BackgroundPrevious research has used RNA sequencing in microdissected kidney tubules or single cells isolated from the kidney to profile gene expression in each type of kidney tubule epithelial cell. However, because proteins, not mRNA molecules, mediate most cellular functions, it is desirable to know the identity and amounts of each protein species to understand function. Recent improvements in the sensitivity of mass spectrometers offered us the ability to quantify the proteins expressed in each of 14 different renal tubule segments from rat.MethodsWe manually dissected kidney tubules from rat kidneys and subjected samples to protein mass spectrometry. We used the “proteomic ruler” technique to estimate the number of molecules of each protein per cell.ResultsOver the 44 samples analyzed, the average number of quantified proteins per segment was 4234, accounting for at least 99% of protein molecules in each cell. We have made the data publicly available online at the Kidney Tubule Expression Atlas website (https://esbl.nhlbi.nih.gov/KTEA/). Protein abundance along the renal tubule for many commonly studied water and solute transport proteins and metabolic enzymes matched expectations from prior localization studies, demonstrating the overall reliability of the data. The site features a “correlated protein” function, which we used to identify cell type–specific transcription factors expressed along the renal tubule.ConclusionsWe identified and quantified proteins expressed in each of the 14 segments of rat kidney tubules and used the proteomic data that we obtained to create an online information resource, the Kidney Tubule Expression Atlas. This resource will allow users throughout the world to browse segment-specific protein expression data and download them for their own research.
T he kidney contains multiple cell types, each with its own characteristic transcriptome and proteome. Discovery of pathophysiological mechanisms, therefore, requires isolation or enrichment of the cell types responsible for disease processes. One experimental approach, allowing highly reliable identification of specific renal tubule cells, is manual microdissection of renal tubules, which has been used for several decades to define the transport processes and
The renal aldosterone-sensitive distal tubule (ASDT) is crucial for sodium reabsorption and blood pressure regulation. The ASDT consists of the late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct. Due to difficulties in isolating epithelial cells from the ASDT in large quantities, few transcriptome studies have been performed on this segment. Moreover, no studies exist on isolated DCT2 and CNT cells (excluding intercalated cells), and the role of aldosterone for regulating the transcriptome of these specific cell types is largely unknown. A mouse model expressing eGFP in DCT2/CNT/initial cortical collecting duct (iCCD) principal cells was exploited to facilitate the isolation of these cells in high number and purity. Combined with deep RNA sequencing technology, a comprehensive catalog of chronic aldosterone-regulated transcripts from enriched DCT2/CNT/iCCD principal cells was generated. There were 257 significantly downregulated and 290 upregulated transcripts in response to aldosterone ( P < 0.05). The RNA sequencing confirmed aldosterone regulation of well-described aldosterone targets including Sgk1 and Tsc22d3. Changes in selected transcripts such as S100a1 and Cldn4 were confirmed by RT-qPCR. The RNA sequencing showed downregulation of Nr3c2 encoding the mineralocorticoid receptor (MR), and cell line experiments showed a parallel decrease in MR protein. Furthermore, a large number of transcripts encoding transcription factors were downregulated. An extensive mRNA transcriptome reconstruction of an enriched CNT/iCCD principal cell population was also generated. The results provided a comprehensive database of aldosterone-regulated transcripts in the ASDT, allowing development of novel hypotheses for the action of aldosterone.
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