Normal distribution and medullary-to-cortical shift of Nestin-expressing cells in acute renal ischemia
2007; Elsevier BV; Volume: 71; Issue: 8 Linguagem: Inglês
10.1038/sj.ki.5002102
ISSN1523-1755
AutoresDaniel Patschan, Tatyana V. Michurina, Haikun Shi, Sebastian Dolff, Sergey V. Brodsky, T. V. Vasilieva, Leona Cohen‐Gould, Josi Winaver, Praveen Chander, Grigori Enikolopov, Michael S. Goligorsky,
Tópico(s)Birth, Development, and Health
ResumoNestin, a marker of multi-lineage stem and progenitor cells, is a member of intermediate filament family, which is expressed in neuroepithelial stem cells, several embryonic cell types, including mesonephric mesenchyme, endothelial cells of developing blood vessels, and in the adult kidney. We used Nestin-green fluorescent protein (GFP) transgenic mice to characterize its expression in normal and post-ischemic kidneys. Nestin-GFP-expressing cells were detected in large clusters within the papilla, along the vasa rectae, and, less prominently, in the glomeruli and juxta-glomerular arterioles. In mice subjected to 30 min bilateral renal ischemia, glomerular, endothelial, and perivascular cells showed increased Nestin expression. In the post-ischemic period, there was an increase in fluorescence intensity with no significant changes in the total number of Nestin-GFP-expressing cells. Time-lapse fluorescence microscopy performed before and after ischemia ruled out the possibility of engraftment by the circulating Nestin-expressing cells, at least within the first 3 h post-ischemia. Incubation of non-perfused kidney sections resulted in a medullary-to-cortical migration of Nestin-GFP-positive cells with the rate of expansion of their front averaging 40 μm/30 min during the first 3 h and was detectable already after 30 min of incubation. Explant matrigel cultures of the kidney and aorta exhibited sprouting angiogenesis with cells co-expressing Nestin and endothelial marker, Tie-2. In conclusion, several lines of circumstantial evidence identify a sub-population of Nestin-expressing cells with the mural cells, which are recruited in the post-ischemic period to migrate from the medulla toward the renal cortex. These migrating Nestin-positive cells may be involved in the process of post-ischemic tissue regeneration. Nestin, a marker of multi-lineage stem and progenitor cells, is a member of intermediate filament family, which is expressed in neuroepithelial stem cells, several embryonic cell types, including mesonephric mesenchyme, endothelial cells of developing blood vessels, and in the adult kidney. We used Nestin-green fluorescent protein (GFP) transgenic mice to characterize its expression in normal and post-ischemic kidneys. Nestin-GFP-expressing cells were detected in large clusters within the papilla, along the vasa rectae, and, less prominently, in the glomeruli and juxta-glomerular arterioles. In mice subjected to 30 min bilateral renal ischemia, glomerular, endothelial, and perivascular cells showed increased Nestin expression. In the post-ischemic period, there was an increase in fluorescence intensity with no significant changes in the total number of Nestin-GFP-expressing cells. Time-lapse fluorescence microscopy performed before and after ischemia ruled out the possibility of engraftment by the circulating Nestin-expressing cells, at least within the first 3 h post-ischemia. Incubation of non-perfused kidney sections resulted in a medullary-to-cortical migration of Nestin-GFP-positive cells with the rate of expansion of their front averaging 40 μm/30 min during the first 3 h and was detectable already after 30 min of incubation. Explant matrigel cultures of the kidney and aorta exhibited sprouting angiogenesis with cells co-expressing Nestin and endothelial marker, Tie-2. In conclusion, several lines of circumstantial evidence identify a sub-population of Nestin-expressing cells with the mural cells, which are recruited in the post-ischemic period to migrate from the medulla toward the renal cortex. These migrating Nestin-positive cells may be involved in the process of post-ischemic tissue regeneration. Plasticity and multipotency of stem cells form the foundation for their therapeutic application. One of the proposed markers of multi-lineage stem and progenitor cells is Nestin.1.Wiese C. Rolletschek A. Kania G. et al.Nestin expression – a property of multi-lineage progenitor cells?.Cell Mol Life Sci. 2004; 61: 2510-2522Crossref PubMed Scopus (520) Google Scholar It was first identified in neuroepithelial stem cells as a member of the family of intermediate filaments.2.Lendahl U. Zimmerman L.B. McKay R.D. CNS stem cells express a new class of intermediate filament protein.Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2685) Google Scholar In embryonic tissues, Nestin is expressed in neuronal and oligodendrocyte precursor cells, developing skeletal and cardiac myocytes, mesonephric mesenchyme and endothelial cells of developing blood vessels, among several other cell types.1.Wiese C. Rolletschek A. Kania G. et al.Nestin expression – a property of multi-lineage progenitor cells?.Cell Mol Life Sci. 2004; 61: 2510-2522Crossref PubMed Scopus (520) Google Scholar These Nestin-expressing cells have a limited (compared to stem cells) proliferation potential and high plasticity. To facilitate the identification and localization of Nestin-expressing cells, Mignone et al.3.Mignone J.L. Kukekov V. Chiang A.S. et al.Neural stem and progenitor cells in Nestin-GFP transgenic mice.J Comp Neurol. 2004; 469: 311-324Crossref PubMed Scopus (514) Google Scholar have generated Nestin-green fluorescent protein (GFP) transgenic mice, which were used as a reporter line for studying neural stem and progenitor cells. In these animals, enhanced GFP cDNA is placed between the promoter and a critical second intron enhancer sequence3.Mignone J.L. Kukekov V. Chiang A.S. et al.Neural stem and progenitor cells in Nestin-GFP transgenic mice.J Comp Neurol. 2004; 469: 311-324Crossref PubMed Scopus (514) Google Scholar and GFP fluorescence colocalizes with the cells expressing endogenous Nestin. As Nestin expression has been observed in the kidneys of rat and mouse during embryogenesis and in the adult mouse kidney under non-ischemic conditions,4.Amoh Y. Yang M. Li L. et al.Nestin-linked green fluorescent protein transgenic nude mouse for imaging human tumor angiogenesis.Cancer Res. 2005; 65: 5352-5357Crossref PubMed Scopus (129) Google Scholar, 5.Zou J. Yaoita E. Watanabe Y. et al.Upregulation of Nestin, vimentin, and desmin in rat podocytes in response to injury.Virchows Arch. 2006; 448: 485-492Crossref PubMed Scopus (113) Google Scholar, 6.Wagner N. Wagner K.D. Scholz H. et al.The intermediate filament protein Nestin is expressed in the developing kidney and heart and might be regulated by the Wilms' tumor suppressor Wt1.Am J Physiol Regul Integr Comp Physiol. 2006; 291: R779-R787Crossref PubMed Scopus (68) Google Scholar, 7.Chen J. Boyle S. Zhao M. et al.Differential expression of the intermediate filament protein Nestin during renal development and its localization in adult podocytes.J Am Soc Nephrol. 2006; 17: 1283-1291Crossref PubMed Scopus (93) Google Scholar we exploited this animal model in an attempt to characterize the behavior of Nestin-GFP-expressing cells in the intact kidney and after renal ischemia. Here, we demonstrate the robust expression of Nestin-GFP-positive cells in the papilla, as well as in the outer medulla and glomeruli and monitor their dynamics in the course of acute ischemic injury. Nestin-GFP-expressing cells were detected in large numbers clustered within the papilla, along the vasa rectae in the outer medulla, and, less prominently, in the glomeruli, juxta-glomerular arterioles, and arterial endothelium (Figures 1, 2 and 3). Glomeruli revealed frequent mesangial pattern of Nestin-GFP expression (Figure 2b). The major ‘niche’ for these cells, the renal papilla, was remarkably similar to that previously described for label-retaining renal stem cells,8.Oliver J.A. Maarouf O. Cheema F.H. et al.The renal papilla is a niche for adult kidney stem cells.J Clin Invest. 2004; 114: 795-804Crossref PubMed Scopus (445) Google Scholar which were also found to express Nestin. Immunohistochemical analysis of Nestin distribution in wild-type animals showed a pattern of expression similar to that seen in Nestin-GFP mice, with the exception that glomerular staining was predominantly in the podocytes, although focal mesangial and endothelial pattern was also present. In general, in control mice, the glomeruli showed moderate Nestin staining. The cortical arteries showed minimal and/or focal staining, whereas the cortical interstitium was moderately positive, as were the inner stripe of the outer medulla and the medullary vascular bundles. Nestin-positive cells were occasionally detectable in the renal capsule and in the pelvic wall (Figure 3).Figure 2(a, c, e) Comparison of Nestin localization patterns in FVB/NJ mice and (b, d, f) Nestin-GFP mice. (e and f) Nestin-GFP-expressing cells were detected in large numbers clustered within the papilla, (c and d) along the vasa rectae in the outer medulla, and, less prominently, (a and b) in the glomeruli and juxta-glomerular arterioles (data not shown) (original magnification: (d) original magnification × 10 and (a–c, e, and f) original magnification × 40).View Large Image Figure ViewerDownload (PPT)Figure 3Distribution of Nestin-GFP in the kidney. (a–c) Normal kidneys show Nestin-GFP (a) in the papilla and in the medullary vascular bundles, (b) in the juxta-glomerular arterioles, and (c) in the glomeruli. Original magnification: (a and b) × 10, (c) × 40. (d–f) In post-ischemic kidneys (24 h), medullary vascular bundles show conspicuous