Proximal Tubule Cells Research Articles

Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles.

Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Donor-specific urinary extracellular vesicles (uEVs) hold potential as biomarkers for assessing allograft status. We aimed to develop a method for identifying donor-specific uEVs based on human leukocyte antigen (HLA) mismatching with the kidney transplant recipients (KTRs). Urine and plasma were obtained from HLA-A2+ donors and HLA-A2- KTRs pre-transplant. CD9 (tetraspanin, EV marker) and HLA-A2 double-positive (CD9+ HLA-A2+) EVs were quantified using isolation-free imaging flow cytometry (IFCM). Healthy individuals' urine was used to investigate CD9+ HLA-class-I+ uEV quantification using IFCM, time-resolved fluoroimmunoassay (TR-FIA), and immunogold staining cryo-electron microscopy (cryo-EM). Culture-derived CD9+ HLA-class-I+ EVs were spiked into the urine to investigate urine matrix effects on uEV HLA detection. Deceased donor kidneys and peritumoral kidney tissue were used for HLA class I detection with histochemistry. The concentrations of CD9+ HLA-A2+ EVs in both donor and recipient urine approached the negative (detergent-treated) control levels for IFCM and were significantly lower than those observed in donor plasma. In parallel, universal HLA class I+ uEVs were similarly undetectable in the urine and uEV isolates compared with plasma, as verified by IFCM, TR-FIA, and cryogenic electron microscopy. Culture supernatant containing HLA class I+ vesicles from B, T, and human proximal tubule cells were spiked into the urine, and these EVs remained stable at 37°C for 8 hours. Immunohistochemistry revealed that HLA class I was predominantly expressed on the basolateral side of renal tubules, with limited expression on their urine/apical side. The detection of donor-specific uEVs is hindered by the limited release of HLA class I+ EVs from the kidney into the urine, primarily due to the polarized HLA class I expression on renal tubules. Identifying donor-specific uEVs requires further advancements in recognizing transplant-specific uEVs and urine-associated markers.

Ethanol extract of galangal (Alpinia Galanga) administration on degeneration, necrosis, and inflammatory cells in the proximal tubules of male mice (Mus musculus) exposed to lead acetate

Introduction: This study aimed to investigate the preventive effect of the ethanolic extract of galangal (Alpinia galanga) on degeneration, necrosis, and inflammatory cells in the kidney proximal tubules of male mice (Mus musculus) exposed to lead acetate. Objective: This was a pure experimental laboratory study using a completely randomized approach/randomized posttest only control group design. Twenty-five male mice (Mus musculus) were divided into five treatment groups: negative control (K-) received 0.5% CMC Na and aquades, positive control (K+) received induction of 20 mg/kgBW lead acetate for 20 days, and P1, P2, and P3 groups received oral administration of galangal ethanol extract from day 1 to day 24 as preventive measures with successive doses of (P1) 200 mg/kgBW, (P2) 400 mg/kgBW, and (P3) 800 mg/kgBW, respectively. Lead acetate administration at a dose of 20 mg/kgBW started from day 4 to day 24. On the 25th day, all groups were sacrificed, and kidney tissues were harvested for histopathological examination using the scoring method. Histopathological data analysis was performed using the Kruskal-Wallis test followed by Mann-Whitney U test to compare differences between groups. Results: The Mann-Whitney U test showed a significant increase in degeneration, necrosis, and infiltration of inflammatory cells in the kidney proximal tubules of male mice (Mus musculus) exposed to lead acetate (p < 0.05). Administration of galangal ethanol extract significantly reduced degeneration, necrosis, and infiltration of inflammatory cells (p < 0.05). The highest dose of galangal ethanol extract (800 mg/kg BW) exhibited a significant decrease in degeneration, necrosis, and infiltration of inflammatory cells (p < 0.05), comparable to the normal condition (K-). Conclusion: Administration of ethanol extract of galangal (Alpinia galanga) effectively prevented lead acetate-induced degeneration, necrosis, and infiltration of inflammatory cells in the kidney proximal tubules of male mice (Mus musculus).

