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.
@article{BeauEtAl2024SGLT5WGCNAHypertension,
title = {[IN-PRINT] Knocking out sodium glucose-linked transporter 5 prevents fructose-induced renal oxidative stress and salt-sensitive hypertension},
author = {Forester, Beau R. and Zhang, Ronghao and Schuhler, Brett and Brostek, Autumn and Gonzalez-Vicente, Agustin and Garvin, Jeffrey L.},
year = {2024},
journal = {Hypertension},
}Proximal tubules (PT) reabsorb fructose via a Na-dependent mechanism. Knocking out the Sodium-Linked Cotransporter 5 (SGLT5, Slc5A10) increases urinary fructose. Elevated dietary fructose increases transport rates in PT, contributing to salt-sensitive hypertension. Whether dietary fructose also elevates transport in other segments of the nephron that do not express known fructose transporters, is a matter of debate. The aldosterone-sensitive distal tubule (ASDT) consists of the late distal convoluted tubule, connecting tubule and collecting duct, and it is essential for regulating fluid volume and blood pressure. We hypothesize that fructose metabolism in PT could affect aldosterone signaling in the distal tubule of rats fed a high-salt diet. We obtained kidney-cortex bulk RNAseq transcriptomes from wild-type and SGLT5 (-/-) Sprague Dawley rats fed a solid diet containing 4 percent NaCl and either 20 percent fructose (FHS) or glucose (GHS). We used Weighted Correlation Network Analysis (WGCNA) to: 1st) create a gene coexpression network of the kidney cortex, and 2nd) identify genes whose expression changes in response to fructose in both wild-type and SGLT5 (-/-). Then, we used open source transcriptomes from mouse ASDT epithelial cells (PMID: 29521601) treated with and without aldosterone to map genes whose expression changes in response to aldosterone to the coexpression modules correlated with dietary fructose. The mouse transcriptome contained 8619 genes that mapped to the rat kidney transcriptome. Of these genes, 454 were significantly changed by aldosterone (ALDO). WGCNA yielded 35 coexpression modules. Five modules were significantly associated with dietary fructose in wild-type. These correlations were blunted in SGLT5 (-/-). Enrichment analysis indicated that 4 of the 35 modules were enriched for ALDO, 2 of which (Paleturquoise: p<5x10-6 and Orange: p<3x10-2) were also correlated with fructose in wild-type (Paleturquoise: p<3x10-2 and Orange: p<4x10-2). Paleturquoise contained 108 genes of which 74 mapped to the mouse transcriptome, and 15 to ALDO. Ontology enrichments in Paleturquoise indicate that it contains genes involved in Na, Cl and HCO3 transport. Importantly, the Amiloride-Sensitive Epithelial Sodium Channel Alpha Subunit (ENaCα; Scnn1a) and the Thiazide-Sensitive Sodium-Chloride Cotransporter (NCC; Slc12a3) belong in this module. Orange contained 148 genes, of which 34 mapped to mouse transcriptomes, and only 5 to ALDO. Orange’s ontology enrichment was largely immune processes, which explain the low number of mapping genes, as ALDO is a tubular epithelial cell signature. We conclude that even on a high-salt diet, kidneys from rats given fructose present higher transcriptional activation of aldosterone-responsive genes than those given glucose. In addition, Orange may be mediating proinflammatory actions of fructose. These effects depend on fructose reabsorption by PTs, as SGLT5 deletion blunts the effects of HSF on ALDO-enriched modules.
