Proximal tubule cell maturation rate and function are controlled by PPARα signaling in kidney organoids.
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ABSTRACT: Kidney organoids derived from human iPSCs have cell heterogeneity of maturation. We used single-cell RNA sequencing (scRNAs-seq) to extract the factor related to proximal tubule maturation. Using the kidney organoids cultured with the factors that accelerate the maturation speed of proximal tubules extracted by the aforementioned method, we examined whether the maturity of the proximal tubules increases within the same induction period.
Project description:Freshly isolated rat kidney proximal tubules were subjected for transcript profiling. Three microarray experiments were done to obtain the kidney proxmial tubule transcriptome.
Project description:Circadian variability in kidney function has long been recognized but is often ignored as a potential confounding variable in in vivo physiological experiments. To provide a guide for physiological studies on the kidney proximal tubule, we have now created a data resource consisting of expression levels for all measurable mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. This approach employs small-sample RNA-sequencing (RNA-seq) applied to microdissected renal proximal tubules including both S1 proximal convoluted tubules (PCTs) and S2 proximal straight tubules (PSTs). The data were analyzed using JTK-Cycle to detect periodicity. The data are provided as a user-friendly web page at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox/. In PCTs, 234 transcripts were found to vary in a circadian manner (3.7 % of total quantified). In PSTs, 334 transcripts were found to vary in a circadian manner (5.3 % of total quantified). Transcripts previously known to be associated with corticosteroid action and transcripts associated with increased flow were found to be overrepresented among circadian transcripts peaking during the “dark” portion of the day (Zeitgeber 14-22), corresponding to the peak levels of corticosterone and glomerular filtration rate in mice.
Project description:Purpose:Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend on the genes expressed.We have used modern RNA-sequencing techniques to identify the gene expression profile of 14 different cell lines plus primary cultures of mouse proximal tubule and compare them to transcriptomes of native kidney proximal tubules. Methods: 14 different proximal tubule cell lines were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures. Results and conclusion: Transcripts expressed in cell lines showed variable match to transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45%) with pig kidney cells (LLC-PK1) close behind (39%). Much lower percentage matches were seen for various human lines including HK-2 cells (26%) and lines from rodent kidneys (18-23%).An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created for interrogation of the data.No cell line closely matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.
Project description:Female sex protects against development of acute kidney injury (AKI). While sex hormones may be involved in protection, the role of differential gene expression is unknown. We conducted gene profiling in male and female mice with or without kidney ischemia-reperfusion injury. Mice underwent bilateral renal pedicle clamping (30 min), and tissues were collected 24 hours after reperfusion. RNA-sequencing was performed on proximal tubules and kidney endothelial cells. Female mice were resistant to ischemic injury compared to males, determined by plasma creatinine, histologic scores, neutrophil infiltration, and extent of apoptosis. Sham mice had sex-specific gene disparities in proximal tubule and endothelium, and male mice showed profound gene dysregulation with ischemia-reperfusion compared to females. After ischemia proximal tubules from females exhibited smaller increases compared to males in injury-associated genes Lcn2, Havcr1, and Krt18, and no upregulation of Sox9 or Krt20. Endothelial upregulation of adhesion molecules and cytokines/chemokines occurred in males, but not females. Upregulated genes in male ischemic proximal tubules were linked to tumor necrosis factor and Toll-like receptor pathways, while female ischemic proximal tubules showed upregulated genes in pathways related to transport. The data suggest that sex-specific gene expression profiles in male and female proximal tubule and endothelium may underlie disparities in susceptibility to AKI.
Project description:Improved specification of metanephric nephron progenitors in conditions that prolong differentiation while simultaneously preventing spontaneous nephron induction resulted in proximal tubule (PT)-enhanced kidney organoids. PT-enhanced organoids exhibited improved PT maturation, with elongated and aligned nephron segments as well as distinct loops of Henle. The striking proximo-distal orientation of nephrons was shown to result from localised WNT antagonism originating from the centre of the organoid.
