In contrast, in animals that received intrarenal injection of hAFSC a decrease of apoptosis was noted, as shown in Figure 7B. The absence of increase in proliferation or apoptosis was demonstrated in animals that received only intrarenal injection of hAFSC without glycerol damage, demonstrating that hAFSC alone do not adversely influence normal tubular cells Figure 7A,B. Animals that did not undergo any treatment control animals did not show any remarkable apoptosis or proliferation data not shown , and were therefore not included in the statistical comparison between ATN groups.
Since the salutary effect of hAFSC injection occurred during the acute phase of ATN, we postulated that this protective effect might involve acute changes in the kidney's cytokine expression.
For relative ease of interpretation, the different cytokines are displayed as five broad functional clusters, based on their principal immunological functions: 1. Interleukins; 2. Activators of B Lymphocytes; 3. Activators of Natural Killers; 4. Chemotactic attractors of Granulocytes and Macrophages; 5. Multiple biological effectors. We did not include a specific category for activators of T Lymphocytes, since in this mouse model activated T Lymphocytes are not expressed.
We analyzed the expression of cytokines in all the experimental groups at different time points up to two weeks. The mouse specific cytokine assay does not cross react with the human cytokine assay. This was confirmed by incubating digested kidney extracts with membranes specific for human cytokines and conversely incubating hAFSC with membranes specific for mouse cytokines data not shown.
Mouse Cytokines were analyzed among all the experimental groups to evaluate changes in the inflammatory milieu.
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Schematic outline representation of mouse cytokine expression over the 14 days among different groups: 1. Mice with ATN only. As a general trend of cytokine expression, in these animals the pro-Inflammatory cytokines increased or remained highly expressed and anti-inflammatory cytokines showed decreased or low expression; 2.
As a general trend of cytokine expression, it is evident that the anti inflammatory cytokines increased over the 14 day study period, while pro-Inflammatory cytokines decreased; 3. As a general trend of cytokine expression, in these animals the pro-Inflammatory cytokines increased or remained highly expressed and anti-inflammatory cytokines showed decreased or low expression; 4. As a general trend of cytokine expression, in these mice the anti-inflammatory cytokines are mildly increased over the 14 days, while pro-inflammatory cytokines are not expressed or show decreased expression over the 14 days.
Mice that were injected with hAFSC, only without inducing ATN, underwent a limited tissue reaction in terms of production of different cytokines, but for most of them this expression was very early after the injection within the first few days and the levels returned to normal by 14 days as shown in Table S1 and Figure 8B. When the human cells are injected both in mice with or without ATN there was a noticeable increase in human cytokine levels, when compared with the cytokines expressed by hAFSC before injection in vitro culture as reported in Table S3 especially during the first days after injection, while most of them returned to normal at 7 days and were not detectable at 14 days.
This model of ATN involves a complex sequence of events wherein myoglobin, released from damaged muscle, damages the epithelial cells of the proximal tubules, producing cast formation, vasoconstriction and decreased glomerular filtration. Peak of the damage represented by high levels of apoptotic cells, increased levels of creatinine and BUN and by histological analysis was confirmed to be between 48—72 after induction of ATN. Nevertheless herein following intrarenal injection after ATN kidney damage, the bioluminescent signal is still present in the kidney after 21 days and hAFSC were found specifically located among the tubules.
In order to determine if the injected cells can differentiate into tubular epithelial cells we performed both immunohistochemistry and RT-PCR using human specific antibodies and human primers. Genes such as PAX2 , NPHS1 and lectins like Dolicholus Biflorus and Peanut Agglutinin are expressed by injected cells, indicating that at least some of the integrated cells are able to commit toward renal differentiation. Furthermore, in some rare instances, injected hAFSC cells could express human Glial Derived Neurotrophic Factor GDNF , which is expressed during very early kidney development; GDNF is not usually detectable in the adult kidney, indicating that injected hAFSC can express also embryonic renal markers, retaining the ability of going through the nephrogenesis.
We have previously shown that hAFSC have the potential to integrate into embryonic kidneys and can participate in key steps of nephrogenesis, indicating that hAFSC can be induced toward to a renal fate when placed in an appropriate environment . However, both in this study as well as in this previous study, the efficiency of integration into kidney lineages was not strikingly high and we did not detect significant clonal expansion of integrated hAFSC.
