Andrea Schramm

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.

Adiponectin upregulates monocytic activin A but systemic levels are not altered in obesity or type 2 diabetes

Adiponectin is an adipocyte-derived protein with atheroprotective and immunoregulatory function. Adiponectin and activin A reduce foam cell formation and adiponectin activates the p38 MAPK pathway that is well described to induce activin A. Therefore, it was analyzed whether adiponectin alters activin A in primary human monocytes. Adiponectin dose- and time-dependently induced activin A in the supernatant, and the maximal amount was observed after 12h of incubation. Adiponectin-stimulated release of activin A was blocked by a p38 MAPK inhibitor. Metformin and pioglitazone are drugs frequently used to treat diabetic patients and metformin slightly reduced monocytic activin A release whereas pioglitazone had no effect. Type 2 diabetes is associated with elevated inflammatory systemic cytokines but activin A serum levels were similar in slim probands, overweight controls and type 2 diabetic patients. Furthermore, activin A did not correlate to systemic adiponectin, body mass index, waist to hip ratio or C-reactive protein. These findings indicate that adiponectin upregulates monocytic activin A release via the p38 MAPK pathway, and this may in part explain the immunoregulatory and antiatherosclerotic effects of this adipokine.

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.

Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies

Nitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein–protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases. Conventionally used methods to detect PPIs exhibit several disadvantages, e.g., co-immunoprecipitations, which depend on suitable high-affinity antibodies. Therefore, we established a cell-based protein-fragment complementation assay (PCA) for the identification of PKG target proteins. Here, a reporter protein (click beetle luciferase) is split into two fragments and fused to two different possible interaction partners. If interaction occurs, the reporter protein is functionally complemented and the catalyzed reaction can then be quantitatively measured. By using this technique, we confirmed the regulator of G-Protein signaling 2 (RGS2) as an interaction partner of PKGIα (a PKG-isoform) following stimulation with 8-Br-cGMP and 8-pCPT-cGMP. Hence, our results support the conclusion that the established approach could serve as a novel tool for the rapid, easy and cost-efficient detection of novel PKG target proteins.

Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca2+ Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons

Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca2+ signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca2+ dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca2+ transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca2+ transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca2+ signaling in CGNs. This cGMP–Ca2+ crosstalk likely affects neurotransmitter-stimulated functions of CGNs.

cGMP-Signaltransduktion in vaskulären Prozessen der Niere

The second messenger cyclic guanosine monophosphate (cGMP) regulates various (patho-)physiological peripheral and central processes. Elucidation of cGMP-mediated mechanisms–the cGMP signalling–is essential to comprehend these diverse functions. Recently, some of these signalling pathways were established in the renovascular system. Furthermore, new drugs were approved which are promising for treatment of thrombotic and hypertensive diseases. Here, we provide an overview regarding cGMP signalling and its future therapeutical options in the renovascular system.

Suppression of kidney fibrosis by cGMP-dependent protein kinase I

Background cGMP is synthesized via nitric oxideor natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. Hence, the integration of cGMP signaling via cGMP-dependent protein kinases (cGK) might play a critical role for renal physiology. Both isozymes were detected in arterioles, mesangium and within the cortical interstitium. In contrast to cGKIa, the b isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts.

Function of cGMP-dependent protein kinase II in volume expansion-induced diuresis

Atrial natriuretic peptide (ANP)/cGMPs cause diuresis and natriuresis. Their downstream effectors beyond cGMP remain unclear. To elucidate a probable function of cGMP-dependent protein kinase II (cGKII), we investigated renal parameters in different conditions (basal, salt diets, starving, water load) using a genetically modified mouse model (cGKII-KO), but did not detect any striking differences between WT and cGKII-KO. Thus, cGKII is proposed to play only a marginal role in the adjustment of renal concentration ability to varying salt loads without water restriction or starving conditions. When WT mice were subjected to a volume load (performed by application of a 10-mM glucose solution (3 % of BW) via feeding needle), they exhibited a potent diuresis. In contrast, urine volume was decreased significantly in cGKII-KO. We showed that AQP2 plasma membrane (PM) abundance was reduced for about 50 % in WT upon volume load, therefore, this might be a main cause for the enhanced diuresis. In contrast, cGKII-KO mice almost completely failed to decrease AQP2-PM distribution. This significant difference between both genotypes is not induced by an altered p-Ser256-AQP2 phosphorylation, as phosphorylation at this site decreases similarly in WT and KO. Furthermore, sodium excretion was lowered in cGKII-KO mice during volume load. In summary, cGKII is only involved to a minor extent in the regulation of basal renal concentration ability. By contrast, cGKII-KO mice are not able to handle an acute volume load. Our results suggest that membrane insertion of AQP2 is inhibited by cGMP/cGKII.

Vascular and renal function of cGMP signalling

Results For the functional analysis of cGMP signalling, transgenic murine mutants were used. cGKI signalling pathways include interaction of the cGKIb-isozyme with the inositol 1,4,5-trisphosphate receptor I (IP3RI) associated protein cGMP kinase substrate (IRAG). NO/cGMP and ANP/cGMP-dependent relaxation of aortic smooth muscle was strongly affected in IRAG-deficient mice. NO/ ANP-dependent inhibition of intracellular calcium release was suppressed in IRAG-deficient vascular smooth muscle cells (VSMC). Furthermore, reduction of store operated calcium entry by cGMP was affected in IRAG-KO VSMC. Basal mean arterial blood pressure, heart rate and activity were not changed in IRAG knockout mice. However, under pathophysiological conditions like sepsis (induced by E. coli lipopolysaccharide application) IRAG knockout mice were resistant to blood pressure reduction.