**5. Background on** *NEDD4* **and** *NEDD4L*/*Nedd4-2*

The current paradigm of *NEDD4L* and salt-sensitive hypertension was confirmed, based on the following—the discovery and naming of *NEDD4L,* ENaC as a binding factor with respect to Liddle mutation, the discovery of KIAA0439 and *NEDD4L* (*Nedd4-2*), and our discovery of the human *NEDD4L* C2 domain and the V13 G/A mutation [45] (Figure 2). These findings provide a basis for our investigation, as described in the following.

Kumar et al. (1992) studied changes in gene expression during the development of neural precursor cells, using subtraction cloning [46]. A group of discovered genes was named the "neural precursor cell-expressed, developmentally, down-regulated" (*NEDD*) and the fourth among the molecules numbered 1 to 10, in order of identification, was *NEDD4*, which Staub et al. (1996) found to be associated with hypertension [40]. Using the yeas<sup>t</sup> 2 hybrid method, they found that NEDD4 bound to

the PY motif where the Liddle mutation of ENaC was concentrated, and that it was a specific ubiquitin ligase of ENaC. Thereafter, *NEDD4* was highlighted, but its significance shifted. With the discovery of the mechanism of ENaC degradation and the failure of retrieval from the cell membrane during the onset of Liddle syndrome, the gene encoding a protein related to this system is notable as a promising candidate associated with salt-sensitive hypertension. However, *NEDD4* was not a potential candidate gene because, new findings came to light, which led to other insights into *NEDD4*/*NEDD4L*.

**Figure 2.** Schematic presentation of exon usage of each human Nedd4L isoform is shown. Exon in dark square shows the newly discovered exons through re-sequencing. Variant 13 (G→A) located at the end of the newly discovered exon 1. Translation start codon (ATG) located in Exon 1 for isoform I, in Exon 1a for isoform II, and newly re-named Exon 6 for isoform III, respectively. (modified from [45]).

Ishikawa et al. (1997) sequenced and published 78 unknown cDNA sequences from a human brain cDNA library [47]. Among these, Harvey and Kumar (2001) identified the KIAA0439 gene as having a Hect domain with ubiquitin ligase activity, while analyzing a Nedd4-like gene in a database [48] and found that it could ubiquitinate ENaC. Chen et al. also located a *NEDD4L* gene on human chromosome 18 with a 67% homology to human *NEDD4* [49]. Around the same time, both Staub and Kamynina et al. discovered *Nedd4-2*, which showed a high homology with *Nedd4* (*Nedd4-1*) in mouse cultured cells [31,50]. A comparison of binding between the WW domain and ENaC revealed that NEDD4-2 showed more ubiquitination activity, and that the endogenous ENaC negative regulator was likely to be *Nedd4-2* [50–52]. Thus, despite species differences between mice and humans, NEDD4-2 (NEDD4L) was essentially established as the main ubiquitinating enzyme in contrast to NEDD4, which was conventionally regarded as the first ubiquitinating enzyme of ENaC.

While the model of the ENaC-NEDD4 system continued to shift towards NEDD4L/NEDD4-2, we clarified significant differences that led to further findings. We and other authors, compared human/mouse *NEDD4*/*Nedd4* with *NEDD4L*/*Nedd4-2* and found that *NEDD4* had a C2 domain unlike *NEDD4L*/*Nedd4-2*, which was a critical difference between the two molecules. However, phylogenetic analyses of gene homology showed that human/mouse *NEDD4L*/*Nedd4-2* (KIAA0439) has a high homology with *NEDD4* in Xenopus, which has only one *NEDD4*, and the *NEDD4L* gene was thought to have appeared early during the evolutionary process. Based on this finding, the apparent difference between the two genes could not be explained.

### **6. Human NEDD4L Is a Causative Gene of Salt-Sensitive Hypertension**

The ENaC-NEDD4L system plays an important role in post-translational modification through ubiquitination, which regulates the ENaC expression on the cell membrane of the terminal nephron. Genetic analyses of the Liddle syndrome and hereditary hypertension found that ENaC, which is restricted to the terminal nephron and regulates sodium reabsorption, and its regulatory protein,

NEDD4L, are promising candidate genes (*NEDD4L*) associated with salt-sensitive hypertension. We focused on the importance of the ENaC-NEDD4L system and selected *NEDD4L* as a target in a genetic analysis of salt-sensitive hypertension.

