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ACE2 is a monocarboxypeptidase which generates Angiotensin (1–7) from Angiotensin II (1–8). Attempts to target the kidney Renin Angiotensin System using native ACE2 to treat kidney disease are hampered by its large molecular size, 100 kDa, which precludes its glomerular filtration and subsequent tubular uptake. Here, we show that both urine and kidney lysates are capable of digesting native ACE2 into shorter proteins of ~60–75 kDa and then demonstrate that they are enzymatically very active. We then truncated the native ACE2 by design from the C-terminus to generate two short recombinant (r)ACE2 variants (1-605 and 1-619AA). These two truncates have a molecular size of ~70 kDa, as expected from the amino acid sequence and as shown by Western blot. ACE2 enzyme activity, measured using a specific substrate, was higher than that of the native rACE2 (1-740 AA). When infused to mice with genetic ACE2 deficiency, a single i.v. injection of 1-619 resulted in detectable ACE2 activity in urine, whereas infusion of the native ACE2 did not. Moreover, ACE2 activity was recovered in harvested kidneys from ACE2-deficient mice infused with 1-619, but not in controls (23.1 ± 4.3 RFU/µg creatinine/h and 1.96 ± 0.73 RFU/µg protein/hr, respectively). In addition, the kidneys of ACE2-null mice infused with 1-619 studied ex vivo formed more Ang (1–7) from exogenous Ang II than those infused with vehicle (AUC 8555 ± 1933 vs. 3439 ± 753 ng/mL, respectively, p < 0.05) further demonstrating the functional effect of increasing kidney ACE2 activity after the infusion of our short ACE2 1-619 variant. We conclude that our novel short recombinant ACE2 variants undergo glomerular filtration, which is associated with kidney uptake of enzymatically active proteins that can enhance the formation of Ang (1–7) from Ang II. These small ACE2 variants may offer a potentially useful approach to target kidney RAS overactivity to combat kidney injury.

BACKGROUND The renin-angiotensin system is a complex regulatory hormonal network with a main biological peptide and therapeutic target, angiotensin (Ang) II (1–8). There are other potentially important Ang peptides that have not been well evaluated. METHODS Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was used for concurrent evaluation of multiple Angs downstream of Ang I (1–10) and Ang II (1–8) in kidney and plasma from wild-type (WT) mice. Angiotensin converting enzyme 2 knockout (ACE2KO) was also used as a way to examine the Angs profile in the absence of ACE2, an enzyme that cleaves both Ang I (1–10) and Ang II (1–8). RESULTS In plasma from both WT and ACE2KO, levels of Ang I (1–10), Ang III (2–8), and Ang (2–10) were the highest of all the renin-angiotensin system (RAS) peptides. The latter two peptides are products of aminopeptidase A cleavage of Ang II (1–8) and Ang I (1–10), respectively. In contrast, plasma levels of Ang II (1–8), and Ang (1–7), the product of Ang II (1–8) cleavage by ACE2, were low. In kidney from both WT and ACE2KO, Ang II (1–8) levels were high as compared to plasma levels. In the ACE2KO mice, a significant increase in either Ang II (1–8) or a decrease in Ang (1–7) was not observed in plasma or in the kidney. CONCLUSION RAS-focused peptidomic approach revealed major differences in Ang peptides between mouse plasma and kidney. These Ang peptide profiles show the dominance of the aminopeptidase A/Ang (2–10) and aminopeptidase A/Ang III (2–8) pathways in the metabolism of Ang I (1–10) and Ang II (1–8) over the ACE2/Ang (1–7) axis. Ang III (2–8) and other peptides formed from aminopeptidase A cleavage may be important therapeutic RAS targets.