The mmu_circ_003062, hsa_circ_0075663/miR-490-3p/CACNA1H axis mediates apoptosis in renal tubular cells in association with endoplasmic reticulum stress following ischemic acute kidney injury

BackgroundWhile recent studies have suggested a potential involvement of circRNAs in acute kidney injury (AKI) after ischemia, mmu_circ_003062 role is undetermined. MethodsThe levels of mmu_circ_003062, miR-490-3p, CACNA1H, GRP78, CHOP and hsa_circ_0075663 were detected by Relative qPCR in Boston University mouse proximal tubule (BUMPT) cells, mouse kidneys, and human renal tubular epithelial (HK-2) cells. Moreover, the levels of hsa_circ_0075663 in serum and urine of patients with AKI following cardiopulmonary resuscitation (CPR) were detected by absolute quantitative PCR. Western blot was used to detect the relative expression of the protein. The function and regulatory mechanism of mmu_circ_003062 and hsa_circ_0075663 were investigated through a series of in vitro and in vivo experiments, including bioinformatic prediction, luciferase reporter assays, FISH, FCM, TUNEL staining, and H&E staining. ResultsIt was found that mmu_circ_003062, hsa_circ_0075663 mediated apoptosis after ischemia/reperfusion (I/R) by interaction with miR-490-3p to enhance CACNA1H expression, thereby leading to the upregulation of endoplasmic reticulum stress (ERS)-relevant proteins GRP78 and CHOP. Ultimately, mmu_circ_003062 downregulation significantly ameliorated ischemic AKI by modulating the miR-490-3p/CACNA1H/GRP78 and CHOP pathway. Furthermore, the plasma and urinary levels of hsa_circ_0075663 in patients with AKI following CPR were significantly higher than non-AKI patients, exhibited a strongly correlation with serum creatinine. ConclusionThe involvement of mmu_circ_003062, hsa_circ_0075663/miR-490-3p/CACNA1H/GRP78 and CHOP axis is significant in the development of ischemic AKI. Moreover, hsa_circ_0075663 has potential as an early diagnostic biomarker.

Protective Effect of Curcumin on D-Galactose-Induced Senescence and Oxidative Stress in LLC-PK1 and HK-2 Cells.

D-galactose has been widely used as an inducer of cellular senescence and pathophysiological processes related to aging because it induces oxidative stress. On the other hand, the consumption of antioxidants such as curcumin can be an effective strategy to prevent phenotypes related to the enhanced production of reactive oxygen species (ROS), such as aging and senescence. This study aimed to evaluate the potential protective effect of curcumin on senescence and oxidative stress and endoplasmic reticulum stress induced by D-galactose treatment in Lilly Laboratories Culture-Porcine Kidney 1 (LLC-PK1) and human kidney 2 (HK-2) proximal tubule cell lines from pig and human, respectively. For senescence induction, cells were treated with 300 mM D-galactose for 120 h and, to evaluate the protective effect of the antioxidant, cells were treated with 5 µM curcumin for 24 h and subsequently treated with curcumin + D-galactose for 120 h. In LLC-PK1 cells, curcumin treatment decreased by 20% the number of cells positive for senescence-associated (SA)-β-D-galactosidase staining and by 25% the expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and increased by 40% lamin B1 expression. In HK-2 cells, curcumin treatment increased by 60% the expression of proliferating cell nuclear antigen (PCNA, 50% Klotho levels, and 175% catalase activity. In both cell lines, this antioxidant decreased the production of ROS (20% decrease for LLC-PK1 and 10 to 20% for HK-2). These data suggest that curcumin treatment has a moderate protective effect on D-galactose-induced senescence in LLC-PK1 and HK-2 cells.

Optimization of mouse kidney digestion protocols for single-cell applications.

