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* Department of Internal Medicine, Medical School Hannover, Hannover, Germany;
Berlin Chemie AG, Berlin, Germany;
Franz-Vollhard Clinic, Klinikum Charité, Berlin, Germany; and
Institute of Clinical Pharmacology, Otto-von-Guericke University, Magdeburg, Germany
1Correspondence: Dept. Int. Med., Medical School Hannover, Carl-Neuberg-Strasse 1, Hannover 30625, Germany. E-mail: janmenne{at}gmx.de
SPECIFIC AIMS
In clinical studies, dihydropyridine channel blockers (CCBs) improve endothelial dysfunction in hypertensive patients and in patients with manifest atherosclerosis. Long-acting third-generation dihydropyridine calcium CCBs improve endothelial dysfunction, but the cellular and molecular mechanisms of tissue protection are not elucidated in detail. We assessed organ (renal) protection by the highly lipophilic CCB lercanidipine in a double-transgenic rat (dTGR) model with overexpression of the human renin and angiotensinogen genes and analyzed protein kinase C (PKC) activation in vitro by life imaging of green fluorescence protein (GFP) tagged PKC molecules.
PRINCIPAL FINDINGS
1. Animal studies and histological finding
Cumulative mortality was 60% in untreated dTGR, whereas no lercanidipine-treated animal or Sprague-Dawley (SD) control rat died. This difference was highly significant (P<0.001). At the end of the study, serum creatinine was significantly lower in SD rats (28±2 µmol/l) and treated dTGR (36±5 µmol/l) as compared with surviving untreated dTGR (66±5 µmol/l; both P<0.05 vs. untreated dTGR). In addition, the albumin excretion rate was 0.24 ± 0.05 mg/24 h in SD rats. It was sevenfold higher in the untreated dTGR (1.70±0.16 mg/24 h), whereas lercanidipine treatment significantly reduced albuminuria in dTGR (0.81±0.1 mg/24 h; P<0.01 vs. untreated dTGR). On histological examination, untreated dTGR had severe renal damage with focal necrosis and arteriolar hyalinosis. Treatment with lercanidipine prevented vascular injury in small renal vessels, inducible NOS (iNOS) activation, monocyte infiltration, and extracellular matrix formation.
2. Asymmetric dimetylarginine and dimethylarginine dimethylaminohydrolase mRNA tissue levels
Mean asymmetric dimetylarginine (ADMA) blood concentration in untreated dTGR was 35% percent higher than in control SD rats; the difference was statistically significant. Administration of lercanidipine significantly reduced ADMA blood levels in treated dTGR. The dimethylarginine dimethylaminohydrolase (DDAH) mRNA levels in kidney tissue were lower in untreated dTGR as compared with SD rats and lercanidipine-treated dTGR but the difference did not reach statistical significance (P=0.055).
3. Effects of lercanidipine on angiotensin II-mediated intracellular and molecular mechanisms
Within 1 s after angiotensin (ANG) II stimulation of HUVECs loaded with fluo 3-acetoxymethyl ester, a marked calcium uptake into the cytoplasm and the nucleus was visible. If the same experiment was performed after preincubation of the cells with lercanidipine, the calcium uptake was markedly reduced and the uptake was delayed. ANG II also induced a strong activation of nuclear PKC-
after 1 min, which decreased after 5 min. Pretreatment with lercanidipine for 90 min not only reduced the initial activation of PKC- but also diminished the nuclear response to ANG II. Lercanidipine not only influenced calcium dependent PKC isoform but also the novel PKC isoforms-
and -
. To visualize PKC movement after exposure with ANG II, we transfected VSMCs cells with a PKC-GFP- or - construct and analyzed translocation of PKC-GFP or fusion proteins using confocal microscopy. Preincubation with lercanidipine for 90 min inhibited this movement substantially. For PKC--GFP, a strong punctuated pattern appeared within a second after cell stimulation with ANG II (Fig. 1
). These aggregations were abolished after preincubation of cells with lercanidipine. To analyze the effects of lercanidipine on PKC translocation in vivo, we performed Western blots from the cytoplasm and particulate/membrane fraction of renal tissue. We found a significantly (P<0.05) lower membrane association of PKC- in lercanidipine-treated dTGR in comparison to untreated dTGR. Administration of potassium-cyanate and desoxy-glucose leads to ATP depletion in the endothelium, which resembles an ischemic condition. After their administration, we observed a marked increase of the albumin flux across the endothelial cell layer, which peaked after 30 min. This increase was reduced by 50% (P<0.05) when lercanidipine was added to the cell culture medium at a concentration of 10–8 M.
