Renal tubular arginase-2 participates in the formation of the corticomedullary urea gradient and attenuates kidney damage in ischemia-reperfusion injury in mice

Aim Arginase 2 (ARG2) is a mitochondrial enzyme that catalyses hydrolysis ofl-arginine into urea andl-ornithine. In the kidney, ARG2 is localized to the S3 segment of the proximal tubule. It has been shown that expression and activity of this enzyme are upregulated in a variety of renal pathologies, including ischemia-reperfusion (IR) injury. However, the (patho)physiological role of ARG2 in the renal tubule remains largely unknown. Methods We addressed this question in mice with conditional knockout ofArg2in renal tubular cells (Arg2(lox/lox)/Pax8-rtTA/LC1 or, cKO mice). Results We demonstrate that cKO mice exhibit impaired urea concentration and osmolality gradients along the corticomedullary axis. In a model of unilateral ischemia-reperfusion injury (UIRI) with an intact contralateral kidney, ischemia followed by 24 hours of reperfusion resulted in significantly more pronounced histological damage in ischemic kidneys from cKO mice compared to control and sham-operated mice. In parallel, UIRI-subjected cKO mice exhibited a broad range of renal functional abnormalities, including albuminuria and aminoaciduria. Fourteen days after UIRI, the cKO mice exhibited complex phenotype characterized by significantly lower body weight, increased plasma levels of early predictive markers of kidney disease progression (asymmetric dimethylarginine and symmetric dimethylarginine), impaired mitochondrial function in the ischemic kidney but no difference in kidney fibrosis as compared to control mice. Conclusion Collectively, these results establish the role of ARG2 in the formation of corticomedullary urea and osmolality gradients and suggest that this enzyme attenuates kidney damage in ischemia-reperfusion injury.

Renal Fanconi Syndrome and Hypophosphatemic Rickets in the Absence of Xenotropic and Polytropic Retroviral Receptor in the Nephron

Camille Ansermet, Dmitri Firsov, Vladimir Katanaev

Tight control of extracellular and intracellular inorganic phosphate (Pi) levels is critical to most biochemical and physiologic processes. Urinary Pi is freely filtered at the kidney glomerulus and is reabsorbed in the renal tubule by the action of the apical sodium -dependent phosphate transporters, NaPi-11a/NaPi-11c/Pit2. However, the molecular identity of the protein(s) participating in the basolateral Pi efflux remains unknown. Evidence has suggested that xenotropic and polytropic retroviral receptor 1 (XPR1) might be involved in this process. Here, we show that conditional inactivation of Xpr1 in the renal tubule in mice resulted in impaired renal Pi reabsorption. Analysis of Pi transport in primary cultures of proximal tubular cells or in freshly isolated renal tubules revealed that this Xpr1 deficiency significantly affected Pi efflux. Further, mice with conditional inactivation of Xpr1 in the renal tubule exhibited generalized proximal tubular dysfunction indicative of Fanconi syndrome, characterized by glycosuria, aminoaciduria, calciuria, and albuminuria. Dramatic alterations in the renal transcriptome, including a significant reduction in NaPi-lla/NaPi-llc expression, accompanied these functional changes. Additionally, Xpr1-deficient mice developed hypophosphatemic rickets secondary to renal dysfunction. These results identify XPR1 as a major regulator of Pi homeostasis and as a potential therapeutic target in bone and kidney disorders.

Amer Soc Nephrology2017

Ablation of the Tumor Suppressor Histidine Triad Nucleotide Binding Protein 1 is Protective Against Hepatic Ischemia/Reperfusion Injury

Gisèle Ferrand

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. Conclusion: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury. (HEPATOLOGY 2011;53:243-252)

2011

Mechanistic studies of DNP and applications of hyperpolarized probes to study renal physiology and metabolism

Alice Radaelli

Dissolution dynamic nuclear polarization (dDNP) is a powerful technique that enhances the magnetic resonance signal of nuclear spins by several orders of magnitude. DNP relies on the principle of cross-relaxation by electron spins driven out of equilibrium to enhance nuclear polarization. When coupled to magnetic resonance spectroscopy/imaging (MRS/I), infusion of 13C-labeled tracers hyperpolarized via DNP in vivo provides real-time estimates of metabolic fluxes, physiological parameters and/or tissue perfusion. The present work focuses on fundamental aspects of DNP, in particular how sample composition affects the DNP of low-gamma nuclei, as well as on in vivo dDNP applications targeting renal metabolism and physiology. The DNP properties of the SA-BDPA radical were studied in a sample of 13C-urea. SA-BDPA, a water-soluble derivative of BDPA, is characterized by a small g-anisotropy and unresolved hyperfine coupling to protons. As a result, at 6.7 T, 1.1 K its ESR linewidth is much smaller than the 13C Larmor frequency, which enabled the observation for the first time of 13C DNP via the solid effect and pure thermal mixing, the latter defined as the process where the electron non-Zeeman reservoir alone provides the energy required for triple-spin flips. The effect of solvent deuteration on 6Li DNP and radical ESR properties was studied in 6LiCl water:glycerol solutions doped with either the nitroxide radical TEMPOL or the trityl radical OX063. Unexpectedly, the TEMPOL-doped samples polarized better than the trityl-doped ones. Across all samples, the relationship between the degree of solvent deuteration with the buildup time constant and polarization level was notably different from what has been reported for 13C. This behavior is indicative of DNP via a combination of the cross effect and thermal mixing mechanisms. The uptake and metabolism of hyperpolarized L-[1-13C]alaninamide was investigated in the rat kidney in vivo. This probe of aminopeptidase N, which can play a role in tumor growth, was previously studied in vitro. Our study showed that alanine production from alaninamide also occurs in vivo, however, with spectral overlap of substrate and product. Alaninamide, having a pKa of 7.9, proved to be sensitive to local pH. Three spectral peaks, corresponding to at least three environments with different pH values, could be observed in the kidney. The two peaks at higher pH were assigned to the blood extra- and (partially) intracellular compartments, while the third one was mainly in the inner part of the kidney. Finally, alaninamide was shown to also be sensitive to dissolved CO2, with the rapid formation of a carbamate adduct following infusion. The renal metabolism of D-[1-13C]alanine by D-amino acid oxidase (DAO) was also studied. Conversion of hyperpolarized D-alanine to pyruvate and further metabolism to lactate and bicarbonate was observed in the kidney only when DAO was not inhibited. DAO activity could also be detected in blood, where leukocytes express the enzyme, but not in the brain and liver, in line with their lower DAO activity. Overall, this thesis provides additional insight into how the experimental conditions can favor a particular DNP mechanism over another. It also significantly expands the scope of in vivo dDNP applications, showing that additional enzyme-catalyzed processes can be detected, along with the potential of amino-acid based hyperpolarized 13C sensors for physiological studies.