Single-cell technologies such as flow cytometry and single-cell RNA sequencing have allowed for comprehensive characterization of the kidney cellulome. However, there is a disparity in the various protocols for preparing kidney single-cell suspensions. We aimed to address this limitation by characterizing kidney cellular heterogeneity using three previously published single-cell preparation protocols. Single-cell suspensions were prepared from male and female C57BL/6 kidneys using the following kidney tissue dissociation protocols: a scRNAseq protocol (P1), a multi-tissue digestion kit from Miltenyi Biotec (P2), and a protocol established in our laboratory (P3). Following dissociation, flow cytometry was used to identify known major cell types including leukocytes (myeloid and lymphoid), vascular cells (smooth muscle and endothelial), nephron epithelial cells (intercalating, principal, proximal, and distal tubule cells), podocytes, and fibroblasts. Of the protocols tested, P2 yielded significantly less leukocytes and type B intercalating cells compared with the other techniques. P1 and P3 produced similar yields for most cell types; however, endothelial and myeloid-derived cells were significantly enriched using P1. Significant sex differences were detected in only two cell types: granulocytes (increased in males) and smooth muscle cells (increased in females). Future single-cell studies that aim to enrich specific kidney cell types may benefit from this comparative analysis.NEW & NOTEWORTHY This study is the first to evaluate published single-cell suspension preparation protocols and their ability to produce high-quality cellular yields from the mouse kidney. Three single-cell digestion protocols were compared and each produced significant differences in kidney cellular heterogeneity. These findings highlight the importance of the digestion protocol when using single-cell technologies. This study may help future single-cell science research by guiding researchers to choose protocols that enrich certain cell types of interest.

Knock-out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para- and transcellular solute transport.

Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase-2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. A knockout of the CN2 gene (CNDP2-KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA-seq analysis, paracellular permeability analysis and ion transport. CNDP2-KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA-seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2-KO cell viability and proliferation, and altered ion and macromolecule transport via trans- and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve invivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis.

Abstract 6944: Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer [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 6944.

Abstract 3500: Refining diagnosis of renal cell carcinoma subtypes through single-cell resolution transcriptomic signatures

Abstract A remaining challenge in the field of renal cell carcinoma (RCC) is diagnosis of rare or mixed subtypes, which hampers the delivery of the most effective healthcare to patients. While a new classification scheme, integrating molecular characteristics of tumors, is emerging, it requires a deep understanding of the tumor heterogeneity and etiopathogenesis of each subtype. We have generated and analyzed single-cell transcriptome profiles of 38 human RCCs and normal kidneys 16 RCCs with diverse histological subtypes and established gene signatures specific to each cell type. We have applied these gene signatures to bulk RNA-sequencing data of 1000 RCC tumors with different histological subtypes and normal kidneys from The Cancer Genome Atlas (TCGA) to characterize different cancer types and cellular origins. Unsupervised clustering analysis of TCGA data shows diagnostic discrepancies for 9.3% cases and subclassifies each major subtype (clear cell, papillary and chromophobe) into clusters with differential clinical outcomes.[KM1]. The pathological marker expression in misdiagnosed tumors is consistent with clustering-based classification, not their original diagnosis. Notably, using unique and subtype-specific gene signature patterns, we identified clear cell papillary renal cell tumor (ccpRCT), which is a newly classified benign entity and the main tumor type representing the mis-diagnosed tumors. These observations have further been confirmed by reviewing histological images of the tumors by an experienced genitourinary pathologist. Interestingly, our gene signatures reveal that ccpRCT does not originate from proximal tubule cells unlike clear cell and papillary RCCs. Taken together, our approach suggests a potential new classification method connected to clinical behaviors and drug responses based on overall transcriptomic differences. Citation Format: Minjun Kim, Ariel Madrigal, Zohreh Mehrjoo, Kate I. Glennon, Madeleine Arseneault, Morag Park, Fadi Brimo, Simon Tanguay, Hamed S. Najafabadi, Yasser Riazalhosseini. Refining diagnosis of renal cell carcinoma subtypes through single-cell resolution transcriptomic signatures [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 3500.

Transcriptomic, epigenomic, and spatial metabolomic cell profiling redefines regional human kidney anatomy

A large-scale multimodal atlas that includes major kidney regions is lacking. Here, we employed simultaneous high-throughput single-cell ATAC/RNA sequencing (SHARE-seq) and spatially resolved metabolomics to profile 54 human samples from distinct kidney anatomical regions. We generated transcriptomes of 446,267 cells and chromatin accessibility profiles of 401,875 cells and developed a package to analyze 408,218 spatially resolved metabolomes. We find that the same cell type, including thin limb, thick ascending limb loop of Henle and principal cells, display distinct transcriptomic, chromatin accessibility, and metabolomic signatures, depending on anatomic location. Surveying metabolism-associated gene profiles revealed non-overlapping metabolic signatures between nephron segments and dysregulated lipid metabolism in diseased proximal tubule (PT) cells. Integrating multimodal omics with clinical data identified PLEKHA1 as a disease marker, and its invitro knockdown increased gene expression in PT differentiation, suggesting possible pathogenic roles. This study highlights previously underrepresented cellular heterogeneity underlying the human kidney anatomy.

