Angiotensin II induced inflammation in the kidney and in the heart of double transgenic rats

Hypertension injures blood vessels and thereby causes end-organ damage. The mechanisms are complicated and, although studied for decades in experimental animal models, are only currently being elucidated [1] The endothelial layer acts as a signal transduction interface for hemo-dynamic forces in the regulation of vascular tone and chronic structural remodeling of arteries. Effects of mechanical forces on signal transduction and gene expression in endothelial cells have been demonstrated [2]. Microvascular endothelium in hypertensive animals has been shown to exhibit increased oxyradical production attributable to xanthine oxidase [3]. Oxyradical production by endothelial cells can result in leukocyte-endothelial adhesion responses that involve transcription-independent and -dependent surface expression of different endothelial cell adhesion molecules. Infiltration of the permeabilized endothelium by leukocytes sets the stage for an inflammatory cascade, involving cytokines, chemokines, growth factors, and matrix metalloproteinases. Altered integrin signaling, the production of tenacin, epidermal growth factor signaling, tyrosine phosphorylation, and activation of downstream pathways culminate in vascular smooth muscle cell proliferation. [4‐6] Evidence is accumulating that matrix molecules provide an environment which decreases the rate of programmed cell death.

We have studied a double-transgenic rat model. The rats harbor both the human renin and human angiotensinogen genes [7, 8]. The rat and human renin-angiotensin systems do not interact, so the hypertension and vasculopathy exhibited by these rats is the result of the overexpression of the human renin-angiotensin system. The rats were developed for the expressed purpose of studying human renin inhibitors that ordinarily do not function in rats. However, we have found that this model, which features renal and cardiac failure by 8 weeks of age in these animals, also provides great utility in studying blood pressure-independent Ang II-related effects. To exclude possible effects of blood pressure, we lowered blood pressure with non-angiotensin-related hypotensive agents. This strategy delayed morbidity and mortality by 1 week but did not ameliorate the inflammatory disease process.

Many inflammation-mediating genes are activated by the transcription factor NF-κB, which resides inactive and bound to the inhibitory protein I-κB in the cytoplasm of T lymphocytes, monocytes, macrophages, endothelial cells, and smooth muscle cells [9]. ANG II stimulates NADPH oxidase, which generates reactive oxygen species (ROS) [10]. ROS may act as signal transduction messengers for several important transcription factors, including NF-κB and AP-1 [11]. Several reports have indicated that angiotensin converting enzyme (ACE) inhibition decreased NF-κB in renal disease [12, 13].

In vitro and in vivo studies showed that pyrrolidine dithiocarbamate (PDTC) is a potent inhibitor of NF-κB, but had no effect on AP-1, CREB, Sp-1, and octamer-binding proteins [14, 15]. The precise mechanism for the biological effects of PDTC is still controversial. Several studies have reported that metal-chelating, thiol modifying and oxygen radical-scavenging antioxidative properties mediate the inhibition of NF-κB [15]. We recently observed that rats harboring both human renin and angiotensinogen genes (dTGR) develop severe renal and cardiac damage, accompanied by increased NF-κB DNA binding activity, MCP-1 production, adhesion molecule expression, inflammation, and die by seven weeks of age [16, 17]. We now investigated the hypothesis that PDTC inhibition of NF-κB prevents inflammation and ameliorates cardiac and renal damage.

DTGR feature hypertension (190 ± 7 mm Hg vs 107 ± 2 mm Hg for SD at week 7, p < 0.001 n = 15, severe renal damage, and focal necrosis in heart and kidney and a mortality of 40 % at 7 weeks. Cardiac hypertrophy is one of the leading features in dTGR (5,9 vs 3,6 heart weigth/body weigth (mg/g). 24 h albuminuria progressively increased in untreated dTGR from 5 to 7 weeks. (57.1 ± 8.7 vs. 0.3 ± 0.1 mg/d week 7). Small vessels showed increased intimal and medial thickness as well as hyaline deposits, tubuls were swollen and filled with proteinaceous material (Fig. 1 and 2). PAI-1 and fibronectin expression were increased in the perivascular space and adventitia of dTGR compared to SD (Fig. 3,4,5,6).

There was significant infiltration in cardiac and renal perivascular space of dTGR compared to SD. Fig. 7 and 8 show a representative immunohistochemistry for ED1, a marker for macophages in the heart and in the kidney (9 and 10). Fig. 24 shows semiquantative scoring for macrophage-monocyte-infiltration of ED-1 in the kidney and in the heart. This was perforned using a computerized cell count programm.

The expression ot the adhesion protein VCAM-1 was increased in the interstitium, intima and adventitia of the small renal and cardiac vessels in dTGR compared to SD animals (Fig. 11,12,13,14). Similar results were obtained for ICAM-1 (see below). The counterreceptors for ICAM-1 and VCAM-1, αLβ2 integrin (LFA-1) and α4β1 (VLA-4) were upregulated in the perivascular space and the adventitia of the heart (Fig. 15 and 16) and the kidney (Fig. 17 and 18) in dTGR compared to SD.