Kidney allotransplantation remains the preferred treatment for end-stage kidney disease, yet donor shortages limit its availability. Xenotransplantation using genetically modified porcine kidneys offers a promising alternative. Here, we report key physiological observations from the first-in-human porcine kidney xenograft over a 51 day postoperative follow-up period. The transplanted kidney maintained essential functions, including waste excretion, electrolyte regulation, and production of concentrated urine, while supporting blood pressure and sodium reabsorption despite reduced activation of the renin-angiotensin-aldosterone system. Sodium retention required diuretic therapy, and mild hypocalcemia and hyperphosphatemia occurred in the setting of pre-existing hypoparathyroidism. Markedly increased urinary uric acid excretion led to hypouricemia without evidence of urate nephropathy. This case demonstrates the ability of a porcine kidney to sustain vital metabolic functions in a living human, while highlighting areas for further research. These findings provide a foundation for optimizing post-transplant care and advancing xenotransplantation as a solution to the critical organ shortage. Kidney shortages limit treatment options for patients with end-stage kidney disease, prompting exploration of xenotransplantation. Here, the authors show that a genetically modified pig kidney sustained essential functions in a living human for 51 days, informing future clinical strategies.

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1–7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na + channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na + reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between 〈ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.

Although microalbuminuria presages diabetic nephropathy, it may be a relatively late sign. Nocturnal hypertension may be an early indicator of diabetic nephropathy. In this study, investigators used 24-hour ambulatory blood-pressure monitoring in a prospective study of 75 subjects with type 1 diabetes mellitus who had normal blood pressure and urinary albumin excretion at enrollment. Over time, microalbuminuria developed in 14 subjects. An increase in nocturnal blood pressure preceded the development of microalbuminuria. The normal nocturnal dip in blood pressure had a negative predictive value of 91 percent for the development of microalbuminuria and was associated with a 70 percent reduction in risk. Among persons with type 1 diabetes mellitus who have normal urinary albumin excretion, the prevalence of hypertension, determined on the basis of blood-pressure readings at office visits, is similar to that in the general population. 1 , 2 Moreover, when microalbuminuria is detected in persons with type 1 diabetes, indicating the presence of incipient nephropathy, hypertension is usually absent, whereas persons with type 2 diabetes usually have overt hypertension when microalbuminuria is first detected. 2 – 4 Thus, if one uses the conventional definition of hypertension, or even the more stringent definition recently proposed for persons with diabetes (i.e., systolic blood pressure that exceeds . . .

ACE and ACE2 Activity in Diabetic Mice Jan Wysocki 1 , Minghao Ye 1 , Maria José Soler 1 , Susan B. Gurley 2 , Hong D. Xiao 3 , Kenneth E. Bernstein 3 , Thomas M. Coffman 2 , Sheldon Chen 1 and Daniel Batlle 1 1 Division of Nephrology/Hypertension, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 2 Division of Nephrology, Duke University Medical Center, Durham, North Carolina 3 Department of Pathology, Emory University, Atlanta, Georgia Address correspondence and reprint requests to Daniel Batlle, MD, Division of Nephrology/Hypertension, The Feinberg School of Medicine, Northwestern University, Searle 10-475, 320 E. Superior, Chicago, IL 60611. E-mail: d-batlle{at}northwestern.edu Abstract ACE-related carboxypeptidase (ACE2) may counterbalance the angiotensin (ANG) II–promoting effects of ACE in tissues where both enzymes are found. Alterations in renal ACE and ACE2 expression have been described in experimental models of diabetes, but ACE2 activity was not assessed in previous studies. We developed a microplate-based fluorometric method for the concurrent determination of ACE and ACE2 activity in tissue samples. Enzymatic activity (relative fluorescence unit [RFU] · μg protein −1 · h −1 ) was examined in ACE and ACE2 knockout mice and in two rodent models of diabetes, the db/db and streptozotocin (STZ)-induced diabetic mice. In kidney cortex, preparations consisting mainly of proximal tubules and cortical collecting tubules, ACE2 activity had a strong positive correlation with ACE2 protein expression (90-kDa band) in both knockout models and their respective wild-type littermates ( r = 0.94, P < 0.01). ACE activity, likewise, had a strong positive correlation with renal cortex ACE protein expression (170-kDa band) ( r = 0.838, P < 0.005). In renal cortex, ACE2 activity was increased in both models of diabetes (46.7 ± 4.4 vs. 22.0 ± 4.7 in db/db and db/m , respectively, P < 0.01, and 22.1 ± 2.8 vs. 13.1 ± 1.5 in STZ-induced diabetic versus untreated mice, respectively, P < 0.05). ACE2 mRNA levels in renal cortex from db/db and STZ-induced diabetic mice, by contrast, were not significantly different from their respective controls. In cardiac tissue, ACE2 activity was lower than in renal cortex, and there were no significant differences between diabetic and control mice ( db/db 2.03 ± 0.23 vs. db/m 1.85 ± 0.10; STZ-induced diabetic 0.42 ± 0.04 vs. untreated 0.52 ± 0.07 mice). ACE2 activity in renal cortex correlated positively with ACE2 protein in db/db and db/m mice ( r = 0.666, P < 0.00 5 ) as well as in STZ-induced diabetic and control mice ( r = 0.621, P < 0.05) but not with ACE2 mRNA ( r = −0.468 and r = −0.522, respectively). We conclude that in renal cortex from diabetic mice, ACE2 expression is increased at the posttranscriptional level. The availability of an assay for concurrent measurement of ACE and ACE2 activity should be helpful in the evaluation of kidney-specific alterations in the balance of these two carboxypeptidases, which are involved in the control of local ANG II formation and degradation. Footnotes ACE2, ACE-related carboxypeptidase; ANG, angiotensin, STZ, streptozotocin. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact Accepted April 6, 2006. Received January 6, 2006. DIABETES

