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Am J Physiol Renal Physiol 294: F229-F235, 2008. First published November 14, 2007; doi:10.1152/ajprenal.00386.2007
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Glial cell line-derived neurotrophic growth factor increases motility and survival of cultured mesenchymal stem cells and ameliorates acute kidney injury

Haikun Shi,1 Daniel Patschan,1 Gunnar P. H. Dietz,2 Mathias Bähr,2 Matthew Plotkin,1 and Michael S. Goligorsky1

1Departments of Medicine and Pharmacology, Renal Research Institute, New York Medical College, Valhalla, New York; and 2Neurologische Universitaetsklinik, Goettingen, Germany

Submitted 14 August 2007 ; accepted in final form 12 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

nestin; apoptosis

GLIAL CELL LINE-DERIVED NEUROTROPHIC growth factor (GDNF), a member of the transforming growth factor family, was described initially as a survival factor of dopaminergic and noradrenergic neurons and spinal motoneurons (reviewed in Ref. 13). More recent clinical trials established its efficacy in treating patients with Parkinson's disease (6). It came as a surprise that GDNF knockout mice showed defects in ureteric bud formation and branching during metanephros development and died at birth due to renal agenesis (8, 11). These observations led to the recognition of nonneuronal functions of GDNF, especially in renal development and in disease. Interestingly, GDNF expression has been found to be enhanced in some models of renal disease (passive Heymann nephritis, puromycin aminonucleoside nephrosis), where GDNF inhibited apoptosis in podocytes (15). Furthermore, studies on partial GDNF depletion showed that it is associated with the reduction in the number of glomeruli without detectable changes in renal blood flow or glomerular filtration rate under basal conditions (3).

Nestin-positive cells, normally abundantly localized to the renal papilla in mice (9), have been implicated in affording renoprotection against ischemic injury (10). We showed that nestin-expressing cells migrated toward the cortex of ischemic kidneys, participated in sprouting angiogenesis ex vivo, and rescued renal function postischemia (10).

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) (17). The promoter region of nestin has consensus binding sites for the midbrain and central nervous system enhancer elements and contains two POU-domain binding sites, with only the downstream site being necessary for central nervous system-specific expression. We hypothesized, therefore, 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. This study was designed to test the above prediction. We demonstrate that GDNF expression is enhanced in the ischemic kidney, administration of GDNF to mice subjected to renal ischemia protects the kidney from developing dysfunction, and that these actions might be attributed to the accelerated migration and/or survival of renal resident MSC.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. MSC (4E clone expressing CD44+, nestin+, and vimentin+, but negative for CD31, CD34, CD117 by FACS analysis) cultures have been previously established and described (12). Cells were cultured in mesenchymal culture medium (MEM) supplemented with 10% horse serum (mesenchymal medium). Alternatively, as indicated in RESULTS, cells were switched to the endothelium-specific medium EGF-2 (Clonetics, Walkersville, MD).

Materials. The following antibodies were used: anti-GDNF (B-8 and D-20, Santa Cruz Biotechnology, Santa Cruz, CA), anti-nestin (RAT 401, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit IgG horseradish peroxidase-linked (Cell Signaling Technology), and goat anti-mouse, goat anti-rabbit Alexa-conjugated, and goat anti-mouse and anti-rabbit FITC-labeled antibodies (Jackson ImmunoResearch Laboratories). GDNF was obtained from Santa Cruz Biotechnology, or it was isolated as previously described (4) and used, where indicated, as an additive to the culture medium. Recombinant human GDNF (due to the conserved sequence, it is widely used in experimental animals) included a His6 tag and the Xpress epitope (Invitrogen, Carlsbad, CA), resulting in an increased total molecular mass of 19 kDa. Thus recombinant GDNF could be readily distinguished from the endogenous form by Western analysis.

