MR Microscopy and microspectroscopy of the intact kidney

Magnetic resonance (MR) techniques have become essential in clinical practice and a broad range of research areas. We begin with a review of the potential and limitations for resolution improvements by MR techniques. The kidney has distinct regional structural, functional and biochemical variability. The isolated perfused rat kidney (IPRK) retains renal function while eliminating movement and susceptibility boundaries which severely limit the potential of MR techniques. The IPRK, with a length of less than 20 mm in the longest axis, will be used to illustrate the potential resolution of different MR techniques and the different: biological information that can be obtained. (C) 2004 Wiley Periodicals, Inc.

The use of simultaneous H-1 & P-31 magic angle spinning nuclear magnetic resonance measurements to characterize energy metabolism during the conversion of muscle to meat

In order to investigate the potential of magic angle spinning nuclear magnetic resonance (MAS NMR) in the elucidation of post-mortem metabolism in muscle biopsies, simultaneous H-1 and (31)p MAS NMR measurements were made continuously on postmortem (20 min to 24 h) muscle longissimus samples from rabbits. The animals had either been or not been given adrenaline (0.5 mg kg(-1) 4 h pre-slaughter) to deplete stores of muscle glycogen. The intracellular pH was calculated from H-1 spectra, and the post-mortem rate of formation of lactate was followed and quantified. Comparison of measurements made on muscle samples from rabbits treated with adrenaline with measurements made on muscle samples from untreated' rabbits revealed significant effects of adrenaline treatment on both pH (pH24 h = 6.42 vs. pH24 It = 5.60) and formation of lactate (16 mmol g(-1) vs. 65 mmol g(-1)). The P-31 NMR spectra were used to follow the rate of degradation of ATP and phosphocreatine. The present study clearly shows that MAS NMR has potential for the study of post-mortem energy metabolism.

Dynamic and regulated association of caveolin with lipid bodies: Modulation of lipid body motility and function by a dominant negative mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cav(DGV)) to study LB formation and to examine its effect on LB function. We now show that the cav(DGV) mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

Characterization of the human cytochrome P450 forms involved in metabolism of tamoxifen to its alpha-hydroxy and alpha,4-dihydroxy derivatives

Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial. cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include alpha-hydroxytamoxifen, alpha,4-dihydroxytamoxifen, and alpha-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing a-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250,mu M) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as alpha-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No alpha,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100,mu M) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of a,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.

Renal endothelial dysfunction and impaired autoregulation after ischemia-reperfusion injury result from excess nitric oxide

Endothelial dysfunction in ischemic acute renal failure (IARF) has been attributed to both direct endothelial injury and to altered endothelial nitric oxide synthase ( eNOS) activity, with either maximal upregulation of eNOS or inhibition of eNOS by excess nitric oxide ( NO) derived from iNOS. We investigated renal endothelial dysfunction in kidneys from Sprague-Dawley rats by assessing autoregulation and endothelium-dependent vasorelaxation 24 h after unilateral ( U) or bilateral ( B) renal artery occlusion for 30 (U30, B30) or 60 min (U60, B60) and in sham-operated controls. Although renal failure was induced in all degrees of ischemia, neither endothelial dysfunction nor altered facilitation of autoregulation by 75 pM angiotensin II was detected in U30, U60, or B30 kidneys. Baseline and angiotensin II-facilitated autoregulation were impaired, methacholine EC50 was increased, and endothelium-derived hyperpolarizing factor ( EDHF) activity was preserved in B60 kidneys. Increasing angiotensin II concentration restored autoregulation and increased renal vascular resistance ( RVR) in B60 kidneys; this facilitated autoregulation, and the increase in RVR was abolished by 100 mu M furosemide. Autoregulation was enhanced by N-omega-nitro-L-arginine methyl ester. Peri-ischemic inhibition of inducible NOS ameliorated renal failure but did not prevent endothelial dysfunction or impaired autoregulation. There was no significant structural injury to the afferent arterioles with ischemia. These results suggest that tubuloglomerular feedback is preserved in IARF but that excess NO and probably EDHF produce endothelial dysfunction and antagonize autoregulation. The threshold for injury-producing, detectable endothelial dysfunction was higher than for the loss of glomerular filtration rate. Arteriolar endothelial dysfunction after prolonged IARF is predominantly functional rather than structural.

