Increased Urinary Angiotensin-Converting Enzyme 2 in Renal Transplant Patients with Diabetes

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be a renoprotective enzyme, since it converts angiotensin (Ang) II to Ang-(1-7). ACE2 has been detected in urine from patients with chronic kidney disease. We measured urinary ACE2 activity and protein levels in renal transplant patients (age 54 yrs, 65% male, 38% diabetes, n = 100) and healthy controls (age 45 yrs, 26% male, n = 50), and determined factors associated with elevated urinary ACE2 in the patients. Urine from transplant subjects was also assayed for ACE mRNA and protein. No subjects were taking inhibitors of the renin-angiotensin system. Urinary ACE2 levels were significantly higher in transplant patients compared to controls (p = 0.003 for ACE2 activity, and p≤0.001 for ACE2 protein by ELISA or western analysis). Transplant patients with diabetes mellitus had significantly increased urinary ACE2 activity and protein levels compared to non-diabetics (p<0.001), while ACE2 mRNA levels did not differ. Urinary ACE activity and protein were significantly increased in diabetic transplant subjects, while ACE mRNA levels did not differ from non-diabetic subjects. After adjusting for confounding variables, diabetes was significantly associated with urinary ACE2 activity (p = 0.003) and protein levels (p<0.001), while female gender was associated with urinary mRNA levels for both ACE2 and ACE. These data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells. Urinary ACE2 could be a marker of renal renin-angiotensin system activation in these patients

Aldosterone Antagonists in Monotherapy Are Protective against Streptozotocin-Induced Diabetic Nephropathy in Rats

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers

The Relationship between Urinary Renin Angiotensin System Markers, Renal and Vascular Function in Adolescents with Type 1 Diabetes

AIMS: The relationship between the renal renin-angiotensin aldosterone system (RAAS) and cardiorenal pathophysiology is unclear. Our aims were to assess (1) levels of urinary RAAS components and (2) the association between RAAS components and HbA1c, urine albumin/creatinine ratio (ACR), estimated glomerular filtration rate (eGFR) and blood pressure in otherwise healthy adolescents with type 1 diabetes mellitus (TID) vs. healthy controls (HC). METHODS: Urinary angiotensinogen and ACE2 levels, activity of ACE and ACE2, blood pressure (BP), HbA1c, ACR and eGFR were measured in 65 HC and 194 T1D from the Adolescent Type 1 Diabetes Cardio-Renal Intervention Trial (AdDIT). RESULTS: Urinary levels of all RAAS components were higher in T1D vs. HC (p<0.0001). Higher HbA1c was associated with higher urinary angiotensinogen, ACE2, and higher activity of ACE and ACE2 (p<0.0001, p=0.0003, p=0.003 and p=0.007 respectively) in T1D. Higher ACR (within the normal range) was associated with higher urinary angiotensinogen (p<0.0001) and ACE activity (p=0.007), but not with urinary ACE2 activity or ACE2 levels. These observations were absent in HC. Urinary RAAS components were not associated with BP or eGFR in T1D or HC. CONCLUSIONS: Otherwise healthy adolescents with T1D exhibit higher levels of urinary RAAS components compared to HC. While levels of all urinary RAAS components correlate with HbA1c in T1D, only urinary angiotensinogen and ACE activity correlate with ACR, suggesting that these factors reflect an intermediary pathogenic link between hyperglycemia and albuminuria within the normal range

Endothelin inhibits vasopressin-stimulated water permeability in rat terminal inner medullary collecting duct.

Renal tubule solute and water transport is subject to regulation by numerous factors. To characterize direct effects of the recently discovered peptide endothelin (ET) on renal tubule transport, we determined signaling mechanisms for ET effects on vasopressin (AVP)-stimulated water permeability (PF) in rat terminal inner medullary collecting duct (IMCD) perfused in vitro. ET caused a rapid, dose-dependent, and reversible fall in AVP- but not cyclic AMP-stimulated PF, suggesting that its effect on PF is by inhibition of cyclic AMP accumulation. Indomethacin did not block ET actions, ruling out a role for prostaglandins in its effect. The protein kinase C (PKC) inhibitor calphostin, or pretreatment of perfused tubules with pertussis toxin, blocked ET-mediated inhibition of AVP-stimulated PF. ET caused a transient increase in intracellular calcium ([Ca2+]i) in perfused tubules, an effect unchanged in zero calcium bath or by PT pretreatment. ET effects on PF and [Ca2+]i desensitized rapidly. Inhibition of PF was transient and largely abolished by 20 min ET preexposure, and repeat exposure to ET did not alter [Ca2+]i. In contrast, PGE2-mediated inhibition of AVP-stimulated PF and increase of [Ca2+]i were sustained and unaltered by prior exposure of IMCD to ET. Thus desensitization to ET is homologous. We conclude that ET is a potent inhibitor of AVP-stimulated water permeability in rat terminal IMCD. Signaling pathways for its effects involve both an inhibitory guanine nucleotide-binding protein and phospholipase-mediated activation of PKC. Since ET is synthesized by IMCD cells, this peptide may be an important autocrine modulator of renal epithelial transport