Animal studies. The animal study protocol was designed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (US Department of Health and Human Services Public Health Services, NIH, NIH Publication no. 86-23, 1985) and approved by the Institutional Animal Care and Use Committee. Male FVB/NJ mice were obtained from Jackson Labs (Bar Harbor, ME). All animals were separately caged with a 12:12-h light-dark cycle and had free access to water and chow throughout the study. FVB/NJ 8-wk-old mice were anesthetized with ketamine and xylazine. Kidneys were exposed through a 1.5-cm midlaparotomy, and clamping of the renal pedicles was performed with microserrefines (Fine Science Tools, Foster City, CA). After 30 min, the clamps were released. The abdominal incision was closed with a 4-0 suture and surgical staples. At time 0, 3 h, and 1, 3, or 7 days after the surgery, the animals were killed. Blood was sampled for creatinine, the kidneys were removed and stored at –80°C for further Western blot analysis (without perfusion-fixation; 4 mice for each time point) or processed after perfusion-fixation for immunofluorescence studies (3 mice each for every time point). For the latter, mice were perfused with 0.9% NaCl followed by 50 ml periodate-lysine-paraformaldehyde solution. A separate group of seven mice received GDNF therapy in combination with renal ischemia. For the GDNF treatment study, 5.0 nmol of recombinant human GDNF dissolved in 0.5 ml saline were injected intraperitoneally in the immediate post-ischemic period.

Western blot analysis. Kidneys were kept on ice and the renal cortex, medulla, and papilla were excised under a dissecting microscope. Five to seven kidneys were used for each experiment, and experiments were repeated at least three times. Pooled tissue samples or cultured MSC were homogenized in RIPA buffer (R0278-50 ml, Sigma) with a complete cocktail of protease inhibitors (Roche, Nutley, NJ) and DNAse. Samples were centrifuged at 14,000 rpm for 30 min, and the supernatants were collected for the GDNF analysis. The pellets were washed with RIPA buffer once and solubilized in 8 M urea at 4°C for 3 h on the shaker. Samples were recentrifuged at 14,000 rpm for 30 min. Protein concentration was determined using a Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of protein were electrophoretically separated in 16–18% Tris-glycine gels for GDNF analysis and in 4–12% Tris-glycine gels for nestin analysis (Invitrogen, Carlsbad, CA) and transferred to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were blocked with PBS containing nonfat dry milk (5% wt/vol), incubated with the primary antibody, anti-rat nestin (Rat-401, 1:100 in PBS-Tween 20, 0.05% containing 1% nonfat milk) or anti-human GDNF (1:2,000 in PBS-Tween 20, 0.05% containing 1% nonfat milk), and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,500 in PBS- Tween 20, 0.05% containing 3% nonfat milk) for 60 min at room temperature. Proteins were detected by SuperSignal West Pico chemiluminescence (Pierce, Rockford, IL). All data presented were obtained in at least three independent experiments.

Immunofluorescence study. Kidney tissue samples from FVB/NJ mice were postfixed in 4% paraformaldehyde solution for 30 min (Electron Microscopy Sciences, Hatfield, PA), followed by incubation in 30% sucrose overnight at 4°C. Embedding was performed in an optimal cutting temperature compound (OCT; Tissue-Tek, Torrance, CA), and samples were stored at –80°C. Frozen samples were cut into 10-µm-thick sections (Cryomicrotom CM 1850, Leica Microsystems, Bannockburn, IL). For nestin and GDNF staining, nonspecific protein binding was blocked by 1-h incubation with 10% goat serum in PBS. For primary incubation, anti-rat nestin or anti-human GDNF (D20, 1:2,000 in PBS 10% goat serum) was applied for 1–2 h at room temperature. Secondary antibody incubation was performed with Alexa 594-conjugated goat anti-mouse IgG or anti-rabbit IgG diluted 1:1,000 in PBS-goat serum (10%, Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Control samples were stained with secondary antibodies only. For staining of cultured MSC, preparations were fixed with 4% paraformaldehyde for 30 min, incubated with 10% goat serum and 0.1% Tween 20 in PBS, followed by incubation with primary antibodies for 1 h at room temperature. After washing with PBS, cells were incubated with FITC- or Alexa-conjugated goat secondary antibodies. Primary antibodies used were mouse monoclonal anti-RAT 401 (1:10; Developmental Studies Hybridoma Bank), mouse anti-GDNF (B-8, 1:100; Santa Cruz Biotechnology). Control sections were prepared by omitting the primary antibody. To visualize the nuclei, tissue sections were counterstained with 4',6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR). Sections and slides were examined under a Nikon compound fluorescence microscope with the appropriate dichroic mirrors (Nikon, Melville, NY), equipped with a Spot Insight camera (Diagnostic Instruments). Images were stored and processed for analysis using Universal Imaging software (MetaMorph suite).

