Jörg Peters

Two different renin isoforms are expressed in extrarenal tissues. The classical renin-a has been associated with detrimental effects, whereas renin-b exerts protective effects during glucose starvation. Glucose starvation selectively increased renin-b mRNA levels. 8Br-cAMP increased renin-a mRNA levels independently of glucose as well as of renin-b in glucose-starved cells. Adenylyl cyclase (AC) stimulation by forskolin increased expression of both renin transcripts, while AC inhibition by SQ22536 produced the opposite effect. PKA inhibition by KT5720 reduced the mRNA levels of both renin transcripts glucose-independently. Forskolin reversed the effect of KT5720 on renin mRNA levels. A23187-mediated increase in [Ca²⁺]i increased renin-b mRNA levels in glucose-starved cells. Ca²⁺ chelator BAPTA decreased renin-a mRNA expression in control cells and renin-b levels glucose-independently. Forskolin reversed the BAPTA-mediated decreases in renin-a but not renin-b expression. While the regulation of renin transcript levels by cAMP and PKA resembled known regulation in the kidney, the effect of intracellular free Ca²⁺ levels were opposite. This supports the existence of a separate renin system in cardiac cells.

ATP6AP2 (ATPase H⁺ Transporting Accessory Protein 2) also known as (pro)renin receptor (P)PR is expressed in the central nervous system (CNS). In humans, variants of ATP6AP2 have been linked to neurodegenerative disorders and mental retardation and to changes in the volume of the hippocampus—a limbic brain structure that is capable of adult hippocampal neurogenesis. Cell proliferation in the adult dentate gyrus (DG) can be positively modulated by voluntary exercise. Somewhat surprisingly, we have recently shown that ATP6AP2 overexpression does not show any significant effects on the number of newly generated cells in the DG in transgene-heterozygous mice. Therefore, we now analyzed the effects of voluntary exercise on cell proliferation in the adult hippocampus in female mice overexpressing ATP6AP2 on both alleles. ATP6AP2 mRNA levels in forebrain and hippocampus were increased dose-dependently compared with wild-type mice, with homozygous exhibiting stronger effects than heterozygous mice. Full-length ATP6AP2 protein levels were also increased dose-dependently in forebrain, but the protein fragment representing the soluble prorenin receptor was not. In total hippocampus extracts, no significant differences in ATP6AP2 protein levels were found. However, in the DG, ATP6AP2 immunoreactivity was markedly increased. While the running pattern of mice with access to a running wheel was similar between the groups, cell proliferation, as indicated by BrdU staining, was increased in wild-type mice as expected, but failed to increase in homozygous ATP6AP2 mice. This finding indicates that ATP6AP2 overexpression does not affect basal adult cell proliferation per se but inhibits plastic changes in the rate of cell proliferation. Thus, ATP6AP2 appears to selectively limit exercise-dependent regulation in the hippocampus rather than baseline neurogenic capacity.

The classical secretory renin-a is known to be involved in angiotensin generation, thereby regulating not only blood pressure, but also promoting oxidative stress as well as apoptotic and necrotic cell death. In contrast, another cytosolic renin isoform named renin-b has been described, exerting protective effects under ischemia-related conditions in H9c2 cardiomyoblasts. Using microarray-based transcriptome analyses, we aimed to identify the signaling pathways involved in mediating cardioprotection in H9c2 cells overexpressing renin-b. By transcriptome profiling, we identified increased gene expression of several genes encoding glycolytic enzymes and glucose transporters, while the transcript levels of TCA-cycle enzymes were decreased. Complementing data from metabolic analyses revealed enhanced glucose consumption and lactate accumulation due to renin-b overexpression. Renin-b overexpression further stimulated AKT/mTOR signaling, where numerous genes involved in this pathway showed altered transcript levels. For AKT, we also detected enhanced phosphorylation levels by means of Western blotting, suggesting an activation of this kinase. Moreover, analysis of the ROS levels identified an increase in ROS accumulation in renin-b-overexpressing cells. Altogether, our data demonstrate that renin-b overexpression induces the metabolic remodeling of H9c2 cells similar to that seen under oxygen deprivation. This metabolic phenotype exerting so-called aerobic glycolysis is also known as the Warburg effect.

A stimulated renin-angiotensin system is known to promote oxidative stress, apoptosis, necrosis and fibrosis. Renin transcripts (renin-b; renin-c) encoding a cytosolic renin isoform have been discovered that may in contrast to the commonly known secretory renin (renin-a) exert protective effects Here, we analyzed the effect of renin-a and renin-b overexpression in H9c2 cardiomyoblasts on apoptosis and necrosis as well as on potential mechanisms involved in cell death processes. To mimic ischemic conditions, cells were exposed to glucose starvation, anoxia or combined oxygen–glucose deprivation (OGD) for 24 h. Under OGD, control cells exhibited markedly increased necrotic and apoptotic cell death accompanied by enhanced ROS accumulation, loss of mitochondrial membrane potential and decreased ATP levels. The effects of OGD on necrosis were exaggerated in renin-a cells, but markedly diminished in renin-b cells. However, with respect to apoptosis, the effects of OGD were almost completely abolished in renin-b cells but interestingly also moderately diminished in renin-a cells. Under glucose depletion we found opposing responses between renin-a and renin-b cells; while the rate of necrosis and apoptosis was aggravated in renin-a cells, it was attenuated in renin-b cells. Based on our results, strategies targeting the regulation of cytosolic renin-b as well as the identification of pathways involved in the protective effects of renin-b may be helpful to improve the treatment of ischemia-relevant diseases.