Degradation of the biologically potent octapeptide angiotensin Ang II-(1-8) is mediated by the activities of several peptidases. The conversion of Ang II to the septapeptide Ang-(1-7) is of particular interest as the latter also confers organ protection. The conversion is catalyzed by angiotensin-converting enzyme 2 and other enzymes that selectively cleave the peptide bond between the proline and the phenylalanine at the carboxyl terminus of Ang II. The contribution of various enzyme activities that collectively lead to the formation of Ang-(1-7) from Ang II, in both normal conditions and in disease states, remains only partially understood. This is largely due to the lack of a reliable and sensitive method to detect these converting activities in complex samples, such as blood and tissues. Here, we report a fluorometric method to measure carboxypeptidase activities that cleave the proline-phenylalanine dipeptide bond in Ang II. This method is also suitable for measuring the conversion of apelin-13. The assay detects the release of phenylalanine amino acid in a reaction with the yeast enzyme of phenylalanine ammonia lyase (PAL). When used in cell and mouse organs, the assay can robustly measure endogenous Ang II and apelin-13-converting activities involved in the renin-angiotensin and the apelinergic systems, respectively.

We studied the non-obese diabetic (NOD) mice model because it develops autoimmune diabetes that resembles human type 1 diabetes. In diabetic mice, urinary albumin excretion (UAE) was ten-fold increased at an \"early stage\" of diabetes, and twenty-fold increased at a \"later stage\" (21 and 40 days, respectively after diabetes diagnosis) as compared to non-obese resistant controls. In NOD Diabetic mice, glomerular enlargement, increased glomerular filtration rate (GFR) and increased blood pressure were observed in the early stage. In the late stage, NOD Diabetic mice developed mesangial expansion and reduced podocyte number. Circulating and urine ACE2 activity were markedly increased both, early and late in Diabetic mice. Insulin administration prevented albuminuria, markedly reduced GFR, blood pressure, and glomerular enlargement in the early stage; and prevented mesangial expansion and the reduced podocyte number in the late stage of diabetes. The increase in serum and urine ACE2 activity was normalized by insulin administration at the early and late stages of diabetes in Diabetic mice. We conclude that the Diabetic mice develops features of early kidney disease, including albuminuria and a marked increase in GFR. ACE2 activity is increased starting at an early stage in both serum and urine. Moreover, these alterations can be completely prevented by the chronic administration of insulin.