We, therefore, postulated that perhaps another benefit could be afforded the injured kidney than simply a structural one by the presence of hAFSC during the acute phase of injury. This beneficial effect with hAFSC was also correlated with significant increases in proliferative activity of tubular epithelial cells, decreased cast formation, and decreased apoptosis of tubular epithelial cells.
We therefore speculate that hAFSC can, when injected early enough in this study contemporaneously with the time of injury attenuate acute renal damage, underscoring the potential protective effects of hAFSC. Moreover, even 14 days after injury hAFSC injection into the kidney still correlated with increased tubular cell proliferation and less tubular damage. Thus, we hypothesize that hAFSC might accelerate the proliferation of epithelial tubular cells that were only partially damaged, while in addition preventing apoptosis.
This mechanism of protection therefore appears to lead to better maintenance of tubular structure, as seen in PAS staining, thus avoiding the increase in BUN and creatinine typically seen in IM-glycerol-induced ATN. During acute renal injury the immune response plays a key role. Damaged kidney endothelial cells attract leukocytes, vasomediators are released with injury, and epithelial cells of the tubule produce pro-inflammatory and chemotactic cytokines .
Bonventre et al. They also speculated that the protection in these animals was not through integration and differentiation of the injected MSC, because of the very short period of time with which a protective response was observed .
The animal model used in these experiments Athymic Nude- Foxn1 nu was chosen in order to be able inject human derived stem cells and evaluate their effect over time, avoiding outright rejection. They have normal B Lymphocytes and they have evidence of increase numbers of NK cells. Thus, even though they are immunodeficient, they do possess some ability to mount and modulate a partial immune response when stimulated .
In addition to the animals that underwent only ATN and mice with no treatment, we introduced mice that were injected with a vehicle saline solution PBS after glycerol damage and compared their respective cytokine levels. Therefore, to further investigate the potential mechanisms by which hAFSC enhance renal protection, we examined intrarenal cytokines, to determine whether there is a general change in inflammatory cytokine pattern in mice that were treated with hAFSC compared to our controls.
One possible explanation for this result may be the increased presence of IL and IL1Ra, both of which are anti-inflammatory cytokines, in the mice treated with hAFSC cells. Moreover, the presence of both of these two anti-inflammatory interleukins appears to have stimulated the production of IL-6, which also possess anti-inflammatory properties in the presence of increased levels of IL and IL1Ra . Therefore we conclude that inflammation can persist over a longer period of time in these non-hAFSC treated mice. Furthermore, mice treated with hAFSC did not show increased levels of important pro-inflammatory cytokines, such as IL27, a Natural Killer cell attractor  or IL12p70, which is known to be inhibited by IL  , .
Furthermore, the injection of the cells prevented an increase in SDF-1 Stromal cell-Derived Factor-1 a potent B chemoattractor, which is produced by several cell types  , thus indicating that many different immune functions can be slowed down or controlled by injection of hAFSC. It is beyond the scope of the current study to define the specific role of each individual cytokine involved in the immune response in kidney injury and repair. Nevertheless, the total effect of the cytokines and chemokines expressed in the ATN kidneys of mice treated with hAFSC evidently lead to a combined action helping to ameliorate the acute phase of injury.
This is also in accordance with the presence of tubular damage in the latter groups at 14 days. This suggests that an important function of hAFSC may be to actually prevent the acute injury. Interestingly, human cytokine levels compared with the basal level secreted in the hAFSC supernatant in vitro before injection are significantly increased early in the course of ATN as shown Table S3 , while the mouse cytokines are only significantly increased after 1 or 2 weeks.
With a few exceptions, most human cytokines are also active on mouse cells  —  , so both the combined effects of human and mouse cytokines may have affected the kidney inflammation and tissue homeostasis milieu. We think that this may be an important concept, and therefore consider that maybe cytokines derived from both the injected hAFSC and the endogenous mouse cytokines are responsible for the observed protective effects.
In addition, it is relevant to underline the decrease in number of injected hAFSC found in the kidney over time, as shown both by the bioluminescence images and by the immunohistochemistry data, as well as by the absence of human cytokine expression at 14 days. Furthermore, after the acute phase, the decrease of creatinine levels, as well as less cast formation and the increase of tubular cell proliferation is still statistically significant at two weeks, at which time damage still persists in the ATN mice treated only with PBS or not treated at all.
