Profiling Cell Heterogeneity and Fructose Transporter Expression in the Rat Nephron by Integrating Single-Cell and Microdissected Tubule Segment Transcriptomes.

Single-cell RNA sequencing (scRNAseq) is a crucial tool in kidney research. These technologies cluster cells based on transcriptome similarity, irrespective of the anatomical location and order within the nephron. Thus, a transcriptome cluster may obscure the heterogeneity of the cell population within a nephron segment. Elevated dietary fructose leads to salt-sensitive hypertension, in part, through fructose reabsorption in the proximal tubule (PT). However, the organization of the four known fructose transporters in apical PTs (SGLT4, SGLT5, GLUT5, and NaGLT1) remains poorly understood. We hypothesized that cells within each subsegment of the proximal tubule exhibit complex, heterogeneous fructose transporter expression patterns. To test this hypothesis, we analyzed rat kidney transcriptomes and proteomes from publicly available scRNAseq and tubule microdissection databases. We found that microdissected PT-S1 segments consist of 81% ± 12% cells with scRNAseq-derived transcriptional characteristics of S1, whereas PT-S2 express a mixture of 18% ± 9% S1, 58% ± 8% S2, and 19% ± 5% S3 transcripts, and PT-S3 consists of 75% ± 9% S3 transcripts. The expression of all four fructose transporters was detectable in all three PT segments, but key fructose transporters SGLT5 and GLUT5 progressively increased from S1 to S3, and both were significantly upregulated in S3 vs. S1/S2 (Slc5a10: 1.9 log2FC, p < 1 × 10-299; Scl2a5: 1.4 log2FC, p < 4 × 10-105). A similar distribution was found in human kidneys. These data suggest that S3 is the primary site of fructose reabsorption in both humans and rats. Finally, because of the multiple scRNAseq transcriptional phenotypes found in each segment, our findings also imply that anatomical labels applied to scRNAseq clusters may be misleading.

Abnormal activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron contributes to fructose-induced salt-sensitive hypertension.

Fructose high-salt (FHS) diets increase blood pressure (BP) in an angiotensin II (Ang II)-dependent manner. Ang II stimulates aldosterone release, which, by acting on the mineralocorticoid receptor (MR), regulates Na + reabsorption by the aldosterone-sensitive distal nephron (ASDN). The MR can be transactivated by glucocorticoids, including those locally produced by 11ß-HSD1. The epithelial sodium channel (ENaC) is a key transporter regulated by MRs. We hypothesized that fructose-induced salt-sensitive hypertension depends in part on abnormal activation of MRs in the ASDN with consequent increases in ENaC expression. We found that aldosterone-upregulated genes in mice ASDN, significantly overlapped with 74 genes upregulated by FHS in the rat kidney cortex (13/74; p≤1x10 -8 ), and that these 74 genes are prominently expressed in rat ASDN cells. Additionally, the average z-score expression of mice-aldosterone-upregulated genes is highly correlated with FHS compared to glucose high-salt (GHS) in the rat kidney cortex (Pearson correlation; r=0.66; p≤0.005). There were no significant differences in plasma aldosterone concentrations between the FHS and GHS. However, 11ß-HSD1 transcripts were upregulated by FHS (log 2 FC=0.26, p≤0.02). FHS increased BP by 23±6 mmHg compared to GHS, and blocking MRs with eplerenone prevented this increase. Additionally, inhibiting ENaC with amiloride significantly reduced BP in FHS from 148±6 to 134±5 mmHg (p≤0.019). Compared to GHS, FHS increased total and cleaved αENaC protein by 89±14 % (p≤0.03) and 47±16 % (p≤0.01) respectively. FHS did not change ß- or γ-subunit expression. These results suggest that fructose-induced salt-sensitive hypertension depends, in part, on abnormal Na + retention by ENaC, resulting from the activation of MRs by glucocorticoids.

Profiling cellular heterogeneity and fructose transporter expression in the rat nephron by integrating single-cell and microdissected tubule segment transcriptomes.

Single-cell RNA sequencing (scRNAseq) is a crucial tool in kidney research. These technologies cluster cells according to transcriptome similarity, irrespective of the anatomical location and ordering within the nephron. Thus, a cluster transcriptome may obscure heterogeneity of the cell population within a nephron segment. Elevated dietary fructose leads to salt-sensitive hypertension, in part by fructose reabsorption in the proximal tubule (PT). However, organization of the four known fructose transporters in apical PTs (SGLT4, SGLT5, GLUT5 and NaGLT1) remains poorly understood. We hypothesized that cells within each subsegment of the proximal tubule exhibit complex, heterogenous fructose transporter expression patterns. To test this hypothesis we analyzed rat and kidney transcriptomes and proteomes from publicly available scRNAseq and tubule microdissection databases. We found that microdissected PT-S1 segments consist of 81±12% cells with scRNAseq-derived transcriptional characteristics of S1, whereas PT-S2 express a mixture of 18±9% S1, 58±8% S2, and 19±5% S3 transcripts, and PT-S3 consists of 75±9% S3 transcripts. The expression of all four fructose transporters was detectable in all three PT segments, but key fructose transporters SGLT5 and GLUT5 progressively increased from S1 to S3, and both were significantly upregulated in S3 vs. S1/S2 (Slc5a10: 1.9 log 2 FC, p<1×10 -299 ; Scl2a5: 1.4 log 2 FC, p<4×10 -105 ). A similar distribution was found in human kidneys. These data suggest that S3 is the primary site of fructose reabsorption in both humans and rats. Finally, because of the multiple scRNAseq transcriptional phenotypes found in each segment our findings also imply that anatomic labels applied to scRNAseq clusters may be misleading.