Nestin-GFP. (e) Occasionally, Nestin-GFP cells are detectable in the kidney capsule (white arrows). (e and f) Nestin-GFP cells decorate endothelial and focally periadventitial layer of arteries (white arrowheads) (original magnification: (d) × 10, (e) × 40, (f) × 60). (g–i) Perivascular adventitial localization of Nestin-GFP-expressing cells in the renal cortex. Note that some cells send long processes in the interstitial space (white arrowheads). Pictures are taken from untreated animals (original magnification × 200).View Large Image Figure ViewerDownload (PPT) Animals were subjected to 30 min of bilateral renal pedicle clamping, as detailed in Materials and Methods, and killed at different times post-ischemia. Expression of Nestin-GFP was examined at 3, 24, 72 h and 7 days post-ischemia using digital image analysis. GFP fluorescence showed enhanced intensity compared to control, which persisted for 1 week (Figures 3 and 5). Glomerular, juxta-glomerular microvasculature, and occasional glomerular epithelial cells displayed increased fluorescence intensity of Nestin-GFP (Figure 4). Interestingly, some endothelial cells of peritubular capillaries showed Nestin expression at 72 h after ischemia (Figure 5). All the findings made in Nestin-GFP transgenic mice were reproducible in wild-type animals subjected to renal ischemia and visualized using anti-Nestin antibodies. At 6 h after ischemia/reperfusion, the glomerular Nestin expression was increased, as judged by quantitative digital image analysis. The endothelial layer of cortical arteries and the cortical interstitium also showed a more intense staining, as did the medullary interstitium and the medullary vascular bundles (Figures 3 and 5). The dynamics of Nestin expression is semiquantitatively summarized in Table 1.Figure 5Post-ischemic enhancement of Nestin-GFP fluorescence in the (a–d) glomeruli and (e, f) peritubular capillaries. (a–c) Representative images of glomeruli in (a) control, (b) 24 h, and (c) 72 h post-ischemia. (d) Summary of Nestin-GFP fluorescence intensity during the course of renal ischemia (n=5). (e) Representative images of renal cortex with Nestin-GFP-positive peritubular capillaries (arrows) after renal ischemia. (f) Summary of Nestin-GFP expression by endothelial cells of peritubular capillaries at different times after ischemia. At 72 h, capillary endothelium was strongly positive for Nestin-GFP (P=0.014, n=3).View Large Image Figure ViewerDownload (PPT)Table 1Summary of fluorescence microscopy and immunohistochemical findings on the distribution of Nestin in control and post-ischemic kidneys of FVB/NJ miceGlomeruliCortical arteriesCortical interstitiumInner stripe of outer medullaMedullary vascular bundlesControl+, podocytes−+++24 h post-ischemia+, endothelial pattern++++, peritubular, more widespread+++ to +++++++ Open table in a new tab These results of immunohistochemical analysis were further corroborated by Western blot analysis of Nestin expression (Figure 6a). Nestin expression was abundant in the papilla and medulla and was barely detectable in the cortex of intact kidneys. After renal ischemia, cortical enrichment with Nestin occurred and was accompanied by the depletion of papillary Nestin expression. These data were in agreement with the results of panoramic side-by-side comparison of kidney images encompassing all layers of the kidney, which showed depletion of Nestin-GFP in the papillae and increased expression in the outer medullae and cortices of post-inschemic kidneys obtained from Nestin-GFP mice (Figure 6b). In an attempt to account for the increased fluorescence intensity of Nestin-GFP in the post-ischemic period, we initially hypothesized that Nestin-GFP-expressing circulating cells engrafted the kidney (although fluorescence-activated cell sorter (FACS) analysis of the circulating cells showed no detectable GFP-positive cells; data not shown). To document possible engraftment, we performed intravital time-lapse videomicroscopy of the kidney. Anesthetized mice were placed on the heated stage of a microscope, the right kidney exposed and immobilized in a kidney cup, and the renal surface visualized using a × 20 water-immersion objective. Time-lapse fluorescence microscopy was performed before ischemia and the same area of the renal surface followed for 3 h after 30-min occlusion of the renal artery by a 120 ms pulses of illumination at the wavelength of 490 nm and simultaneous recording of the emitted light at wavelength of 530 nm. In seven separate experiments, there was no detectable de novo appearance of Nestin-GFP fluorescence, thus, strongly suggesting that the post-ischemic rise in the Nestin-GFP fluorescence intensity was not owing to the engraftment by the circulating Nestin-GFP cells, at least during the first 3 h of reperfusion. As the above time-lapse videomicroscopy data were obtained from the population of