This is also in accordance with the data on cytokine levels that are still significantly elevated at 1 and 2 weeks in ATN mice treated with PBS only or not treated at all, as compared to ATN mice treated with hAFSC in which cytokine levels had resolved by 1—2 weeks. In these mice, injected only with hAFSC there was no alteration of creatinine or BUN, no increase in apoptosis or proliferation or tubular damage; thus indicating that hAFSC per se do not alter the normal physiology and morphology of the kidney.
In addition, the human cytokines produced by the injected cells in mice that did not undergo ATN disappear quickly and with no apparent adverse consequences. In conclusion, we have demonstrated that early direct injection of hAFSC into the kidney strongly ameliorates ATN injury, as reflected by more rapid resolution of tubular structural damage, by tubular cell proliferation and by normalized creatinine and BUN levels. In addition, our data show evidence of potent immunomodulatory effects of hAFSC that appear to control the local immune response in favor of a tissue cytokine and cellular milieu that promotes prevention or resolution of tissue damage.
Taken together these findings suggest that hAFSC may have therapeutic potential in ATN and, by extrapolation, perhaps in other kidney diseases. Samples of human amniotic fluid from male fetuses 12—18 week of gestation were provided to our laboratory by Genzyme Genetics Corporation Mongovria, CA, USA after karyotyping analysis. No written or verbal consent was required since samples were not identified and information obtained about the samples was limited to karyotype and fetal health status. The stem cell population was separated from the general human amniotic cellular milieu using standard Magnetic Sorting MACS techniques [Miltenyi Biotech] against the cell surface marker, c-kit, as described by Atala et al.
Pluripotential characteristics of the clonal and subclonal groups were tested according to protocols also outlined by Atala et al. The isolation and characterization of pluripotency of human and mouse AFSC is a very well established protocol in our laboratory, and clones used in these experiments are the same as used for our previous publications  , .
Before injection, a clonal hAFSC population at passage 40 was trypsinized in 0. Controlled intramuscular injection of glycerol was performed under anesthesia by surgically exposing the caudal thigh muscle and slowly injecting the glycerol solution prior to delivery of cells. This suggests that the T-deficient mice are relatively protected as compared to wild type mice against glycerol-rhabdomyolysis-induced ATN .
The mice were carefully anesthetized using isofluorane inhalation. Once satisfactory anesthesia was achieved, the mice were prepared for surgery using chlorhexidine. The kidneys were then replaced into the retroperitoneum, the incision closed with polypropylene suture and the mice were allowed to recover from anesthesia. The animals were maintained on a heating pad throughout the period of anesthesia. The animals were sterilely draped to prevent contact of the kidneys with the skin of the animal to reduce risk of peritonitis.
As control, mice were injected with saline vehicle solution PBS 2 hours after glycerol damage. In addition, another animal group was injected only with hAFSC without previous glycerol damage using the same technique described above for the cell suspension and surgery. At different times points from 24 hours to 3 weeks , the injected and the control mice were sacrificed.
The kidneys were extracted, washed in PBS, and processed in one of the following ways depending on the analysis performed. The kidneys were minced in small pieces and the RNA extracted using Qiagen RNeasy kit [Qiagen] according to the manufacturer's instructions.
Briefly, with the use of silica-gel columns RNA is separated from DNA through centrifugation after lysis and homogenization of the samples. Ethanol addition allows RNA to bind the silica-gel before the centrifugation step. Amplification of the resulting cDNA was carried out using only specific human primers not coding for mouse sequences. Specific human primer sequences, predicted sizes of amplicons and specific annealing temperatures are shown in Table 1. The slides were immersed in Periodic Acid Solution [Sigma-Aldrich] for 5 minutes at room temperature; rinsed in several changes of distilled water, then stained with Schiffs reagent [Sigma-Aldrich] for 15 minutes at room temperature, washed in running tap water for 5 minutes and finally counterstained with hematoxylin solution Gill [Sigma-Aldrich] for 90 seconds.
In addition, some kidneys were frozen in liquid nitrogen using Tissue-Tek O. The Vector Core performed the transduction, the titration of the virus and the multiplicity of infection. Briefly, two cycles of transduction were performed by removing old medium and adding new virus supernatant and medium. Before in vivo injections, a simple in vitro test was employed to determine the minimum amount of hAFSC detectable by bioluminescence.
