A Protein Kinase A–Independent Pathway Controlling Aquaporin 2 Trafficking as a Possible Cause for the Syndrome of Inappropriate Antidiuresis Associated with Polycystic Kidney Disease 1 Haploinsufficiency

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1^+/− mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1^+/− mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca²⁺ levels, promotes AQP2 trafficking independent of the AVP–PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance.In most mammals, regulation of water balance is critically dependent on water intake and excretion, which is under control of the antidiuretic hormone arginine vasopressin (AVP). In the kidney, AVP binds to the V2 vasopressin (V2R) receptor, activating the cAMP/protein kinase A (PKA) signal transduction cascade, promoting the fusion of intracellular vesicles containing aquaporin 2 (AQP2) to the apical plasma membrane, and increasing luminal permeability.^1–3 This translocation is accompanied by AVP-dependent phosphorylation of AQP2 at serine-256 (pS256).Mice in which S256 could not be phosphorylated (AQP2-S256L) develop polyuria and hydronephrosis because of a defect in AQP2 trafficking to the plasma membrane.⁴ Interestingly, it connects to polycystic kidney disease (PKD). Mutations in polycystin-1 (Pkd1^+/−) gene cause PKD, whereas PKD1 haplo-insufficient mice (Pkd1^+/−), showing an inappropriate antidiuresis, display significantly higher levels of pS256 compared with wild-type (WT) littermates; the prominent expression at the apical plasma membrane of collecting duct principal cells, despite normal V2R expression and normal cAMP levels, is associated with unchanged AVP expression in the brain, despite chronic hypo-osmolality.⁵These observations underscore the crucial role of AQP2 phosphorylation at S256 in controlling the cellular distribution and fate of AQP2.^1,6,7 As for many proteins, the function and the trafficking of AQP2 are modulated by a balance of reversible phosphorylation and dephosphorylation. Preventing dephosphorylation of AQP2 with okadaic acid, inhibitor of phosphatase 1 (PP1), inhibitor of phosphatase 2A (PP2A), and inhibitor of phosphatase 2B (PP2B) significantly increased AQP2-pS256.⁸ Proteomic analysis of inner medulla collecting duct identified PP2A as a phosphoprotein isolated from inner medullary collecting duct samples treated with either calyculin-A, a specific PP2A inhibitor, or vasopressin,⁹ suggesting the possible participation of this phosphatase in cellular events triggered by physiologic stimulus, such as vasopressin in renal collecting duct cells.The complexity of AQP2 regulation was further increased by phosphoproteomics studies showing that, other than S256, vasopressin modulates the phosphorylation status of three other sites within the C terminus (S261, S264, and S269). Although vasopressin increases S264 and S269 phosphorylation, it decreases S261 phosphorylation.^9–12 Regarding the potential kinases responsible for the phosphorylation of these sites, c-Jun N-terminal kinase, p38, and cyclin-dependent kinases (cdks) cdk1 and cdk5 can phosphorylate AQP2 peptides at S261 in vitro.^13,14 Here, in the attempt to investigate the potential involvement of cdks in AQP2 regulation, we discovered a new PKA-independent signal transduction pathway regulating AQP2 phosphorylation and localization. We found that selective inhibition of cdks with R-roscovitine is associated with a decrease of intracellular Ca²⁺ levels and a significant downregulation of the phosphatase PP2A activity, resulting in an increase of AQP2 phosphorylation at S256 and targeting to the apical membrane. Physiologically, this novel regulatory mechanism might be of clinical interest, because it better elucidates the molecular bases of pathologic states characterized by disturbances in water balance.     

Integrated analytical techniques with high sensitivity for studying brain translocation and potential impairment induced by intranasally instilled copper nanoparticles

Health impacts of inhalation exposure to engineered nanomaterials have attracted increasing attention. In this paper, integrated analytical techniques with high sensitivity were used to study the brain translocation and potential impairment induced by intranasally instilled copper nanoparticles (CuNPs). Mice were exposed to CuNPs in three doses (1, 10, 40 mg/kg bw). The body weight of mice decreased significantly in the 10 and 40 mg/kg group (p < 0.05) but recovered slightly within exposure duration. Inductively coupled plasma mass spectrometry (ICP-MS) analysis showed that CuNPs could enter the brain. Altered distribution of some important metal elements was observed by synchrotron radiation X-ray fluorescence (SRXRF). H&E staining and immunohistochemical analysis showed that CuNPs produced damages to nerve cells and astrocyte might be the one of the potential targets of CuNPs. The changes of neurotransmitter levels in different brain regions demonstrate that the dysfunction occurred in exposed groups. These data indicated that CuNPs could enter the brain after nasal inhalation and induced damages to the central nervous system (CNS). Integration of effective analytical techniques for systematic investigations is a promising direction to better understand the biological activities of nanomaterials.     

