**9. AGE-induced peptidase**

Our recent studies assessed the potential role of Ang-(1-7) in the progression of epithelial-tomesenchymal transition (EMT) in the renal epithelial NRK-52 cell line by either advanced glycation end products (AGEs) or the cytokine TGF-β [100]. Ang-(1-7) essentially abolished the cellular indices for EMT in the NRK-52 cells likely through the inhibition of the noncanonical ERK 1/2-signaling pathway stimulated by AGE (100). Interestingly, AGE exposure reduced the intracellular levels of Ang-(1-7) but did not alter the cellular content of Ang II [100]. Processing of Ang I to Ang-(1-7) by thimet oligopeptidase tended to be reduced by AGE; however, the intracellular metabolism of Ang-(1-7) to Ang-(1-4) was significantly increased by AGE exposure [100]. These data suggest that AGE-induced EMT may reflect lower Ang-(1-7) tone in the renal epithelial cells that may be permissive for the progression of EMT and the increase in tissue fibrosis. Our preliminary data suggest that DPP 3 is not responsible for the AGE-induced metabolism of Ang-(1-7) in these cells and that another peptidase activity may participate in the cellular metabolism of the peptide. As shown in **Figure 4A**, the NRK-52 cytosolic fraction readily hydrolyzed the quenched Ang-(1-7) fluorescent substrate Abz1 -Ang- (1-7)-Tyr7 (NO2) to Abz-Ang-(1-4). By contrast, DPP 3 in the HK-2 renal cells did not cleave the Ang-(1-7) fluorescent peptide (**Figure 4B**). The failure of DPP 3 to cleave the fluorescent Ang- (1-7) substrate is consistent with the inability of DPP 3 to hydrolyze blocked N-terminal residues of peptides such as the Abz moiety on the Ang-(1-7) substrate. Moreover, hydrolysis of the N-terminally blocked Ang-(1-7) fluorescent substrate by the cytosolic fraction of the NRK-52 cells suggests the peptidase is likely an endopeptidase. Apart from the identification of the Ang-(1-7)-degrading activity in the NRK-52E cells, it remains to be determined whether intracellular levels of Ang-(1-7) influence the EMT process.

**Figure 4.** Metabolism of Abz1 -Ang-(1-7)-Tyr7 (NO2). (A) HPLC chromatograph reveals that the cytosolic fraction of the NRK-52E cells cleaves the Ang-(1-7)-fluorescent substrate Abz-Ang-(1-7) to Abz-Ang-(1-4). (B) By contrast, HK-2 cytosolic fraction does not hydrolyze the Ang-(1-7)-fluorescent substrate suggesting an endopeptidase activity distinct from DPP 3. The large void peak is absorbance from the DMSO solvent to dissolve the fluorescent substrate. Substrate concentration and incubation conditions are identical for both cell types. The HK-2 cell data are adapted from Wilson et al. [57].

### **10. Characterization of peptidase pathways**

Ang-(1-7) (**Figure 2**) [99]. The preferred hydrolysis of Ang-(3-7) by DPP 3 would account for our inability to demonstrate the accumulation of Ang-(3-7) following the initial metabolism of Ang-(1-7). In regard to an *in vivo* role for DPP 3 to modulate Ang-(1-7), human HK-2 cells were treated with varying doses of the JMV-390 inhibitor and we assessed both the endogenous content of Ang-(1-7) and the intracellular DPP-3/Ang-(1-7)-degrading activity in the cells. As shown in **Figure 3**, 20- and 200-nM JMV-390 reduced DPP3 activity by >30 and >80%, respectively, as compared to control suggesting that the inhibitor effectively penetrates the cells [99]. The lower dose of JMV increased the cellular content Ang-(1-7) approximately twofold, although this did not reach statistical significance. The higher dose of JMV, however, significantly reduced the intracellular levels of the peptide [99]. We interpret the latter results that the high dose may spill over to block other peptidases including thimet oligopeptidase that may be involved in the generation of Ang-(1-7) in the renal cells [57]. Thus, the blockade of Ang-(1-7)-forming enzymes by the high-dose JMV may override any protective effects of DPP 3 inhibition. We are currently assessing alternative approaches to block DPP 3 within the HK-2 cells and assess both secreted and intracellular levels of Ang-(1-7), as well as Ang-(1-7)-

