By using a potential-dependent J-aggregate-forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1), we find that membrane potentials across mitochondria in a living cell can be heterogeneous. Remarkably, even within a long contiguous mitochondrion, regional heterogeneity in membrane potentials appears to be possible.

Programmed cell death, or apoptosis, is important in homeostasis of the immune system: for example, non-functional or autoreactive lymphocytes are eliminated through apoptosis. One member of the tumour necrosis factor receptor (TNFR) family, Fas (also known as CD95 or Apo-1), can trigger cell death and is essential for lymphocyte homeostasis. FADD/Mort1 is a Fas-associated protein that is thought to mediate apoptosis by recruiting the protease caspase-8. A dominant-negative mutant of FADD inhibits apoptosis initiated by Fas and other TNFR family members. Other proteins, notably Daxx, also bind Fas and presumably mediate a FADD-independent apoptotic pathway. Here we investigate the role of FADD in vivo by generating FADD-deficient mice. As homozygous mice die in utero, we generated FADD-/- embryonic stem cells and FADD-/- chimaeras in a background devoid of the recombination activating gene RAG-1, which activates rearrangement of the immunoglobulin and T-cell receptor genes. We found that thymocyte subpopulations were apparently normal in newborn chimaeras. Fas-induced apoptosis was completely blocked, indicating that there are no redundant Fas apoptotic pathways. As these mice age, their thymocytes decrease to an undetectable level, although peripheral T cells are present in all older FADD-/- chimaeras. Unexpectedly, activation-induced proliferation is impaired in these FADD-/- T cells, despite production of the cytokine interleukin (IL)-2. These results and the similarities between FADD-/- mice and mice lacking the beta-subunit of the IL-2 receptor suggest that there is an unexpected connection between cell proliferation and apoptosis.

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Na + -K + -2Cl − cotransporters (NKCCs), including NKCC1 and renal-specific NKCC2, and the Na + -Cl − cotransporter (NCC) play pivotal roles in the regulation of blood pressure (BP) and renal NaCl reabsorption. Oxidative stress-responsive kinase-1 (OSR1) is a known upstream regulator of N(K)CCs. We generated and analyzed global and kidney tubule-specific (KSP) OSR1 KO mice to elucidate the physiological role of OSR1 in vivo, particularly on BP and kidney function. Although global OSR1 −/− mice were embryonically lethal, OSR1 +/− mice had low BP associated with reduced phosphorylated (p) STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK) and p-NKCC1 abundance in aortic tissue and attenuated p-NKCC2 abundance with increased total and p-NCC expression in the kidney. KSP-OSR1 −/− mice had normal BP and hypercalciuria and maintained significant hypokalemia on a low-K + diet. KSP-OSR1 −/− mice exhibited impaired Na + reabsorption in the thick ascending loop on a low-Na + diet accompanied by remarkably decreased expression of p-NKCC2 and a blunted response to furosemide, an NKCC2 inhibitor. The expression of total SPAK and p-SPAK was significantly increased in parallel to that of total NCC and p-NCC despite unchanged total NKCC2 expression. These results suggest that, globally, OSR1 is involved in the regulation of BP and renal tubular Na + reabsorption mainly via the activation of NKCC1 and NKCC2. In the kidneys, NKCC2 but not NCC is the main target of OSR1 and the reduced p-NKCC2 in KSP-OSR1 −/− mice may lead to a Bartter-like syndrome.Bartter syndrome | electrolytes | hormone | knockout mice | volume R ecent studies have shown that Na + -K + -2Cl − cotransporters (NKCCs) and the Na + -Cl − cotransporter (NCC) play very important roles in the regulation of blood pressure (BP) and extracellular volume. NKCCs consist of ubiquitous NKCC1 and renal-specific NKCC2. NKCC1 can modulate BP through vascular and renal effects (1-4). NKCC2 and NCC are two renal Na + cotransporters expressed in the thick ascending limbs (TALs) and distal convoluted tubules (DCTs) of the kidney, respectively, accounting for 20% and 10% of filtered Na + reabsorption (5). In human essential hypertension and salt-sensitive or spontaneously hypertensive animal models, activation of NKCC1 and NKCC2 has also been reported to play a pivotal role in the pathogenesis of hypertension (6, 7). In addition, activation of NCC by gene mutations in WNK1 and WNK4 leads to an autosomal dominant salt-sensitive hypertension known as pseudohypoaldosteronism type II (PAHII) (8). On the other hand, loss-of-function mutations in the SLC12A1 and SLC12A3 genes encoding NKCC2 and NCC can lead to renal salt-wasting hypotension with hypokalemic metabolic alkalosis, known as Bartter syndrome (BS) (9) and Gitelman syndrome (GS) (10), respectively.In vitro studies have shown that posttranscriptional phosphorylation of NKCC1/2 and NCC plays a crucial role in the regulation of normal transport activity. Oxidative stress-responsive kinase-1 (OSR1) (11) and STE20 (s...

Inflammatory processes can stimulate renal epithelial cells to release cytokines, chemoattractants and matrix proteins into the interstitium, thus contributing to interstitial injury during acute allograft rejection. To test the role of interleukin 17 (IL-17) in this process, cultured human renal epithelial cells (hRECs) were first established and treated with or without human IL-17 (hIL-17) for 2, 4, 8 and 10 h in vitro. Significant elevations of IL-6 and IL-8 levels were noted in the supernatants in a dose-dependent and time-dependent manner, as also for IL-6 mRNA expression. Secondly, using a rat acute allograft rejection model, the correlation between IL-17 expression and histopathological changes was serially studied. The results demonstrated that increased expression of IL-17 protein on infiltrating mononuclear cells (MNCs) was detectable on day 2. This corresponds to the borderline change of acute rejection according to the Banff classification, and it increased progressively to day 5. Serial study of IL-6, IL-8 and IL-17 mRNA expression of the renal allograft confirmed IL-17 mRNA expression in the allograft early on post-transplant day 2, whereas IL-6 and IL-8 expression started on day 3. Thirdly, IL-17 expression was observed in human renal allograft and urinary sediment. IL-17 protein expression was found in human subclinical (borderline) rejection renal allograft biopsy tissue and none in biopsy tissue not showing any evidence of rejection. There was also a 100% detectable rate of IL-17 mRNA expression in the MNCs of urinary sediment of patients with subclinical borderline rejection. These results demonstrate that hRECs exposed to IL-17 can produce inflammatory mediators with the potential to stimulate early alloimmune responses, which may also serve to give warning of acute renal allograft rejection.