A schematic diagram in Fig. 19 summarizes the Ang II signaling in the dTGR model, resulting in oxidative stress, participation of endothelin, and transcription factor activation. These transcription factors activate a host of genes involved in inflammation including surface adhesion molecules, thrombogenic factors, chemokines, and inducible NO synthase

Subsequently, we used the dTGR model for a pharmacological intervention with PDTC to prevent NF-κB signalling. In week 7 mortality was reduced to 0%, cardiac hypertrophy reduced to 4.90 ± 0.1 (vs. 5.77 ± 0.1 mg/g, p < 0.001), albuminuria to 2.5 ± 0.8 mg/d and blood pressure to 162 ± 8 mm Hg, respectively. ICAM-1 expression in the kidney was increased in the intima, adventitia, and in the perivascular space of the small vessels in untreated dTGR. Glomeruli and tubules also showed increased ICAM-1 expression. Expression of ICAM-1 was markedly reduced by treatment with PDTC and was similar to the constitutive ICAM-1 expression in control animals at week 7 (Fig. 20a,b,c). Furthermore we showed significantly reduced macrophage-monocyte-infiltration in the kidney and in the heart by semiquantative cell count analysis (Fig. 24) in PDTC treated rats compared to untreated dTGR (6 ± 1 vs. 22 ± 1, p < 0.0001). These results demonstrate that PDTC treatment reduced mononuclear cell infiltration and inhibited the expression of adhesion molecules.

Next, we investigated the effects of PDTC on iNOS expression. We observed a strong increase in iNOS expression in the glomeruli in the vessel walls of renal arterioles (Fig. 21d,e,f). Treatment with PDTC greatly reduced the iNOS immunoreactivity both in the blood vessels and the glomeruli. Furthermore, we investigated the activation of the transcription factor NF-κB, which is one of the main regulator of ICAM-1 and iNOS gene expression. Immunohistochemistry (phase contrast resolution) shows the localization of the subunit p65 of NF-κB in a cardiac vessel. The p65 expression was increased in the endothelium, smooth muscle cells (Fig. 22), as well as in the vessel wall, infiltrated cells, glomeruli, and tubules (data not shown) of dTGR kidneys. PDTC markedly reduced p65 expression (Fig. 22). No immunoreaction was observed in non-transgenic SD rats (Fig. 22). The antibody recognizes an epitope overlapping the nuclear location signal of p65 subunit and therefore selectively stains released, activated NF-κB after dissociation from its inhibitor IκBa [24]. We used EMSA for the detection of NF-κB DNA binding activity in the kidney (Fig. 23A) and heart (Fig. 23D). NF-κB DNA binding activity in the kidney was markedly reduced by PDTC treatment and even more in the heart. Each lane represents a separate animal. Renal homogenates were incubated with antibodies against the NF-κB subunits anti-p50, anti-p65, anti-c-Rel, and anti-Rel B (23 B). Binding specificity was demonstrated by competition of excess unlabeled oligonucleotides containing the kB site from the MHC-enhancer (H2K) (23 C). AP-1 activity was increased in dTGR kidneys compared with SD (data not shown). However, PDTC treatment did not reduce AP-1 binding activity (data not shown).

As the growing role of "oxidative stress" in the pathogenesis of hypertension becomes more appreciated, our impression of hypertension as a rather indolent, solely hemo-dynamic process is being revised [25]. Reactive oxygen species are the end result of univalent reductions in oxygen, resulting in the production of superoxide anion, hydrogen peroxide, and water. Reactive oxygen species influence both normal and abnormal cellular processes, including cellular growth, hypertrophy, remodeling, lipid oxidation, modulation of vascular tone, and inflammation [26]. Reactive oxygen species can also act as intracellular signaling molecules in vascular cells controlling growth, survival, and apoptosis [4]. An important source of reactive-oxygen species are the NADH/NADPH oxidases, which are regulated in part by Ang II [10]. Touyz and Schiffrin recently showed that in vascular smooth muscle cells from human peripheral resistance arteries, Ang II increased H₂O₂ generation through phospholipase D-dependent, NADH/NADPH oxidase-sensitive pathways [27]. A role for reactive oxygen species in Ang II-mediated transcription factor activation has been established. The potential targets of reactive oxygen species in endothelial and vascular smooth muscle cells are extracellular signal-related kinases, stress-activated protein kinases, caspases, and NF-κB [4]. NF-κB activation has been associated with vascular inflammation [28]. The activation of NF-κB involves the phosphorylation and subsequent proteolytic degradation of the inhibitory protein IκB by specific IκB kinases. The free NF-κB, a heterodimer consisting of 2 proteins, p50 and p65, passes into the nucleus, where it binds to kB sites in the promoter region of numerous genes involved in inflammation [29]. Many of these genes code for cytokines, chemokines, enzymes, proteins involved in coagulation, receptors, protein-ases, and adhesion molecules. These molecules contribute to the alterations in structure and mechanical properties responsible for the remodeling of resistance arteries in hypertension [30].