@inproceedings{ZhangEtAl2023Abs_AldoFruc,
title = {[ABSTRACT] Addition of fructose to a high-salt diet increases the expression of aldosterone-response genes},
author = {Zhang, Ronghao and Gonzalez-Vicente, Agustin},
booktitle = {Physiology},
year = {2023},
month = may,
volume = {38},
url = {https://journals.physiology.org/doi/abs/10.1152/physiol.2023.38.S1.5733301},
}Sexual dimorphism is a key biological variable in different organ systems. Although a growing body of evidence at single cell-resolution highlights sex differences in the kidneys of humans and mice, information regarding rats is limited. We integrated publicly available datasets to construct a single-cell RNA sequencing atlas of the rat kidney, incorporating data from 3 females and 3 males. Transcriptomes from the tubular epithelial cell clusters, exhibited strong correlation with those from rat-kidney microdissected tubule segments and single-cell RNA sequencing of human kidney biopsies. We hypothesize that transcriptomes of rat tubular epithelial cells present sexual dimorphism. To test this hypothesis, we identified genes that were differentially expressed between sexes (DEGs: log2FC >= 1; adjusted-p <= 0.05) in proximal tubules (PT), thick ascending limbs (TAL), distal convoluted tubules (DCT), principal cells (PC) and intercalated cells A and B (IC-A and IC-B, respectively). Gene enrichments of PT DEGs were conducted using WikiPathways. The number of genes positively enriched in males was as follows: 43 PT, 9 TAL, 16 DCT, 23 PC, 12 I-CA and 19 I-CB. While females presented 44 PT, 4 TAL, 4 DCT, 3 PC, 1 IC-A and 3 IC-B. Four transcripts were upregulated in all male segments, 1) glutathione peroxidase 3 (Gpx3), 2) lysozyme 2 (Lyz2), 3) locus A1 (RT1-A1) which enables beta-2-microglobulin and peptide binding activity, and 4) AABR07060872.1 an Ig-like domain-containing protein. Only one long non-coding RNA (AABR07039356.2) was upregulated in all female segments. The three most enriched pathways in male PT were: 1) Proximal tubule transport (WP4917; p < 1x1e-14), 2) Glycolysis and gluconeogenesis (WP534; p < 2x1e-12) and 3) Metabolic reprograming in colon cancer (WP4290; p < 8x1e-9). In females, the most enriched pathways were: 1) Trans-sulfuration and one-carbon metabolism (WP2525; p < 6x1e-9), 2) One-carbon metabolism related pathways (WP3940; p < 1x10-8) and 3) Amino acid metabolism (WP3925; p < 7x1e-8). Our results show the presence of sex-related transcriptional differences across the rat nephron, but predominantly in PT. These differences impact key metabolic functions of PT such amino acids and carbohydrates metabolism and transport processes.
@inproceedings{KramerEtAl2023Abs_SexDimorphicTubeEpiCell,
title = {[ABSTRACT] Sexually dimorphic transcriptional phenotypes in tubular epithelial cells of the rat kidney},
author = {Kramer, Benjamin and Zhang, Ronghao and Gonzalez-Vicente, Agustin},
booktitle = {Hypertension},
year = {2023},
month = oct,
volume = {80},
}Proximal tubule fructose metabolism is key to fructose-induced hypertension, but the roles of sex and stress are unclear. We hypothesized that females are resistant to the salt-sensitive hypertension caused by low amounts of dietary fructose compared to males and that the magnitude of the increase in blood pressure (BP) depends, in part, on amplification of the stress response of renal sympathetic nerves. We measured systolic BP (SBP) in rats fed high salt with either no sugar (HS), 20 percent glucose (GHS) or 20 percent fructose (FHS) in the drinking water for 7-8 days. FHS increased SBP in both males (Δ22±9 mmHg; p<0.046) and females (Δ16±3 mmHg; p<0.0007), while neither GHS nor HS alone induced changes in SBP in either sex. The FHS-induced increase in SBP as measured by telemetry in the absence of added stress (8±2 mmHg) was significantly lower than that measured by plethysmography (24±5 mmHg) (p<0.014). However, when BP was measured by telemetry simulating the stress of plethysmography, the increase in SBP was significantly greater (15±3 mmHg) than under low stress (8±1 mmHg) (p<0.014). Moderate-stress also increased telemetric diastolic (p < 0.006) and mean BP (p<0.006) compared to low-stress in FHS-fed animals. Norepinephrine excretion was greater in FHS-fed rats than HS-fed animals (Male: 6.4±1.7 vs.1.8±0.4 nmole/Kg/day; p<0.02. Female 54±18 vs. 1.2±0.6; p<0.02). We conclude that fructose-induced salt-sensitive hypertension is similar in males and females unlike other forms of hypertension, and the increase in blood pressure depends in part on an augmented response of the sympathetic nervous system to stress.
@article{BrostekEtAl2022PR,
title = {Independent effects of sex and stress on fructose-induced salt-sensitive hypertension},
author = {Brostek, A. and Hong, N. J. and Zhang, R. and Forester, B. R. and Barmore, L. E. and Kaydo, L. and Kluge, N. and Smith, C. and Garvin, J. L. and Gonzalez-Vicente, A.},
year = {2022},
month = oct,
volume = {10},
journal = {Physiological Reports},
publisher = {John Wiley & Sons Inc.},
doi = {10.14814/phy2.15489},
}