In addition, the expression of the luciferase gene was confirmed by PCR after 20 passages in culture. As control, to exclude background signal, 5 mice were injected only with luciferin and no cells. The imaging system consists of a cooled, back-thinned charge-coupled device CCD camera to capture both a visible light photograph of the animal taken with light-emitting diodes and a luminescent image.
Frozen and paraffin slides were stained for immunofluorescence. The slides were then placed in working solution of Vector Antigen Retrieval as described in the data sheet. Afterwards, the slides were washed in PBS for 5 minutes for 3 times. All the reagents were furnished by the kit. In all the experimental groups, the apoptotic nuclei were counted as a fraction of the total number of nuclei present in the section using consecutive, non-overlapping fields of TUNEL-stained specimens.
The percentage of apoptotic cells was estimated without knowledge of the experimental group. The number of proliferative cells was determined using the PCNA Staining Kit Invitrogen, c 93— at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks after injections in every experimental group above described as suggested by the kit data sheet. Briefly, after deparaffination and rehydration and blocking, the slides were incubated with a biotinylated mouse anti-PCNA primary antibody for 1 hour at room temperature followed with incubation with Streptevidin-HRP and revealed with DAB substrate.
In all the experimental groups the proliferative nuclei were counted as a fraction of the total number of nuclei present in the section using consecutive, non-overlapping fields of PCNA-stained specimens. The percentage of proliferative cells was estimated without knowledge of the experimental group.
The facial vein was lanced with a 5 mm animal lancet and blood collected using standard protocols approved by the Animal Core Facility at Childrens Hospital of Los Angeles and Saban Research Institute. Animals were divided into different groups per each time point as follow 24 hours, 48 hours, 72 hours, 1 week and 2 weeks : 1. Post-damage measurements were obtained every 24 hours.
The blood samples were used to monitor renal function, by analyzing creatinine and BUN levels. The kidney sections were divided as follow: 1. Sections obtained from mice that underwent ATN with no injection of hAFSC sacrificed at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks; 2.
Section obtained from mice that underwent ATN and injection of hAFSC sacrificed at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks; 3. Sections obtained from mice that underwent ATN with and injection of PBS sacrificed at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks; 4. Sections obtained from mice that underwent only intrarenal injection of hAFSC and sacrificed at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks.
Tubular injury was evaluated based on different parameters using PAS staining: including disruption of tubular membranes and brush borders, but mainly relaying on cast formation since it is the most evident sign of damage in stained sections. In the experimental groups, the tubular injury was counted as a fraction of the total number of tubules present in the section using consecutive, non-overlapping fields of PAS-stained specimens.
The percentage of damaged tubules was estimated without knowledge of the experimental group. To examine if the injection of hAFSC would interfere with the modulation of inflammation after acute kidney injury human and mouse cytokines levels were measured in digested mouse kidneys at 24 hours, 48 hours, 72 hours, 1 week and 2 weeks in every experimental groups 1. The sample is centrifuged at The supernatant is then transferred to a clean tube and the protein concentration measured by UV-vis spectroscopy. After 1 minute incubation the membranes were exposed for 20 seconds to x-ray film. A P value less than 0.
In the table are reported the P values for the cytokine analysis at 1,2, 3, 7 and 14 days. Blank cells in the table indicate no statistically significant change in cytokine expression. In the table are reported the P values for the mouse cytokine analysis at 1, 2, 3, 7 and 14 days. In the table are reported the P values for the human cytokine analysis at 1, 2, 3, 7 and 14 days. We would like to thanks Dr. Francesco Boldrin for help with the molecular biology experiments, Casey Brewer for the immunostaining, Dr. Wrote the paper: LP.
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Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Acute Tubular Necrosis ATN causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Introduction Acute Tubular Necrosis ATN is characterized by acute tubular cell injury and renal dysfunction  , . Download: PPT. Figure 2. However, in animal facilities that exclude pathogens to which they are susceptible, athymic mice exhibit a life span nearly as long as that of their euthymic counterparts ILAR, It is therefore essential that the housing provided for these animals creates a barrier between the sus- ceptible host and infectious organisms.
The Guide for the Care and Use of Laboratory Animals NRC, states: "The caging or housing system is one of the most important elements in the physical and social environment of research animals" p.
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How- ever, equally important in the maintenance of a barrier are proper husbandry procedures, personnel management, and equipment maintenance. To ensure the health of pathogen-free animals, programs should be designed to monitor the colony by using clinical, serologic, pathologic microbiologic, and par- asitologic techniques Hsu et al. Because the well-being of pathogen-free animals is so strongly influenced These systems are described below. All components can be sterilized separately by autoclaving.
