biomedicines

Article

Blood Pressure Regulation Evolved from BasicHomeostatic Components

Alon Botzer 1, Yoram Finkelstein 2 and Ron Unger 1,*

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Citation: Botzer, A.; Finkelstein, Y.;

Unger, R. Blood Pressure Regulation

Evolved from Basic Homeostatic

Components. Biomedicines 2021, 9,

469. https://doi.org/10.3390/

biomedicines9050469

Academic Editor: Juan Sahuquillo

Received: 7 March 2021

Accepted: 22 April 2021

Published: 25 April 2021

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4.0/).

1 The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel;alon.botzer@gmail.com

2 Neurology and Toxicology Service and Unit, Shaare Zedek Medical Center, Jerusalem 9103102, Israel;yoram.finkelstein@gmail.com

* Correspondence: ron@biomodel.os.biu.ac.il

Abstract: Blood pressure (BP) is determined by several physiological factors that are regulated by arange of complex neural, endocrine, and paracrine mechanisms. This study examined a collectionof 198 human genes related to BP regulation, in the biological processes and functional prisms, aswell as gene expression in organs and tissues. This was made in conjunction with an orthologyanalysis performed in 19 target organisms along the phylogenetic tree. We have demonstrated thattransport and signaling, as well as homeostasis in general, are the most prevalent biological processesassociated with BP gene orthologs across the examined species. We showed that these genes andtheir orthologs are expressed primarily in the kidney and adrenals of complex organisms (e.g., highorder vertebrates) and in the nervous system of low complexity organisms (e.g., flies, nematodes).Furthermore, we have determined that basic functions such as ion transport are ancient and appearin all organisms, while more complex regulatory functions, such as control of extracellular volumeemerged in high order organisms. Thus, we conclude that the complex system of BP regulationevolved from simpler components that were utilized to maintain specific homeostatic functions thatplay key roles in existence and survival of organisms.

Keywords: blood pressure; homeostasis; evolution; gene orthologs

1. Introduction

The multifactorial nature of BP regulation involves many genes with a widespreaddistribution across numerous cellular subsystems, posing significant challenges in theeffort to decipher its complex mechanisms [1]. Here we implement a comparative approachto review the phylogenetic history of the BP system via an analysis of BP associated genesand their orthologs in 19 organisms, enabling us to gain an evolutionary perspective of thedevelopment of the system.

Current evidence proposes that the blood vascular system initially emerged in anancestor of the tripoblasts over 600 million years ago, as a means to withstand the time-distance constraints of diffusion [2]. This has advanced over the course of evolution andexpanded to a more complex “machinery” to support the functional requirements of highorder organisms.

We suggest that the most fundamental organismal function of this machinery is topreserve the internal milieu, namely, to maintain a stable internal environment concerningtemperature, electrolytes and water concentrations, as external conditions perpetuallychange. Internal environment, or “milieu intérieur” is a concept formulated by ClaudeBernard, postulating that “the stability of the internal environment (the milieu intérieur)is the condition for the free and independent life”. This is the fundamental principle ofhomeostasis, a term coined later by Walter Bradford Cannon.

Claude Bernard also stated: “The constancy of the environment presupposes a perfec-tion of the organism such that external variations are at every instant compensated and

Biomedicines 2021, 9, 469. https://doi.org/10.3390/biomedicines9050469 https://www.mdpi.com/journal/biomedicines

Biomedicines 2021, 9, 469 2 of 18

brought into balance. In consequence, far from being indifferent to the external world, thehigher animal is on the contrary in a close and wise relation with it, so that its equilibriumresults from a continuous and delicate compensation established as if the most sensitive ofbalances” [3].

This work has utilized bioinformatic tools and data mining techniques to investigatethe BP system and elucidate the major homeostatic roles it played in the physiology of thecorresponding organisms. In fact, we went back, tracing the evolution of genes that areinvolved in human BP regulation.

2. Materials and Methods2.1. BP Genes, Orthologous Proteins and Target Organism Selection

This study investigated a set of 198 genes associated with the BP regulation system.These genes were obtained and identified by means of bioinformatic analyses performedpreviously by Botzer et al. [4]. A set of orthologous proteins pertaining to these genes,from 19 organisms (see Figure 1) were obtained from two different sources—STRING andInParanoid.

STRING—Search Tool for the Retrieval of Interacting Genes/Proteins, (http://string-db.org/ (accessed on May 2017)) is a meta-resource that aggregates most of the availableinformation on protein–protein associations and includes both direct physical interactionsand protein interactions derived from literature. Since version 9.1, it contains pre-computedorthology relations imported from the eggNOG database [5]. eggnog—evolutionary geneal-ogy of genes: Non-supervised Orthologous Groups, (http://eggnogdb.embl.de (accessedon May 2017)) is a public resource that provides Orthologous Groups (OG’s) of proteinsat different taxonomic levels. It provides pairwise orthology relationships within OG’sbased on analysis of phylogenetic trees. Protein sequences from the selected organismswere extracted and used to compute an all-against-all pair-wise similarity matrix. Thecomparison uses Smith-Waterman alignments and computational adjustments of the scores,as in BLAST, to prevent spurious hits between low-complexity sequence regions [6]. In-Paranoid (http://InParanoid.sbc.su.se (accessed on February 2018))—gathers proteomesof completely sequenced eukaryotic species and calculates pairwise ortholog relationshipsamong them. This tool implements a two-pass BLAST approach that makes use of high-precision compositional score matrix adjustment, but avoids the alignment truncation thatsometimes follows [7]. Both tools demonstrated similar results, but we decided to pursueanalyses with data retrieved from STRING, which included a more comprehensive andupdated coverage.

We then composed an organism-to-protein similarity map, consisting of similarityscores between the 198 human BP protein sequences and their ortholog proteins in the19 target organisms, as obtained from STRING. This map was produced by calculationof pairwise sequence alignment, using EMBOSS Needle Global Alignment tool (https://www.ebi.ac.uk/Tools/psa/emboss_needle/ (accessed on February 2018)). This toolcreates an optimal global alignment of two sequences using the Needleman-Wunschalgorithm, returning a value representing the percent of similarity between sequences,enabling homology to be inferred and the evolutionary relationship between the sequencesstudied [8].

Target organism selection was made with the intention to create a span of a wide vari-ety of complexities—starting with the multicellular organism T. adhaerens as the simplest,going through flies, nematodes, fish, amphibians and eventually complex organisms as pri-mates and other mammals (see Figure 1 for a complete list of organisms). We gave emphasisto well-researched model organisms, displaying sufficient and reliable orthology data, aswell as extended annotations derived from established biological and genetic knowledge.

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Figure 1. Cont.

Biomedicines 2021, 9, 469 4 of 18Biomedicines 2021, 9, x FOR PEER REVIEW 4 of 19

Figure 1. Heatmap displays the hierarchical clustering of similarity percentage scores between human BP genes (vertical axis) and their orthologs in 19 organisms (horizontal axis). Green color denotes the more conserved genes across the 19 organisms, aligned at the top of the map (see conservation scale arrow to the right of map). A phylogenetic tree describing the developmental order is placed above the map.

STRING—Search Tool for the Retrieval of Interacting Genes/Proteins, (http://string-db.org/ (accessed on May 2017)) is a meta-resource that aggregates most of the available information on protein–protein associations and includes both direct physical interactions and protein interactions derived from literature. Since version 9.1, it contains pre-com-puted orthology relations imported from the eggNOG database [5]. eggnog—evolution-ary genealogy of genes: Non-supervised Orthologous Groups, (http://eggnogdb.embl.de (accessed on May 2017)) is a public resource that provides Orthologous Groups (OG’s) of proteins at different taxonomic levels. It provi

des pairwise orthology relationships within OG’s based on analysis of phylogenetic trees. Protein sequences from the selected organisms were extracted and used to compute an all-against-all pair-wise similarity matrix. The comparison uses Smith-Waterman alignments and computational adjustments of the scores, as in BLAST, to prevent spuri-ous hits between low-complexity sequence regions [6]. InParanoid (http://InPara-noid.sbc.su.se (accessed on February 2018))—gathers proteomes of completely sequenced eukaryotic species and calculates pairwise ortholog relationships among them. This tool implements a two-pass BLAST approach that makes use of high-precision compositional score matrix adjustment, but avoids the alignment truncation that sometimes follows [7]. Both tools demonstrated similar results, but we decided to pursue analyses with data re-trieved from STRING, which included a more comprehensive and updated coverage.