Based on a draft sequence of human chromosome 18 determined using genomic cDNA expression sequence tag (EST) data such as KIAA0439, Xenopus Nedd4 cDNA extracted from GenBank, Human and Mouse EST, and other databases, we performed Basic Local Alignment Search Tool (BLAST) and cross-match analyses. We searched for SNP by analyzing common polymorphisms determined by re-sequencing the genomic DNA of 48 normotensive persons whose DNA samples were registered in the HyperGEN network. We also analyzed human *NEDD4L* gene transcripts using RT-PCR, 5-RACE, and quantitative PCR targeting human RNA. In addition to known exon-introns, including the ATG codon, two exons (1a and 1) for which the splice site on the genomic DNA sequence exon-end was noted and an exon (exon 2) common to the transcription products from both, were discovered in chromosome 18. Exon 2 had a high homology with the C2 domain of the ancestral Nedd4, such as that in Xenopus and fugu and was thought to be a shared C2 domain that was conserved during molecular evolution [45] (Figure 3).

The start codon ATG of KIAA0439 was found in exon 6 of the conventional gene and in exon 7 of the gene that we discovered. Genomic DNA re-sequencing revealed 38 SNP. Among these, the 13th gene mutation (Variant 13 G/A) was found just before the exon 1 splice site. We spliced cDNA synthesized from human RNA through RT-PCR, immediately after the G base in those with a G allele, and the full-length C2 domain after exon 2 was encoded. In contrast, a frame shift occurred in those possessing an A allele because the splice site moved downstream by 10 bases, ending at the stop codon at exon 2. As a result, the gene product of exon 1 was knocked out (Figure 2). The results of the 5-RACE analyses of mRNA from human kidney and adrenal tissues revealed six transcripts. Among these, isoform I was a transcriptional product of exon 1, isoform II was a transcriptional product of exon 1a, and isoform III was located between exons 2 and 3. Exon 2a encoded the transcription initiation site of KIAA0439. Further analysis using quantitative PCR showed isoform I and II expression in the kidney and lung, respectively, suggesting that it might be under tissue-specific transcriptional control [45].

These results showed that the human *NEDD4L* gene expresses two isoforms each with a C2 domain and another without a C2 domain. Among these, isoform I is a gene product resulting from a G/A mutation, a common variant, and isoform I was hypothesized to be associated with salt-sensitive hypertension. Therefore, we studied variant 13 (G/A) in 367 Japanese persons. An analysis based on genotype, alleles, and three inheritance patterns revealed that the GG and G alleles significantly correlated with essential hypertension. We then assessed the significance of the two types of C2 domains in the human Nedd4L gene and six types of transcriptional diversity. The results of a related study of Japanese patients revealed that a variant 13 (G/A) mutation correlated with essential hypertension [53–55]. In summary, the human NEDD4L gene is likely to be a causative gene of salt-sensitive hypertension in humans [56–58].

### **7. NEDD4L Is the Causative Protein of Salt-Sensitive Hypertension**

We investigated the *Nedd4L* gene function in salt-sensitive hypertensive Dahl rats [59]. We initially analyzed the transcriptomes of rat RNA using the 5 'RACE method, to determine whether or not the *Nedd4L* gene in rats has a C2 domain. The results revealed an isoform with a C2 domain in rats and a novel exon on rat genomic DNA. The C2 domain of rat *Nedd4L* was 100% identical to the human C2 domain. Northern blotting using a novel "isoform A" possessing a C2 domain and a probe of the WW domain common to other isoforms, revealed the tissue-specific expression of *Nedd4L* gene transcription products, especially of isoform A, the expression of which was localized in the kidneys, lung, brain, and heart [59].

Changes in salt loading were examined by quantitative PCR and in situ hybridization. The results showed that salt intake increased *Nedd4L* expression in normal DR rats, but did not alter *Nedd4L* expression in salt-sensitive DS rats. The trend of *Nedd4L* expression determined by in situ hybridization was similar in kidney tissues [59]. A study of human kidney tissues obtained under written informed consent, showed that *NEDD4L* expression was restricted to late DCT and lower CNT, CCD, and collecting renal tubules, as in rats, and this distribution was consistent with that of ENaC expression, suggesting that *NEDD4L* is involved in the regulation of ENaC expression [60].