Significance: Primary aldosteronism (increased production of the adrenal steroid hormone aldosterone) is the most common cause of secondary hypertension. We here generated a mouse model of familial hyperaldosteronism type IV with a heterozygous gain-of-function mutation in a calcium channel gene (Cacna1h^M1560V/+).Cacna1h^M1560V/+mice have about twofold elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism) and elevated blood pressure, with an overall mild phenotype. Elevated adrenal aldosterone synthase expression inCacna1h^M1560V/+mice is associated with increased intracellular calcium concentrations in glomerulosa cells. This model allows for the ex vivo analysis of calcium signaling in aldosterone-producing glomerulosa cells of the adrenal gland.Cacna1h^−/−mice have normal aldosterone synthase expression, with implications for the evaluation of CACNA1H as a therapeutic target.

The renin-angiotensin system is known to regulate blood pressure as well as water- and electrolyte balance. An activated RAS is involved in the development of hypertension and hypertension-related organ damage. Thus, inhibitors of the RAS are protective and markedly increasing the life span of patients. In contrast, renin transcripts have been discovered encoding a cytoplasmatic renin isoform, termed renin-b, which is not harmful but may be even protective. Here we demonstrate that depletion of renin-b encoding transcripts by small interference RNA decreased ATP levels and increased basal necrosis as well as apoptosis rates. Furthermore, renin-b depletion potentiated the anoxia-induced increase of necrosis rates. Vice versa, overexpression of renin-b prevented the anoxia-induced increase of caspase-mediated apoptosis rates. Besides, cells overexpressing renin-b exhibited even reduced mitochondrial mediated apoptosis rates under anoxia, when compared with normoxic conditions, as indicated by Annexin V labeling. However, whereas the protective effect of renin-b on caspase-mediated apoptosis was completely blocked by the renin inhibitor CH732, the effect on mitochondrial-mediated apoptosis was not affected by CH732 at all. From these data we conclude that renin-b overexpression mediates cardioprotective effects under anoxia with respect to mitochondrial induced apoptosis angiotensin-independently, but with respect to caspase induced apoptosis likely in an angiotensin-dependent manner.

Secretory renin promotes hypertrophy, apoptosis, necrosis and fibrosis through angiotensin generation. It has been claimed that local expression of renin contributes to the deleterious effects of the renin-angiotensin system in the heart. Besides the classic renin transcript (renin-a), encoding for secretory renin, a putative brain-specific (renin-b) and a putative lung-specific (renin-c) transcript may exist in human. In contrast to secretory renin, renin-b cannot be secreted, remains within the cytosol and is imported into mitochondria. In contradiction to renin-a, renin-b exerts cardioprotective effects in hearts and in cardiac cells of the rat under ischemia related conditions. To date, available data on cardiac renin expression remain inconsistent. Nobody has yet investigated which renin transcripts are expressed in the human heart. We systematically analyzed the levels of renin transcripts using specific and sensitive nested reverse-transcriptase polymerase chain reactions (RT-PCR) in human ventricular biopsies obtained from patients with heart diseases. In the 33 biopsies available, neither the expression of classic renin-a, nor of the alternative renin-c was detected. In contrast, the renin-b transcript, which was previously classified as brain-specific, was found in 11/33 ventricular biopsies. Our data exclude the expression of secretory renin and indicate that local expression of cytosolic renin but not of secretory renin can play a functional role in the human heart.

Although the renin-angiotensin system usually promotes oxidative stress and cell death, renin transcripts have been discovered, whose transcription product may be cardioprotective. These transcripts encode a non-secretory renin isoform that is localized in the cytosol and within mitochondria. Here we tested the hypotheses that cytosolic renin [ren(2-9)] expression promotes cell survival under hypoxia and glucose depletion by preserving the mitochondrial membrane potential (∆Ψ_m) and mitigating the accumulation of ROS. To simulate ischemic insults, we exposed H9c2 cells to glucose deprivation, anoxia or to combined oxygen-glucose deprivation (OGD) for 24 hours and determined renin expression. Furthermore, H9c2 cells transfected with the empty pIRES vector (pIRES cells) or ren(2-9) cDNA-containing vector [ren(2-9) cells] were analyzed for cell death, ∆Ψ_m, ATP levels, accumulation of ROS, and cytosolic Ca²⁺ content. In pIRES cells, expression of ren(1A-9) was stimulated under all three ischemia-related conditions. After OGD, the cells lost their ∆Ψ_m and exhibited enhanced ROS accumulation, increased cytosolic Ca²⁺ levels, decreased ATP levels as well as increased cell death. In contrast, ren(2-9) cells were markedly protected from these effects. Ren(2-9) appears to represent a protective response to OGD by reducing ROS generation and preserving mitochondrial functions. Therefore, it is a promising new target for the prevention of ischemia-induced myocardial damage.