Collecting duct PGE2 responses reduce water loss with empagliflozin in mice with type 2 diabetes mellitus

Introduction: Sodium-glucose cotransporter 2 inhibitors such as empagliflozin (EMPA) protect against diabetic kidney disease. Prostaglandin E2 (PGE2) the main renal product of cyclooxygenase-2, inhibits vasopressin (AVP)-water reabsorption in the collecting duct (CD). The novelty of this study is that for the first time, we examined if EMPA affects the renal PGE2/EP receptor system and determined if CD responses to EMPA prevent water loss. Methods: Four groups of adult male mice were studied after 6 weeks of treatment: control (db/m), db/m+EMPA (10 mg/kg/day in chow), type 2 diabetic diabetic/dyslipidemia (db/db), and db/db+EMPA. Tubules were microdissected for quantitative polymerase chain reaction (qPCR) and CD water transport was measured in response to AVP, with or without PGE2. Results: Hyperglycemia and albuminuria were attenuated by EMPA. Renal mRNA expression for COX, PGE synthase, PGE2 (EP) receptor subtypes, CD AVP V2 receptors and aquaporin-2 was elevated in db/db mice, but unchanged by EMPA. Urine PGE2 levels increased in db/db but were unchanged by EMPA. AVP-water reabsorption was comparable in db/m and db/m+EMPA, and equally attenuated to 50% by PGE2. In db/db mice, AVP-water reabsorption was reduced by 50% compared to non-diabetic mice, and this reduction was unaffected by EMPA. In db/db mice, AVP-stimulated water transport was more significantly attenuated with PGE2 (62%), compared to non-diabetic mice, but this attenuation was reduced in response to EMPA, to 28%. Conclusion: In summary, expression of renal PGE2/EP receptors is increased in db/db mice, and this expression is unaffected by EMPA. However, in diabetic CD, PGE2 caused a greater attenuation in AVP-stimulated water reabsorption, and this attenuation is reduced by EMPA. This suggests that EMPA attenuates diabetes-induced excess CD water loss

Angiotensin-(1-7) activates a tyrosine phosphatase and inhibits glucose-induced signalling in proximal tubular cells

<b>Background.</b> In the diabetic kidney, stimulation of mitogen-activated protein kinases (MAPKs) leads to extracellular matrix protein synthesis. In the proximal tubule, angiotensin-(1–7) [Ang-(1–7)] blocks activation of MAPKs by angiotensin II. We studied the effect of Ang-(1–7) on signalling responses in LLC-PK1 cells in normal (5 mM) or high (25 mM) glucose.<p></p> <b>Methods.</b> The p38 MAPK was assayed by immunoblot, Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity was measured after immunoprecipitation, cell protein synthesis was determined by [3H]-leucine incorporation and transforming growth factor-β1 (TGF-β1), fibronectin and collagen IV were assayed by immunoblots and/or ELISA.<p></p> <b>Results.</b> High glucose stimulated p38 MAPK. This response was inhibited by Ang-(1–7) in a concentration-dependent fashion, an effect reversed by the receptor Mas antagonist A-779. Ang-(1–7) increased SHP-1 activity, via the receptor Mas. An inhibitor of tyrosine phosphatase, phenylarsine oxide, reversed the inhibitory effect of Ang-(1–7) on high glucose-stimulated p38 MAPK. Ang-(1–7) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-β1. Conversely, Ang-(1–7) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV.<p></p> <b>Conclusions.</b> These data indicate that in proximal tubular cells, binding of Ang-(1–7) to the receptor Mas stimulates SHP-1, associated with the inhibition of glucose-stimulated p38 MAPK. Ang-(1–7) selectively inhibits glucose-stimulated protein synthesis and TGF-β1. In diabetic nephropathy, Ang-(1–7) may partly counteract the profibrotic effects of high glucose

Urinary Ang II and Ang-(1-7).

<p>(A) Graph shows box plots of RIA for Ang II in urine specimens from transplant recipients without diabetes (no Diabetes) or with diabetes (Diabetes). For each box plot, median values are indicated by the line within the box, with value shown above the line. The box represents 50% of the values (25^th and 75^th percentiles), with the upper bar representing the 90^th percentile and the lower bar representing the 10^th percentile. Open circles indicate outliers. * p = 0.027, Diabetes vs. No Diabetes. (B) Graph shows box plots of EIA for Ang-(1-7) in urine specimens from transplant recipients without or with diabetes. There was no significant difference between the two groups (p = 0.126).</p