We tested the hypothesis that inhibition of NF-κB prevents inflammatory responses and ameliorates cardiac and renal damage. We found that NF-κB inhibition prevented inflammatory responses, ameliorated cardiac and renal damage, and reduced mortality completely. Chronic PDTC treatment decreased blood pressure and cardiac hypertrophy slightly, but reduced albuminuria almost completely. The modest decrease in blood pressure was probably related to amelioration of renal failure rather than the result of a vasodilator effect.

We showed earlier that ANG II production in the kidneys and elsewhere is responsible for this severe vasculopathy [31]. ANG II stimulates various signaling pathways leading to NF-κB activation. PDTC is a potent inhibitor of NF-κB [32]. Schreck et al. and Liu et al. demonstrated that PDTC inhibited NF-κB activation of various stimulants, but had no effect on AP-1, CREB, Sp-1, and octamer-binding proteins in several cell lines and in vivo [14, 15]. In our study, PDTC markedly decreased NF-κB binding activity in the heart and kidney, while AP-1 was not decreased. Nakamura et al. found that administration of antioxidants inhibited cardiac hypertrophy [33]. In our model, chronic treatment with PDTC partially reduced cardiac hypertrophy.

NF-κB plays a central role in various cardiovascular diseases. Ruiz-Ortega et al. reported that NF-κB activation and MCP-1 expression in the renal cortex was reduced by ACE inhibition in experimental immune complex nephritis [13]. Morrissey and Klahr showed that ACE inhibition decreased NF-κB in kidneys with ureteral obstruction [34]. Recently, Rangan et al. demonstrated that NF-κB inhibition by PDTC reduced inflammation and tubulointerstitial injury in nonimmune proteinuric rats [19]. Myocardial and renal reperfusion injury develops to a large extent subsequent to the complex interaction of multiple cytokines and activated adhesion molecules [20, 35, 36]. Morishita et al. demonstrated that NF-κB inhibition by a decoy technique reduced the extent of myocardial infarction following reperfusion [36]. Hernandez-Presa have shown that ACE inhibition prevents arterial NF-κB activation, chemokine expression, and macrophage infiltration in early accelerated atherosclerosis [12]. We performed immunohistochemical analysis for NF-κB subunit p65, because in contrast to the p50 subunit, p65 contains the transcription activation domain. The antibody recognizes an epitope overlapping the nuclear location signal of p65 subunit and therefore selectively stains released activated NF-κB after dissociation from its inhibitor IκBα [24]. The NF-κB subunit p65 was increased in the endothelium, smooth muscles cells of damaged small vessels, and infiltrated cells in dTGR. The staining pattern resembled the localization of the p65 expression in atherosclerotic lesions.

Untreated dTGR show a 150-fold increased albuminuria. Remuzzi and coworkers demonstrated that increased glomerular permeability is followed by increased filtration of macromolecules, followed by excessive tubular protein reabsorption [37, 38]. This process leads to abnormal accumulation of proteins in endolysosomes and endoplasmatic reticulum. Altogether, these processes foster the activation of NF-κB dependent and independent cytokines resulting in renal inflammation. Chronic inhibition of NF-κB by PDTC probably inhibited both the direct activation of NF-κB by ANG II as well as the subsequent activation induced by the increased glomerular permeability in our model, thereby breaking the self-amplifying loop.

We also examined inducible NO synthase (iNOS). Promoter deletion and mutation studies in cultured cells demonstrated that NF-κB plays a critical role in transcriptional regulation of the iNOS gene induced by various cytokines [39] NO regulates numerous physiological and pathophysiological processes, including smooth muscle contractility, platelet reactivity, and the cytotoxic activity of leukocytes. However, inappropriate release of this mediator has been linked to the pathogenesis of a number of disease states [40]. While endothelial NO serves beneficial roles as a messenger and host defense molecule, excessive NO production by inducible NOS can be cytotoxic. Indeed, peroxynitrite anion formation, protein tyrosine nitration, and hydroxyl radical production may contribute to the evolution of several commonly encountered renal diseases, including postischemic renal failure, obstructive nephropathy, and renal allograft rejection, among others [41]. Our data show that inhibition of NF-κB by PDTC markedly reduces inflammation, iNOS expression in the dTGR most likely leading to decreased cytotoxicity, and cell proliferation. Thus, NF-κB activation plays an important role in ANG II-induced end-organ damage.