In vitro effects of GDNF. Subconfluent MSC were deprived of serum for 20 h, and GDNF was added at the concentration of 100 ng/ml. At different times, as indicated in RESULTS, cells were harvested and prepared for GDNF and nestin analysis. For dose-response studies, cells were incubated in the presence of different concentrations of GDNF (0, 20, 40, 80, and 160 ng/ml) at the end of 20 h of serum starvation. To emulate an hypoxic microenvironment, cells were cultured in MEM with 10% horse serum under 2% O2-5% CO2-93% N2 using a controlled-atmosphere chamber and O2 analyzer (ProOx 110, Reming Bioinstruments, Redfield, NY).

Wound-healing assay. MSC were grown to confluence in MEM with 10% horse serum supplemented with 100 ng/ml GDNF or in the endothelial EGM2 medium supplemented with 2% FBS with 100 ng/ml GDNF. After standard wounds were inflicted with a needle tip, images of wounded monolayers were obtained at 0, 3, 6, 12, and 24 h. The size of the wounds was measured using digital morphometric analysis. The initial wounded area was considered 100%, and the reduction of the wound size was computed from the consecutive images.

Statistical analysis. The results were expressed as means ± SE. The means of two populations were compared by Student's t-test. For multiple comparisons, analysis of variance was employed. Differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In vitro studies of effects of GDNF on MSC. Using cultured kidney-derived MSC, which abundantly express nestin (12), we examined the expression of GDNF and its actions. MSC express endogenous GDNF with electrophoretic mobility similar to that expressed in the brain (Fig. 1A). Serum starvation resulted in the downregulation of GDNF expression in MSC (Fig. 1B). MSC also exhibited the GDNF-induced expression of GDNF. Figure 2, A and B, summarizes the dose-response and time course of the observed positive GDNF feedback regulation. Due to the higher molecular weight of recombinant GDNF compared with the endogenously expressed form, the observed upregulation of GDNF could be attributed solely to the enhanced expression of the endogenous GDNF, without overlap with the added recombinant product. This response could be elicited with as low as 20 ng/ml GDNF and was observable already after 60-min exposure. 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 (Fig. 2, C and D). In view of the previous demonstration of upregulation of nestin expression in the cortex of ischemic kidneys, further studies were performed in FVB/NJ mice under baseline and ischemic conditions to gain insights into in vivo relevance of these observations.


Figure 1
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  		Fig. 1. Expression of glial cell line-derived neurotrophic growth factor (GDNF) in mesenchymal stem cells (MSC). A: electrophoretic mobility of immunodetectable GDNF in MSC is similar to that detectable in the brain. B: serum starvation leads to the reduced expression of GDNF (top, representative results; bottom, densitometric summary of n = 3 experiments). *P < 0.05.

 

Figure 2
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  		Fig. 2. Feedback upregulation of GDNF expression and parallel changes in nestin expression. Shown are dose-response (A) and time course (B) of GDNF expression, time course of nestin expression after application of GDNF (C), and comparison of GDNF and nestin expression (D). Note the concomitant increase in GDNF and nestin expression after application of GDNF; n = 3 experiments.

