Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes.

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that key cytoskeletal proteins in DBD kidneys were proteolytically cleaved, generating peptide fragments, predominantly in grafts with suboptimal posttransplant function. Interestingly, α-actinin-4 and talin-1 proteolytic fragments were detected in brain death but not in circulatory death or living donor kidneys with similar donor characteristics. As talin-1 is a specific proteolytic target of calpain-1, we investigated a potential trigger of calpain activation and talin-1 degradation using human ex vivo precision-cut kidney slices and in vitro podocytes. Notably, we showed that activation of calpain-1 by transforming growth factor-ß generated proteolytic fragments of talin-1 that matched the degradation fragments detected in DBD preimplantation kidneys, also causing dysregulation of the actin cytoskeleton in human podocytes; events that were reversed by calpain-1 inhibition. Our data provide initial evidence that brain death donor kidneys are more susceptible to cytoskeletal protein degradation. Correlation to posttransplant outcomes may be established by future studies.

Cold acclimation preserves hindgut reabsorption capacity at low temperature in a chill-susceptible insect, Locusta migratoria.

Cold acclimation increases cold tolerance of chill-susceptible insects and the acclimation response often involves improved organismal ion balance and osmoregulatory function at low temperature. However, the physiological mechanisms underlying plasticity of ion regulatory capacity are largely unresolved. Here we used Ussing chambers to explore the effects of cold exposure on hindgut KCl reabsorption in cold- (11 °C) and warm-acclimated (30 °C) Locusta migratoria. Cooling (from 30 to 10 °C) reduced active reabsorption across recta from warm-acclimated locusts, while recta from cold-acclimated locusts maintained reabsorption at 10 °C. The differences in transport capacity were not linked to major rearrangements of membrane phospholipid profiles. Yet, the stimulatory effect of two signal transduction pathways were altered by temperature and/or acclimation. cAMP-stimulation increased reabsorption in both acclimation groups, with a strong stimulatory effect at 30 °C and a moderate stimulatory effect at 10 °C. cGMP-stimulation also increased reabsorption in both acclimation groups at 30 °C, but their response to cGMP differed at 10 °C. Recta from warm-acclimated locusts, characterised by reduced reabsorption at 10 °C, recovered reabsorption capacity following cGMP-stimulation at 10 °C. In contrast, recta from cold-acclimated locusts, characterised by sustained reabsorption at 10 °C, were unaffected by cGMP-stimulation. Furthermore, cold-exposed recta from warm-acclimated locusts were insensitive to bafilomycin-α1, a V-type H+-ATPase inhibitor, whereas this blocker reduced reabsorption across cold-exposed recta from cold-acclimated animals. In conclusion, bafilomycin-sensitive and cGMP-dependent transport mechanism(s) are likely blocked during cold exposure in warm-acclimated animals while preserved in cold-acclimated animals. These may in part explain the large differences in rectal ion transport capacity between acclimation groups at low temperature.

Earlier springs enable high-Arctic wolf spiders to produce a second clutch.

Spiders at southern latitudes commonly produce multiple clutches, but this has not been observed at high latitudes where activity seasons are much shorter. Yet the timing of snowmelt is advancing in the Arctic, which may allow some species to produce an additional clutch. To determine if this is already happening, we used specimens of the wolf spider Pardosa glacialis caught by pitfall traps from the long-term (1996-2014) monitoring programme at Zackenberg, NE Greenland. We dissected individual egg sacs and counted the number of eggs and partially developed juveniles, and measured carapace width of the mothers. Upon the discovery of a bimodal frequency distribution of clutch sizes, as is typical for wolf spiders at lower latitudes producing a second clutch, we assigned egg sacs to being a first or second clutch depending on clutch size. We tested whether the median capture date differed among first and second clutches, whether clutch size was correlated to female size, and whether the proportion of second clutches produced within a season was related to climate. We found that assigned second clutches appeared significantly later in the season than first clutches. In years with earlier snowmelt, first clutches occurred earlier and the proportion of second clutches produced was larger. Likely, females produce their first clutch earlier in those years which allow them time to produce another clutch. Clutch size for first clutches was correlated to female size, while this was not the case for second clutches. Our results provide the first evidence for Arctic invertebrates producing additional clutches in response to warming. This could be a common but overlooked phenomenon due to the challenges associated with long-term collection of life-history data in the Arctic. Moreover, given that wolf spiders are a widely distributed, important tundra predator, we may expect to see population and food web consequences of their increased reproductive rates.

Parasitoids indicate major climate-induced shifts in arctic communities.

