Elisabeth Schinner

Cyclic cytidine 3′,5′-monophosphate (cCMP) signals via cGMP kinase I

We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane‐permeable cCMP analogue dibutyryl‐cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP‐dependent protein kinase (cGK) Iα and Iβ and stimulated cGK in aorta lysates. cCMP‐induced relaxation was abolished in cGKI‐knockout tissue. Additionally, deletion of inositol–trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP‐mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP‐mediated inhibition of platelet aggregation was absent in cGKI‐ and IRAG‐deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.

Cyclic Nucleotide Signalling in Kidney Fibrosis

Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure.

The cyclic GMP–dependent protein kinase Iα suppresses kidney fibrosis

Cyclic guanosine monophosphate (cGMP) is synthesized by nitric oxide or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions in the kidney. Hence, integration of cGMP signaling by cGMP-dependent protein kinases (cGKs) might play a critical role in renal physiology; however, detailed renal localization of cGKs is still lacking. Here, we performed an immunohistochemical analysis of cGKIα and cGKIβ isozymes in the mouse kidney and found both in arterioles, the mesangium, and within the cortical interstitium. In contrast to cGKIα, the β-isoform was not detected in the juxtaglomerular apparatus or medullary fibroblasts. Since interstitial fibroblasts play a prominent role in interstitial fibrosis, we focused our study on cGKI function in the interstitium, emphasizing a functional differentiation of both isoforms, and determined whether cGKIs influence renal fibrosis induced by unilateral ureter obstruction. Treatment with the guanylyl cyclase activators YC1 or isosorbide dinitrate showed stronger antifibrotic effects in wild-type than in cGKI-knockout or in smooth muscle-cGKIα-rescue mice, which are cGKI deficient in the kidney except in the renal vasculature. Moreover, fibrosis influenced the mRNA and protein expression levels of cGKIα more strongly than cGKIβ. Thus, our results indicate that cGMP, acting primarily through cGKIα, is an important suppressor of kidney fibrosis.

cGMP becomes a drug target

Cyclic guanosine 3′,5′-monophosphate (cGMP) serves as a second messenger molecule, which regulates pleiotropic cellular functions in health and disease. cGMP is generated by particulate or soluble guanylyl cyclases upon stimulation with natriuretic peptides or nitric oxide, respectively. Furthermore, the cGMP concentration is modulated by cGMP-degrading phosphodiesterases. Several targets of cGMP are utilized to effect its various cellular functions. These effector molecules comprise cGMP-dependent protein kinases, ion channels, and phosphodiesterases. During the last decade, it emerged that cGMP is a novel drug target for the treatment of pulmonary and cardiovascular disorders. In this respect, several drugs were developed, which are now in clinical phase studies for, e.g., pulmonary hypertension or cardiovascular diseases. These new drugs act NO-independently with/without heme on soluble guanylyl cyclases or induce subtypes of particular guanylyl cyclases and thereby lead to new therapeutic concepts and horizons. In this regard, the fifth cGMP meeting held in June 2011 in Halle, Germany, comprised the new therapeutic challenges with the novel functional and structural concepts of cGMP generating and effector molecules. This report summarizes the new data on molecular mechanisms, (patho)physiological relevance, and therapeutic potentials of the cGMP signaling system that were presented at this meeting.

Signaling via IRAG is essential for NO/cGMP-dependent inhibition of platelet activation

Platelet activation is strongly affected by nitric oxide/cyclic GMP (NO/cGMP) signaling involving cGMP-dependent protein kinase I (cGKI). Previously it was shown that interaction of the cGKI substrate IRAG with InsP3RI is essential for NO/cguanosine monophosphate (GMP)-dependent inhibition of platelet aggregation in vitro and in vivo. However, the role of Inositol-trisphosphate receptor associated cGMP kinase substrate (IRAG) for platelet adhesion or granule secretion was unknown. Here, we analysed the functional role of IRAG for platelet activation. Murine IRAG-deficient platelets displayed enhanced aggregability towards several agonists (collagen, thrombin and TxA2). NO- or cGMP-dependent inhibition of agonist induced ATP- or 5-HT secretion from dense granules, and P-selectin secretion from alpha granules was severely affected in IRAG-deficient platelets. Concomitantly, the effect of NO/cGMP on platelet aggregation was strongly reduced in IRAG-deficient platelets. Furthermore, GPIIb/IIIa-mediated adhesion of platelets to fibrinogen could only weakly be inhibited in IRAG-deficient mice contrary to wild-type (WT) mice. Our results suggest that signaling via IRAG is essential for NO/cGMP-dependent inhibition of platelet activation regarding granule secretion, aggregation and adhesion. This platelet disorder might cause that the bleeding time of IRAG-deficient mice was reduced.

Interaction of cCMP with the cGK, cAK and MAPK Kinases in Murine Tissues

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-32P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.