 
GDNF expression after renal ischemia and GDNF-induced renoprotection. Immunohistochemical analysis of kidneys obtained from control mice showed a faint cortical and medullary fluorescence (Fig. 3). In contrast, already 15-h postischemia expression of GDNF was much expanded, occupying the entire cortex, but did not change appreciably in the medulla. Similar results were obtained using Western blot analysis of kidney lysates. Cortical expression of GDNF, faintly detectable in control samples, already showed a robust upregulation already 3 h after the ischemic episode (Fig. 4). Medullary expression of GDNF showed no appreciable dynamics (Fig. 5). These data demonstrated that GDNF is induced early postischemia in the renal cortex and may theoretically serve as a guidance cue for migrating MSC. The above findings lead to the next question: Could supplemental GDNF administered early after ischemia ameliorate postischemic renal injury?


Figure 3
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  		Fig. 3. Immunohistochemical detection of GDNF in control and ischemic kidneys. A: panoramic view of the kidneys stained with antibody against GDNF. B: detailed view of glomeruli and cortical tubules in control and postischemic kidneys.

 

Figure 4
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  		Fig. 4. Renal cortical expression of GDNF after acute ischemia. A: typical results of Western blot analysis of GDNF expression in postischemic renal cortex at 3 h and 1, 3, and 7 days. STD, GDNF standard. B: densitometric summary of GDNF expression in n = 3 experiments. *P < 0.05.

 

Figure 5
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  		Fig. 5. Renal medullary expression of GDNF after acute ischemia. A: typical results of Western blot analysis of GDNF expression in postischemic renal medulla at 3 h and 1, 3, and 7 days. B: densitometric summary of GDNF expression in n = 3 experiments.

 
To address this question, mice were subjected to 30-min bilateral renal ischemia and received GDNF postoperatively. As summarized in Fig. 6, GDNF-treated mice showed significant renoprotection against acute ischemia. This was associated with the enhanced GDNF expression in the kidneys of GDNF-treated mice, but no further elevation above that seen in nontreated ischemic control, and the appearance of a faint 19-kDa band indicative of a nonmetabolized fraction of injected recombinant GDNF (Fig. 6B). The fraction of exogenous GDNF represented only ~7% of the total GDNF band density. Nestin expression was also increased in GDNF-treated ischemic kidneys, but the differences did not reach statistical significance compared with nontreated ischemic kidneys (Fig. 6B). This indicates that the observed renoprotection could be attributed to GDNF's effects on susceptible cell survival and/or motility, rather than to its amplification of GDNF expression. To address potential mechanisms of the observed renoprotection, additional in vitro studies were performed.


Figure 6
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  		Fig. 6. Renoprotective action of GDNF against renal ischemia. A: administration of GDNF to animals with renal ischemia resulted in the reduction in serum creatinine levels after renal ischemia. *P < 0.05. B: administration of GDNF to animals with renal ischemia did not result in its increased expression above the already elevated level seen in ischemic kidneys. The level of nestin, however, was slightly elevated above that seen in ischemic kidneys. Neither of these differences were statistically significant.

 
Effects of hypoxia and GDNF on mesenchymal cell motility and survival in vitro. Oliver et al. (9) and Patschan et al. (10) have previously demonstrated high motility of renal nestin-expressing MSC. We asked a question: Is GDNF a credible motogenic factor for these cells? This was addressed by further testing the ability of GDNF to induce motility in a wound-healing assay. Confluent monolayers were wounded, as detailed in MATERIALS AND METHODS, and the rate of wound closure was examined microscopically. As shown in Fig. 7, cultured MSC rapidly reduced the surface area of the wound, so that by 24 h wounds were uniformly closed (data not shown). This process was further accelerated in the presence of 100 ng/ml GDNF, but only in the endothelial-selective culture medium containing VEGF (EGM-2 medium), whereas no further acceleration was detectable in MEM.