TNIK depletion induces inflammation and apoptosis in injured renal proximal tubule epithelial cells.

In the aftermath of acute kidney injury (AKI), surviving proximal tubule epithelia repopulate injured tubules to promote repair. However, a portion of cells fail to repair [termed failed-repair proximal tubule cells (FR-PTCs)] and exert ongoing proinflammatory and profibrotic effects. To better understand the molecular drivers of the FR-PTC state, we reanalyzed a mouse ischemia-reperfusion injury single-nucleus RNA-sequencing (snRNA-seq) atlas to identify Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in FR-PTCs but not in healthy or acutely injured proximal tubules after AKI (2 and 6 wk) in mice. We confirmed expression of Tnik protein in injured mouse and human tissues by immunofluorescence. Then, to determine the functional role of Tnik in FR-PTCs, we depleted TNIK with siRNA in two human renal proximal tubule epithelial cell lines (primary and immortalized hRPTECs) and analyzed each by bulk RNA-sequencing. Pathway analysis revealed significant upregulation of inflammatory signaling pathways, whereas pathways associated with differentiated proximal tubules such as organic acid transport were significantly downregulated. TNIK gene knockdown drove reduced cell viability and increased apoptosis, including differentially expressed poly(ADP-ribose) polymerase (PARP) family members, cleaved PARP-1 fragments, and increased annexin V binding to phosphatidylserine. Together, these results indicate that Tnik upregulation in FR-PTCs acts in a compensatory fashion to suppress inflammation and promote proximal tubule epithelial cell survival after injury. Modulating TNIK activity may represent a prorepair therapeutic strategy after AKI.NEW & NOTEWORTHY The molecular drivers of successful and failed repair in the proximal tubule after acute kidney injury (AKI) are incompletely understood. We identified Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in failed-repair proximal tubule cells after AKI. We tested the effect of siTNIK depletion in two proximal tubule cell lines followed by bulk RNA-sequencing analysis. Our results indicate that TNIK acts to suppress inflammatory signaling and apoptosis in injured renal proximal tubule epithelial cells to promote cell survival.

Distribution of the SLC2A9 genotype and its association with the uric acid level in patients with arterial hypertension and atrial fibrillation

Asymptomatic hyperuricemia (HU) is widespread in the population and is one of the main risk factors for cardiovascular diseases. Evidence of numerous studies supports the association between increased uric acid (UA) levels and the risk of arterial hypertension (HTN) and/or atrial fibrillation (AF). One of the causes for an increase in UA levels and a significant risk factor for HU is the polymorphism of the SLC2A9 gene, a highly specific urate transporter in proximal renal tubule cells, encoding the GLUT9 protein.The aim of the study is to investigate the frequency of genotypes and alleles of the SLC2A9 gene rs734553 polymorphic variant and its association with the UA level in patients with HTN and AF, as well as in healthy individuals. 141 patients, including 50 healthy patients (group 1), 68 – with HTN and AF (group 2), 23 – with HTN (group 3) were examined.In the overall group of patients, the frequencies of the SLC2A9 gene rs734553 polymorphism were as follows: AA – 46.1 % (n = 65), AC – 36.2 % (n = 51), CC – 17.7 % (n = 25). According to the genotyping results of patients, in group 1 the AA genotype was 64 % (n = 32), AC – 22 % (n = 11), CC – 14 % (n = 7); in group 2 – 32.4 % (n = 22), 48.5 % (n = 33) and 19.1 % (n = 13); in group 3 47.8 % (n = 11), 30.4 % (n = 7) and 21.7 % (n = 5), respectively. The CC genotype patients (420 [413; 424] μmol/l) and the AC genotype patients (330 [284; 412] μmol/l) had higher uricemia values than those with the AA genotype (310 [281; 341] μmol/l) ( p = 0,003). HU with the AA genotype in group 2 was diagnosed in 2 (2.9 %) patients, with the AC genotype in 12 (17.6 %), and with the CC genotype in 10 (14.7 %) patients; in group 3 – 1 (4.3 %), 1 (4.3 %) and 3 (13 %), respectively. One patient (2 %) with HU in group 1 had the CC genotype.The distribution of genotype and allele frequencies of the SLC2A9 gene rs734553 polymorphic variant and its association with the UA level in patients with HTN and AF, as well as in healthy individuals were established. In patients with HTN and AF, the AC genotype occurred 3 times more often and the CC genotype occurred 2 times more often than in healthy patients. HU occurred significantly more often ( р = 0.003) in the group of patients with HTN and AF and the CC genotype, as well as in patients with the CC genotype.

