Hypertension is a complex disease influenced by sex, and genetic and environmental factors. Blood pressure (BP) is a continuous trait that is heritable in primates, including humans and baboons. The kidneys play a role in systemically regulating BP. Sex differences in BP onset and control with antihypertensive drug therapies have been observed in humans and rodents. Hypertension studies in nonhuman primates (NHP) to date have focused on males. We hypothesized that there are differences in renal molecular networks associated with BP in female and male primates. Sodium-naïve female (n=8) and male (n=9) baboons were fed a low-sodium chow diet prior to and during the study. Implantable telemetry devices continuously monitored heart rate and blood pressure over 24-hours, and ultrasound-guided kidney biopsies were collected for RNA-Seq. Serum 17 beta-estradiol concentration correlated BP in females. BP in males correlated with Na+ intake, blood urea nitrogen, and glucose. Cell type composition of renal biopsies was consistent between females and males. Sex differences were observed in the kidney transcriptomes by principal components analysis and weighted gene co-expression network analysis. Network analysis revealed HNF4A, ESR1, ESR2, SMARCA4, TP53, and NR3C1 as BP regulators in males. Our results demonstrate sex differences in primate kidney molecular networks and provide evidence of a novel link between renal transcription factors and BP regulation in males. Understanding sex differences and transcriptome variation in primate kidneys correlated with BP and clinical measures associated with BP will inform better therapies towards the goal of precision medicine for women and men.

Blood pressure (BP) is influenced by genetic variation and sodium intake with sex-specific differences; however, studies to identify renal molecular mechanisms underlying the influence of sodium intake on BP in nonhuman primates (NHP) have focused on males. To address the gap in our understanding of molecular mechanisms regulating BP in female primates, we studied sodium-naïve female baboons (n=7) fed a high sodium (HS) diet for 6 weeks. We hypothesized that in female baboons variation in renal transcriptional networks correlates with variation in BP response to a high sodium diet. BP was continuously measured for 64-hour periods throughout the study by implantable telemetry devices. Sodium intake, blood samples for clinical chemistries, and ultrasound-guided kidney biopsies were collected before and after the HS diet for RNA-Seq and bioinformatic analyses. We found that on the LS diet but not the HS diet, sodium intake and serum 17 beta-estradiol concentration correlated with BP. Furthermore, kidney transcriptomes differed by diet - unbiased weighted gene co-expression network analysis revealed modules of genes correlated with BP on the HS diet but not the LS diet. Our results showed variation in BP on the HS diet correlated with variation in novel kidney gene networks regulated by ESR1 and MYC, i.e., these regulators have not been associated with BP regulation in male humans or rodents. Validation of the mechanisms underlying regulation of BP associated gene networks in female NHP will inform better therapies towards greater precision medicine for women.

Traditional bulk RNA-Seq pipelines do not assess cell-type composition within heterogeneous tissues. Therefore, it is difficult to determine whether conflicting findings among samples or datasets are the result of biological differences or technical differences due to variation in sample collections. This report provides a user-friendly, open source method to assess cell-type composition in bulk RNA-Seq datasets for heterogeneous tissues using published single cell (sc)RNA-Seq data as a reference. As an example, we apply the method to analysis of kidney cortex bulk RNA-Seq data from female (N=8) and male (N=9) baboons to assess whether observed transcriptome sex differences are biological or technical, i.e., variation due to ultrasound guided biopsy collections. We found cell-type composition was not statistically different in female versus male transcriptomes based on expression of 274 kidney cell-type specific transcripts, indicating differences in gene expression are not due to sampling differences. This method of cell-type composition analysis is recommended for providing rigor in analysis of bulk RNA-Seq datasets from complex tissues. It is clear that with reduced costs, more analyses will be done using scRNA-Seq; however, the approach described here is relevant for data mining and meta analyses of the thousands of bulk RNA-Seq data archived in the NCBI GEO public database.

Hypertension is a complex disease influenced by sex, and genetic and environmental factors. Blood pressure (BP) is a continuous trait that is heritable in primates, including humans and baboons. The kidneys play a role in systemically regulating BP. Sex differences in BP onset and control with antihypertensive drug therapies have been observed in humans and rodents. Hypertension studies in nonhuman primates (NHP) to date have focused on males. We hypothesized that there are differences in renal molecular networks associated with BP in female and male primates. Sodium-naïve female (n=8) and male (n=9) baboons were fed a low-sodium chow diet prior to and during the study. Implantable telemetry devices continuously monitored heart rate and blood pressure over 24-hours, and ultrasound-guided kidney biopsies were collected for RNA-Seq. Serum 17 beta-estradiol concentration correlated BP in females. BP in males correlated with Na+ intake, blood urea nitrogen, and glucose. Cell type composition of renal biopsies was consistent between females and males. Sex differences were observed in the kidney transcriptomes by principal components analysis and weighted gene co-expression network analysis. Network analysis revealed HNF4A, ESR1, ESR2, SMARCA4, TP53, and NR3C1 as BP regulators in males. Our results demonstrate sex differences in primate kidney molecular networks and provide evidence of a novel link between renal transcription factors and BP regulation in males. Understanding sex differences and transcriptome variation in primate kidneys correlated with BP and clinical measures associated with BP will inform better therapies towards the goal of precision medicine for women and men.

OBJECTIVES/GOALS: The goal of this study was to understand the impact of a high sodium diet on gene networks in the kidney that correlate with blood pressure in female primates, and translating findings to women. METHODS/STUDY POPULATION: Sodium-naïve female baboons (n=7) were fed a low-sodium (LS) diet for 6 weeks followed by a high sodium (HS) diet for 6 weeks. Sodium intake, serum 17 beta-estradiol, and ultrasound-guided kidney biopsies for RNA-Seq were collected at the end of each diet. Blood pressure was continuously measured for 64-hour periods throughout the study by implantable telemetry devices. Weighted gene coexpression network analysis was performed on RNA-Seq data to identify transcripts correlated with blood pressure on each diet. Network analysis was performed on transcripts highly correlated with BP, and in silico findings were validated by immunohistochemistry of kidney tissues. RESULTS/ANTICIPATED RESULTS: On the LS diet, Na+ intake and serum 17 beta-estradiol concentration correlated with BP. Cell type composition of renal biopsies was consistent among all animals for both diets. Kidney transcriptomes differed by diet; analysis by unbiased weighted gene co-expression network analysis revealed modules of genes correlated with BP on the HS diet. Network analysis of module genes showed causal networks linking hormone receptors, proliferation and differentiation, methylation, hypoxia, insulin and lipid regulation, and inflammation as regulators underlying variation in BP on the HS diet. Our results show variation in BP correlated with novel kidney gene networks with master regulators PPARG and MYC in female baboons on a HS diet. DISCUSSION/SIGNIFICANCE: Previous studies in primates to identify molecular networks dysregulated by HS diet focused on males. Current clinical guidelines do not offer sex-specific treatment plans for sodium sensitive hypertension. This study leveraged variation in BP as a first step to identify correlated kidney regulatory gene networks in female primates after a HS diet.