CACNA2D1 99.9 67.4 94.1 96.2 95.3 79.3 83.8 94.2 77 84.5 81.8 75 28.3 45.7 43 44.5 42.4 38.9 41.1KCNK2 100 57.5 97.7 98.8 99.5 96 99.5 95.5 95.1 93.9 89 84.6 39.6 35.9 35.8 17.1 39.2 40.5 31.4NR3C2 99.7 67.7 91.4 95.3 96.2 94.8 97.8 94 93 88.8 76.1 63.2 20.5 26 16.1 25.3 21.1 17.2 5.8LPL 100 88.3 96.2 95.6 94.8 96.2 95.6 90 88 81 83.3 71.8 18.8 46 32.4 39.6 40.4 0 42.4PTGS2 99.7 98.5 93 93.2 95 94.2 94.7 91.9 92.5 90.9 86.8 85.2 57.9 62.7 6.4 17.1 33.8 15.4 31PTGS1 94.7 93.7 92.9 94 94 94.8 95.3 88.1 79.5 84.2 84.4 79.1 58.1 55.1 25.5 32.2 33.1 14.2 30.4NR3C1 99.9 97.4 47.9 92.3 95.9 96.3 95.6 89.7 88.9 84.9 75.8 63.4 24.4 29.5 23.3 29.1 25.5 22.7 19.8ADRA1B 97.9 99 54 96.9 87.5 97.7 74.8 85.6 81.7 63 64.2 60.7 44.2 34.9 40.8 33.8 37.4 33 30.1SCNN1G 90 93 92.9 91.9 92.2 91.4 93.6 86.6 87.1 80.4 74.5 33.4 38.7 32 39 33.9 28.9 34.7 35.1SCNN1B 87.9 98.8 90.8 90.9 94.1 91.3 93.9 87.3 87.4 76 75 32.9 38.4 33 38.3 33.7 29.4 33.1 38.6EDNRB 99.4 96.1 77.3 77.5 77.3 76.4 80.7 72 61.3 67.3 60.9 61 36.1 29.8 32.9 33.6 35.7 30.4 27.2DRD5 99 97.1 87.2 87.2 91.2 89 80.9 81.5 47.6 77 73.7 64 45.6 34.6 43.7 39 42.3 36.7 35.1F2R 99.8 98.4 81.2 87.9 88.6 87.4 91.6 82.4 43.6 71 70.2 65.4 35.5 33.7 37.1 29.9 33.8 32.8 38.6WNK4 99.5 89 52.4 88.8 53.4 90.1 83.7 80.9 31.9 53.3 37.3 39 17.5 14.8 22.8 23.2 14.7 32.3 19.3WNK1 99.3 97.7 77.6 77.3 92.3 86.9 84.3 83.5 59.9 72.5 62.2 53.1 9.6 8 36.7 35.5 10.3 35.9 10.1PDX1 99.3 98.6 90.1 89.4 93.6 93 95.4 78.3 62.4 66.8 59.6 55.9 24.7 28.8 37.5 23.3 18.5 28.5 33.1SELE 99.5 97.7 75 81.4 86.1 70.9 88 42.3 72.1 45.4 48.1 43.1 36.8 13.4 32.3 22.5 23.5 23.3 16SCNN1A 62.4 95.9 83.5 82.8 77 75.6 81.1 68.2 67.8 64.1 57.4 35.4 33.8 33.2 35.5 28.4 29.1 31.2 30.3GIPR 60.2 85.7 83.4 83.4 88.8 89.7 90.6 72.8 52.3 53.8 57.3 59.4 45.2 45.1 37.5 37.5 37.4 39.8 24.9HP 81.3 89.9 77.3 76.4 77.1 85.7 77.1 72.4 27.7 30.7 43.1 26.9 43 33.1 25.4 30.1 30.4 28.5 28.8PTGER3 86.8 80.9 74 76.6 44.8 77.7 85.7 65.6 44.3 65.1 68.1 66.4 47.4 31.9 34 34 28.7 0 34.5PTGIR 97.9 97.4 77.2 78.1 88.1 87.4 80.1 61.9 45.3 43.4 57.8 57.2 43.8 33.4 30.4 30.6 31 0 32.8AGT 99 95.5 73.7 71.5 77.4 60 80.6 67.3 62.4 56.2 51.9 46.2 32.2 34.4 34.5 32.4 32.6 0 32.7HSD11B1 100 97.9 7.4 87.7 71.2 91.8 91.9 43.8 70.7 72.8 69.5 60.9 56.2 52.8 49.5 44.1 40.1 43.5 40.9CLCNKB 99.1 91.7 88.8 90 92.4 91.6 87.5 42.2 72.2 73.5 55.3 58.5 47.3 53.5 27.5 32.6 35.1 39.8 50.6CLCNKA 97.2 86.9 89.4 90.8 93 91.7 86.6 42.2 71.8 73.1 54.9 49.8 47.2 52.7 27.5 32.8 35 40.3 51.6LRP8 99.2 97.6 89.4 82.7 87.4 78.2 87.5 64.6 54.1 70.3 60.7 62.9 44.7 35.9 10.8 42.4 44.2 42.2 39.4CMA1 98.8 98.4 80.2 77.3 86.3 80.5 66.4 71.4 64.8 56.7 51.6 54.3 47.2 48.7 10.4 44.5 45.1 39.8 47.7CTSG 98.4 92.2 78.1 77.6 75 72.4 72.6 70 62.6 58 49.8 56.5 45.4 49.8 6 37 14.3 36.4 46.3SERPINA4 99.5 98.8 73.3 59.4 81.2 70 63.1 60.3 66.6 63.9 61.2 54.6 47.4 46.3 47.3 45 44.2 42.1 40.5CYP11B2 79.2 97.4 83.1 83 85.5 84.1 88.1 60.7 66.7 57.3 56.6 48.6 47.2 37.9 43.2 44.5 41.4 43.3 45.9CYP11B1 82.1 95.8 81.5 81.2 84.5 82.1 86.3 60.5 66.3 56.4 58 48 48.9 38.4 42.3 44.6 42.2 42.8 40.6HSD11B2 100 97.3 90.4 88.1 70.6 88.9 73.1 48.9 64 55.9 59.3 54.1 40.3 38.3 37.2 35.2 33.9 40.6 39.7AVPR2 99.7 93 89.8 89.8 89.8 87.6 86.5 54.6 65.4 58 56.3 56.3 46.5 41.1 42.1 34.8 30.8 43.9 35CA4 99.4 91.1 74.4 71.8 83.1 79.6 80.8 43.9 38.2 53.7 46.7 52.5 47.6 43.9 44.8 43.9 36.6 42.9 38.5ADRA2C 54.7 69.6 93.7 93.5 90 58.3 57.7 71.7 37.4 71.5 69.7 65.8 43.4 48.3 39.3 38.4 41.6 45.2 24.7ADRA1D 70.1 46.9 88.6 88.1 62 83.8 47.6 80.1 56.4 60.2 48.9 64 37.7 29.5 39.7 32.7 36.7 33.5 25.4DRD4 70.2 92.6 77 77.3 47.5 48.9 45.7 53.9 43.4 58 52.7 62.9 37.2 34.9 36.5 37.3 37.2 43.9 27.3KLK1 54.2 73.7 78.2 81.7 25.6 85.9 80.9 59.7 59.9 55.8 56.1 56.5 50.8 52.7 39 51.3 47.8 43.4 50.2GOSR2 86.2 96.3 82.5 