Crosstalk with renal proximal tubule cells drives acidosis-induced inflammatory response and dedifferentiation of fibroblasts via p38-singaling

BackgroundTubulointerstitial kidney disease associated microenvironmental dysregulation, like acidification, inflammation and fibrosis, affects tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Cell–cell communication in turn modulates the interstitial microenvironment. We assessed the impact of acidosis on inflammatory and fibrotic responses in proximal tubule cells and fibroblasts as a function of cellular crosstalk. Furthermore, cellular signaling pathways involved were identified.MethodsHK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression of inflammation markers (TNF, TGF-ß and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin and vimentin), fibrosis markers (collagen III and fibronectin) and phospho- as well as total MAPK levels were determined by western blot. Secreted collagen III and fibronectin were measured by ELISA. The impact of MAPK activation was assessed by pharmacological intervention. In addition, necrosis, apoptosis and epithelial permeability were determined.ResultsIndependent of culture conditions, acidosis caused a decrease of COX-2, vimentin and fibronectin expression in proximal tubule cells. Only in monoculture, ß-Catenin expression decreased and collagen III expression increased in tubule cells during acidosis. By contrast, in coculture collagen III protein expression of tubule cells was reduced. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin protein expression and a decrease of TGF-ß expression exclusively in coculture. In monoculture, expression of COX-2 and fibronectin was reduced. Collagen III expression of fibroblasts was reduced by acidosis independent of culture conditions.In coculture, acidosis enhanced phosphorylation of ERK1/2, JNK1/2 and p38 transiently in proximal tubule cells. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. ERK1/2 and JNK1/2 were not affected in fibroblasts. Inhibition of JNK1/2 and p38 under coculture conditions reduced acidosis-induced changes in fibroblasts significantly.ConclusionsOur data show that the crosstalk between proximal tubule cells and fibroblasts is crucial for acidosis-induced dedifferentiation of fibroblasts into an inflammatory phenotype. This dedifferentiation is at least in part mediated by p38 and JNK1/2. Thus, cell–cell communication is essential for the pathophysiological impact of tubulointerstitial acidosis.

Multiomics Analyses Identify AKR1A1 as a Biomarker for Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. As many genes associate with DKD, multi-omics approaches were employed to narrow the list of functional genes, gene products and related pathways providing insights into the pathophysiological mechanisms of DKD. The Kidney Precision Medicine Project human kidney single-cell RNA-sequencing (scRNAseq) dataset and Mendeley Data on human kidney cortex biopsy proteomics were utilized. R package Seurat was used to analyze scRNAseq and subset proximal tubule cells. PathfindR was applied for pathway analysis in cell type-specific differentially expressed genes and R limma package was used to analyze differential protein expression in kidney cortex. A total of 790 differentially expressed genes were identified in proximal tubule cells, including 530 upregulated and 260 downregulated transcripts. Compared with differentially expressed proteins, 24 genes/proteins were in common. An integrated analysis combining protein quantitative trait loci (pQTL), GWAS hits (estimated glomerular filtration rate) and a plasma metabolomics analysis was performed using baseline metabolites predictive of DKD progression in our longitudinal Diabetes Heart Study samples. Aldo-keto reductase family 1 member A1 gene (AKR1A1) was revealed as a potential molecular hub for DKD cellular dysfunction in several cross-linked pathways featured by deficiency of this enzyme.

A transfer learning framework to elucidate the clinical relevance of altered proximal tubule cell states in kidney disease

The application of single-cell technologies in clinical nephrology remains elusive. We generated an atlas of transcriptionally defined cell types and cell states of human kidney disease by integrating single-cell signatures reported in the literature with newly generated signatures obtained from 5 patients with acute kidney injury. We used this information to develop kidney-specific cell-level information ExtractoR (K-CLIER), a transfer learning approach specifically tailored to evaluate the role of cell types/states on bulk RNAseq data. We validated the K-CLIER as a reliable computational framework to obtain a dimensionality reduction and to link clinical data with single-cell signatures. By applying K-CLIER on cohorts of patients with different kidney diseases, we identified the most relevant cell types associated with fibrosis and disease progression. This analysis highlighted the central role of altered proximal tubule cells in chronic kidney disease. Our study introduces a new strategy to exploit the power of single-cell technologies toward clinical applications.

