*3.1. KO Models of SHR and SHRSP*

SHR (spontaneously hypertensive rat) is a representative genetically hypertensive model that was established by selective breeding of rats with relatively high blood pressure in an outbred colony of Wistar rats that had been maintained in Kyoto University. SHRSP (stroke-prone SHR) is a substrain of SHR that genetically develops more severe hypertension and stroke. Despite the fact that both strains have been widely used for clarifying the responsible genes and the underlying mechanisms of hypertension and its complications [103–105], the literature evaluating cardiovascular phenotypes by using KO/KI models are still scant (Table 5).

SHR is a useful model for hypertensive cardiac hypertrophy [106]. A quantitative trait locus (QTL) related to the left ventricular hypertrophy was previously mapped on chromosome (Chr) 8 by phenotyping of congenic strains between SHR and normotensive Brown Norway (BN) rats [107]. Liška et al. identified promyelocytic leukemia zinc finger (*Plzf*) as a candidate gene on the cardiac QTL and showed that the deletion in SHR did not alter the BP but ameliorated cardiac hypertrophy and fibrosis [108].

Complement 3 (C3) that is overexpressed in aortic smooth muscle cells of SHR has been proposed as a candidate gene responsible for the development of hypertension in this model [109]. Mechanistically, C3-C3a receptor signaling accelerates a change in the characteristic of VSMC and glomerular mesangial cells from contractile to synthetic phenotype via activation of Krüppel-like factor 5 (KLF5) that is a transcription factor to induce the synthetic phenotype of mesenchymal cells [110,111]. Negishi et al. revealed that the C3 deficiency mitigated a salt-sensitive BP elevation and renal injury with decreased renal Ang II level and urinary catecholamine excretion [112].

Rubattu et al. previously identified a QTL on Chr 1 responsible for the susceptibility to salt-induced stroke by a linkage analysis F2 cross between SHR and SHRSP [113]. They identified NADH dehydrogenase (ubiquinone) 1 subunit C2 (*Ndufc2*), encoding a component of the electron transport chain, as a plausible candidate gene in the stroke QTL, then proved that the heterozygous deletion in SHR by ZFN strongly exacerbated the stroke susceptibility with increased oxidative stress and inflammation both in vitro and in vivo [114].

Besides the above, we recently created peroxiredoxin 2 (*Prdx2*) KO SHR to investigate whether the deletion of an antioxidant gene exacerbates cerebro-cardiovascular phenotypes of SHR [115]. Consequently, *Prdx2* KO SHR had greater basal BP compared with WT SHR. Furthermore, the lifespan of *Prdx2* KO SHR under a salt loading condition was shorter than that of WT SHR despite no difference in BP after salt loading between the KO and the WT. No apparent inter-strain differences were found in histopathological evaluation for brain, heart, and kidney lesions, and therefore, the reason for the short life span of *Prdx2* KO SHR under the salt loading condition remains fully unknown.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is an endothelial scavenger receptor that is closely involved in the pathogenesis of atherosclerosis [116]. Recently, Liang et al. reported that LOX-1 deficiency had a protective role in spontaneous brain damage in SHRSP with no significant change of BP [117].

We previously found a QTL on Chr1 that affected exaggerated sympathetic responses to the stress of SHRSP by genetic analysis of congenic lines between SHRSP and normotensive Wistar-Kyoto rat (WKY) [118]. Among the genes in the QTL region, stromal interaction molecule 1 (*Stim1*) with a nonsense mutation in SHRSP was identified as a promising candidate ([47], see also Section 2.2 Vasculatures). As STIM1 plays a key role in Ca2+ homeostasis in the body, we expected that the *Stim1* mutation was a genetic determinant responsible for cerebro-cardiovascular traits; however, no significant differences were observed in the sympathetic stress responses as well as age-dependent changes in BP between *Stim1* KI SHRSP and SHRSP, i.e., with WT and mutant allele for *Stim1*, respectively [49]. Phenotyping of the *Stim1* KI SHRSP is currently in progress, and the results will be described elsewhere.

#### *3.2. KO Models of Dahl SS*

Dahl salt-sensitive (SS) rats originate from a closed colony of Sprague-Dawley (SD) rats and are widely used as a salt-sensitive hypertension model that develop severe hypertension (>200 mmHg) and the complications such as hypertensive kidney injury and heart failure when fed high-salt diets [119]. SS/Jr and DSS/N strains have been separately established by Rapp and Iwai, respectively. Compared with SHR and SHRSP, multiple KO/KI models with SS/Jr genetic backgrounds have been actively created (Table 5).

In 2011, Moreno et al. first reported the phenotype of renin KO SS/Jr, in which a severe decrease in basal BP and abnormal kidney morphologies were observed [120]. Thereafter, a growing literature has shown pathophysiological implications of multiple genes on cardiorenal disease traits in SS/Jr [121–133]. Among them, pleckstrin homology domain containing family A member 7 (*Plekha7*) is a plausible candidate gene for essential hypertension identified by GWAS. A risk variation on *Plekha7*, encoding an adherence junction protein [134], for elevated systolic BP has been found in multiple human populations [135–139]. In this context, Endres et al. created SS/Jr lacking the functional domain of *Plekha7* by ZFN and revealed that the *Plekha7* functional KO SS/Jr had significantly lower BP and renal and cardiac damage under the 8% high-salt diet condition [123]. This is a meaningful study that verified a direct effect of the GWAS gene on the hypertensive phenotype in a genetic rat model with salt sensitivity.



**5.**moleculesinrat

#### **4. Conclusions**

Hypertension is a multifactorial disease; nevertheless, the majority of previous research has focused on monogenic effects under inducible hypertensive conditions such as Ang II infusion and DOCA-salt in mice. Recent advances in genome-editing techniques, however, have made it possible to create knock-out and knock-in animals more easily, efficiently, and rapidly in rats as well as in mice [140–143]. Accordingly, it is necessary to create knock-out and knock-in models with multiple mutations in different loci to mimic complex genetic backgrounds of hypertensive patients and to uncover how the genetic interactions cause hypertension. As in the case of Dahl SS [144], translation of the findings in the experimental model into human hypertension remains highly challenging. However, translational approaches to bridge the gap between humans and rodent models would be necessary for understanding genetic and molecular mechanisms of essential hypertension in the post-GWAS era. A goal of basic hypertension research using experimental models may reconstruct 'genetically hypertensive mice/rats' from normotensive strains, and vice versa.

**Author Contributions:** H.O.: conceptualized and drafted the manuscript; H.O. and T.N.: revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partly supported by JSPS KAKENHI (Grant Number 21H03374) to T.N.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**