80.3 82.1 83.4 83 86 0 78.6 76.4 83.2 69.1 65.8 51.3 53.8 0 52.7 55.2RENBP 99.5 95.1 89.1 89.6 90.5 86.1 90.6 81.3 63.8 0 74.8 70.8 67.4 60.5 66.5 0 0 0 0PNMT 99.6 83.1 89.8 86.1 89.8 92.6 78.9 87.2 78 67.3 70.7 53.1 49.5 0 0 0 0 40.3 0MAS1 99.7 99.4 95.4 95.1 45.4 92.9 53.9 92.9 81 74.5 54 44.7 35.4 39.7 37.3 0 0 0 0PTGER2 98.9 98 88.7 91.2 88.9 89.7 81.6 75.3 72.3 39.7 64.5 55 32.4 37.9 32.1 0 0 0 36.8TBXA2R 99.3 90.2 68.8 68.3 77.1 75.9 46.3 70.1 65.1 43.7 58.3 44.4 43.4 30 29.8 0 0 0 0IGF1 99 99 71.9 72.4 88.3 87.8 59.2 61.5 64.3 64.5 62.4 57.1 17.6 26.9 20.8 0 0 0 0PLA2G1B 98.6 96.6 85.1 85.8 89.9 48 87.2 73.2 62 70.3 74.7 65.8 51.2 49.4 56 16.9 12.2 48.1 34.6COMT 99.6 96.3 87.2 87.6 88.9 38 73.4 70.1 70.3 73.1 69.6 65.1 62.9 59.9 51.2 0 0 0 0NPR3 99.4 99.1 93 93 93.7 95.6 96.5 83 40.3 83.4 74 65 17.3 40.2 15 18.6 19.3 21.2 17.7VEGFC 99 96.9 91.2 91.7 94.8 92.2 83.1 73.9 48.8 83.8 73.1 68.1 32.4 0 0 25 0 0 0CACNG1 100 90.1 87.9 90.6 91.1 87.9 94.2 88.8 86.2 71.4 58.1 80.5 22.1 0 0 0 0 0 0NPY 99 100 94.9 95.9 99 99 100 84.5 90.7 91.8 88.7 77.3 0 0 0 0 0 0 0AVP 96.3 60.2 84.5 85.1 91 91.6 68.3 80 73.1 68.3 67.9 61.1 39.7 0 33.2 0 0 0 0APOA1 100 32.5 75.7 80.5 92.5 87.3 87.6 66.7 45.8 66.3 61.3 47.6 0 0 0 0 0 0 0PTHLH 84.1 83.3 0 87.6 93.3 93.8 93.8 87 84.2 80.9 60.3 50 0 0 0 0 0 0 0GNAS 99.5 83.1 31.8 72.4 80.6 33.4 33.7 31.9 30.9 34.1 34.5 34.2 29.2 28.9 30.9 30.6 31.1 28.9 30.7SCNN1D 57.4 66.7 43.3 41 55 53.5 50.5 45.2 35.4 42.6 42.7 28 28.6 29.1 29.1 26.7 25.8 28.1 27.2IGF2 78.8 23.3 70.8 69.5 81.6 67.1 65.8 24.8 28 53.7 54 51.9 25.8 28.2 17.3 0 0 0 0GH2 55.3 57.1 44.5 44.5 44.8 41.6 45.5 42.8 0 40.7 48.7 32.7 0 0 0 0 0 0 0INS 99.1 83.6 87.3 85.5 30.1 81.8 88.2 0 0 68.2 64.6 61.8 13.3 0 0 0 0 0 0SLC14A2 99.2 97.9 90.3 91.6 93.3 88.4 91.9 37.2 34.8 72.5 34.5 19.5 23.2 0 22.2 0 0 0 0LEPR 99.2 92.1 85.8 85.2 91 89.5 90.8 59.4 64.4 64.1 59.3 41.3 22.4 0 0 0 0 0 0CALCA 95.7 92.9 79 79 84.4 70.8 82.3 43.8 60.4 62.2 47 57.1 0 0 0 0 0 0 0EDN2 99.4 97.2 76.4 78.1 72.5 79.4 61.8 66 41.6 56.9 40.1 48.9 0 0 0 0 0 0 0ADM 100 97.3 76.6 72.6 86.5 85.4 55.1 73.4 51.7 64.4 54.4 46.3 0 0 0 0 0 0 0EDN1 98.1 94.8 76.1 77.5 77 78.4 80.8 68 62.2 55.4 53.2 50.7 0 0 0 0 0 0 0GH1 95.4 89.9 76.6 78 78.4 76.7 78.9 74.1 47.8 73.6 66.4 52.2 0 0 0 0 0 0 0GCG 100 95.1 95 93.9 95.6 63.9 95.6 90.7 34.7 77.2 53.2 53.1 0 0 0 0 0 0 0IAPP 97.8 96.6 73.1 75.3 89.9 79.1 87.6 76.4 52.1 48.9 0 45 0 0 0 0 0 0 0GAL 100 75.6 80.6 82.3 76.8 85.4 87 76.6 44.8 72.4 0 69.1 0 0 0 0 0 0 0VEGFB 100 98.6 60.1 90.3 45.5 87.4 54.8 45.3 46.2 39.1 35.9 40.6 18 0 0 0 34.2 0 0APOE 95.9 76 81.2 82.4 82.2 83.9 23.8 62 57 37.5 50 50.9 0 0 0 0 0 0 0VEGFA 21.6 93.2 85.2 84.7 71.3 60.5 50 64 38.4 44.8 29.5 34.2 29.9 0 0 0 0 0 0NPPA 98.7 94.8 85 84.3 86.1 88.2 89.5 78.3 74 50 0 0 0 0 0 0 0 0 0LEP 100 95.8 89.8 91 72.1 90.4 94 80.2 68.9 0 0 0 0 0 0 0 0 0 0NPPC 99.2 98.4 94.4 92.9 96 93.7 74.6 83.1 27.8 0 49 53.8 0 0 0 0 0 0 0APOA2 99 98 74.5 78.4 92 85 90 80 0 0 0 0 0 0 0 0 0 0 0ICAM3 98.9 92.7 35.7 35.1 61.9 72.7 70.5 48 52.4 0 34.5 23.5 20 0 0 0 0 0 0ICAM1 98.3 34.8 64.5 67.2 76.4 69.7 73.4 49.4 52.2 0 35.4 24.6 0 0 0 0 0 0 0KNG1 62 95.7 69.1 67.7 50.8 80.2 82 52.2 12.6 37.2 30.3 23.2 8.9 0 0 0 0 0 0TRH 97.5 93.4 61.9 61.9 83.5 79.5 85.2 51.7 24 44.1 36.1 44.5 0 0 0 0 0 0 0EDN3 99.6 94.1 51.2 58.3 60.2 47.6 49.8 51.9 27.7 31.4 35.7 25.2 0 0 0 0 0 0 0ICAM2 98.9 89.8 71.2 70.7 65.4 61.9 66.9 62.4 20 21.4 21.8 32.9 0 0 0 0 0 0 0BSND 90.9 95 76.9 76.9 82.3 80.7 82 22.8 16.8 0 0 29.6 0 0 0 0 0 0 0APOC4 95.3 89.8 71.7 69.3 68.5 72.4 74 0 0 0 0 0 0 0 0 0 0 0 0APOC2 99 91.1 76.2 76.2 51.9 80.2 81.2 0 58.2 0 0 0 0 0 0 0 0 0 0APOC3 82.1 90.9 70.3 74.7 81 73.3 0 59.2 0 0 0 0 0 0 0 0 0 0 0NPPB 98.5 67.9 0 0 61.7 67.2 67.2 0 46.5 0 45.7 0 0 0 0 0 0 0 0