Interferon-induced, antiviral human MxA protein localizes to a distinct subcompartment of the smooth endoplasmic reticulum

Human MxA protein belongs to the superfamily of dynamin-like large GTPases that are involved in intracellular membrane trafficking. MxA is induced by interferons-alpha/beta (IFN-alpha/beta) and is a key component of the antiviral response against RNA viruses. Here, we show that MxA localizes to membranes that are positive for specific markers of the smooth endoplasmic reticulum, such as Syntaxin17, but is excluded from other membrane compartments. Overexpression of MxA leads to a characteristic reorganization of the associated membranes. Interestingly, Hook3, mannose-6-phosphate receptor, and Lamp-1, which normally accumulate in cis-Golgi, endosomes, and lysosomes, respectively, also colocalized with MxA, indicating that these markers were redistributed to the MxA-positive compartment. Functional assays, however, did not show any effect of MxA on endocytosis or the secretory pathway. The present results demonstrate that MxA is an IFN-induced antiviral effector protein that resembles the constitutively expressed large GTPase family members in its capacity to localize to and reorganize intracellular membranes.

Pro-fibrotic polymorphisms predictive of advanced liver fibrosis in the severely obese

Background/Aims: Insulin resistance and systemic hypertension are predictors of advanced fibrosis in obese patients with non-alcoholic fatty liver disease (NAFLD). Genetic factors may also be important. We hypothesize that high angiotensinogen (AT) and transforming growth factor-beta1 (TGF-beta1) producing genotypes increase the risk of liver fibrosis in obese subjects with NAFLD. Methods: One hundred and five of 130 consecutive severely obese patients having a liver biopsy at the time of laparoscopic obesity surgery agreed to have genotype analysis. Influence of specific genotype or combination of genotypes on the stage of hepatic fibrosis was assessed after controlling for known risk factors. Results: There was no fibrosis in 70 (67%), stages 1-2 in 21 (20%) and stages 3-4 fibrosis in 14 (13%) of subjects. There was no relationship between either high AT or TGF-beta1 producing genotypes alone and hepatic fibrosis after controlling for confounding factors. However, advanced hepatic fibrosis occurred in five of 13 subjects (odds ratio 5.7, 95% confidence interval 1.5-21.2, P = 0.005) who inherited both high AT and TGF-beta1 producing polymorphisms. Conclusions: The combination of high AT and TGF-beta1 producing polymorphisms is associated with advanced hepatic fibrosis in obese patients with NAFLD. These findings support the hypothesis that angiotensin II stimulated TGF-beta1 production may promote hepatic fibrosis. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Angiotensin II facilitates autoregulation in the perfused mouse kidney: An optimized in vitro model for assessment of renal vascular and tubular function

Background and Aims: We have optimized the isolated perfused mouse kidney (IPMK) model for studying renal vascular and tubular function in vitro using 24-28 g C57BL6J mice; the wild type controls for many transgenic mice. Methods and Results: Buffer composition was optimized for bovine serum albumin concentration (BSA). The effect of adding erythrocytes on renal function and morphology was assessed. Autoregulation was investigated during stepped increases in perfusion pressure. Perfusion for 60 min at 90-110 mmHg with Krebs bicarbonate buffer containing 5.5% BSA, and amino acids produced functional parameters within the in vivo range. Erythrocytes increased renal vascular resistance (3.8 +/- 0.2 vs 2.4 +/- 0.1 mL/min.mmHg, P < 0.05), enhanced sodium reabsorption (FENa = 0.3 +/- 0.08 vs 1.5 +/- 0.7%, P < 0.05), produced equivalent glomerular filtration rates (GFR; 364 +/- 38 vs 400 +/- 9 muL/min per gkw) and reduced distal tubular cell injury in the inner stripe (5.8 +/- 1.7 vs 23.7 +/- 3.1%, P < 0.001) compared to cell free perfusion. The IPMK was responsive to vasoconstrictor (angiotensin II, EC50 100 pM) and vasodilator (methacholine, EC50 75 nM) mediators and showed partial autoregulation of perfusate flow under control conditions over 65-85 mmHg; autoregulatory index (ARI) of 0.66 +/- 0.11. Angiotensin II (100 pM) extended this range (to 65-120 mmHg) and enhanced efficiency (ARI 0.21 +/- 0.02, P < 0.05). Angiotensin II facilitation was antagonized by methacholine (ARI 0.76 +/- 0.08) and papaverine (ARI 0.91 +/- 0.13). Conclusion: The IPMK model is useful for studying renal physiology and pathophysiology without systemic neurohormonal influences.

