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1.
Am J Physiol Renal Physiol ; 294(1): F229-35, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18003856

ABSTRACT

Glial cell line-derived neurotrophic growth factor (GDNF), a member of the transforming growth factor family, is necessary for renal organogenesis and exhibits changes in expression in models of renal disease. Nestin is an intermediate filament protein originally believed to be a marker of neuroepithelial stem cells and recently proposed as a marker of mesenchymal stem cells (MSC). Having demonstrated the participation of nestin-expressing cells in renoprotection during acute renal ischemia, we hypothesized that growth factors and transcription factors similar to those operating in the nervous system should be also operant in the kidney and may be induced after noxious stimuli, such as an ischemic episode. Using cultured kidney-derived MSC, which abundantly express nestin, we confirmed expression of GDNF by these cells and demonstrated the GDNF-induced expression of GDNF. The cellular expression of nestin paralleled that of GDNF: serum starvation decreased the expression, whereas application of GDNF resulted in a dose-dependent increase in nestin expression. Immunohistochemical and Western blot analyses of kidneys obtained from control and postischemic mice showed that expression of GDNF was much enhanced in the renal cortex, a pattern similar to the previously reported expression of nestin. Based on the observed GDNF-induced GDNF expression, we next explored the effect of supplemental GDNF administered early after ischemia on renal function postischemia. GDNF-treated mice were protected against acute ischemia. To address potential mechanisms of the observed renoprotection, in vitro studies showed that GDNF accelerated MSC migration in a wound-healing assay. Hypoxia did not accelerate, but rather slightly reduced, the motility of MSC and reduced the expression of GDNF in MSC by approximately twofold. Furthermore, GDNF was cytoprotective against oxidative stress-induced apoptotic death of MSC. Collectively, these data establish 1) an autoregulatory circuit of GDNF-induced GDNF expression in renal MSC; 2) induction of GDNF expression in postischemic kidneys; 3) the ability of exogenous GDNF to ameliorate ischemic renal injury; and 4) a possible contribution of GDNF-induced motility and improved survival of MSC to renoprotection.


Subject(s)
Cell Movement/physiology , Glial Cell Line-Derived Neurotrophic Factor/physiology , Ischemia/prevention & control , Kidney Cortex/blood supply , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Animals , Apoptosis/drug effects , Cell Hypoxia/physiology , Cell Movement/drug effects , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Intermediate Filament Proteins/metabolism , Ischemia/metabolism , Ischemia/pathology , Kidney Cortex/metabolism , Kidney Cortex/pathology , Male , Mice , Mice, Inbred Strains , Nerve Tissue Proteins/metabolism , Nestin , Oxidative Stress/physiology
3.
Am J Physiol Renal Physiol ; 293(5): F1512-7, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17881464

ABSTRACT

Ischemic acute kidney injury in experimental diabetes mellitus (DM) is associated with a more severe deterioration in renal function than shown in nondiabetic animals. We evaluated whether the early recovery phase from acute kidney injury is associated with a more prolonged and sustained decrease in renal perfusion in diabetic mice, which could contribute to the impaired recovery of renal function. Perfusion to the renal cortex and medulla was evaluated by laser-Doppler flowmetry in 10- to 12-wk-old anesthetized mice with type 2 DM (db/db), heterozygous mice (db/m), and nondiabetic (control) mice (C57BL/6J). After baseline measurements were obtained, the right renal artery was clampedfor 20 min followed by reperfusion for 60 min. The data demonstrated that, in all three groups studied, the reperfusion phase was characterized by a significant increase in the medullary-to-cortical blood flow ratio. Moreover, during recovery from ischemia, there was a marked prolongation in the time (in min) required to reach peak reperfusion in the cortex (db/db: 20.7 +/- 4.0, db/m: 12.92 +/- 1.9, C57BL/6J: 9.3 +/- 1.3) and the medulla (db/db: 20.8 +/- 3.2, db/m: 12.88 +/- 1.89, C57BL/6J: 11.2 +/- 1.2). Additionally, the slope of the recovery phase was lower in db/db mice (cortex: 61.9 +/- 23.1%/min, medulla: 16.3 +/- 3.6%/min) than in C57BL/6J mice (cortex: 202.2 +/- 41.6%/min, medulla: 42.1 +/- 7.2%/min). Our findings indicate that renal ischemia is associated with a redistribution of blood flow from cortex to medulla, not related to DM. Furthermore, renal ischemia in db/db mice results in a marked impairment in reperfusion of the renal cortex and medulla during the early postischemic period.


Subject(s)
Diabetes Complications , Ischemia/complications , Ischemia/physiopathology , Kidney/blood supply , Renal Circulation , Acute Disease , Animals , Kidney Cortex/blood supply , Kidney Medulla/blood supply , Laser-Doppler Flowmetry , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Recovery of Function , Time Factors
4.
J Mol Cell Cardiol ; 41(4): 724-31, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16887142

ABSTRACT

Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.