Conservationro

wIV

row

III

Figure 1. Heatmap displays the hierarchical clustering of similarity percentage scores between human BP genes (vertical axis) andtheir orthologs in 19 organisms (horizontal axis). Green color denotes the more conserved genes across the 19 organisms, aligned at thetop of the map (see conservation scale arrow to the right of map). A phylogenetic tree describing the developmental order is placedabove the map.

2.2. Creation of a Similarity Map and Hierarchical Clustering

The organism-to-protein similarity map enabled us to implement a two-way hierarchi-cal clustering on the similarity matrix scores, also known as hierarchical cluster analysis.

Hierarchical clustering is an algorithm that recursively merges objects based on theirpair-wise distance. Neighboring objects are merged first, while objects farthest apart aremerged last. The ultimate result is a set of clusters, where each cluster is distinct fromeach other cluster, and the objects within each cluster are considerably similar to eachother. The main output of hierarchical clustering is a dendrogram, which shows thehierarchical relationship between the clusters. This calculation provided us with clusters oforganisms on one axis, and of BP proteins in the other axis, suggesting many interestingconclusions relating to the evolutionary order by which the circulatory system has evolved.Hierarchical clustering was performed using the Broad Institute Morpheus tool (https://software.broadinstitute.org/morpheus/ (accessed on May 2018)), implementing Euclidiandistance metric and the Complete linkage method on both axes, emphasizing the maximumof the between-cluster dissimilarities [9].

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2.3. Functional and Tissue Expression Enrichment Analyses

For a detailed in-depth analysis, we chose five organisms that essentially reflect theentire span of complexities of the 19 organisms that appear in the heatmap—Homo sapiens,Mus musculus, Danio rerio, Drosophila melanogaster, and C. elegans.

For each organism, we investigated the resulting clusters of orthologous proteins thatwere obtained from the hierarchical cluster analysis. These analyses consisted of functionalGO term annotations (biological processes) as well as organ/tissue expression enrichmentpatterns. This was carried out by a set of tools, some of which specialize in human andmouse data, while others focus on other organisms. Table 1 describes the list of the toolswe utilized.

Table 1. Tissue enrichment and functional annotation tools utilized in this work.

Tool Link to URL Content

GeneAnalytics https://ga.genecards.org/ (accessedon February 2018)

Expression-based matching algorithm, provides geneannotations and enrichment in each specific tissue or organ inhuman or mouse.

GeneOntology geneontology.org/ (accessed onFebruary 2018)

Provides a computational representation regarding thefunctional enrichment of genes (proteins produced by genes)from a variety of organisms from human to bacteria.

g:Profiler https://biit.cs.ut.ee/gprofiler/gost(accessed on October 2018)

Finds biological categories enriched in gene lists, functionality,conversions between gene identifiers and mappings to theirorthologs.

TissueEnrichhttps://tissueenrich.gdcb.iastate.edu/(accessed on October 2018)

Defines tissue-specific genes using RNA-Seq data from theHuman Protein Atlas, GTEx, and mouse ENCODE.

Bgee https://bgee.org/ (accessed onNovember 2018)

A database used to retrieve and compare gene expressionpatterns in multiple animal species.

TSEAhttp://genetics.wustl.edu/jdlab/tsea/(accessed on October 2018)

Tissue specific expression analysis for human and mouse.

MouseMinehttp://www.mousemine.org/mousemine/begin.do (accessed onNovember 2018)

Provides queries for anatomy and gene ontology enrichmentsas well as other analyses for mouse.

ZebrafishMinehttp://www.zebrafishmine.org/begin.do(accessed on November 2018)

Searches for zebrafish related biological data including genes,proteins, tissues, and more.

ZEOGShttp://zeogs.scienceontheweb.net/index.html (accessed on November2018)

Predicts sites of preferential expression of zebrafish gene sets.

FlyMine http://www.flymine.org/ (accessedon November 2018)

Integrates many types of data for drosophila and otherorganisms. runs flexible queries for genes, proteins, ontologyterms, and tissue enrichments.

FlyBase http://flybase.org/convert/id(accessed on December 2018)

Curates and organizes a diverse array of genetic, molecular,genomic, and developmental information in drosophila.

WormBasehttps://www.wormbase.org/tools/enrichment/tea/tea.cgi (accessed onDecember 2018)

Provides information concerning the genetics, genomics, andbiology of C. elegans and related nematodes.

WormExphttp://wormexp.zoologie.uni-kiel.de/wormexp/ (accessed onDecember 2018)

Integrates all published expression data for C. elegans, runsflexible queries for gene enrichment analyses on selectedgene sets.

GO term enrichment tools (primarily GeneOntology) are used to evaluate characteris-tics of sets of genes by comparing the frequency of GO terms in the sample gene set withthe frequency of the same set of GO terms in a reference set, usually a whole genome. Thetools apply the binomial test to identify over or under-represented terms in the sample geneset compared to the reference genome set. The default parameters also apply a Bonferronicorrection for multiple comparisons. A similar principle is implemented in the tissueenrichment analyses tools utilized for the different organisms, as specified in Figure 2.

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Since GO is roughly hierarchical, with “child” terms being more specialized than their“parent” terms, we were compelled to elect the terms in the 4–5 orders of the hierarchy ineach cluster-species, in order to obtain broad definitions that enable comparison betweenthe vast span of organisms in the analysis. As a control, the same GO term functionalanalysis was performed 30 times on randomly selected sets of 200 genes. This randomanalysis has not yielded any significant enrichment results, emphasizing the validity of theresults for the set of blood pressure genes.

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Figure 2. Demonstrates a summary of functional analysis results overlaid on heatmap of Figure 1. In each box, results

pertain to the enriched biological processes for the respective genes-organisms of the cluster. For ease of interpretation,

the colored text represents biological process families, reflecting their distribution throughout the map. For full functional

analysis results please refer to Supplementary Table S1.

Tissue and organ expression enrichment analyses reveal that by and large genes in

column I clusters are abundant in the kidney and somewhat in the adrenal gland, pan-

creas, and liver. In column II, gene expression is evident mainly in the kidney and nervous

system, and slightly in the pancreas. This expression pattern changes significantly for col-

umn III, where gene expression is clearly noticeable exclusively in nervous system tissues.

Figure 3 demonstrates a summary of tissue expression for the various organisms investi-

gated. For full expression analysis results please refer to Supplementary Table S2. Alto-

gether, the functional and expression results of the analysis in zebrafish display the sum-

mation of results of analyses in the higher and lower order species. This reflects the unique

spot of zebrafish in ontogeny that well supports the rationale to exploit zebrafish as a

model organism in biological research [10].

Figure 2. Demonstrates a summary of functional analysis results overlaid on heatmap of Figure 1. In each box, resultspertain to the enriched biological processes for the respective genes-organisms of the cluster. For ease of interpretation,the colored text represents biological process families, reflecting their distribution throughout the map. For full functionalanalysis results please refer to Supplementary Table S1.

3. Results

The heatmap representing the results of the two-way clustered similarity map ispresented in Figure 1. This map has yielded three clusters in the organism axis (columns Ito III) and four clusters in the gene axis (rows I to IV).

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The order by which the organism axis has aligned as a result of the clustering processis consistent with the natural known developmental order: (1) column I—high complexitymammals—from Pan troglodytes to Equus caballus, (2) column II—medium complexity—from O. anatinus to Danio rerio, (3) column III—low complexity—B. floridae to T. adhaerens.This order, presented in Figure 1 as a phylogenetic tree, is consistent with the devel-opmental order of the blood circulatory mechanism in respect to the affiliated genes;4—chamber heart system in high-order mammals and avians, 3 and 2—chamber heartin fish and amphibians, diffusion and hemolymph-based systems in insects and simplemulti-cellular organisms.

