibrosis in rat kidneys. This gene signature also showed a potential for classifying human kidney samples. Our multiomics approach provides fresh insights into the possible mechanisms underlying HTN-associated kidney disease, the target cell types, the protective effects of steroidal and nonsteroidal MRAs, and amiloride.

New insights in the renal distribution profile of TRPC3 – Of mice and men

Several reports previously investigated the Transient Receptor Potential Canonical subfamily channel 3 (TRPC3) in the kidney. However, most of the conclusions are based on animal samples or cell cultures leaving the door open for human tissue investigations. Moreover, results often disagreed among investigators. Histological description is lacking since most of these studies focused on functional aspects. Nevertheless, the same reports highlighted the potential key-role of TRPC3 in renal disorders. Hence, our interest to investigate the localization of TRPC3 in human kidneys. For this purpose, both healthy mouse and human kidney samples that were originated from tumor nephrectomies have been prepared for immunohistochemical staining using a knockout-validated antibody. A blocking peptide was used to confirm antibody specificity. A normalized weighted diaminobenzidine (DAB) area score between 0 and 3 comparable to a pixelwise H-score was established and employed for semiquantitative analysis. Altogether, our results suggest that glomeruli only express little TRPC3 compared to several segments of the tubular system. Cortical and medullary proximal tubules are stained, although intracortical differences in staining exist in mice. Intermediate tubules, however, are only weakly stained. The distal tubule was studied in three localizations and staining was marked although slightly varying throughout the different subsegments. Finally, the collecting duct was also immunolabeled in both human and mouse tissue. We therefore provide evidence that TRPC3 is expressed in various localizations of both human and mouse samples. We verify results of previous studies and propose until now undescribed localizations of TRPC3 in the mouse but especially and of greater interest in the human kidney. We thereby not only support the translational concept of the TRPC3 channel as key-player in physiology and pathophysiology of the human kidney but also present new potential targets to functional analysis.

STING/ACSL4 axis-dependent ferroptosis and inflammation promote hypertension-associated chronic kidney disease

Hypertension is a primary modifiable risk factor for cardiovascular diseases, which often induces renal end-organ damage and complicates chronic kidney disease (CKD). In the present study, histological analysis of human kidney samples revealed that hypertension induced mtDNA leakage and promoted the expression of stimulator of interferon genes (STING) in renal epithelial cells. We used angiotensin II (AngII)- and 2K1C-treated mouse kidneys to elucidate the underlying mechanisms. Abnormal renal mtDNA packing caused by AngII promoted STING-dependent production of inflammatory cytokines, macrophage infiltration, and a fibrogenic response. STING knockout significantly decreased nuclear factor-κB activation and immune cell infiltration, attenuating tubule atrophy andextracellular matrix accumulation invivo and invitro. These effects delayed CKD progression. Immunoprecipitationassays and liquid chromatography-tandem mass spectrometry showed that STING and ACSL4 were directly combined at the D53 and K412 amino acids of ACSL4. Furthermore, STING induced renal inflammatory response and fibrosis through ACSL4-dependent ferroptosis. Last, inhibition of ACSL4 using small interfering RNA, rosiglitazone, or Fer-1 downregulated AngII-induced mtDNA-STING-dependent renal inflammation. These results suggest that targeting the STING/ACSL4 axis might represent a potential strategy for treating hypertension-associated CKD.

