Circadian gene expression in mouse renal proximal tubule

Abstract

Circadian variability in kidney function is well recognized but is often ignored as a potential confounding variable in physiological experiments. Here, we have created a data resource consisting of expression levels for mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. Small-sample RNA sequencing was applied to microdissected S1 proximal convoluted tubules and S2 proximal straight tubules. After stringent filtering, the data were analyzed using JTK-Cycle to detect periodicity. The data set is provided as a user-friendly webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox2/. In proximal convoluted tubules, 234 transcripts varied in a circadian manner (4.0% of the total). In proximal straight tubules, 334 transcripts varied in a circadian manner (5.3%). Transcripts previously known to be associated with corticosteroid action and with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day [zeitgeber time (ZT)14-22], corresponding to peak levels of corticosterone and glomerular filtration rate in mice. To ask whether there is a time-of-day dependence of protein abundances in the kidney, we carried out LC-MS/MS-based proteomics in whole mouse kidneys at ZT12 and ZT0. The full data set (n = 6,546 proteins) is available at https://esbl.nhlbi.nih.gov/Databases/Circadian-Proteome/. Overall, 293 proteins were differentially expressed between ZT12 and ZT0 (197 proteins greater at ZT12 and 96 proteins greater at ZT0). Among the regulated proteins, only nine proteins were found to be periodic in the RNA-sequencing analysis, suggesting a high level of posttranscriptional regulation of protein abundances.NEW & NOTEWORTHY Circadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-sequencing transcriptomics and LC-MS/MS-based proteomics to identify gene products expressed in a periodic manner. The data were used to construct user-friendly web resources.

Keywords: RNA sequencing; circadian variation; proteomics; proximal tubule; transcriptomics.

Graphical abstract

Figure 1.

Two examples of time-course data for transcripts using images downloaded from the data-sharing webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox/Circadian_Amplitude_PCT.html. Transcripts are indicated by official gene symbols. “S1” refers to the proximal convoluted tubule (PCT). “ZT” is zeitgeber time, representing the time point in hours since the lights on time. A: the transcript coding for solute carrier family 30 (zinc transporter) member 5 (Slc30a5) in the PCT has a phase of 8 h, corresponding to a peak amplitude 8 h after lights on. B: the transcript coding for zinc finger and BTB domain containing 43 (Zbtb43) in the PCT has a phase of 18 h (peak amplitude ∼6 h after lights off). TPM, transcripts per million.

Figure 2.

Two examples of time-course data for transcripts using images downloaded from the data-sharing webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox/Circadian_Amplitude_PST.html. Transcripts are indicated by official gene symbols. “S2” refers to the proximal straight tubule (PST). ZT is zeitgeber time, representing the time point in hours since the lights on time. A: the transcript coding for inter-α-trypsin inhibitor heavy chain H5 (Itih5) in the PST has a phase of 4 h, with a peak amplitude about 4 h after lights on. B: the transcript coding for zinc finger and BTB domain containing 41 (Zbtb41) in the PST has a phase of 20 h, peaking ∼8 h after lights off. TPM, transcripts per million.

Figure 3.

Volcano plot of proteomic data for 6,546 proteins in the mouse whole kidney. The horizontal axis shows the log₂(ZT12/ZT0 protein abundance ratio) for individual proteins (where ZT is zeitgeber time). P values are from the t statistic calculated from a 3 vs. 3 comparison of log₂(abundance) values. The horizontal dashed line indicates P = 0.05. The vertical dashed lines indicate 95% confidence limits for changes in control vs. control comparisons.

Figure 4.

Nonproximal proteins showing differences in log₂(ZT12/ZT0 protein abundance ratio) values in whole kidney samples (where ZT is zeitgeber time). Transcripts per million (TPM) values are from a prior paper reporting transcript abundances in microdissected mouse renal tubule segments. Values are highlighted with different shades of yellow to emphasize differences in gene expression. PTS1, initial segment of the proximal tubule; PTS2, proximal straight tubule in cortical medullary rays; PTS3, last segment of the proximal straight tubule; DTL1, short descending limb of the loop of Henle; DTL2, long descending limb of the loop of Henle in the outer medulla; DTL3, long descending limb of the loop of Henle in the inner medulla; ATL, thin ascending limb of the loop of Henle; MTAL, medullary thick ascending limb of the loop of Henle; CTAL, cortical thick ascending limb of the loop of Henle; DCT, distal convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct; OMCD, outer medullary collecting duct; IMCD, inner medullary collecting duct.