Gesundheitsförderung älterer türkischer Migrantinnen

Background

Elderly persons with an immigration background often face health problems due to specific living and working conditions. Focussing on this target group, health courses which concentrate on nutrition and exercise have been developed and tried out in three interventions within the “Sa?lik” project (Turkish: health).

Purpose of this study (question)

Based on instruments for evaluation which are immanent within the courses, the following questions are answered: which knowledge is transferred subject to what kind of conditions of intervention? What kinds of learning targets can be achieved? What are the limits of the interventions?

Material and methods

Health courses are characterized by a subjective data collection. While courses are undertaken data is gathered on account of documenting personal goals and achievements. Moreover, a status of course-related knowledge or behavior is queried at the end of the intervention.

Results and discussion

It is crucial that the interventions cause changes in behavior. Within the given project’s perspective, however, these changes could only be followed up to the end of the intervention. Apart from this it became evident that the health courses undertaken can only reach persons who implicitly intend or are prepared for changes in behavior. Therefore, with respect to the perspective of the project it can be asked how behavior promoting health can be constantly supported. Furthermore, it remains to be clarified whether and how people can be reached if they do not give preference to healthy behavior.     

Early identification of peritonitis by peritoneal cytokine measurement

PURPOSE: The assessment of plasma cytokine levels adds a useful tool to the diagnostic measures in severe inflammatory diseases. Proinflammatory cytokine levels in abdominal fluid after abdominal surgery have been shown to far exceed plasma cytokine levels. Thus, we investigated the local release of interleukin 1, interleukin 6, and tumor necrosis factor- in patients after colorectal surgery during the early postoperative period to evaluate whether it may serve as an indicator of evolving peritonitis. METHOD: In a prospective, observational pilot study, the first 12 consecutive patients who did not develop any postoperative complications (Group I), and the first 12 patients with secondary peritonitis caused by an anastomotic leakage (Group II), were included in the study. Interleukin 6, interleukin 1, and tumor necrosis factor- levels were determined in the abdominal exudate and compared between the groups within the first four days after colorectal surgery. RESULTS: Abdominal fluid interleukin 6 levels in Group II patients were higher (162,500 ± 105,800 pg/ml) as early as the first postoperative day compared with Group I (27,940 ± 13,860 pg/ml; P < 0.0001); this lasted for the whole observation period. The same applies to tumor necrosis factor- levels (461.4 ± 167.8 pg/ml vs. 175.8 ± 178.6 pg/ml on day 1; P = 0.0007). The difference in interleukin 1 cytokine levels became statistically significant on the third postoperative day. Moreover, abdominal fluid cytokine levels rose in Group II, whereas they remained virtually unchanged or even tended to decrease over time in Group I. CONCLUSION: We suggest that the estimation of the peritoneal cytokine levels might be an additional diagnostic tool that can support the early recognition of peritonitic complications in colorectal surgery.     

Cell-based resurfacing of large cartilage defects: long-term evaluation of grafts from autologous transgene-activated periosteal cells in a porcine model of osteoarthritis

OBJECTIVE: To investigate the potential of transgene-activated periosteal cells for permanently resurfacing large partial-thickness cartilage defects. METHODS: In miniature pigs, autologous periosteal cells stimulated ex vivo by bone morphogenetic protein 2 gene transfer, using liposomes or a combination of adeno-associated virus (AAV) and adenovirus (Ad) vectors, were applied on a bioresorbable scaffold to chondral lesions comprising the entire medial half of the patella. The resulting repair tissue was assessed, 6 and 26 weeks after transplantation, by histochemical and immunohistochemical methods. The biomechanical properties of the repair tissue were characterized by nanoindentation measurements. Implants of unstimulated cells and untreated lesions served as controls. RESULTS: All grafts showed satisfactory integration into the preexisting cartilage. Six weeks after transplantation, AAV/Ad-stimulated periosteal cells had adopted a chondrocyte-like phenotype in all layers; the newly formed matrix was rich in proteoglycans and type II collagen, and its contact stiffness was close to that of healthy hyaline cartilage. Unstimulated periosteal cells and cells activated by liposomal gene transfer formed only fibrocartilaginous repair tissue with minor contact stiffness. However, within 6 months following transplantation, the AAV/Ad-stimulated cells in the superficial zone tended to dedifferentiate, as indicated by a switch from type II to type I collagen synthesis and reduced contact stiffness. In deeper zones, these cells retained their chondrocytic phenotype, coinciding with positive staining for type II collagen in the matrix. CONCLUSION: Large partial-thickness cartilage defects can be resurfaced efficiently with hyaline-like cartilage formed by transgene-activated periosteal cells. The long-term stability of the cartilage seems to depend on physicobiochemical factors that are active only in deeper zones of the cartilaginous tissue.     