#6111 FIRST IDENTIFICATION AND CHARACTERIZATION OF MICROPLASTICS IN HUMAN KIDNEY AND URINE

Abstract Background and Aims Microplastics (MPs), plastic fragments less than 5mm in diameter, have been recognized as a global environmental issue. Various studies have shown their ubiquitous presence and their toxicity on marine and terrestrial fauna. Currently in humans, the presence of MP has been evidenced in lungs, placenta, blood, and liver. However, there is still very little knowledge about their presence in different organs and tissues and their prospective toxicity. The objective of this study was to investigate the presence of microplastics in human kidneys and urine. Method We obtained 10 urine samples from healthy individuals and 10 kidney healthy tissue samples from nephrectomies in renal cancer The detection and characterization of microplastics was performed by a light microscope (Leica, total magnification of 500x) coupled to a Raman spectrometer (Renishaw System 2000). To this purpose, specimens were digested by a 10% KOH solution at 60°C and subsequently filtered on membrane filters with micropores of 0.2 μm to retain possible particles. This procedure was performed adopting a “plastic-free” protocol. Procedural blanks were performed as controls using the same protocol. To determine the nature of the detected particles, Raman spectra were compared to the ones present in the Renishaw spectrometer database (inorganic materials, polymers and forensic materials) [i], in the SLOPP Library of Microplastics [ii], and in the IRUG Spectral Database (pigments section) [iii]. A home-developed software was employed to compare spectra. Data analysis was performed by using the statistical software package Prism6 (Graphpad Software) Chi-square test, Student's t-test were performed to compare data accordingly. The significance threshold was set at p &amp;lt; 0.05. Results 17 fragments (mean 1,7±2,11/sample) were identified and characterized on 7 out of 10 human kidney samples. The result was significant (p.value 0.041) compared with controls (mean 0,33±0,49/blank). 9 fragments were identified and characterized on 7 out of 10 human urine sample (mean 1.28±0,49/sample) highly significant (p.value 0.0002). Spectra analyzed by microRaman showed the presence polymers, polymers additives and pigments associated to polymers such as: hematite, Cu- phthalocyanine blue, Cerulean Blue, Polystyrene, Styrene-Isoprene and polyethylene. Conclusion We first demonstrated the presence of MPs in human kidneys, and we also confirm their presence in urine assuming the presence of a kidney clearance mechanism. Using Raman Microspectroscopy, it was also possible to determine the nature and quantity of MPs. The remarkable relevance of this identification, potentially concern much of the humans, deserve a widespread attention of the medical community for its potential implications. Further studies are urgently needed to investigate the possible nephrotoxicity of MPs, mechanisms of kidney clearance and tissue accumulation.

Meta-analysis of single-cell and single-nucleus transcriptomics reveals kidney cell type consensus signatures

While the amount of studies involving single-cell or single-nucleus RNA-sequencing technologies grows exponentially within the biomedical research area, the kidney field requires reference transcriptomic signatures to allocate each cluster its matching cell type. The present meta-analysis of 39 previously published datasets, from 7 independent studies, involving healthy human adult kidney samples, offers a set of 24 distinct consensus kidney cell type signatures. The use of these signatures may help to assure the reliability of cell type identification in future studies involving single-cell and single-nucleus transcriptomics while improving the reproducibility in cell type allocation.

Single cell G-protein coupled receptor profiling of activated kidney fibroblasts expressing transcription factor 21.

Activated fibroblasts deposit fibrotic matrix in chronic kidney disease (CKD) and G-protein coupled receptors (GPCRs) are the most druggable therapeutic targets. Here, we set out to establish a transcriptional profile that identifies activated kidney fibroblasts and the GPCRs that they express. RNA sequencing and single cell qRT-PCR were performed on mouse kidneys after unilateral ureteral obstruction (UUO). Candidate expression was evaluated in mice with UUO or diabetes or injected with adriamycin or folic acid. Intervention studies were conducted in mice with diabetes or UUO. Correlative histology was performed in human kidney tissue. Transcription factor 21 (Tcf21)+ cells that expressed 2 or 3 of Postn, Acta2 and Pdgfra were highly enriched for fibrogenic genes and were defined as activated kidney fibroblasts. Tcf21+ α-smooth muscle actin (α-SMA)+ interstitial cells accumulated in kidneys of mice with UUO or diabetes or injected with adriamycin or folic acid, whereas renin-angiotensin system blockade attenuated increases in Tcf21 in diabetic mice. Fifty-six GPCRs were up-regulated in single Tcf21+ kidney fibroblasts, the most up-regulated being Adgra2 and S1pr3. Adenosine receptors, Adora2a/2b, were up-regulated in Tcf21+ fibroblasts and the adenosine receptor antagonist, caffeine decreased Tcf21 upregulation and kidney fibrosis in UUO mice. TCF21, ADGRA2, S1PR3 and ADORA2A/2B were each detectable in α-SMA+ interstitial cells in human kidney samples. Tcf21 is a marker of kidney fibroblasts that are enriched for fibrogenic genes in CKD. Further analysis of the GPCRs expressed by these cells may identify new targets for treating CKD.

Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process.

Introduction: The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remain to be elucidated. Methods: Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm, and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Results and Discussion: Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed 'cell junctions,' 'actin binding,' and 'cytoskeleton organization' as top terms. Known foot process components were identified, and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed that Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and support the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.

Unbiased Human Kidney Tissue Proteomics Identifies Matrix Metalloproteinase 7 as a Kidney Disease Biomarker.

Although gene expression changes have been characterized in human diabetic kidney disease (DKD), unbiased tissue proteomics information for this condition is lacking. The authors conducted an unbiased aptamer-based proteomic analysis of samples from patients with DKD and healthy controls, identifying proteins with levels that associate with kidney function (eGFR) or fibrosis, after adjusting for key covariates. Overall, tissue gene expression only modestly correlated with tissue protein levels. Kidney protein and RNA levels of matrix metalloproteinase 7 (MMP7) strongly correlated with fibrosis and with eGFR. Single-cell RNA sequencing indicated that kidney tubule cells are an important source of MMP7. Furthermore, plasma MMP7 levels predicted future kidney function decline. These findings identify kidney tissue MMP7 as a biomarker of fibrosis and blood MMP7 as a biomarker for future kidney function decline. Diabetic kidney disease (DKD) is responsible for close to half of all ESKD cases. Although unbiased gene expression changes have been extensively characterized in human kidney tissue samples, unbiased protein-level information is not available. We collected human kidney samples from 23 individuals with DKD and ten healthy controls, gathered associated clinical and demographics information, and implemented histologic analysis. We performed unbiased proteomics using the SomaScan platform and quantified the level of 1305 proteins and analyzed gene expression levels by bulk RNA and single-cell RNA sequencing (scRNA-seq). We validated protein levels in a separate cohort of kidney tissue samples as well as in 11,030 blood samples. Globally, human kidney transcript and protein levels showed only modest correlation. Our analysis identified 14 proteins with kidney tissue levels that correlated with eGFR and found that the levels of 152 proteins correlated with interstitial fibrosis. Of the identified proteins, matrix metalloprotease 7 (MMP7) showed the strongest association with both fibrosis and eGFR. The correlation between tissue MMP7 protein expression and kidney function was validated in external datasets. The levels of MMP7 RNA correlated with fibrosis in the primary and validation datasets. Findings from scRNA-seq pointed to proximal tubules, connecting tubules, and principal cells as likely cellular sources of increased tissue MMP7 expression. Furthermore, plasma MMP7 levels correlated not only with kidney function but also associated with prospective kidney function decline. Our findings, which underscore the value of human kidney tissue proteomics analysis, identify kidney tissue MMP7 as a diagnostic marker of kidney fibrosis and blood MMP7 as a biomarker for future kidney function decline.