superficial cells, we sought to generalize it to the whole kidney. This was accomplished with FACS analysis of dispersed cells. After obtaining a single-cell suspension, as detailed in Materials and Methods, GFP-positive cells were analyzed by FACS. Cells were also co-stained for detection of markers of hematopoietic lineage and endothelial progenitor cells. The results are shown in Figure 7. In the post-ischemic period, there was no significant change in the total number of Nestin-GFP-expressing cells up to 7 days, but there was a mild increase in fluorescence intensity of individual cells (Figure 7). Approximately 20% of Nestin-GFP-positive cells co-expressed markers of hematopoietic stem cells (CD34+, CD150+) or endothelial progenitor (Flk-1+, Tie-2+) cells and their proportion remained stable in all experimental groups. These data provide an independent confirmation to the videomicroscopy findings and bring to the fore the question of intensity of GFP fluorescence as a measure of Nestin expression by individual cells. Owing to the observed redistribution of Nestin-expressing cells in the post-ischemic kidneys, we pursued this finding using ex vivo imaging studies. In a separate series of experiments, cross-sections of kidneys obtained from Nestin-GFP mice were incubated in a basal cell culture medium on the stage of a compound inverted microscope and GFP fluorescence monitored for the subsequent 3 h. Although this experimental protocol could rule out any engraftment of the kidney by Nestin-GFP expressing cells, it also offered the possibility to follow the behavior of a large number of the resident Nestin-GFP-positive cells in the non-perfused kidney sections. A remarkable finding obtained in three separate experiments was the medulla-to-cortex migration of Nestin-GFP-positive cells along the papillo-cortical gradient (Figure 8). The rate of expansion of the front of Nestin-GFP-positive cells averaged initially 40 μm/30 min and was detectable already after 30 min of ex vivo incubation of kidney sections. To examine the properties of this gradient in more detail and to correlate it with the post-ischemic migration of Nestin-GFP-expressing cells in the course of renal ischemia/reperfusion, we performed line-scan analysis of low-magnification images obtained from kidney sections of Nestin-GFP mice at different time points post-ischemia. Images were aligned using the edge of cortical surfaces and consecutive images screened. The data showed a consistent medullary peak of fluorescence intensity (corresponding to the high concentration of Nestin-GFP-positive cells along vascular bundles), which normally declined from the medulla to the cortex of the kidney. However, in post-ischemic kidneys this gradient showed a time-dependent shift of the peak toward the cortex, consistent with the observed ex vivo relatively rapid migration of Nestin-GFP-expressing cells (Figure 8c). Yet, the contribution of tissue swelling to the observed expansion of Nestin-expressing cells in ex vivo experiments and in fixed kidney sections, presented in Figure 8, is difficult to assess quantitatively, thus prompting the next series of in vitro studies. To further examine the migratory capacity of Nestin-expressing cells, we employed in vitro matrigel angiogenesis assay.9.Nicosia R.F. Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.Lab Invest. 1990; 63: 115-122PubMed Google Scholar,10.Gealekman O. Brodsky S.V. Zhang F. et al.Endothelial dysfunction as a modifier of angiogenic response in Zucker diabetic fat rat: amelioration with Ebselen.Kidney Int. 2004; 66: 2337-2347Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Aortic and renal tissue explants were obtained from FVB/NJ wild-type mice and embedded in matrigel, as detailed in Materials and Methods. After 3–12 days in culture, depending on the source of the explants, capillary-like cords expressing Nestin sprouted from the explants (Figure 9). Co-staining of these cells for endothelial markers (Tie-2) and for Nestin showed a remarkable colocalization of both markers. These findings may suggest the participation of Nestin-expressing cells in post-ischemic angiogenesis and offered an explanation to the finding of post-ischemic Nestin expression in endothelial cells of peritubular capillaries (Figure 5). The data presented herein establish the utility of a model developed for studies of neural stem and progenitor cells, transgenic Nestin-GFP mouse, for investigations into the renal injury and repair. Although many questions related to the differentiation potential of Nestin-expressing cells residing in the kidney remain unanswered, the data obtained provide evidence for the robust expression and distribution of these cells in the normal mouse kidney and their dynamics after acute ischemia. The major findings (1) establish the renal papilla and medullary vascular bundles as the ‘niche’ for Nestin-expressing cells with the gradient sharply declining toward the cortex, (2) demonstrate increased Nestin expression in a sub-population of renal resident cells in the post-ischemic period, despite the lack of increase in the actual number of Nestin-expressing cells and engraftment from extrarenal sources, and (3) document migration of Nestin-expressing cells after ischemia from the papilla and outer medulla toward the renal cortex. The identity of Nestin-expressing cells has been elusive. In the previous studies, these cells were proposed to represent a pool of neural progenitors,11.Frederiksen K. McKay R.D. Proliferation and differentiation of rat neuroepithelial precursor cells in vivo.J Neurosci. 