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The committee recommends the use of transparent plastic cages to facilitate routine animal observation without the need to open the cage more frequently than is necessary for sanitation and experimentation. These units represent the simplest solution to housing pathogen-free rodents, but they require rigid discipline and operating procedures if they are to be used as a primary barrier against disease Sedlacek et al. HEPA- Filtered Laminar-Air- Flow Systems These units are composed of modular chambers, hoods, and racks that place cages under a positive flow of filtered air, independent of the room ventilation system.
They can either be used to house rodents or to hold rodents while cages are changed. Transportable units are commercially avail- able Figure ; filters for whole-room application usually must be custom designed. Typically, these devices use high-efficiency particulate air HEPA filters that are capable of removing A plenum and blower fan in the unit distribute the ultra- filtered air evenly over the cages in a horizontal laminar fashion. When planning an animal room with laminar-air-flow devices.
Low-velocity vertical mass air flow may be a successful adjunct to horizontal laminar air flow; however, the effectiveness of vertical mass air flow by itself is controversial Thigpen and Ross, Regardless of the manufacturer or type of HEPA- filtered air supply device used in a room housing immunodeficient rodents, there are three essential components of management: 1. Figure courtesy of Lab Products.
Attempts should be made to minimize dust in rooms equipped with these units to reduce the potential for loaded filters and failing systems. Laminar-air-flow benches or cabinets, preferably on casters for easy man- agement, can be used as a work surface for cage changing and experimental manipulation of animals. The principle applied here is the same as that for housing units. The committee considers this technique to be a useful adjunct to proper husbandry procedures, especially in facilities where barriers are not available or animal use is less restricted.
The most widely used isolators are made of flexible laminated vinyl plastic ILAR, , pp. They can be chemically sterilized and easily adapted to specific needs. Isolators are also available in stainless steel, nylon,. All such units have a filtered air supply and exhaust. Food and supplies must be sterilized and passed into the unit through a sterile entry chamber. Although isolators are an ideal housing system for keeping immunodeficient rodents pathogen-free, they are labor intensive, limit the number of animals that can be cared for in a given space, and seriously impair the ability to manipulate the animals.
Depending on the intended use, these cage racks can supply HEPA-filtered air or animal room air. The efficacy of these systems in protecting immunodeficient animals from infectious agents has not been evaluated. The degree of protection provided should be dictated by the goals of the intended research.
Precautions essential to gerontological studies, for example, might not be necessary in studies lasting only 1 or 2 months. However, all infections, including inapparent ones, are a constant threat to successful experimentation. Inapparent infections can, in a sense, be even more harmful than overt infections because they might go undetected and their impact on the study might be overlooked.
Care must be taken to ensure that the results of an experiment in which immunodeficient rodents are used reflect the parameters measured and not concomitant infection. Environmental Conditions General rules that should be followed to ensure efficient husbandry are presented below. Temperature Immunodeficient rodents have been satisfactorily maintained in rooms where the temperature is regulated between It has been shown that the thermoneutral zone for nude mice is above that for haired mice Weihe, and that thyroid function in nude mice is.
As a practical matter, these steps are rarely taken. Furthermore, excessively high room temperatures create husbandry problems, such as fermentation of feed and bedding, excess bacterial growth in watering systems, and an unpleasant work environment. Most managers find that a typical rodent room temperature of Room temperature should be graphically monitored, and an alarm should be installed to signal a malfunction of the temperature control.
Readers should refer to that document for details. Food and Water Food. Only sterilized or pasteurized diets should be fed because many commercial diets are contaminated with Enterobacteriaceae Williams and Habermann, Sterilization of food reduces this potential source of infection. Vitamin-fortified, sterilizable diets are available and should be processed just prior to feeding. When diets are sterilized, the autoclave function must be carefully monitored by qualified personnel to ensure that sterilization has been achieved. It is also important that the vitamin content of the feed remains at recommended levels and that the diet is not excessively hard or clumped.
Diets decontaminated by irradiation are also commercially available and can be considered as alternatives to steam-sterilized or pas- teurized diets. Acidification of the drinking water with HCl to a pH of 2. McPherson, However, this pro- cedure may impair the action of antibiotics and vitamins placed in the drinking water.
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