The gene axis was aligned in four clusters, highlighting the more conserved genesacross the 19 organisms at the top of the map (row I). The least conserved genes are shownat the bottom of the map (row IV), depicting a majority of zero value similarity scores,due to absence of orthologues genes in low complexity organisms. It is also amenable todiscern that row I harbors genes associated with more basal functions, as ion transport,which is assumed to be ancient, hence appears in all organisms; while genes that are linkedto a more complex regulatory functions, as control of extracellular volume or endothelialvasoconstriction appear in row IV, hence emerge in more recent organisms only.

Functional gene ontology (GO) term annotation (a statement regarding the functionof genes) analysis demonstrates that homeostasis is the most prevalent biological processacross most heatmap clusters. Transport processes are significant in row I, while bloodcirculation and vasculature structure are prevalent in columns I and II. Signaling processesappear most in row IV of columns I and II, rows I—III of columns II and III. Responseto stimulus is mainly displayed in column II and slightly in columns I and III. Figure 2demonstrates a summary of the functional analysis results for the various organisms inves-tigated, displaying the enriched biological processes for each cluster. For full functionalanalysis results please refer to Supplementary Table S1.

Tissue and organ expression enrichment analyses reveal that by and large genes incolumn I clusters are abundant in the kidney and somewhat in the adrenal gland, pancreas,and liver. In column II, gene expression is evident mainly in the kidney and nervous system,and slightly in the pancreas. This expression pattern changes significantly for column III,where gene expression is clearly noticeable exclusively in nervous system tissues. Figure 3demonstrates a summary of tissue expression for the various organisms investigated. Forfull expression analysis results please refer to Supplementary Table S2. Altogether, thefunctional and expression results of the analysis in zebrafish display the summation ofresults of analyses in the higher and lower order species. This reflects the unique spotof zebrafish in ontogeny that well supports the rationale to exploit zebrafish as a modelorganism in biological research [10].

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Figure 3. Demonstrates a summary of expression analysis results overlaid on heatmap of Figure 1. In each box, results

pertain to the enriched organ/tissue expression for the respective genes-organisms of the cluster. For ease of interpretation,

colored text represents tissue/organ types, reflecting their distribution throughout the map. For full functional analysis

results please refer to Supplementary Table S2.

4. Discussion

Regulation of BP is a complex systemic mechanism due to numerous physiological

elements, involving: pressure-volume regulation, which is tightly related to pressure-na-

triuresis [11], rapid control of vessel resistance by the central nervous system (CNS), spe-

cifically modulated by both sympathetic and parasympathetic nervous system—the two

branches of the autonomic nervous system (ANS) [12], neurotransmitters (e.g., noradren-

aline (NA), adrenaline) and hormones (e.g., angiotensin) as well as the long-term activity

of the renin-angiotensin-aldosterone-system (RAAS). These mechanisms are efficient at

maintaining BP within a normal physiological range at Systolic BP of <120 mmHg and

Diastolic BP of <80 mmHg [13].

This study examined a collection of 198 human genes related to BP regulation in the

biological processes and functional prisms, as well as gene expression in organ and tissue

perspective. In this context, it is unavoidable that the question of homeostasis will arise.

This observation, in conjunction with the orthology analysis we performed in 19 target

organisms along the phylogenetic tree, evokes numerous interesting conclusions and fur-

ther questions.

Homeostasis sustains the “internal milieu” of an organism’s cells, tissues, organs,

and whole body, within limits that are compatible with survival. This internal milieu re-

flects the composition of the primordial ocean, first and foremost its electrolyte and water

Figure 3. Demonstrates a summary of expression analysis results overlaid on heatmap of Figure 1. In each box, resultspertain to the enriched organ/tissue expression for the respective genes-organisms of the cluster. For ease of interpretation,colored text represents tissue/organ types, reflecting their distribution throughout the map. For full functional analysisresults please refer to Supplementary Table S2.

4. Discussion

Regulation of BP is a complex systemic mechanism due to numerous physiologicalelements, involving: pressure-volume regulation, which is tightly related to pressure-natriuresis [11], rapid control of vessel resistance by the central nervous system (CNS),specifically modulated by both sympathetic and parasympathetic nervous system—thetwo branches of the autonomic nervous system (ANS) [12], neurotransmitters (e.g., no-radrenaline (NA), adrenaline) and hormones (e.g., angiotensin) as well as the long-termactivity of the renin-angiotensin-aldosterone-system (RAAS). These mechanisms are effi-cient at maintaining BP within a normal physiological range at Systolic BP of <120 mmHgand Diastolic BP of <80 mmHg [13].

This study examined a collection of 198 human genes related to BP regulation inthe biological processes and functional prisms, as well as gene expression in organ andtissue perspective. In this context, it is unavoidable that the question of homeostasiswill arise. This observation, in conjunction with the orthology analysis we performed in19 target organisms along the phylogenetic tree, evokes numerous interesting conclusionsand further questions.

Homeostasis sustains the “internal milieu” of an organism’s cells, tissues, organs,and whole body, within limits that are compatible with survival. This internal milieu

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reflects the composition of the primordial ocean, first and foremost its electrolyte and waterbalance [14,15]. Homeostasis in homeothermic land-dwelling organisms is profoundlydeveloped since their fluid balance, blood pH, oxygen tension and particularly their bodytemperature must be maintained within tight boundaries amid all conditions of their lifecycle and in all their habitats. Homeostasis will tend to stabilize BP, maintaining it at asteady state.

The term stress defines any stimulus or succession of stimuli of such magnitude asto tend to disrupt the homeostasis of the organism. When mechanisms of adjustment failor become incoordinate or disproportionate, the stress should be referred to as an insult.The response of the organism to stress depends largely on biochemical and physiologicalhomeostatic mechanisms [16]. One of the most important mechanisms for maintaininghomeostasis is the negative feedback system; if a physiological disturbance occurs, thebody will counteract the disturbance via a negative feedback mechanism and attempt toreturn the body to its normal steady state.

The more complex the organism, the more complex its adaptation to the ever-changingenvironmental conditions. Biological systems may undergo functional plasticity in theeffort to adapt. This is the mainstay of evolutionary progression [17].

The results of this study may elucidate the diversity of the modulating responsemechanisms during phylogenesis: from the response of primitive organisms to physicalstimuli (such as osmotic or heat stressors) to the complex endocrine and neurobehavioralstress response to both physical, mental, and cognitive challenges in human.

4.1. The Function of the CNS in the Maintenance of Homeostasis

The CNS is the organ that orchestrates the response to stressful stimuli through theautonomic, neuroendocrine, and immune systems, as well as through neurobehavioralresponses such as fight-or-flight response. Adaptation to stress and to changing envi-ronmental conditions involves neural and humoral mediators: neurotransmitters andneuromodulators, hormones and cytokines of the immune system. The goal of this adapta-tion is to maintain homeostasis and promote survival of the organism [18].

The stress responses include primarily the activation of the mesolimbic system, thehypothalamo-pituitary-adrenal (HPA) axis and the ANS. The dopaminergic (DA-ergic)activity in the septo-hippocampus is a mainstay of the mesolimbic stress response, due to itsclose relationship with the hypothalamus (autonomic control), midbrain reticular formation(arousal) and multiple sensory pathways (attention). A DA-ergic pathway originates inthe ventro-medial tegmentum of the midbrain and projects to the lateral septal nuclei withabundant indirect interconnections with the cholinergic nucleus of the medial septum,forming a final common pathway of the neural, psychic and endocrine stress response.Indeed, time honored studies corroborate the major importance of catecholamines in theANS and of the glucocorticoids (GCs) in the adrenal cortex, as well as the CNS septo-hippocampal DA-ergic and cholinergic systems in the stress response. These complexmechanisms of adaptation are critical in the control of homeostasis [17,19]. Additionally,the mesolimbic pathway transports dopamine (DA) from the ventro-medial tegmentumto the nucleus accumbens and amygdala. The nucleus accumbens is found in the ventralmedial portion of the striatum and is believed to play a role in reward, desire, and theplacebo effect. The amygdala is a key component of the limbic system and is associatedwith emotion. Unabated DA-ergic stimulation (the absence of negative feedback) has beenpostulated to be associated with disorders such as binge eating or drug addiction [20,21].