Figure 7
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  		Fig. 7. GDNF accelerates in vitro wound healing under normoxic (A) and hypoxic (B) conditions. Mesenchymal stem cells were cultured in the specifically formulated MEM in a regular 95% air-5% CO2 incubator or under hypoxic conditions (2% O2-5% CO2-93% N2) using a controlled-atmosphere chamber and O2 analyzer (ProOx 110), as detailed in MATERIALS AND METHODS. C: dynamics of wound healing by MSC cultured in EGM-2 medium under normoxic and hypoxic conditions (n = 3 independent experiments). Note that hypoxia decelerated wound healing and application of GDNF to hypoxic cultures restores the normal rate of wound healing.

 
Exposure of cells to an hypoxic environment (hypoxic chamber) for 20 h showed a similar acceleration of wound healing in the presence of GDNF in the endothelial-specific (EGM-2 medium), but not in MEM. Interestingly, hypoxia per se did not accelerate, but rather slightly reduced the motility of MSC. Thus we next examined the expression of GDNF under hypoxic conditions. As shown in Fig. 8, hypoxia reduced the expression of GDNF in MSC by approximately twofold. These findings provided a tentative explanation for the beneficial action of exogenous GDNF observed in vivo.


Figure 8
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  		Fig. 8. Hypoxia suppresses GDNF expression in MSC. Analysis of GDNF abundance was performed as detailed in MATERIALS AND METHODS. Experiments were repeated 3 times.

 
Next, the effects of GDNF on the oxidative stress-induced survival of MSC were tested. MSC were exposed to a range of concentrations of hydrogen peroxide (100, 150 and 200 µM) for 24 h in the absence and presence of GDNF. The number of apoptotic MSC, as detected using antibodies against activated caspase-3, was dose dependently increased by exposure to hydrogen peroxide (Fig. 9). The addition of 100 ng/ml of GDNF to the culture medium significantly blunted the hydrogen peroxide-induced apoptosis resulting in the increased proportion of surviving cells.


Figure 9
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  		Fig. 9. Survival of MSC in the presence of GDNF. Studies were performed as detailed in MATERIALS AND METHODS. MSC were exposed to hydrogen peroxide (100, 150, and 200 µM) in the presence or absence of GDNF. Experiments were repeated 3 times. *P < 0.05 compared with respective controls not containing GDNF.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The data presented herein 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 functional protection against ischemia.

In adult rats, various noxious stimuli induced the expression and secretion of GDNF by astrocytes, while this response was attenuated in stroke-prone spontaneously hypertensive rats (18). Our studies suggest that GDNF induction represents a local and, perhaps adaptive, renal response to ischemia-reperfusion. In fact, further supplementation with GDNF ameliorated postischemic renal dysfunction. The observed parallel dynamics of GDNF and nestin in MSC and in the ischemic kidney is of special interest. This phenomenon, although not typical of the regulation of nestin in the central nervous system, has been previously observed in neural cell-derived precursors, where nestin is upregulated after GDNF treatment (2). Reactive astroglia reexpress nestin after injury and GDNF. In our study, kidney-derived MSC behaved similarly: serum starvation or hypoxia, resulting in downregulation of GDNF, led to the decline, whereas GDNF-induced GDNF upregulation led to the elevation in nestin expression. Notably, both responses occurred with a similar time course. In addition, GDNF upregulation during the postischemic period was accompanied by the enhanced expression of nestin, both occurring in the kidney cortex. It is unlikely that upregulation of GDNF in the postischemic period was due to hypoxia per se of renal MSC because in vitro studies convincingly demonstrated that hypoxia in cultured MSC suppressed rather than induced GDNF expression. That does not exclude, however, a possibility that other cell types become effectors of hypoxia-inducible GDNF synthesis. Alternatively, it is not excluded that other mechanisms are responsible for of GDNF induction. One of such contender mechanisms could be oxidative stress of ischemia-reperfusion. This assumption was tested in vitro but was rejected as a valid explanation of the observed surge in GDNF expression during the post-ischemic period (data not shown). Most probably, hypoxia inducible factor-1-induced VEGF expression leading to upregulation of GDNF is one of the key mechanisms for its induction postischemia. This assumption is based on the following reasons. It is intriguing that the effects of GDNF on MSC motility and survival were most obvious in the presence of VEGF, the major constituent of the endothelial-selective culture medium. Gao et al. (5) demonstrated that in the developing mesenchyme of the embryonic kidney, Flk-1 activation by VEGF-A is necessary to maintain the expression of Pax2, which in turn stimulates expression of GDNF. In this context, postischemic induction of VEGF has been previously demonstrated (16), and such an upregulation of VEGF expression may be necessary for the later surge in GDNF expression. This speculation will need to be further explored in future studies.