Subject(s)
Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Myosin Heavy Chains/metabolism , Receptors, Transforming Growth Factor beta/physiology , Alleles , Animals , Cell Lineage , Crosses, Genetic , Female , Gene Expression Regulation, Developmental , Homozygote , Integrases/genetics , Male , Mice , Mice, Transgenic , Microfilament Proteins/genetics , Muscle Proteins/genetics , Muscle, Smooth, Vascular/blood supply , Muscle, Smooth, Vascular/embryology , Myosin Heavy Chains/genetics , Receptors, Transforming Growth Factor beta/genetics , Recombination, Genetic , Signal Transduction , Testis/embryology , Testis/metabolism
5.
J Mol Cell Cardiol ; 40(1): 148-56, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16288910

ABSTRACT

Transforming growth beta-1 (TGF-beta1) appears to play a critical role in the regulation of arterial intimal growth and the development of atherosclerosis. TGF-beta1 is expressed at increased levels in diseased arteries; however, its role in disease development remains controversial. Experiments in which TGF-beta1 is overexpressed in the artery wall of transgenic mice could clarify the role of TGF-beta1 in the development or prevention of vascular disease. However, constitutive overexpression of a TGF-beta1 transgene in the mouse artery wall is embryonically lethal. Therefore, to overexpress TGF-beta1 in the artery wall of adult mice, we generated mice that were transgenic for a conditional, tetracycline operator (tetO)-driven TGF-beta1 allele. These mice were viable, and when crossed with mice expressing a tetracycline-regulated transactivator (tTA) in the heart, expressed the TGF-beta1 transgene in a cardiac-restricted and doxycycline-dependent manner. Nevertheless, breeding of the tetO-TGF-beta1 transgene into three lines of mice transgenic for a smooth muscle-targeted tTA (SM22alpha-tTA mice; reported elsewhere to transactivate tetO-driven alleles in smooth muscle cells of large arteries) did not yield expression of the TGF-beta1 transgene. Moreover, tTA expression was not detected in aortae of the SM22alpha-tTA mice. Transgenic mice that express tTA at high levels in vascular smooth muscle and reliably transactivate tetO-driven transgenes would be useful for deciphering the role of TGF-beta1 (or other proteins) in normal arterial physiology and in the development of arterial disease. Currently available SM22alpha-tTA mice were not useful for this purpose. Generation of higher-expressing lines of SM22alpha-tTA mice appears warranted.


Subject(s)
Gene Expression Regulation , Microfilament Proteins/genetics , Muscle Proteins/genetics , Transforming Growth Factor beta/genetics , Alleles , Animals , Doxycycline/pharmacology , Gene Expression Regulation/drug effects , Heart/physiology , Mice , Mice, Transgenic , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Smooth, Vascular/physiology , Transcriptional Activation , Transforming Growth Factor beta/drug effects , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1 , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
7.
Eur J Biochem ; 269(18): 4551-8, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12230567

ABSTRACT

A number of findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). A recent study has demonstrated that the C tails of the beta and gamma subunits of ENaC are subject to phosphorylation by at least three protein kinases [Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R. & Garty, H. (2002) J. Biol. Chem. 277, 13539-13547]. One of them was identified as ERK which phosphorylates betaT613 and gammaT623 and affects the channel interaction with Nedd4. The current study identifies a second protein kinase as casein kinase 2 (CK2), or CK-2-like kinase. It phosphorylates betaS631, a well-conserved serine on the beta subunit. Such phosphorylation is observed both in vitro using glutathione-S-transferase-ENaC fusion proteins and in vivo in ENaC-expressing Xenopus oocytes. The gamma subunit is weakly phosphorylated by this protein kinase on another residue (gammaT599), and the C tail of alpha is not significantly phosphorylated by this kinase. Thus, CK2 may be involved in the regulation of the epithelial Na+ channel.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Sodium Channels/metabolism , Amino Acid Sequence , Animals , Casein Kinase II , Epithelial Sodium Channels , Glutathione Transferase/metabolism , Molecular Sequence Data , Oocytes , Phosphorylation , Protein Binding , Sequence Alignment , Xenopus
8.
J Biol Chem ; 277(16): 13539-47, 2002 Apr 19.
Article in English | MEDLINE | ID: mdl-11805112

ABSTRACT

Phosphorylation of the epithelial Na(+) channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the beta and gamma subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of beta and gammaENaC, have been identified by an in vitro assay. One of these phosphorylates betaThr-613 and gammaThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of gammaThr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating betaThr-613 and gammaThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions beta613 or gamma623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on betaThr-613 and gammaThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of betaThr-613 and gammaThr-623 down-regulates the channel by facilitating its interaction with Nedd4.


Subject(s)
Calcium-Binding Proteins/metabolism , Ligases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Sodium Channels/chemistry , Ubiquitin-Protein Ligases , Amino Acid Sequence , Animals , Biosensing Techniques , Blotting, Western , CHO Cells , Cricetinae , Cytosol/metabolism , Down-Regulation , Endosomal Sorting Complexes Required for Transport , Epithelial Sodium Channels , Glutathione Transferase/metabolism , Insulin/metabolism , Kinetics , Molecular Sequence Data , Nedd4 Ubiquitin Protein Ligases , Oocytes/metabolism , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , RNA, Complementary/metabolism , Rats , Recombinant Fusion Proteins/metabolism , Surface Plasmon Resonance , Threonine/chemistry , Time Factors , Xenopus , Xenopus Proteins
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