Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% (P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (-31%, P < 0.05) as well as in cells exposed to acid (-90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH(4)Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.     

Renal function and cortical (Na(+)+K(+))-ATPase activity, abundance and distribution after ischaemia-reperfusion in rats.

The effects of ischaemic injury and reperfusion on renal function, cortical ATP content, alkaline phosphatase activity and (Na(+)+K(+))-ATPase activity and abundance in cortical homogenates and isolated basolateral and apical membranes were examined. Rats were submitted to 5 or 40 min of right renal artery occlusion and 60 min of reperfusion. Renal function of the ischaemic-reperfused kidney was studied by conventional clearance techniques. Our results show that 1 h of reperfusion after a short period of renal ischaemia (5 min) allows the complete restoration of the biochemical features of cortical cells and functional properties of the injured kidney. A longer period of ischaemia, such as 40 min, followed by 1 h of reperfusion showed functional and biochemical alterations. ATP recovered from 26% after 40 min of ischaemia to 50% of control values after 1 h reperfusion. However, renal function was strongly impaired. Brush border integrity was compromised, as suggested by AP excretion and actin appearance in urine. Although total cortical (Na(+)+K(+))-ATPase activity was not different from controls, its distribution in isolated apical and basolateral membranes was abnormal. Remarkably, we detected an increase in alpha-subunit protein abundance that may suggest that (Na(+)+K(+))-ATPase synthesis is promoted by ischaemia-reperfusion. This increase may play an important role in the pathophysiology of ischaemic acute renal failure.     

Integrated gut/liver microphysiological systems elucidates inflammatory inter‐tissue crosstalk

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut‐liver tissue interactions under normal and inflammatory contexts, via an integrative multi‐organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long‐term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut‐liver crosstalk. Moreover, significant non‐linear modulation of cytokine responses was observed under inflammatory gut‐liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA‐seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut‐liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut‐liver interaction also negatively affected tissue‐specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi‐tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648–2659. © 2017 Wiley Periodicals, Inc.     

Effect of acetaminophen on the membrane anchoring of Na+, K+ATPase of rat renal cortical cells

In previous works we reported that the administration of a toxic dose of acetaminophen (APAP) induces acute renal failure (ARF) and promotes changes on Na(+), K(+)ATPase distribution in renal proximal plasma membranes. In the present work, we analyzed if APAP could promote the dissociation of Na(+), K(+)ATPase from its membrane anchorage. The participation of calpain activation was also evaluated. We analyzed the Triton X-100 extractability of Na(+), K(+)ATPase in freshly isolated cortical cell suspensions incubated with different APAP concentrations (0.1, 1, 10 and 100 mM). Both alpha(1) and beta(1) subunits were studied by Western blot. APAP promoted the increment of both subunits abundance in the Triton-soluble fraction. Calpain activation was detected in the membrane fractions of cells incubated with APAP. Incubation with APAP 0.1, 1 and 10 mM did not promote an increment in LDH release compared with controls, while APAP 100 mM promoted an increased LDH release. Our results show that incubation of proximal cells with sublethal and lethal APAP concentrations promotes the detachment of Na(+), K(+)ATPase from its membrane anchoring. Inhibition of calpain activation by SJA 7029 protected against APAP-induced membrane damage but not against APAP-induced increase of the Triton X-100 extractability of Na(+), K(+)ATPase.     

Modelling pest population resurgence due to recolonization of fields following an insecticide application

1. To investigate the effect of insect recolonization on the insecticide-induced resurgence of crop pests, a modified Lotka–Volterra predator–prey model was partitioned into two areas, 'sprayed' and 'unsprayed'.
2. The unsprayed area provided a source of insects to recolonize the sprayed area, resulting in a change in pest dynamics in both areas following an insecticide application.
3. Model sensitivity to insecticide selectivity and rates of predation, insect dispersal and pest population increase were examined.
4. Resurgence risk in the sprayed area increased with increasing pest dispersal rate, but decreased with increasing predator dispersal rate.
5. Pest resurgence could also occur in the unsprayed area, especially when prey dispersal rates were low. The extent of resurgence in the unsprayed area could in some circumstances be worse than in the sprayed area itself.
6. The more efficient and longer-lived the predators, the greater the level of pest resurgence in both areas following insecticide use.
7. More selective insecticides, killing the pest, but not the predator, reduced resurgence provided that the average life-span of the predators was reasonably long. Even highly selective insecticides could cause resurgence of the pest in the unsprayed area.
8. When the prey carrying capacity of the unsprayed area was increased relative to that of the sprayed area, resurgent effects in the unsprayed area were reduced, but could still be significant under some circumstances.