GDNF's effect on MSC survival under stress conditions is not unique to this cell type. GDNF was found to improve survival of neuronal (7, 14, 19) and nonneuronal cells, like podocytes (15). This rescue from apoptotic cell death has been attributed to the activation of the phosphatidylinositol 3-kinase/Akt pathway (1).

The finding that hypoxia or oxidative stress (hydrogen peroxide experiments) suppressed expression of GDNF in MSC may explain the observed therapeutic effect of GDNF treatment: it could be attributable to the local induction of GDNF synthesis by renal MSC and/or other renal resident or even extrarenal cell types. This initial boost could potentially initiate the GDNF-induced GDNF expression, as detected in our in vitro and in vivo studies, and eventually lead to the mobilization of nestin-expressing cells with their earlier demonstrated renoprotective properties.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52783 (M. S. Goligorsky), Fellowship Grant PA 1530/1-1 from the Deutsche Forschungsgemeinschaft (DFG) to D. Patschan, DFG Research Center for Molecular Physiology of the Brain (G. P. H. Dietz and M. Bähr), and the Westchester Artificial Kidney Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. S. Goligorsky, Depts. of Medicine and Pharmacology, Renal Research Institute, New York Medical College, Valhalla, NY (e-mail: Michael_goligorsky{at}nymc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anitha M, Gondha C, Sutliff R, Parsadanian A, Mwangi S, Sitaraman S, Srinivasan S. GDNF rescues hyperglycemia-induced diabetic enteric neuropathy through activation of the PI3K/Akt pathway. J Clin Invest 116: 344–356, 2006.[CrossRef][Web of Science][Medline]
  2. Chalazonitis A, Rothman T, Chen J, Gershon M. Age-dependent differences in the effects of GDNF and NT-3 on the development of neurons and glia from neural crest-derived precursors immunoselected from the fetal rat gut: expression of GFRalpha-1 in vitro and in vivo. Dev Biol 204: 385–406, 1998.[CrossRef][Web of Science][Medline]
  3. Cullen-McEven L, Kett M, Dowling J, Anderson W, Bertram J. Nephron number, renal function and arterial pressure in aged GDNF heterozygous mice. Hypertension 41: 335–340, 2003.[Abstract/Free Full Text]
  4. Dietz GPH, Valbuena PC. Application of a blood-brain barrier-penetrating form of GDNF in a mouse model for Parkinson's Disease. Brain Res 1082: 61–66, 2006.[CrossRef][Web of Science][Medline]
  5. Gao X, Chen X, Taglienti M, Rumballe B, Little M, Kreidberg J. Angioblast-mesenchyme induction of early kidney development is mediated by Wt1 and VEGF-A. Development 132: 5437–5449, 2005.[Abstract/Free Full Text]
  6. Gill S, Patel N, Hotton G, O'Sullivan K, McCarter R, Bunnage M, Brooks D, Svendesen C, Heywood P. Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat Med 9: 589–595, 2003.[CrossRef][Web of Science][Medline]
  7. Katsuragi S, Ikeda T, Date I, Shingo T, Yasuhara T, Ikenoue T. Grafting of glial cell line-derived neurotrophic factor secreting cells for hypoxic-ischemic encephalopathy in neonatal rats. Am J Obstet Gynecol 192: 1137–1145, 2005.[CrossRef][Web of Science][Medline]