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CONCLUSIONS AND SIGNIFICANCE
In the present study, we explored cellular and molecular mechanisms by which lipophilic CCBs may affect endothelial function, prevent tissue injury, and hence improve survival in a transgenic animal model of ANG II-mediated organ damage. We could demonstrate that administration of lercanidipine significantly reduced tissue inflammation and tubulo-interstitial fibrosis. These changes in renal histology were accompanied by preserved renal function and reduced albuminuria in lercanidipine-treated dTGR, resulting in an improved outcome.
We provide first in vitro evidence that the beneficial effects of long-acting lipophilic CCBs may result, at least in part, from inhibition of PKC. Among the various signaling pathways that mediate intracellular effects of ANG II, PKC isoforms have been shown to be activated in several cell types, e.g., VSMCs and endothelial and mesangial cells. We have analyzed the effect of lercanidipine on the classical PKC isoform- and the novel PKC isoform- translocation using GFP-tagged PKC- and PKC- isoforms in transfected living cells and confocal laser scanning fluorescent microscope. We have monitored the movement of this fusion protein after addition of lercanidipine and could clearly demonstrate an inhibition of the ANG II-mediated translocation of PKC- and PKC-. That inhibition of PKC activation and translocation to cellular membranes by lercanidipine might also be relevant in vivo is supported by our observation that less PKC- is found in the particulate/membrane fraction of renal tissue of lercanidipine-treated dTGR. This effect of lercanidipine on PKC activation and translocation may explain some of the observed clinical findings in our animal model of ANG II-mediated organ damage. For example, increased PKC- activity is thought to be responsible for the enhanced leakage of the endothelial cell layer for albumin. Indeed, we were able to show that administration of lercanidipine reduces the albumin permeability of ischemic endothelial cells. Furthermore, it has been demonstrated that PKC- activation is involved in the development of albuminuria, cardiac hypertrophy and fibrosis, and heart failure. In vitro PKC- interacts with L-selectin and stimulates ICAM mRNA expression. PKC- enhances ICAM and vascular cell adhesion molecule expression in vitro. Finally, some of the beneficial renal effects of ANG-converting enzyme (angiotensin 1-converting enzyme) inhibitors seem to result from PKC inhibition in vivo.
In the present experimental setting, we have also unfolded a novel mechanism by which lipophilic dihydropyridine CCBs may exert organ protection. Treatment with lercanidipine reduced ADMA blood levels in dTGR. This endogenous NOS inhibitor has attracted much attention in cardiovascular medicine recently, and its role in endothelial dysfunction, atherosclerosis, and cardiovascular mortality has been studied in various clinical conditions such as hypertension, renal disease, insulin resistance, peripheral vascular disease, and acute coronary syndrome. Our data support the notion that dihydropyridine CCBs may influence the NO pathway by modulation of ADMA blood levels and thus the inhibitory effect of ADMA on NOS. We provide further evidence that lercanidipine treatment modulates DDAH activity. This intracellular enzyme is a key regulator of ADMA metabolism. Interestingly, we were able to show that lercanidipine also reduces iNOS expression in tissue of the dTGR. Results from experimental studies have revealed that DDAH activity can be directly regulated by S-nitrosylation of its active site by NO, thereby creating a regulatory feedback loop of NO production, DDAH activity, and ADMA blood levels. The implication of this finding is that under conditions of increased NO production such as in inflammation, where iNOS generates abundant NO, S-nitrosylation diminishes DDAH activity, and this in turn would lead to accumulation of ADMA and to NOS inhibition. Lercanidipine may interrupt this vicious cycle by inhibiting iNOS and thus increasing DDAH activity or by directly inhibiting DDAH, or both (Fig. 2
).
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In summary, we present data that the highly lipophilic third generation dihydropyridine CCB lercanidipine prevents ANG II-induced renal injury and improves survival by reducing tissue inflammation and fibrosis. These beneficial effects may result from important intracellular actions of lercanidipine such as inhibition of PKC activation and modulation of DDAH activity.
FOOTNOTES
2 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4087fje
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  T. Rosenthal, E. Rosenmann, D. Tomassoni, and F. Amenta Effect of Lercanidipine on Kidney Microanatomy in Cohen-Rosenthal Diabetic Hypertensive Rats Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2007; 12(2): 145 - 152. [Abstract] [PDF]
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