1988; 8: 1144-1151Crossref PubMed Google Scholar stem cells of the hair follicles capable of differentiating into blood vessels,12.Amoh Y. Li L. Yang M. et al.Nascent blood vessels in the skin arise from Nestin-expressing hair-follicle cells.Proc Natl Acad Sci USA. 2004; 101: 13291-13295Crossref PubMed Scopus (186) Google Scholar mesenchymal stem cells,13.Frojdman K. Pelliniemi L.J. Lendahl U. et al.The intermediate filament protein Nestin occurs transiently in differentiating testis of rat and mouse.Differentiation. 1997; 61: 243-249Crossref PubMed Google Scholar angiogenic endothelial cells,14.Mokry J. Nemecek S. Angiogenesis of extra- and intraembryonic blood vessels is associated with expression of Nestin in endothelial cells.Folia Biol (Praha). 1998; 44: 155-161PubMed Google Scholar and pericytes.7.Chen J. Boyle S. Zhao M. et al.Differential expression of the intermediate filament protein Nestin during renal development and its localization in adult podocytes.J Am Soc Nephrol. 2006; 17: 1283-1291Crossref PubMed Scopus (93) Google Scholar In the kidney, as presented in this study, Nestin-positive cells did not show co-expression of stem cell markers (c-kit), and only a small population expressed hematopoietic stem cell markers (CD 34 and 150) or endothelial progenitor cell markers (Flk-1 and Tie-2). They did not express neuronal nitric oxide synthase (data not shown) or α−smooth muscle actin. From the morphologic standpoint, Nestin-positive cells were localized to or were in close vicinity to the blood vessels such as vasa rectae, juxta-glomerular arterioles, and glomerular and peritubular capillaries and arteries; the latter, predominantly in the periadventitial location and focally in the intimal surface. Functionally, these cells were capable of fast migration toward the cortex after renal ischemia. Collectively, these properties are consistent with Nestin-expressing cells representing a population of mural cells: their perivascular location, lack of endothelial and smooth muscle cell markers, and migration properties argue in favor of this assignment. Recent studies by Nicosia and co-workers, in part, favor this conclusion by demonstrating that postnatal aorta contains multipotent mural cells expressing Nestin. An increase in the overall fluorescence intensity without upward change in the number of Nestin-GFP-expressing cells argues in favor of the increased Nestin expression, and consequently GFP fluorescence, in individual pre-existing cells. The promoter region of Nestin has consensus binding sites for the midbrain and central nervous system enhancer elements and contains two Pit-1, Oct-1, Unc-86 homology region (POU) domain binding sites, with only the downstream site being necessary for central nervous system-specific expression,16.Josephson R. Muller T. Pickel J. et al.POU transcription factors control expression of CNS stem cell-specific genes.Development. 1998; 125: 3087-3100PubMed Google Scholar all of which have been preserved in the construct driving GFP expression in transgenic mice. It would be reasonable to predict, therefore, that the regulatory transcription factors similar to the nervous system should be operant in the kidney, and induced after an ischemic episode. The existing data on the expression of several neuronal markers in the kidney (glial cell line-derived neurotrophic factor, synaptophysin, etc.) also conform to this conclusion.17.Durbec P. Marcos-Gutierrez C.V. Kilkenny C. et al.GDNF signalling through the Ret receptor tyrosine kinase.Nature. 1996; 381: 789-793Crossref PubMed Scopus (700) Google Scholar, 18.Moore M.W. Klein R.D. Farinas I. et al.Renal and neuronal abnormalities in mice lacking GDNF.Nature. 1996; 382: 76-79Crossref PubMed Scopus (1047) Google Scholar, 19.Pichel J.G. Shen L. Sheng H.Z. et al.Defects in enteric innervation and kidney development in mice lacking GDNF.Nature. 1996; 382: 73-76Crossref PubMed Scopus (966) Google Scholar, 20.Sanchez M.P. Silos-Santiago I. Frisen J. et al.Renal agenesis and the absence of enteric neurons in mice lacking GDNF.Nature. 