Climatic impacts are especially pronounced in the Arctic, which as a region is warming twice as fast as the rest of the globe. Here, we investigate how mean climatic conditions and rates of climatic change impact parasitoid insect communities in 16 localities across the Arctic. We focus on parasitoids in a widespread habitat, Dryas heathlands, and describe parasitoid community composition in terms of larval host use (i.e., parasitoid use of herbivorous Lepidoptera vs. pollinating Diptera) and functional groups differing in their closeness of host associations (koinobionts vs. idiobionts). Of the latter, we expect idiobionts-as being less fine-tuned to host development-to be generally less tolerant to cold temperatures, since they are confined to attacking hosts pupating and overwintering in relatively exposed locations. To further test our findings, we assess whether similar climatic variables are associated with host abundances in a 22 year time series from Northeast Greenland. We find sites which have experienced a temperature rise in summer while retaining cold winters to be dominated by parasitoids of Lepidoptera, with the reverse being true for the parasitoids of Diptera. The rate of summer temperature rise is further associated with higher levels of herbivory, suggesting higher availability of lepidopteran hosts and changes in ecosystem functioning. We also detect a matching signal over time, as higher summer temperatures, coupled with cold early winter soils, are related to high herbivory by lepidopteran larvae, and to declines in the abundance of dipteran pollinators. Collectively, our results suggest that in parts of the warming Arctic, Dryas is being simultaneously exposed to increased herbivory and reduced pollination. Our findings point to potential drastic and rapid consequences of climate change on multitrophic-level community structure and on ecosystem functioning and highlight the value of collaborative, systematic sampling effort.

Does the route matter? A preclinical review of mesenchymal stromal cell delivery to the kidney.

Mesenchymal stromal/stem cell (MSC) therapy has been thoroughly tested in preclinical animal models and holds great promise for the treatment of kidney diseases. It is becoming increasingly evident that the efficacy of MSC therapy is dependent on several factors including dosage, the tissue source of MSCs, the route of delivery and timing of administration. In a time where MSC therapy is moving from preclinical research to clinically therapeutic use, the importance of choice of delivery method, modality, and administration route increases. In this review, we provide an overview of the different MSC delivery routes used in preclinical kidney disease models, highlight the recent advances in the field, and summarize studies comparing delivery routes of MSCs to the kidney.

An animal-free preclinical drug screening platform based on human precision-cut kidney slices.

OBJECTIVE: Renal fibrosis is one of the main pathophysiological processes underlying the progression of chronic kidney disease and kidney allograft failure. In the past decades, overwhelming efforts have been undertaken to find druggable targets for the treatment of renal fibrosis, mainly using cell- and animal models. However, the latter often do not adequately reflect human pathogenesis, obtained results differ per strain within a given species, and the models are associated with considerable discomfort for the animals. Therefore, the objective of this study is to implement the 3Rs in renal fibrosis research by establishing an animal-free drug screening platform for renal fibrosis based on human precision-cut kidney slices (PCKS) and by limiting the use of reagents that are associated with significant animal welfare concerns. RESULTS: Using Western blotting and gene expression arrays, we show that transforming growth factor-ß (TGF-ß) induced fibrosis in human PCKS. In addition, our results demonstrated that butaprost, SC-19220 and tamoxifen - all putative anti-fibrotic compounds - altered TGF-ß-induced pro-fibrotic gene expression in human PCKS. Moreover, we observed that all compounds modulated fairly distinct sets of genes, however they all impacted TGF-ß/SMAD signaling. In conclusion, this study revealed that it is feasible to use an animal-free approach to test drug efficacy and elucidate mechanisms of action.

EP1 receptor antagonism mitigates early and late stage renal fibrosis.

AIM: Renal fibrosis is a major driver of chronic kidney disease, yet current treatment strategies are ineffective in attenuating fibrogenesis. The cyclooxygenase/prostaglandin system plays a key role in renal injury and holds great promise as a therapeutic target. Here, we used a translational approach to evaluate the role of the PGE2 -EP1 receptor in the pathogenesis of renal fibrosis in several models of kidney injury, including human (fibrotic) kidney slices. METHODS: The anti-fibrotic efficacy of a selective EP1 receptor antagonist (SC-19220) was studied in mice subjected to unilateral ureteral obstruction (UUO), healthy and fibrotic human precision-cut kidney slices (PCKS), Madin-Darby Canine Kidney (MDCK) cells and primary human renal fibroblasts (HRFs). Fibrosis was evaluated on gene and protein level using qPCR, western blot and immunostaining. RESULTS: EP1 receptor inhibition diminished fibrosis in UUO mice, illustrated by a decreased protein expression of fibronectin (FN) and α-smooth muscle actin (αSMA) and a reduction in collagen deposition. Moreover, treatment of healthy human PCKS with SC-19220 reduced TGF-ß-induced fibrosis as shown by decreased expression of collagen 1A1, FN and αSMA as well as reduced collagen deposition. Similar observations were made using fibrotic human PCKS. In addition, SC-19220 reduced TGF-ß-induced FN expression in MDCK cells and HRFs. CONCLUSION: This study highlights the EP1 receptor as a promising target for preventing both the onset and late stage of renal fibrosis. Moreover, we provide strong evidence that the effect of SC-19220 may translate to clinical care since its effects were observed in UUO mice, cells and human kidney slices.