The neuroendocrine system integrates the functions of two major control systems: thenervous system and the endocrine system. Thus, both internal and external fluctuationsare monitored by the nervous sense organs, the neural signals of which are processed bythe CNS and converted to endocrine outputs.

Furthermore, associated with the neuroendocrine system are distinct circumventricu-lar fenestrations in the blood-brain barrier, mainly the chemoreceptor trigger zone (CTZ)in the area postrema of the medulla oblongata. The CTZ allows hormones and neurotrans-

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mitters to enter directly from the blood to the cerebrospinal fluid (CSF), hence enabling thefeedback mechanisms with the neurosecretory system itself.

The hypothalamus plays a principal role in the neuroendocrine control system, whichtogether with its connections to the pituitary gland comprise the hypothalamic pituitarysystem—a neurosecretory system that produces releasing hormones (or releasing factors),emitting them to the pituitary gland. Important pituitary hormones are the adrenocorti-cotropic hormone (ACTH), which controls the adrenal cortex as well as growth hormone-inhibiting hormone (GHIH), which regulates the endocrine system and affects neurotrans-mission. Moreover, upper neurons of the ANS are located in the hypothalamus and controlthe adrenal medulla, an additional crucial endocrine gland. These roles of the CNS in themaintenance of homeostasis are dynamic, empowering the organism to function efficiently.

As seen in the Results section and in Figure 3, the adrenal gland and pancreas arestrongly pronounced in human and mouse in rows II–IV, while the CNS and brain manifestrows I–III in fruitfly and roundworm. Interestingly, in the zebrafish gene enrichment inthe CNS and the brain are prominent in all clusters, whereas the pancreas appears in rowsIII–IV, emphasizing the clear homology of these anatomic properties among the variousspecies. This may suggest that organs that are enriched in expression of highly conservedgenes across species reflect shared ancestry.

4.2. The Role of the Adrenal Glands

The adrenal gland comprises two endocrine tissues that differ in function and em-bryonal origin: The adrenal cortex, a steroidogenic tissue that evolved from the coelomicepithelium; and the adrenal medulla, a catecholamine producing tissue composed of chro-maffin cells. The medulla derived from the neuroectoderm, which migrated to the adrenalgland blastema.

The secretion of the adrenal cortex hormones is controlled by ACTH in a closed loopfeedback system. It produces numerous steroids secreted in widely varying amounts; theseare classified as mineralocorticoids (MCs) and GCs including sex steroids. The main MCis aldosterone, which is the most potent steroid affecting active transport of sodium ionsacross membranes and thus is crucially important in maintaining electrolyte balance andas a result, in BP homeostasis. Cortisol is the principal GC in human and most mammalianprimates and is quantitatively the main secretory product of the adrenal cortex.

The medulla is basically a modified sympathetic ganglion that converts tyrosine intoDA, NA, and adrenaline, which are secreted in postsynaptic response to direct neuralinputs. These inputs originate in the CNS (cortical cerebral areas) and are transmitted tothe adrenal medulla via cholinergic preganglionic sympathetic neurons [22].

As noted, the primordium of the adrenal cortex coincides with the mesonephricblastema. It develops within the kidney in fish and on the kidney in amphibians. Thekidneys of both fish and amphibians are of mesonephric origin [23].

In fish, chromaffin and cortical adrenal cells are not placed in proximity, but ratherintermingle. The interrenal cells, that correspond to the adrenal cortex in mammals, fromwhich GCs and MCs are secreted, do not encapsulate the chromaffin cells, which are thefunctional equivalent of the adrenal medulla. NA is the main catecholamine stored by thechromaffin cells, albeit variable quantities of adrenaline can also be present.

Amphibians show a closer relationship between cortical and chromaffin cells. Theadrenals of amphibians lay on the ventral side of the kidneys, as the mesonephros—theanalogue of the mammalian kidney, preserving the contact between the two organs duringontogenesis [24].

In mammals, where the evolution of the excretory system leads to the developmentof the metanephros, adrenal cortical cells completely surround the chromaffin cell mass—namely the medulla—and are grouped together to form the adrenal gland. Traces for thiscan be seen in our detailed expression analysis results (Supplementary Table S2), whererow I–II are expressed in the adrenal glands of mammals, and in chromaffin cells andmesonephros of the zebrafish.

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4.3. Nervous Regulation of the Cardiovascular System

The circulation is regulated partly by intrinsic and local mechanisms in each tissueand prominently by the nervous system, the action of which is extremely rapid and com-prehensive. All the vascularized tissues in vertebrates are supplied with sympathetic nervefibers. Sympathetic stimulation to the arterioles increases the resistance, thus changing theblood flow through the tissues. Sympathetic stimulation of the larger vessels decreasestheir blood volume. In a similar manner, cardiac output and BP are remarkably increasedby increased sympathetic activity. Both heart rate and stroke volume (the two componentsof cardiac output) alongside vascular resistance are the main determinants of BP.

The internal environment is monitored by sense organs and organelles: chemore-ceptors sensitive to the partial oxygen pressure in the arterial blood, mechanoreceptorssensitive to blood pressure, and chemoreceptors within the central nervous system itselfsensitive to hydrogen ion concentration or to various hormones. The input from the senseorgans and organelles is transmitted to the CNS, in which it is processed and from whichthe appropriate outputs are sent to the effectors—muscles and glands. Above all, the vaso-motor center (VMC)—in fact a network of neurons within the medulla oblongata, regulatesBP and other homeostatic functions by a dual action: vasoconstriction and vasodilation.

Tonic adrenergic discharge to the arterioles sustains arterial pressure, while fluctua-tions in this tonic discharge constitute the mechanism affecting the feedback regulation ofBP carried out by the carotid sinus (baroreceptor) and the carotid body (chemoreceptorthat modulates the cardiovascular and respiratory systems via sympathetic tone). Theeffects of this discharge are of substantial importance in the preparation of the organism toendure emergency.

4.4. Catecholamines and BP Homeostasis

Dopamine (DA), noradrenaline (NA), and adrenaline (known as catecholamines)are physiologically active molecules, acting both as hormones and neurotransmitters,crucial for the sustainment of homeostasis via the ANS. This work considered DA receptorgenes (DRs), (DR1-like subtype—DRD1, DRD5, and DR2-like—DRD2, DRD3, DRD4)and adrenergic receptor genes (ARs), (adrenoceptors type alpha—ADRA1A, ADRA1B,ADRA1D, ADRA2A, ADRA2B, ADRA2C, and beta—ADRB1, ADRB2, ADRB3) appearingin rows II–III, all which play key physiological roles in the nervous and cardiovascularsystems, specifically in BP regulation.

Almost all vasomotor nerves are adrenergic. The alpha-adrenoceptors are preeminentin innervation of vascular smooth muscles and also in the lower urinary tract. In themyocardium, beta1-adrenoceptors predominate and stimulate the rate and contractility.In both cases, neurotransmitter NA exerts its physiologic effects by binding to alphaARs, whereas adrenaline reacts with both alpha and beta ARs, causing vasoconstrictionand vasodilation, respectively. Generally, it is the alpha1-AR subtype, which is situatedpostsynaptically in smooth muscles, that causes vasoconstriction of blood vessels whenstimulated. Sympathetic overactivity in hypertension results in an excess stimulation ofpostsynaptic alpha1 ARs [25].