  8. Moore MW, Klein RD, Farinas I, Sauer H, Armanini M, Phillips H, Reichardt L, Ryan A, Carver-Moore K, Rosenthal A. Renal and neuronal abnormalities in mice lacking GDNF. Nature 382: 76–79, 1996.[CrossRef][Medline]
  9. Oliver J, Maarouf O, Cheema F, Al-A wqati Q. The renal papilla is a niche for adult kidney stem cells. J Clin Invest 114: 795–804, 2004.[CrossRef][Web of Science][Medline]
  10. Patschan D, Michutina T, Shi H, Dolff S, Brodsky S, Vasilieva T, Cohen-Gould L, Winaver J, Chander P, Enikolopov G, Goligorsky MS. Normal distribution and medullary-to-cortical shift of nestin-expressing cells in acute renal ischemia. Kidney Int 71: 744–754, 2007.[CrossRef][Web of Science][Medline]
  11. Pichel J, Shen L, Sheng H, Granholm A, Drago J, Grinberg A, Lee E, Huang S, Saarma M, Hoffer B, Sariola H, Westphal H. Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382: 73–76, 1996.[CrossRef][Medline]
  12. Plotkin M, Goligorsky MS. Mesenchymal cells from adult kidney support angiogenesis and differentiate into multiple interstitial cell types including erythropoietin producing fibroblasts Am J Physiol Renal Physiol 291: F902–F912, 2006.[Abstract/Free Full Text]
  13. Sariola H, Saarma M. Novel functions and signaling pathways for GDNF. J Cell Sci 116: 3855–3862, 2003.[Abstract/Free Full Text]
  14. Sharma H. Post-traumatic application of brain-derived neurotrophic factor and glial-derived neurotrophic factor on the rat spinal cord enhances neuroprotection and improves motor function. Acta Neurochir Suppl 96: 329–334, 2006.[Medline]
  15. Tsui C, Shankland S, Pierchala B. Glial cell line-derived neurotrophic factor and its receptor Ret is a novel ligand-receptor complex critical for survival response during podocyte injury. J Am Soc Nephrol 17: 1543–1552, 2006.[Abstract/Free Full Text]
  16. Villanueva S, Cespedes C, Vio C. Ischemic acute renal failure induces the expression of a wide range of nephrogenic proteins. Am J Physiol Regul Integr Comp Physiol 290: R861–R870, 2006.[Abstract/Free Full Text]
  17. Wiese C, Rolletschek A, Kania G, Blyszczuk P, Tarasov K, Tarasova Y, Wersto R, Boheler K, Wobus A. Nestin expression–a property of multi-lineage progenitor cells? Cell Mol Life Sci 61: 2510–2522, 2004.[CrossRef][Web of Science][Medline]
  18. Yamagata K, Tagami M, Ikeda K, Tsumagari S, Yamori Y, Nara Y. Differential regulation of GDNF mRNA expression during hypoxia and reoxygenation in astrocytes isolated from stroke-prone spontaneously hypertensive rats. Glia 37: 1–7, 2002.[CrossRef][Web of Science][Medline]
  19. Zhao Z, Alam S, Oppenheim R, Prevette D, Evenson A, Parsadanian A. Overexpression of glial cell line-derived neurotrophic factor in the CNS rescues motoneurons from programmed cell death and promotes their long-term survival following axotomy. Exp Neurol 190: 263–275, 2004.[CrossRef][Web of Science][Medline]



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