Subsequently, we performed in vitro functional experiments using heterologous gene expression systems using Xenopus oocytes. Initially, we successfully cloned three isoforms of human *NEDD4L* both with and without the C2 domain. A significant reduction in the amiloride-sensitive ENaC current by isoform II and III with ENaC cRNA injected was observed, when either isoform II or III cRNAs were injected into the Xenopus oocyte. The current was significantly restored when isoform I cRNAs were coinjected with other isoforms, indicating the dominant negative e ffects of the isoform I product against the downregulation of cell surface ENaC by isoforms II and III. Such interactions might abnormally increase sodium reabsorption in ASDN, suggesting that the human *NEDD4L* gene, especially the evolutionarily new isoform I, is a candidate gene for salt-sensitive hypertension in human [53,54,60,61].

### **8. Generation of Nedd4-2 C2 KO Mice and Discovery of Salt-Sensitive Hypertension with Potential Contributions to Cardio-Renal Involvements**

Lastly, we decided to develop genetic engineered model of salt-sensitive hypertension such as *Nedd4-2* (*NEDD4L* in human) using knockout mice and examined the detailed phenotypic manifestations to determine the critical roles of *Nedd4-2* in salt-sensitive hypertension. First, we determined genetic variations of mice *Nedd4-2* using *in silico* exploration of the transcriptional start site of the gene. As reported previously, both human and rodent *NEDD4L*/*Nedd4-2* showed molecular diversity, with and without a C2 domain in their N-terminal. The *NEDD4L*/*Nedd4-2* isoforms with a C2 domain were hypothesized to be related closely to ubiquitination of ENaCs. *In silico* gene identification analysis of mice *Nedd4-2* C2 domain coding exon was performed using the BLAST and cross-match program. Then, the sequences of EST and cDNA in the GenBank database (nr) were aligned and compared with the sequence of mice *Nedd4-2* exon 4, which was already known as the transcription start site of mice *Nedd4-2* without a C2 domain. Cross-match analyses were performed repeatedly between the cDNA/EST sequences and genomic sequences, and the results were parsed with chromosome 18 of C57Bl6/J, to form a consistent assembly of EST, cDNA, and a genomic sequence. Thus, the expressed sequence tag and cDNA alignment and the results of the *in silico* bioinformatic database analysis of *Nedd4-2* on mouse chromosome 18q showed a newly identified exon2 coding the C2 domain of mice *Nedd4-2* [62].

Subsequently, we started to create a targeting vector for the newly discovered "exon 2", which codes the C2 domain of NEDD4-2 and generated genetically engineered mice without a C2 domain of NEDD4-2 [62]. Mice without a NEDD4-2 C2 domain did not show any growth retardation or infertility. We performed a detailed metabolic balance study and continuous blood pressure monitoring analyses, using metabolic cages for individual mice and unrestrained telemetry systems. Under normal oral salt intake, both the wild littermates and *Nedd4-2* C2 KO mice did not show any phenotypic di fferences, according to blood pressure, urinary sodium excretion, urinary osmotic pressure, urine volume, and water intake. However, under high oral salt intake, *Nedd4-2* C2 KO mice showed blood pressure elevation with reduced urinary sodium excretion [62]. Detailed quantitative analyses for the mRNA expressions levels along laser-captured urinary tubules showed a significant step-wise elevation of ENaC mRNA expressions, in accordance with both their genetic background and oral salt intake, which was abolished into the normal, through amiloride treatment [62]. ENaC mRNA expressions were paradoxically enhanced, despite a high oral salt intake, with a condition of single exon ablation for *Nedd4-2* gene. These results suggested that ENaC itself act as a "sensor" of intra-tubular salt with intra-tubular epithelial "excite-transcription coupling", which might regulate ENaC gene expressions paradoxically without the NEDD4-2 C2 domain [62]. This could be a pathological molecular mechanism underlying the salt-sensitive hypertension.

As NEDD4-2 with C2 domain is expressed not only in renal tubular cells but also in cardiomyocytes working as ubiquitinating enzyme for voltage-gated sodium-channel, SCN5a, we performed subsequent experiments examining the electrophysiological change of the heart after myocardial infarction, in mice. Additionally, to determine detailed characteristics of salt-sensitive hypertension of the mice, we tried a mineral corticoid receptor antagonist treatment for *Nedd4-2* C2 KO mice. Finally, us and other authors [63,64], have previously found that *Nedd4-2* C2 KO mice showed eplerenone-resistant salt-sensitive hypertension [65] and enhanced electrophysiological abnormalities, after myocardial infarction [66], suggesting a pivotal role of the *Nedd4-2* isoform with C2 domain for cardio-renal association, with regards to target-organ damages of the subjects with salt-sensitive hypertension.