1996; 382: 70-73Crossref PubMed Scopus (1003) Google Scholar In fact, the deletion of glial cell line-derived neurotrophic factor provides a dramatic example of the ensuing defect exclusively in the nervous and renal systems, further strengthening this link. Moreover, the Nestin promoter and enhancer elements are regulated by POU-domain transcription factors,21.Lothian C. Lendahl U. An evolutionarily conserved region in the second intron of the human Nestin gene directs gene expression to CNS progenitor cells and to early neural crest cells.Eur J Neurosci. 1997; 9: 452-462Crossref PubMed Scopus (137) Google Scholar some members of which are expressed in the early development of brain and kidney22.Lan L. Liu M. Liu Y. et al.Expression of qBrn-1, a new member of the POU gene family, in the early developing nervous system and embryonic kidney.Dev Dyn. 2006; 235: 1107-1114Crossref PubMed Scopus (10) Google Scholar and mutations of the POU 6F2 gene have been found in Wilms tumors.23.Perotti D. De Vecchi G. Testi M.A. et al.Germline mutations of the POU6F2 gene in Wilms tumors with loss of heterozygosity on chromosome 7p14.Hum Mutat. 2004; 24: 400-407Crossref PubMed Scopus (27) Google Scholar In addition, POU-domain transcription factors have been found to be differentially expressed in experimental diabetic nephropathy.24.Wada J. Zhang H. Tsuchiyama Y. et al.Gene expression profile in streptozotocin-induced diabetic mice kidneys undergoing glomerulosclerosis.Kidney Int. 2001; 59: 1363-1373Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Together with nuclear hormone receptors like retinoid X receptor and retinoic acid receptor, which serve as regulators of Nestin expression, these factors present in the neural and renal tissues may exert the observed Nestin-enhancing effect in renal ischemia. Considering the functional plasticity of Nestin-expressing cells in the kidney, it is conceivable that a sub-population of these cells represent a pool of perivascular angiogenic progenitors. Several lines of evidence argue in favor of such a conclusion. The localization of Nestin-expressing cells in proximity to vasa rectae, juxta-glomerular arterioles, glomeruli, and peritubular capillaries places them in the category of pericytes. Yet, these cells do not express α−smooth muscle actin, thus ruling out their true pericytic nature. On the other hand, only a small proportion of the Nestin-expressing cells stain positively with endothelial or endothelial progenitor markers, arguing against their differentiated endothelial identity. However, these cells actively participate in in vitro angiogenesis from the aortic and renal explants and, in the process, gain expression of endothelial marker Tie-2, supporting the notion that they represent angiogenic progenitor cells. The fact that terminal differentiation into mature endothelial cells and/or pericytes is accompanied by the loss of Nestin expression,21.Lothian C. Lendahl U. An evolutionarily conserved region in the second intron of the human Nestin gene directs gene expression to CNS progenitor cells and to early neural crest cells.Eur J Neurosci. 1997; 9: 452-462Crossref PubMed Scopus (137) Google Scholar,25.Zimmerman L. Parr B. Lendahl U. et al.Independent regulatory elements in the Nestin gene direct transgene expression to neural stem cells or muscle precursors.Neuron. 1994; 12: 11-24Abstract Full Text PDF PubMed Scopus (480) Google Scholar makes it difficult to monitor their transformation. However, the finding that a small proportion of Nestin-expressing cells co-expresses endothelial or pericytic markers may buttress the view that the residual Nestin expression remains detectable at the early stages of such a transformation. Theoretically, it cannot be ruled out that Nestin is re-expressed in some cells during their further differentiation. Taken together, our findings suggest that Nestin expression is abundant in the adult kidney, Nestin-expressing cells are localized to the papilla, outer medullary vascular bundles, glomeruli, juxta-glomerular microvasculature. Medullo-cortical migration of these cells and their ability to acquire endothelial phenotypical markers and participate in angiogenesis are indicative of their plasticity and may suggest the role for these Nestin-expressing cells in the process of tissue regeneration after ischemic damage. 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. FVB/NJ and C57/BL6 mice were obtained from Jackson Labs (Bar Harbor, ME, USA). Generation of Nestin-GFP transgenic mice was described in detail by Mignone et al.3.Mignone J.L. Kukekov V. Chiang A.S. et al.Neural stem and progenitor cells in Nestin-GFP transgenic mice.J Comp Neurol. 