Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia-reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, irondependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the firstin-class compound ferrostatin-1 (Fer-1). Even in conditions with extraordinarily severe IRI, 16-86 exerts strong protection to an extent which has not previously allowed survival in any murine setting. In addition, 16-86 further potentiates the strong protective effect on IRI mediated by combination therapy with necrostatins and compounds that inhibit mitochondrial permeability transition. Renal tubules thus represent a tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. Finally, ferroptosis mediates postischemic and toxic renal necrosis, which may be therapeutically targeted by ferrostatins and by combination therapy.

Loss of kidney function in renal ischemia/reperfusion injury is due to programmed cell death, but the contribution of necroptosis, a newly discovered form of programmed necrosis, has not been evaluated. Here, we identified the presence of death receptor-mediated but caspase-independent cell death in murine tubular cells and characterized it as necroptosis by the addition of necrostatin-1, a highly specific receptor-interacting protein kinase 1 inhibitor. The detection of receptor-interacting protein kinase 1 and 3 in whole-kidney lysates and freshly isolated murine proximal tubules led us to investigate the contribution of necroptosis in a mouse model of renal ischemia/reperfusion injury. Treatment with necrostatin-1 reduced organ damage and renal failure, even when administered after reperfusion, resulting in a significant survival benefit in a model of lethal renal ischemia/reperfusion injury. Unexpectedly, specific blockade of apoptosis by zVAD, a pan-caspase inhibitor, did not prevent the organ damage or the increase in urea and creatinine in vivo in renal ischemia/reperfusion injury. Thus, necroptosis is present and has functional relevance in the pathophysiological course of ischemic kidney injury and shows the predominance of necroptosis over apoptosis in this setting. Necrostatin-1 may have therapeutic potential to prevent and treat renal ischemia/reperfusion injury.

The paracellular claudin channel of the thick ascending limb (TAL) of Henle is critical for Ca++ reabsorption in the kidney. Genome‐wide association studies (GWASs) have identified claudin‐14 associated with hypercalciuric nephrolithiasis. Here, we show that claudin‐14 promoter activity and transcript are exclusively localized in the TAL. Under normal dietary condition, claudin‐14 proteins are suppressed by two microRNA molecules (miR‐9 and miR‐374). Both microRNAs directly target the 3′‐UTR of claudin‐14 mRNA; induce its mRNA decay and translational repression in a synergistic manner. Through physical interaction, claudin‐14 blocks the paracellular cation channel made of claudin‐16 and ‐19, critical for Ca++ reabsorption in the TAL. The transcript and protein levels of claudin‐14 are upregulated by high Ca++ diet, while downregulated by low Ca++ diet. Claudin‐14 knockout animals develop hypermagnesaemia, hypomagnesiuria, and hypocalciuria under high Ca++ dietary condition. MiR‐9 and miR‐374 transcript levels are regulated by extracellular Ca++ in a reciprocal manner as claudin‐14. The Ca++ sensing receptor (CaSR) acts upstream of the microRNA‐claudin‐14 axis. Together, these data have established a key regulatory role for claudin‐14 in renal Ca++ homeostasis.

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid-base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H + -selective microelectrodes and the pHsensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH e and pH i ) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO 2 conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO 2 . Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH e whenever seawater pH changes. However, measurements of pH i demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na + and HCO 3 − , suggesting a bicarbonate buffer mechanism involving secondary active Na + -dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH i enables calcification to proceed despite decreased pH e . However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage. pH microelectrode | Strongylocentrotus droebachiensis | acid-base regulation | Na + -HCO 3 − transport | epithelial transport S ea urchin larvae have been shown to react with particular sensitivity to CO 2 -induced reductions in seawater pH (1-4). When larvae are chronically exposed to elevated seawater pCO 2 of >0.1 kPa, e.g., as is predicted to occur during the next century in response to anthropogenic CO 2 emissions or through upwelling of low-pH deep water, this sensitivity is reflected in reduced growth and developmental rates (5, 6). Echinoderm larvae are considered to be especially vulnerable to seawater pH reduction and to the associated changes in calcium carbonate saturation state of seawater (Ω Cal ) because their internal skeleton is composed of high magnesium calcite, a highly soluble form of CaCO 3 (7, 8). However, long-term reductions in growth and development might just as well be evoked by other physiological mechanisms that are also sensitive to hypercapnia and the related acid-base disturbances. Recent studies conducted on several marine taxa including mollusks (9) and echinoderms (10) demonstrated increased metabolic rates in response to elevated seawater pCO 2 . It was concluded that reductions in somatic growth and rate of development were caused by a sh...

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