PD17-06 WILMS TUMOR: INSIGHTS INTO THE ROLE OF INTEGRINS DRIVING CANCER STEM CELLS SELF-RENEWAL VS. DIFFERENTIATION

You have accessJournal of UrologyCME1 Apr 2023PD17-06 WILMS TUMOR: INSIGHTS INTO THE ROLE OF INTEGRINS DRIVING CANCER STEM CELLS SELF-RENEWAL VS. DIFFERENTIATION Astgik Petrosyan, Valentina Villani, Paola Aguiari, Min Madhi, Roger De Filippo, Gregory Shackleford, Anat Erdreich-Epstein, Stefano Da Sacco, and Laura Perin Astgik PetrosyanAstgik Petrosyan More articles by this author , Valentina VillaniValentina Villani More articles by this author , Paola AguiariPaola Aguiari More articles by this author , Min MadhiMin Madhi More articles by this author , Roger De FilippoRoger De Filippo More articles by this author , Gregory ShacklefordGregory Shackleford More articles by this author , Anat Erdreich-EpsteinAnat Erdreich-Epstein More articles by this author , Stefano Da SaccoStefano Da Sacco More articles by this author , and Laura PerinLaura Perin More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Wilms Tumor (WT) is the most common pediatric renal cancer. Growing evidence links WT to aberrant nephrogenesis. While studies highlighted the genetic complexity of WT, little is known about the molecular mechanisms that regulate WT development. Here we report that uncommitted nephrogenic progenitors (NPs) expressing SIX2 and CITED1 (the master regulators of renal development) present characteristics of cancer stem cells (CSCs) and are the ones driving WT. We have also studied the role of integrins in these NPs in regulating WT development. METHODS: WT and human fetal kidney (hFK) samples were histologically analyzed, digested to single-cell suspension, incubated with Smartflare-probe to isolate SIX2+CITED1+ cells, and processed for RNA-seq, single-cell RNA-seq and spatial transcriptomics. Xenografts of WT-NPs and hFK-NPs were generated and tumor formation was assessed. Analyses of mechanisms that regulate self-renewal vs. differentiation were performed in vitro and in vivo. Knockdown with miREs against SIX2 and CITED1 was performed on WT-NPs and processed for RNA-seq. RESULTS: By comparing NPs from different WT subtypes and NPs from hFK we identified that cells expressing SIX2 and CITED1 fulfill CSC criteria, reliably recapitulating WT in transplantation studies. We showed that self-renewal vs. differentiation of SIX2+CITED1+ WT CSCs is regulated by the interplay between integrins ITGB1 and ITGB4. WT transplantation studies show that blocking ITGB1 or ITGB4 leads to higher number of SIX2+CITED1+ cells in the xenografts. Knockdown of SIX2 and CITED1 increased expression of kidney differentiation markers LHX1, WNT7B, PODXL, MECOM, reduced expression of nephrogenic markers MEOX1, TMEM100, EYA1, MAYFB, and increased expression of ITGB1, ITGB4, and LAMA5. CONCLUSIONS: These studies define SIX2+CITED1+ cells as the nephrogenic CSCs of WT, where ITGB1 and ITGB4 interplay may play a role in self-renewal vs. differentiation and serve as a potential target for new strategies to treat WT. Source of Funding: GOFARR Fund © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e497 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Astgik Petrosyan More articles by this author Valentina Villani More articles by this author Paola Aguiari More articles by this author Min Madhi More articles by this author Roger De Filippo More articles by this author Gregory Shackleford More articles by this author Anat Erdreich-Epstein More articles by this author Stefano Da Sacco More articles by this author Laura Perin More articles by this author Expand All Advertisement PDF downloadLoading ...

Treatment effects of soluble guanylate cyclase modulation on diabetic kidney disease at single-cell resolution

Diabetic kidney disease (DKD) is the most common cause of renal failure. Therapeutics development is hampered by our incomplete understanding of animal models on a cellular level. We show that ZSF1 rats recapitulate human DKD on a phenotypic and transcriptomic level. Tensor decomposition prioritizes proximal tubule (PT) and stroma as phenotype-relevant cell types exhibiting a continuous lineage relationship. As DKD features endothelial dysfunction, oxidative stress, and nitric oxide depletion, soluble guanylate cyclase (sGC) is a promising DKD drug target. sGC expression is specifically enriched in PT and stroma. In ZSF1 rats, pharmacological sGC activation confers considerable benefits over stimulation and is mechanistically related to improved oxidative stress regulation, resulting in enhanced downstream cGMP effects. Finally, we define sGC gene co-expression modules, which allow stratification of human kidney samples by DKD prevalence and disease-relevant measures such as kidney function, proteinuria, and fibrosis, underscoring the relevance of the sGC pathway to patients.