Increased mononuclear cell activation and apoptosis early after human liver transplantation is associated with a reduced frequency of acute rejection

Experimental models of orthotopic liver transplantation (OLT) have shown that the very early events post-OLT are critical in distinguishing immunogenic and tolerogenic reactions. In rodents, increased leukocyte apoptosis and cytokine expression have been demonstrated in tolerogenic strain combinations. Information from human OLT recipients is less abundant. The aim of this study was to determine the amount of early leukocyte activation and apoptosis following human OLT, and to correlate this with subsequent rejection status. Peripheral blood mononuclear cells (PBMC) were isolated from 76 patients undergoing OLT - on the day prior, 5 hrs after reperfusion (day 0), and 18-24 hrs post-OLT (day 1). The mean level of apoptotic PBMCs on post OLT day 1 was higher than healthy recipients (0.9% +/- 0.2 vs. 0.2% +/- 0.1, p = 0.013). Apoptosis was greater in nonrejecting (NR) (1.1% +/- 0.3) compared with acutely-rejecting (R) (0.3% +/- 0.1, p = 0.021) patients. On day 1, PBMC from NR patients had increased expression of IFN-gamma (p = 0.006), IL-10 (p = 0.016), and CD40 ligand (p = 0.02) compared with R. Donor cell chimerism on day 1 did not differ between the groups indicating that this was unlikely to account for increased PBMC apoptosis in the NR group. Interestingly, the level of chimerism on day 0 was significantly higher in NR (3.8% +/- 0.6) compared with R (1.2% +/- 0.4, p = 0.004) patients and there was a close correlation between chimerism on day 0 and cytokine expression on day 1. These results imply that similar mechanisms are occurring in the human liver to promote graft acceptance as in the experimental models of liver transplantation and suggest that strategies that promote liver transplant acceptance in rodents might be applicable to humans.

Effect of naloxone-precipitated morphine withdrawal on c-fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing, hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 It for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis. however these were in CRH negative neurons. (C) 2004 Published by Elsevier Ireland Ltd.

The rat Na+-sulfate cotransporter rNaS2: functional characterization, tissue distribution, and gene (slc13a4) structure

Inorganic sulfate is essential for numerous functions in mammalian physiology. In the present study, we characterized the functional properties of the rat Na+-sulfate cotransporter NaS2 (rNaS2), determined its tissue distribution, and identified its gene (slc13a4) structure. Expression of rNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by phosphate, thiosulfate, tungstate, selenate, oxalate, and molybdate, but not by citrate, succinate, or DIDS. Transport kinetics of rNaS2 determined a K-M for sulfate of 1.26 mM. Na+ kinetics determined a Hill coefficient of n=3.0 +/- 0.7, suggesting a Na+:SO42- stoichiometry of 3:1. rNaS2 mRNA was highly expressed in placenta, with lower levels found in the brain and liver. slc13a4 maps to rat chromosome 4 and contains 17 exons, spanning over 46 kb in length. This gene produces two alternatively spliced transcripts, of which the transcript lacking exon 2 is the most abundant form. Its 5' flanking region contains CAAT- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, SP1, and AP-2 consensus sequences. This is the first study to characterize rNaS2 transport kinetics, define its tissue distribution, and resolve its gene (slc13a4) structure and 5' flanking region.