DA, a major neurotransmitter in both CNS and PNS holds an essential function in thehomeostatic control of BP, by regulation of vascular smooth muscle contraction, epithelialsodium transport, and reactive oxygen species (ROS) production. It is considered a majorplayer in homeostatic regulation of extra-cellular fluid (ECF) volume and BP due to the vasteffect it induces on renal hemodynamics as well as humoral agents such as catecholamines,aldosterone, renin, vasopressin and endothelin (e.g., DA inhibits NA release and acts as avasodilator at normal concentrations by activation of D2 receptors; decreases aldosteronesecretion via activation of D3 receptors, while significantly enhancing renin secretion byactivation of D1 receptors).

DA also adjusts sodium and fluid intake by means of activities within the gastroin-testinal tract and CNS, primarily by regulation of the cardiovascular control centers in

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the brain stem [26]. Each DA receptor subtype participates in BP regulation by specificmechanisms for the subtype; DRD2 and DRD5 act within the CNS and PNS.

The mesolimbic DA system is implicated in diurnal profiles of the mean BP. A cleardip in the mean BP and heart rate occurs during the resting period (e.g., nocturnal dipin human) [27]. An increase in arterial BP can be seen during the transition from non-REM to REM sleep, displaying phasic surges throughout REM sleep that derive fromthe physiological phase of paradoxical sleep. Furthermore, the mesolimbic DA system isinvolved in the increases in REM-associated BP fluctuations [28], a homeostatic adaptationto the fluctuating states of the different sleep and arousal phases. Since the mesolimbic DAsystem surges are involved in REM-associated increases in BP, the homeostatic effect is theresult of a greater ability to deal with emotional stress. The pineal gland translates lightsignals received by the retina to the rest of the body, for example through the synthesis ofthe hormone melatonin, which is produced and released at night and helps to regulate thebody’s metabolic activity during sleep. NA is involved in regulating this synthesis andrelease of melatonin in the pineal gland. DA through ADRA1B-DRD4 and ADRB1-DRD4receptor heteromers inhibits the effects of NE, resulting in a decrease in the production andrelease of melatonin [29].

Although there are two classes of DA receptors, DR1-like and DR2-like, the natriureticeffect of DA is primarily mediated by DR1-like receptors [30]. During sodium loading,DR2-like receptors may contribute to the natriuresis [31]. Of the three DR2-like receptors itis likely that it is the DRD3 receptor that interacts with the DRD1 receptor because it is themajor DR2-like receptor expressed in the renal proximal tubule and the thick ascendinglimb of Henle [32]. The DRD1, DRD3, DRD4 receptors interact with the renin-angiotensinsystem (RAAS), affecting epithelial transport and control of secretion of multiple humoralagents and their receptors [33].

From the comparative perspective, our results show a high degree of conservation(>60%) for catecholamines in vertebrate species analyzed, especially in mammals, as wellas in invertebrates such as C. elegans, Drosophila melanogaster, and Ciona intestinalis (~45%).

The vertebrate DR1-like receptors are most closely related to the DOP1 group membersof C. elegans and Drosophila melanogaster, both responsible for upregulation of intracellularcAMP in the presence of DA. The vertebrate DR2-like receptors share the most homologywith the C. elegans CeDOP2 and the Drosophila melanogaster DD2R, sharing a similar responsein the decrease of intracellular cAMP levels when treated with DA [34]. In Drosophilamelanogaster, expression patterns of DOP1 (an ortholog of the vertebrate D1-type DAreceptors) were observed in mushroom body neurons, subesophageal ganglion, and inunpaired abdominal ganglia [35], while DD2R is expressed in the larval and adult nervoussystems, and in cell groups that include peptidergic neurons [36]. In C. elegans it was shownthat CeDOP1 is expressed in mechanosensory neurons, motor neurons and the ventral nervecord, as well as sensory support cells, which are glial-like cells that surround the sensilla(simple invertebrate sense organs that may take the form of a hair or bristle) [37], whileCeDOP2 is expressed in DA-nergic neurons, suggesting it may act as an autoreceptor [38].

4.5. The Kidney’s Role in Maintaining Homeostasis

Notably, genes in clusters of rows I–III are expressed in the kidney of zebrafish, mouse,and human. Their functional analysis demonstrated that homeostasis and transport are theprimary biological processes that characterize these clusters, while signaling and stimulusare secondary.

The cells of the kidney contain many specialized ion channels and transporters, whichact in concert to regulate volume and ionic concentration by absorption or secretion of ionsinto the urine [39]. The ion channels and transporters play essential roles in organelle towhole organism function. These roles range from regulation of cell volume, membraneexcitability and pH, to control of systemic salt and water balance and behavior [40].

Our work specifically relates to several prominent gene families—solute carriers (pri-marily electroneutral potassium chloride cotransporters, glucose transporters, bicarbonate

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transporter proteins), calcium voltage-gated channels, sodium channels, epithelial chlo-ride voltage-gated channels, potassium voltage-gated channels, DRs, and ARs, which areconserved across most species examined (similarity > 50%).

It is especially important to underscore the largest gene family we analyzed in thiswork (21 members)—Solute Carriers (SLCs), which are responsible for the regulation ofvarious types of substances over the cell membrane. They typically rely on an ion gradientover the cell membrane as a mechanism for transportation [41]. Interestingly, all SLCs thatare found in human are also found in C. elegans and Drosophila melanogaster and even somein T. adhaerens (SLC8A2, SLC12A3, SLC12A4, SLC4A4, SLC6A2 >50% similarity), indicatingthat this superfamily is ancient and was present before the divergence of Bilateria (animalswith bilateral symmetry).

Another noteworthy gene family we analyzed is the voltage-gated ion channel super-family, comprising calcium, potassium, and sodium ion channels. As can be seen from theheatmap in Figure 1, KCNJ1, KCNJ11, KCNJ6 are well conserved (>45%) in all 19 species,including T. adhaerens, along with CACNA1C, CACNA1H, CACNA1A, CACNA2D1 (>40%)in all 19 species. Yu et al. suggested that this family has evolved from a bacterial ancestorchannel and accumulated regulatory domains for ligand binding in the course of evolution.The resulting signaling mechanisms control most aspects of cell physiology, complex pro-cesses in the brain and movements of muscles, all of which allowed the development ofcomplex multicellular organisms [42].

Multicellular organisms control their internal environment by altering either theelectrolytes concentration (osmolality) or the extracellular volume, maintaining the acid-base balance. In mammals, the main site of this complex regulation is the kidney.

The role of human kidney is maintaining the fragile equilibrium within the internalmilieu, namely the ECF and the intra-cellular fluid (ICF). From the evolutionary pointof view, the human kidney evolved from primitive mechanisms of the ionic and osmotichomeostasis in fish. The data presented in this work provide evidence that the genes thatare implicated in these mechanisms appear in fish, but not in earlier stages of phylogeny.

The ion composition of the ECF is essentially identical in all animal species (includ-ing fish, amphibians, reptiles, and mammals) where the kidneys are utilized to regulatehomeostasis [15]. Since salt concentration of the ocean increased over time, saltwater fishbegan to contain hypo-osmolar internal milieu and constantly exhausted water to thehyperosmolar environment. To endure this challenge, saltwater fish consume considerableamounts of water [43]; hence the acquired capability of fish to actively preserve the internalmilieu is remarkable.

The zebrafish pronephros, which consists of two nephrons with glomeruli, con-tains two tubules that are analogous in many ways to the segments of the mammaliannephron [44]. The proximal straight segment of this nephric tubule displays a brush border-like columnar epithelial cells [45]. This segment plays a crucial role in reabsorption ofelectrolytes and small molecules through a glomerular filtration barrier [46]. This is thephysiological principle that underlies the mammalian renal function.

Kidney functions are achieved both independently and in conjunction with otherorgans, particularly the endocrine system via various endocrine hormones, including,among others: renin (REN), angiotensin II, aldosterone, antidiuretic hormone (ADH),and two atrial natriuretic peptides (NPPA, NPPB) which are evolutionarily conserved.These natriuretic peptide hormones are secreted by atrial myocytes in response to stretch,angiotensin II stimulation, endothelin, and sympathetic (beta adrenergic) stimulation [47].