2004; 469: 311-324Crossref PubMed Scopus (514) Google Scholar All animals were separately caged with a 12:12-h light–dark cycle and had free access to water and chow throughout the study. A subcutaneous injection of 250 U/kg heparin was given 15 min before the operation. After a 1.5-cm mid-laparotomy, both kidneys were exposed and clamping of the renal pedicle was performed with microserrefines (Fine Science Tools, Foster City, CA, USA). After 25 min, the clamps were released. The abdominal incision was closed with a 4-0 suture and surgical staples. At different time points after surgery (3 and 6 h, 1, 2, and 7 days), the animals were killed for further analyses. Kidneys were harvested from FVB/NJ mice at 24 h after bilateral renal ischemia of 30 min. Cortices, medullae, and papillae were excised under a dissecting microscope. Four to five kidneys were used for each experiment, and experiments were repeated at least three times. Pooled tissue samples were homogenized in radioimmunoprecipitation buffer (20 mM Tris, pH 7.8, 140 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1 mM NaF, and 1 mM orthovanadate) with Complete Mini protease inhibitors (Roche, Nutley, NJ, USA). Samples were centrifuged at 14 000 r.p.m. for 30 min. Pellets were washed with radioimmunoprecipitation buffer and solubilized in 8 M urea at 4°C for 3 h. Protein concentration was determined using a Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were electrophoretically separated in 4–20% Tris-glycine gels (Invitrogen, Carlsbad, CA, USA) and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). After blocking with phosphate-buffered saline (PBS) containing non-fat dry milk (5% w/v), membranes were incubated with the primary antibody anti-rat Nestin (Rat-401, 1:10 in PBS-bovine serum albumin (BSA) 1%) (Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA, USA). After washes with PBS-Tween 20 (0.1% v/v), membranes were incubated with horse-reddish peroxidase-conjugated secondary antibodies (Amersham, Piscataway, NJ, USA) for 60 min at room temperature. Membranes were washed and protein was detected by SuperSignal West Pico chemiluminescence (Pierce, Rockford, IL, USA). For preparation of cell suspension, kidneys were placed in 2 ml of ice-cold Roswell's Park Memorial Institute medium 1640 (Invitrogen, Carlsbad, CA, USA) and minced using a sterile scalpel (BD Biosciences, Rockville, MD, USA). Digestion of the tissue was performed in collagenase II (Invitrogen, Carlsbad, CA, USA) solution (1 mg in 1 ml of Roswell's Park Memorial Institute medium 1640 ml) for 30 min at 37°C in 5% CO2. Cell suspensions were passed through a 35 μm sieve. Repeated digestions were performed until microscopic evaluation showed a suspension of single cells. Finally, cells were washed in PBS-BSA 1% (w/v), counted, and kept on ice in the dark until further analysis. To quantify total renal Nestin-GFP-positive cells and the expression of various markers by these cells using FACS analysis, 106 to 3 × 106 cells from the respective single-cell suspensions were incubated with different primary antibodies for 1 h at 4°C in the dark. The following antibodies were used for primary incubation: fluorescein isothiocyanate-conjugated anti-mouse CD34 (RAM34), phycoerythrin-conjugated anti-mouse Flk-1 (Avas12α1) (BD Biosciences, Rockville, MD, USA), phycoerythrin-conjugated anti-mouse CD150 (eBioscience, San Diego, CA, USA), and anti-human Tie-2 (sc-9026) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibody for Tie-2 staining was Alexa Fluor 594 donkey anti-rabbit IgG (A21207) (Molecular Probes, Eugene, OR, USA). Incubation with secondary antibodies was performed for 30 min at 4°C in the dark. After each incubation step, cells were washed with PBS-BSA 1% (w/v) and finally fixed in 4% paraformaldehyde. Data were acquired using a FACScan cytometer equipped with a 488 nm argon laser and a 635 nm red diode laser and analyzed using CellQuest software (Becton Dickinson, San Jose, CA, USA). The set-up of FACScan was performed using unstained and single secondary antibody-stained cells. To determine the gating parameters for the quantification of Nestin-GFP-positive cells, kidneys of wild-type animals (C57/BL6 mice) were analyzed first. Kidney tissue samples from FVB/NJ mice were fixed in 4% paraformaldehyde solution (Electron Microscopy Sciences, Hatfield, PA, USA), followed by incubation in 30% sucrose overnight at 4°C. Embedding was performed in an optimal cutting temperature (OCT) compound (Tissue-Tek, Torrance, CA, USA), and embedded samples were stored at -80°C. Frozen samples were cut into 10 μm thick sections (Cryomicrotom CM 1850, Leica Microsystems, Bannockburn, IL, USA). For Nestin staining (kidneys from FVB/NJ mice), nonspecific protein binding was blocked by 1 h incubation with 1% PBS-BSA. For primary incubation, anti-rat Nestin (Rat-401, 1:10 in PBS-BSA 1%) (Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA, USA) was applied overnight at 4°C. Secondary antibody incubation was performed with fluorescein isothiocyanate-conjugated anti-mouse IgG (1:100 in PBS-BSA 1%) (Jackson Immunoresearch, West Grove, PA, USA) for 1 h at room temperature. Control