High resolution spatial profiling of kidney injury and repair using RNA hybridization-based in situ sequencing

Emerging spatially resolved transcriptomics technologies allow for the measurement of gene expression in situ at cellular resolution. We apply direct RNA hybridization-based in situ sequencing (dRNA HybISS, Cartana part of 10xGenomics) to compare male and female healthy mouse kidneys and the male kidney injury and repair timecourse. A pre-selected panel of 200 genes is used to identify cell state dynamics patterns during injury and repair. We develop a new computational pipeline, CellScopes, for the rapid analysis, multi-omic integration and visualization of spatially resolved transcriptomic datasets. The resulting dataset allows us to resolve 13 kidney cell types within distinct kidney niches, dynamic alterations in cell state over the course of injury and repair and cell-cell interactions between leukocytes and kidney parenchyma. At late timepoints after injury, C3+ leukocytes are enriched near pro-inflammatory, failed-repair proximal tubule cells. Integration of snRNA-seq dataset from the same injury and repair samples also allows us to impute the spatial localization of genes not directly measured by dRNA HybISS.

Sensing Dying Cells in Health and Disease: The Importance of Kidney Injury Molecule-1.

Kidney injury molecule-1 (KIM-1), also known as T-cell Ig and mucin domain-1 (TIM-1), is a widely recognized biomarker for AKI, but its biological function is less appreciated. KIM-1/TIM-1 belongs to the T-cell Ig and mucin domain family of conserved transmembrane proteins, which bear the characteristic six-cysteine Ig-like variable domain. The latter enables binding of KIM-1/TIM-1 to its natural ligand, phosphatidylserine, expressed on the surface of apoptotic cells and necrotic cells. KIM-1/TIM-1 is expressed in a variety of tissues and plays fundamental roles in regulating sterile inflammation and adaptive immune responses. In the kidney, KIM-1 is upregulated on injured renal proximal tubule cells, which transforms them into phagocytes for clearance of dying cells and helps to dampen sterile inflammation. TIM-1, expressed in T cells, B cells, and natural killer T cells, is essential for cell activation and immune regulatory functions in the host. Functional polymorphisms in the gene for KIM-1/TIM-1, HAVCR1 , have been associated with susceptibility to immunoinflammatory conditions and hepatitis A virus-induced liver failure, which is thought to be due to a differential ability of KIM-1/TIM-1 variants to bind phosphatidylserine. This review will summarize the role of KIM-1/TIM-1 in health and disease and its potential clinical applications as a biomarker and therapeutic target in humans.

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.

Retinoic acid receptor α activity in proximal tubules prevents kidney injury and fibrosis

Chronic kidney disease (CKD) is characterized by a gradual loss of kidney function and affects ~13.4% of the global population. Progressive tubulointerstitial fibrosis, driven in part by proximal tubule (PT) damage, is a hallmark of late stages of CKD and contributes to the development of kidney failure, for which there are limited treatment options. Normal kidney development requires signaling by vitamin A (retinol), which is metabolized to retinoic acid (RA), an endogenous agonist for the RA receptors (RARα, β, γ). RARα levels are decreased in a mouse model of diabetic nephropathy and restored with RA administration; additionally, RA treatment reduced fibrosis. We developed a mouse model in which a spatiotemporal (tamoxifen-inducible) deletion of RARα in kidney PT cells of adult mice causes mitochondrial dysfunction, massive PT injury, and apoptosis without the use of additional nephrotoxic substances. Long-term effects (3 to 4.5 mo) of RARα deletion include increased PT secretion of transforming growth factor β1, inflammation, interstitial fibrosis, and decreased kidney function, all of which are major features of human CKD. Therefore, RARα's actions in PTs are crucial for PT homeostasis, and loss of RARα causes injury and a key CKD phenotype.

Mitophagy regulates mitochondrial number following pharmacological induction of mitochondrial biogenesis in renal proximal tubule cells.