Our recent studies assessed the potential role of Ang-(1-7) in the progression of epithelial-tomesenchymal transition (EMT) in the renal epithelial NRK-52 cell line by either advanced glycation end products (AGEs) or the cytokine TGF-β [100]. Ang-(1-7) essentially abolished the cellular indices for EMT in the NRK-52 cells likely through the inhibition of the noncanonical ERK 1/2-signaling pathway stimulated by AGE (100). Interestingly, AGE exposure reduced the intracellular levels of Ang-(1-7) but did not alter the cellular content of Ang II [100]. Processing of Ang I to Ang-(1-7) by thimet oligopeptidase tended to be reduced by AGE; however, the intracellular metabolism of Ang-(1-7) to Ang-(1-4) was significantly increased by AGE exposure [100]. These data suggest that AGE-induced EMT may reflect lower Ang-(1-7) tone in the renal epithelial cells that may be permissive for the progression of EMT and the increase in tissue fibrosis. Our preliminary data suggest that DPP 3 is not responsible for the AGE-induced metabolism of Ang-(1-7) in these cells and that another peptidase activity may participate in the cellular metabolism of the peptide. As shown in **Figure 4A**, the NRK-52 cytosolic fraction readily hydrolyzed the quenched Ang-(1-7) fluorescent substrate Abz1

(NO2) to Abz-Ang-(1-4). By contrast, DPP 3 in the HK-2 renal cells did not cleave the

Ang-(1-7) fluorescent peptide (**Figure 4B**). The failure of DPP 3 to cleave the fluorescent Ang- (1-7) substrate is consistent with the inability of DPP 3 to hydrolyze blocked N-terminal residues of peptides such as the Abz moiety on the Ang-(1-7) substrate. Moreover, hydrolysis of the N-terminally blocked Ang-(1-7) fluorescent substrate by the cytosolic fraction of the NRK-52 cells suggests the peptidase is likely an endopeptidase. Apart from the identification of the Ang-(1-7)-degrading activity in the NRK-52E cells, it remains to be determined whether

intracellular levels of Ang-(1-7) influence the EMT process.


dependent actions on these cells.

12 Enzyme Inhibitors and Activators

**9. AGE-induced peptidase**

(1-7)-Tyr7

In the review of the peptidases of the Ang-(1-7) axis, a brief discussion of the various biochemical approaches to characterize these enzymatic components is warranted. Ideally, utilization of the endogenous peptide substrates including Ang I, Ang II, and Ang-(1-7) should be assessed to identify the peptidase activities involved in the RAS [101]. One advantage is that the contribution of various peptidases for a given peptide is directly comparable to determine the predominant pathway in a particular tissue or treatment condition. Peptidase activities derived by different synthetic substrates are not comparable unless standardized to the enzyme concentration. Moreover, the use of endogenous peptide substrates may reveal novel peptidase activities involved in angiotensin processing [102, 103]. Peptidase assays developed in our laboratory typically utilize 125I-radiolabled peptides coupled to highperformance liquid chromatography (HPLC)-based separation and automated in-line γdetection. Advantages of this are that only microliter amounts of serum or microgram quantities of tissue are normally required that reflects detection sensitivity in the fmol range and the lack of detector interference or quenching [33].

More recent studies have incorporated mass spectroscopy (MS) detection of angiotensin metabolism in tissues, cells, and plasma, as well as the derivation of processing networks [104]. Velez and colleagues applied HPLC-MS analysis of Ang I processing in rat glomeruli to reveal the predominant processing of Ang I to Ang-(1-7) catalyzed by neprilysin [104]. Interestingly, the authors could not demonstrate an Ang I to Ang II pathway even following the blockade of the Ang-(1-7) pathway with a neprilysin inhibitor. Hildesbrand et al. [103] utilized a HPLCtandem quadrupole system (HPLC-MS/MS) to reveal multiple metabolism pathways from Ang I to its N-terminal metabolites Ang-(5-10) and Ang-(4-10), as well as Ang II and Ang-(1-7) in immobilized proteins from human plasma. Suski et al. [105] reported that Ang I was primarily converted to Ang-(1-7) in vascular smooth muscle cells (VSMCs) as characterized by HPLC-MS/MS and confirms our earlier study that thimet oligopeptidase directly processed Ang I to Ang-(1-7) in rat VSMC [106]. Grobe and colleagues have applied "in situ" MALDI to characterize both renal and cardiac metabolism of exogenous Ang II [107, 108]. Ang-(1-7) was the primary product from Ang II in the renal cortex while Ang III was the major metabolite in the medulla [108]. In the heart, Ang III and Ang-(1-7) were products of Ang II metabolism catalyzed by APA and ACE2, respectively [107]. These data confirm earlier HPLC-based studies on the contribution of ACE2 to Ang-(1-7) formation in the mouse and human heart [66, 109]. Although this approach cannot distinguish intracellular versus membrane or extracellular processing and requires relatively high-substrate concentrations, it is likely that these systems will develop the required sensitivity and resolution to detect peptides *in situ*, as well as characterize the extent of enzymatic processing.