samples were stained with secondary antibodies only. To visualize the nuclei, tissue sections were counterstained with 4′,6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR, USA). Sections were examined with either a Nikon compound fluorescence microscope with the appropriate dichroic mirrors (Nikon, Walt Whitman, NY, USA) or with a Zeiss LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY, USA). For analysis of the expression pattern of medullary Nestin under prolonged ischemia, kidneys from 3-month-old Nestin-GFP transgenic mice were collected and longitudinally cut. Sections were placed in a culture dish in a basal cell culture medium supplemented with 10 mM N−2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and 2% fetal bovine serum. During the time course of 3 h without blood supply, the medullary Nestin expression pattern, as judged by GFP fluorescence, was continuously monitored at low magnification (× 4 or × 10 objectives) using a Nikon compound fluorescence microscope with the appropriate dichroic mirror (Nikon, Walt Whitman, NY, USA). In a second series of experiments, fixed kidney sections obtained from Nestin-GFP mice at different time points post-ischemia were aligned using the cortical surface as a common starting point. The cortico-medullary line-scan was performed in the direction perpendicular to the kidney surface using MetaVue software routines (Universal Devices Corporation, Downingtown, PA, USA). Fluorescence intensity along a cortico-medullary axis was profiled. Ten line-scans per kidney were obtained and data analyzed. Mice were anesthetized by intraperitoneal injection with 2% α−chloralose and 10% urethane in PBS (6 ml/kg) and a polyethylene tube was inserted via tracheotomy to facilitate spontaneous respiration. Animals were kept on the temperature-controlled plate (Tokai Hit, Nikon Inc., Melville, NY, USA) for the duration of experiments. A Nikon intravital MM-40 microscope equipped with a × 20, × 40, and × 60 1.2 NA Plan Apo water-immersion objectives, a telescopic tube, and a Cascade 512F digital camera (Photometrics, Tucson, AZ, USA) was used to monitor kidney surface. Images were collected, recorded, and analyzed using MetaMorph software. The explants of freshly isolated thoracic aorta and renal cortex from FVB/NJ wild-type mice (about 0.5 mm3) were placed in the ice-cold sterile endothelial basal medium (EBM)-2 medium supplemented with human-epidermal growth factor, vascular endothelial growth factor, insulin-like growth factor, and heparin and containing 2% fetal bovine serum. All tissue samples were rinsed three times with a fresh EBM-2 medium. The explants were embedded in growth factor-depleted Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) in culture chambers (Nalge Nunc Int., Rochester, NY, USA), as described previously.9.Nicosia R.F. Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.Lab Invest. 1990; 63: 115-122PubMed Google Scholar,26.Brodsky S. Chen J. Lee A. et al.Plasmin-dependent and -independent effects of plasminogen activators and inhibitor-1 on ex vivo angiogenesis.Am J Physiol Heart Circ Physiol. 2001; 281: H1784-H1792PubMed Google Scholar Sprouting cells were previously identified as predominantly endothelial, based on their staining with endothelial cell markers, thus allowing this widely used technique to be employed as an angiogenic assay.9.Nicosia R.F. Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.Lab Invest. 1990; 63: 115-122PubMed Google Scholar,27.Brodsky S.V. Smith M. Kashgarian M. et al.A model for ex vivo renal angiogenesis.Nephron Exp Nephrol. 2003; 93: e46-e52Crossref PubMed Scopus (9) Google Scholar After 7–14 days in matrigel culture, cells were fixed with 4% paraformaldehyde and stained for the expression of Tie-2 (anti-human Tie-2 (sc-9026) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)) and Nestin (anti-rat Nestin (Rat-401, 1:10 in PBS-BSA 1%) (Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA, USA). Secondary incubation for Tie-2 was performed with fluorescein isothiocyanate-conjugated AffiniPure goat anti-rabbit (Jackson Immunoresearch, West Grove, PA, USA), secondary antibody for Nestin was Alexa Fluor 594 donkey anti-mouse IgG (Molecular Probes, Eugene, OR, USA). Analysis of Nestin-expressing cells participating in sprouting angiogenesis in explant cultures was performed under an inverted fluorescence microscope (Nikon) equipped with a CCD camera (Diagnostic Instruments, Sterling Heights, MI, USA) relaying images to a monitor. The results were expressed as mean±s.e.m. 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. These studies were supported in part by the NIH grants DK 52783 (MSG), by The Seraph Foundation (GE), Fellowship grant from the Deutsche Forschungsgemeinschaft (DFG) PA 1530/1-1 (DP), and Westchester Artificial Kidney Foundation.
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