Oxidative Stress Promotes Uptake,Accumulation, and Oligomerization of Extracellular α-Synuclein in Oligodendrocytes

The accumulation and aggregation of α-synuclein (α-Syn) in glial cytoplasmic inclusions originating in oligodendrocytes is a characteristic hallmark of multiple system atrophy, a progressive adult onset neurodegenerative disorder. The origin of α-Syn deposition in oligodendrocytes in multiple system atrophy is still unclear, but the uptake of α-Syn from the environment after neuronal secretion has been discussed. The present study was undertaken to investigate the consequences of α-Syn uptake from the environment in cultured oligodendroglial cells and its localization and potential to form intracellular aggregates in the absence or presence of the microtubule-associated protein tau, which has been demonstrated to act synergistically with α-Syn. Primary rat brain oligodendrocytes and clonal oligodendroglial OLN-93 cells were incubated with human recombinant soluble and pre-aggregated α-Syn. The data show that oligodendrocytes are capable to take up and internalize soluble and pre-aggregated α-Syn from their growth medium. In a time-dependent manner, α-Syn oligomerizes and small intracellular aggregates are formed. These do not exert cytotoxic responses or mitochondrial impairment. Oxidative stress exerted by hydrogen peroxide further promotes α-Syn oligomer formation and leads to an enlargement of the aggregates. This process is not affected or modified by the presence of tau in OLN-93 cells. Furthermore, membrane lipid modification by docosahexaenoic acid promotes α-Syn uptake and oligomerization, indicating that changing the membrane lipid composition and structure contributes to the protein aggregation process and pathological events. Hence, although α-Syn taken up by oligodendrocytes from the environment is not toxic per se, under conditions of oxidative stress, which might occur during chronic disease progression and aging, aggregates are enlarged and eventually may contribute to cytotoxicity and cellular death.     

Increased Osteopontin Contributes to Inhibition of Bone Mineralization in FGF23‐Deficient Mice

Excessive FGF23 has been identified as a pivotal phosphaturic factor leading to renal phosphate‐wasting and the subsequent development of rickets and osteomalacia. In contrast, loss of FGF23 in mice (Fgf23^?/?) leads to high serum phosphate, calcium, and 1,25‐vitamin D levels, resulting in early lethality attributable to severe ectopic soft‐tissue calcifications and organ failure. Paradoxically, Fgf23^?/? mice exhibit a severe defect in skeletal mineralization despite high levels of systemic mineral ions and abundant ectopic mineralization, an abnormality that remains largely unexplained. Through use of in situ hybridization, immunohistochemistry, and immunogold labeling coupled with electron microscopy of bone samples, we discovered that expression and accumulation of osteopontin (Opn/OPN) was markedly increased in Fgf23^?/? mice. These results were confirmed by qPCR analyses of Fgf23^?/? bones and ELISA measurements of serum OPN. To investigate whether elevated OPN levels were contributing to the bone mineralization defect in Fgf23^?/? mice, we generated Fgf23^?/?/Opn^?/? double‐knockout mice (DKO). Biochemical analyses showed that the hypercalcemia and hyperphosphatemia observed in Fgf23^?/? mice remained unchanged in DKO mice; however, micro‐computed tomography (µCT) and histomorphometric analyses showed a significant improvement in total mineralized bone volume. The severe osteoidosis was markedly reduced and a normal mineral apposition rate was present in DKO mice, indicating that increased OPN levels in Fgf23^?/? mice are at least in part responsible for the osteomalacia. Moreover, the increased OPN levels were significantly decreased upon lowering serum phosphate by feeding a low‐phosphate diet or after deletion of NaPi2a, indicating that phosphate levels contribute in part to the high OPN levels in Fgf23^?/? mice. In summary, our results suggest that increased OPN is an important pathogenic factor mediating the mineralization defect and the alterations in bone metabolism observed in Fgf23^?/? bones. © 2014 American Society for Bone and Mineral Research.