Project description:Global gene expression in the primary cultured mouse kidney proximal tubule cells treated either DMSO or 1uM GW4064 (a FXR agonist) was compared. Results provide insight into mechanisms underlying effects of FXR activation on gene expression in mouse kidney proximal tubule cells. Male C57/BJ mice aged 6 weeks were sacrificed under anesthesia and kidney proximal tubule cells were cultured until confluent. Cells were treated with either GW4064 (1uM) or equal amount of DMSO and incubated for 24 hours. 4 total RNA samples per group were analyzed and gene expression was compared between the groups.
Project description:Pax2 and Pax8 are homologous transcription factors required for kidney development and medullary urine concentration. However, their function in proximal tubule homeostasis and response to acute kidney injury is unknown. Mice with proximal tubules consisting of a mosaic of wild-type and Pax2/8 mutant proximal tubules cells were generated. Gene expression of mutant and wild-type proximal tubule cells was compared under homeostatic conditions using single-nucleus RNA sequencing.
Project description:Epigenetic mechanisms are considered to contribute to diabetic nephropathy by maintaining memory of poor glycemic control during the early stages of diabetes. However, DNA methylation changes in the human kidney are poorly characterized, because of the lack of cell type-specific analysis. We examined DNA methylation in proximal tubules purified from diabetic nephropathy patients and identified differentially methylated CpG sites, given the critical role of proximal tubules in the kidney injury. Hypermethylation was observed at CpG sites annotated to genes responsible for proximal tubule functions, including gluconeogenesis, nicotinamide adenine dinucleotide synthesis, transporters of glucose, water, phosphate, and drugs, in diabetic kidneys, while genes involved in oxidative stress and the cytoskeleton exhibited demethylation. Methylation levels of CpG sites annotated to ACTN1, BCAR1, MYH9, UBE4B, AFMID, TRAF2, TXNIP, FOXO3, and HNF4A were correlated with the estimated glomerular filtration rate, while methylation of the CpG site in RUNX1 was associated with interstitial fibrosis and tubular atrophy. Hypermethylation of G6PC and HNF4A was accompanied by decreased expression in diabetic kidneys. Proximal tubule-specific hypomethylation of metabolic genes related to HNF4A observed in control kidneys was compromised in diabetic kidneys, suggesting a role for aberrant DNA methylation in the dedifferentiation process. Multiple genes with aberrant DNA methylation in diabetes overlapped genes with altered expressions in maladaptive proximal tubule cells, including transcription factors PPARA and RREB1. In conclusion, DNA methylation derangement in the proximal tubules of patients with diabetes may drive phenotypic changes, characterized by inflammatory and fibrotic features, along with impaired function in metabolism and transport.
Project description:Acute kidney injury (AKI) is a major health care concern. There are no therapies for the treatment of AKI. The primary site of damage during AKI is the proximal tubule, which are highly metabolically active cells that rely upon fatty acids for energy. Proximal tubules are notably mitochondria- and peroxisomes-rich cell type that mediate fatty acid oxidation (FAO). Sirtuins reverse post-translational lysine acylation and control many biological processes including FAO. Sirtuin1 and sirtuin3 are protective against AKI. However, the role of the mitochondrial Sirtuin5 (Sirt5) during AKI has yet to be determined. We found Sirt5 to be highly expressed in the proximal tubule. At baseline Sirtuin5 knockout (Sirt5-/-) animals had modestly decreased mitochondrial function but significantly increased FAO, which was localized to the peroxisome. While no overt kidney phenotype was observed in SIRT5-/- mice, following ischemia reperfusion injury Sirt5-/- animals had significantly improved kidney function and less tissue damage. This coincided with increased peroxisomal activity in the Sirt5-/- proximal tubules in preference to the mitochondria. Here we have identified a novel mechanism driving protection of kidneys from ischemic injury. If this can be harnessed it may prove to be an effective therapeutic.