Background: Mitochondrial biogenesis (MB) induction through the activation of the 5-Hydroxytriptamine (5-HT) 1F receptor (HTR1F) is a promising mechanism for the treatment of diseases characterized by mitochondrial dysfunction, such as acute kidney injury (AKI). While several studies report pharmacological activation of MB in the proximal tubule, it is unclear how the proximal tubule regulates itself once the pharmacological activation is removed. Mitophagy is the process of selective mitochondria degradation. We hypothesize that mitophagy decreases mitochondrial number after pharmacological stimulation and restore mitochondrial homeostasis. Methods: Renal proximal tubules were treated at time 0hr with LY344864 or vehicle for 24h and then removed. LY344864, a selective HTR1F agonist, induces MB in renal proximal tubules as previously reported (Gibbs et al., Am J Physiol Renal Physiol, 2018, 314(2), F260-F268). Vehicle and pharmacological reagents were added at the 24h time point. Electron microscopy was used to assess mitochondrial morphology, number, and autolysosomes. Seahorse Bioscience XF-96 extracellular flux analyzer was used to measure maximal mitochondrial oxygen consumption rates (FCCP-OCR), a functional marker of MB. Results: LY344864 treatment increased FCCP-OCR, phosphorylation of protein kinase B (AKT), peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α), and mitochondrial number after 24h. These endpoints decreased to baseline 24h after LY344864 removal. Treatment with ROC-325, an autophagy inhibitor, increased Sequestosome-1 (SQSTM1/P62) and microtubule-associated protein-1 light chain 3 (LC3B) after 24h of treatment. Also, ROC-325 treatment sustained the elevated mitochondrial number after LY344864 pre-treatment and removal. Conclusion: These data revealed that inhibition of autophagy extends elevated mitochondrial number and function by preventing the lysosomal degradation of mitochondria after the removal of LY344864.

Distinct concentration-dependent oxidative stress profiles by cadmium in a rat kidney proximal tubule cell line

Levels and chemical species of reactive oxygen/nitrogen species (ROS/RNS) determine oxidative eustress and distress. Abundance of uptake pathways and high oxygen consumption for ATP-dependent transport makes the renal proximal tubule particularly susceptible to cadmium (Cd2+)-induced oxidative stress by targeting ROS/RNS generation or antioxidant defence mechanisms, such as superoxide dismutase (SOD) or H2O2-metabolizing catalase (CAT). Though ROS/RNS are well-evidenced, the role of distinct ROS profiles in Cd2+ concentration-dependent toxicity is not clear. In renal cells, Cd2+ (10–50 µM) oxidized dihydrorhodamine 123, reaching a maximum at 2–3 h. Increases (up to fourfold) in lipid peroxidation by TBARS assay and H2O2 by Amplex Red were evident within 30 min. ROS and loss in cell viability by MTT assay with 50 µM Cd2+ could not be fully reversed by SOD mimetics Tempol and MnTBAP nor by SOD1 overexpression, whereas CAT expression and α-tocopherol were effective. SOD and CAT activities were attenuated below controls only with >6 h 50 µM Cd2+, yet augmented by up to 1.5- and 1.2-fold, respectively, by 10 µM Cd2+. Moreover, 10 µM, but not 25–50 µM Cd2+, caused 1.7-fold increase in superoxide anion (O2•−), detected by dihydroethidium, paralled by loss in cell viability, that was abolished by Tempol, MnTBAP, α-tocopherol and SOD1 or CAT overexpression. H2O2-generating NADPH oxidase 4 (NOX4) was attenuated by ~50% with 10 µM Cd2+ at 3 h compared to upregulation by 50 µM Cd2+ (~1.4-fold, 30 min), which was sustained for 24 h. In summary, O2•− predominates with low–moderate Cd2+, driving an adaptive response, whereas oxidative stress by elevated H2O2 at high Cd2+ triggers cell death signaling pathways.HighlightsDifferent levels of reactive oxygen species are generated, depending on cadmium concentration.Superoxide anion predominates and H2O2 is suppressed with low cadmium representing oxidative eustress.High cadmium fosters H2O2 by inhibiting catalase and increasing NOX4 leading to oxidative distress.Superoxide dismutase mimetics and overexpression were less effective with high versus low cadmium.Oxidative stress profile could dictate downstream signalling pathways.