Inhibition of the TGFβ signalling pathway by cGMP and cGMP‐dependent kinase I in renal fibrosis

Agents that enhance production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) ameliorate the progression of renal fibrosis. However, the molecular mechanism of this process is not fully understood. We hypothesize that the antifibrotic effects of cGMP and cGMP‐dependent kinase I (cGKI) are mediated via regulation of the TGFβ signalling pathway, both via ERK and the Smad‐dependent route. Kidney fibrosis was induced by unilateral ureter obstruction (UUO) in wild‐type and cGKI‐deficient (cGKI‐KO) mice. The cGMP/cGKI signalling pathway was activated by application of the soluble guanylate cyclase (sGC) stimulator BAY 41‐8543 (BAY), beginning 1 day after UUO. After 7 days, the antifibrotic effects of BAY were analysed by measuring mRNA and protein expression of characteristic fibrotic biomarkers. The effects of cGMP/TGFβ on cultured fibroblasts were also analysed in vitro. BAY application influenced the activity of the extracellular matrix (ECM)‐degrading matrix metalloproteases (MMP2 and MMP9) and their inhibitor tissue inhibitors of metalloproteinase‐1, the secretion of cytokines (e.g. IL‐6) and the expression pattern of ECM proteins (e.g. collagen, fibronectin) and profibrotic mediators (e.g. connective tissue growth factors and plasminogen‐activator inhibitor‐1). Activation of the cGMP/cGKI signalling pathway showed protective effects against fibrosis which were mediated by inhibition of P‐Erk1/2 and translocation of P‐smad3. The elucidation of these signalling mechanisms might support the development of new therapeutic options regarding cGMP/cGKI‐mediated antifibrotic actions.

Involvement of Cyclic Guanosine Monophosphate-Dependent Protein Kinase I in Renal Antifibrotic Effects of Serelaxin

Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/cGMP to inhibit transforming growth factor-β (TGF-β) signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin. Methods and Results: Kidney fibrosis was induced by unilateral ureteral obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagen1A1, total collagen, and fibronectin. The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO. Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1-dependent TGF-β signaling and increased PDE5a phosphorylation.

Regulation of the Na+‐K+‐2Cl− cotransporter by cGMP/cGMP‐dependent protein kinase I after furosemide administration

Sodium chloride reabsorption in the thick ascending limb of the loop of Henle is mediated by the Na+‐K+‐2Cl− cotransporter (NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, less is known about the mechanism regulating the electrolyte transporter. Considering the well‐established effects of nitric oxide on NKCC2 activity, cGMP is likely involved in this regulation. cGMP‐dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates different substrates after activation through cGMP. We investigated the potential correlation between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild‐type (wt) and cGKIα‐rescue mice with furosemide. cGKIα‐rescue mice expressed cGKIα only under the control of the smooth muscle‐specific transgelin (SM22) promoter in a cGKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in cGKIα‐rescue mice compared to that in wt mice. We analyzed the phosphorylation of NKCC2 by western blotting and immunostaining using the phosphospecific antibody R5. The administration of furosemide significantly increased the phosphorylated NKCC2 signal in wt but not in cGKIα‐rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether cGKI was involved in this process, we analyzed vasodilator‐stimulated phosphoprotein, which is phosphorylated by cGKI. Furosemide injection resulted in increased vasodilator‐stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI‐mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2.

Antifibrotic function of cGKI in the kidney.

Results The cGKIa isozyme is expressed in the renal medullary interstitium. Unilateral ureter kidney was taken as model for the analysis of interstitial kidney fibrosis in wild type, cGKI-KO and cGKIa rescue mice (which express cGKIa solely in smooth muscle in a cGKI-KO background). We tested whether the pharmacological stimulation of the cGMP/cGKI signalling pathway affects the induction of interstitial kidney fibrosis. For this purpose we used a) NO-independent sGC stimulators (YC-1, Bay41-8543) and b) the pregnancy hormone serelaxin which was shown to induce cGMP levels and is currently tested for treatment of acute heart failure. sGC stimulators effectively suppressed TGFb levels, myofibroblast differentiation (SMA) and deposition of extracellular matrix (ECM) (collagen, fibronectin) in wild type mice involving RhoA/ROCK signalling in contrast to cGKI-KO and/or cGKIa rescue mice. Serelaxin treatment by osmotic pumps continuously enhanced cGMP concentrations in the kidney. Serelaxin also strongly reduced interstitial kidney fibrosis via diminished cytokines (TGFb, CTGF), myofibroblasts and ECM and by regulation of matrix metalloproteases (MMP-2, MMP-9) dependent on the presence of cGKI. However, our results indicated that serelaxin might exert different signalling pathways e.g. via MAPK. Conclusion Our results suggest that pharmacological treatment with sGC stimulators or serelaxin enhancing cGMP suppresses interstitial kidney fibrosis via cGMP/cGKI signalling.