Blocking ion channels is an important part of anti-hypertensive pharmacotherapy.This is exemplified by the commonly used drugs—nifedipine, amlodipine, diltiazem, andverapamil which block the voltage-gated calcium channels of vascular smooth muscle cells,thus lowering BP; and amiloride that blocks the epithelial sodium channels at the distalconvoluted tubule and the collecting duct, thereby inhibiting sodium-potassium exchange,while lowering BP independent of aldosterone [48,49].

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4.6. Sensing the Environment in Order to Maintain Homeostatic Balance

An extensive network of sensory organs and organelles play an important role inmaintaining homeostatic balance and monitoring the internal environment. These include:mechanoreceptors sensitive to BP, chemoreceptors that react to the partial oxygen pressurein the arterial blood and chemoreceptors within the CNS, which are sensitive to pH andto numerous hormones. Ion channels in the hypothalamus gauge the ECF osmolalityand sodium concentration. Baroreceptors situated in numerous circulatory beds alongwith ion channels and various receptors within the distal tubule in the kidney nephron,regulate blood volume. The input from the sense organs and organelles is transmittedfor processing to the CNS, from which appropriate outputs are forwarded to the effectortissues—muscles and glands. The volume effectors are the renin–angiotensin–aldosteronesystem (RAAS), alongside sympathetic nerve activation (SNA) as well as glomerular fil-tration rate and physical gradients along the nephron. Homeostatic control mechanismsmaintain the balance between fluid gain and fluid loss. Body water homeostasis is mainlyregulated through fluid ingestion. Sensory osmoreceptors, found primarily in the hypotha-lamus, detect changes in plasma osmolarity and contribute to fluid-balance regulationin the body. If the body is fluid deficient, increased plasma osmolarity is sensed by theosmoreceptors. When osmoreceptors detect high plasma osmolarity (a sign of low bloodvolume/dehydration), they send signals to the hypothalamus, which creates the sensationof thirst. This result also triggers an increase in ADH secretion, causing fluid retention bythe kidneys and urine output to be reduced. Macula densa cells in the nephron’s ascendingloop of Henle are another type of osmoreceptor. If the macula densa is stimulated byhigh osmolarity, the juxtaglomerular apparatus (JGA) releases renin into the bloodstream.The subsequent production of angiotensin II acts on the hypothalamus to cause thirst.Angiotensin II causes vasoconstriction and aldosterone release. Aldosterone increases ex-pression of the nephron’s ATPase pumps resulting in increased water reabsorption throughsodium cotransport [50].

4.7. RAAS Evolution

The RAAS is the primary volume-regulating pathway in mammals, acting as the cen-tral controller of blood pressure homeostasis in humans. The renin-angiotensin-aldosteronesystem emerged approximately 450 million years ago, in the Paleozoic era, as marine or-ganisms moved to the land and endured a strong selection pressure to conserve salt andmaintain volume homeostasis, which were crucial for survival [51]. In this work we haveexamined the sequence homologs for 20 human genes associated with RAAS pathwayin 19 model organisms. Interestingly, homologs for all genes were present in all speciesfrom Danio rerio (Zebrafish) up to Pan troglodytes (Chimpanzee), with lowest similarityscore of 46% for angiotensinogen (AGT). Since our data do not provide evidence for thepresence of AGT homologs prior to Danio rerio, and due to its central role in the RAAS, thiscould indicate that the pathway emerged approximately with the appearance of zebrafish.This observation, in conjunction with the fact that zebrafish is the taxon with the mostprimitive juxtaglomerular apparatus [52], agrees with the physiological evidence regardingits emergence in bony fishes [53]. Our work reveals several orthologs for ACE and ACE2from B. floridae to T. adhaerens, representing primitive chordates, as well as insects andmulticellular organisms, which lack most RAAS components and are characterized byan open circulatory system. No ortholog sequence was found in the C. elegans, a factthat challenges the advent and initial roles of the enzyme. Recent genomic data revealedthe presence of orthologs in yet additional remote phyla, as placozoa and proteobacteria,suggesting that it emerged early in evolution. Thus, by and large, ACE exists from bacteriaup to mammals, exhibiting conserved features such as structure, as well as molecular andbiochemical characteristics. In higher order organisms, ACE exerts its effect mainly in thecapillaries of the lungs, whereas the evolution of the respiratory system was a key step inthe transition of the animal world from marine life to land habitation. Characteristics of

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mammalian ACE could therefore be the outcome of an extended course of evolutionaryspecialization of an ancient protease, the functions of which are not yet known [54].

Adrenal steroids, both MCs and GCs, influence almost all aspects of vertebrate devel-opment, as well as regulation of an extensive selection of physiological processes, includingdevelopment, differentiation, reproduction, and homeostasis [24]. Their receptors belongto the nuclear receptor super-family of proteins, which are ligand-activated transcriptionfactors, implicated in steroid activity in a myriad of physiological, morphological, andbehavioral processes [55].

GRs and MRs encoded in human by the NR3C1 and NR3C2 genes respectively, arerepresentatives of the two principal functional families of corticosteroid receptors in ver-tebrates. These have not been found in invertebrates, as confirmed in our work, whereorthologs for NR3C1 and NR3C2 appear first only in Danio rerio with similarity >63%. TheMR and the GR are known to have descended from the early corticosteroid receptor [56],which first appeared in jawless fish, at the base of the vertebrate lineage, while distinctMR first appeared in cartilaginous fish [57]. Interestingly, the ancient GR evolved throughduplication, to develop into a GR due to two gene mutations [58]. It is hypothesized thatthis evolutionary process would have alleviated the difficulty to adapt to blood pressureelevation caused by increased gravitational effects of land habitation [59].

5. Conclusions

To conclude this study, it appears that the genes that are involved in BP regulationare primarily implicated in mechanisms associated with the maintenance of homeostasis.Evidence for this can be seen in the conserved genes across the entire range of organismsinvestigated, performing functions that are notably related to homeostasis, e.g., ion trans-port, response to stimulus and more, as well as expression patterns in tissues and organsthat support homeostatic processes in the respective species.

This work is merely an attempt to cover the broad scope of the evolutionary dissectionof the genetic machinery behind BP regulation; nonetheless it highlights the importance ofthe comparative approach that presupposes that the comprehension of our evolutionarypast can highly enlighten our understanding of the genetic background of common disease.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/biomedicines9050469/s1, Table S1: GO term biological processes enrichment per cluster genesand organisms, Table S2: tissue/organ enrichment per cluster genes and organisms.

Author Contributions: Conceptualization, A.B. and R.U.; methodology, A.B. and R.U.; formalanalysis, A.B.; investigation, A.B.; resources, R.U.; data curation, A.B.; writing—original draftpreparation, A.B.; writing—review and editing, Y.F. and R.U.; visualization, A.B.; supervision, Y.F.and R.U.; project administration, A.B. All authors have read and agreed to the published version ofthe manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Data supporting reported results of this study can be found in publiclyarchived datasets as specified in links throughout the manuscript.

Acknowledgments: Special thanks to John Moult for constructive discussions and a critical perspec-tive. The authors are also indebted to Gil Kotton, Tirza Doniger and Orit Adato for their valuableassistance throughout this work.

Conflicts of Interest: The authors declare no conflict of interest.

Biomedicines 2021, 9, 469 16 of 18

Abbreviations

CNS Central Nervous SystemANS Autonomous Nervous SystemRAAS Renin Angiotensin Aldosterone SystemDA DopamineNA NoradrenalineCTZ Chemoreceptor Trigger ZoneACTH Adrenocorticotropic HormoneGC GlucocorticoidGR Glucocorticoid ReceptorAR Adrenergic ReceptorECF Extracellular FluidSLC Solute CarrierGO Gene Ontology

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