BACKGROUND
mesenchymal stromal cells  are pluripotent cells that differentiate into various mature cell types  <cit> .
a distinctive property of msc is that they are not immunogenic  <cit> , and inhibit cell and antibody-mediated immunity in several ways, including the induction of t regulatory cell differentiation  <cit> .
in a rat model of kidney transplantation we found that msc injected in the graft improved its function and attenuated renal injury, reducing significantly tubulitis, vasculitis, glomerulitis and immune cell infiltration.
furthermore, we traced msc in the recipient tissues and found an irrelevant number of them in the kidney.
the last finding suggests that mediators account for the protection provided by msc to the renal graft  <cit> .
a renal protective effect of msc was shown also in a mouse model of renal transplantation, in which msc suppressed rejection when they were infused before transplantation, while post-transplant infusion worsened graft outcome  <cit> .
interestingly, msc injected before transplantation were found in lymphoid organs and did not localize in the graft, confirming the need for some mediation of msc effects.
indeed a mediated action of msc has been proved in diverse experimental disease models  <cit> , and cytokines have been proposed to play the role of effectors  <cit> .
however, as yet no evidence has been given that msc modify the cytokine network in the setting of renal graft, so that we have felt it interesting to test the hypothesis in the present study.
in addition, we have thought of the scatter factors as a system that could be reset by msc and provide protection in the renal graft model.
the scatter factor  system consists of hepatocyte growth factor  and macrophage stimulating protein  and their receptors met and ron respectively.
there are several reasons to think that msc operate through the sf system.
in fact, the sfs are expressed in normal kidney  <cit>  and participate in the regulation of cell growth and inflammation in various renal diseases including autoimmune forms, e.g.
they stimulate renal cell growth and modulate monocyte traffic in the kidney  <cit> .
furthermore, we demonstrated that in experimental anti thy- <dig> nephritis msc improve renal injury by modulating sfs  <cit> .
in summary, we investigated whether msc injected into the renal graft modify the cytokine network and sfs in a way that fits with the concept that these systems are the effectors of msc-induced graft protection.
RESULTS
characterization of rat msc
as detailed elsewhere the msc used in the present study were isolated from sprague dawley egfp rats and differentiated into osteogenic and adipogenic cells.
flow fluorocytometry analysis showed that msc were positive for cd <dig>  and cd <dig>  and were negative for cd <dig> , cd11b , cd <dig> , cd <dig> .
tissue and serum levels of cytokines
the expression in renal tissue of ifn-γ, il- <dig>  il- <dig>  il- <dig>  il- <dig> was significantly increased in grafted kidneys not injected with msc compared with native kidneys.
msc injection attenuated significantly the rise of ifn-γ and caused a further increase of il- <dig>  msc did not modify tissue levels of any else cytokine.
il- <dig> tissue levels were similar in all groups .
in msc untreated rats grafting was associated with a rise in serum levels of all tested cytokines, except for il- <dig>  msc treatment reduced serum ifn-γ and il- <dig> and increased il- <dig> compared to untreated rats .table  <dig>
cytokine levels in renal tissue
pg/mg
a
b
c
b vs c
p
inf-γ
<dig>  ±  <dig>
§
<dig>  ±  <dig> °
<dig>  ±  <dig>
↓
§
p <  <dig>  vs b, c; °p <  <dig>  vs c
il-10
<dig>  ±  <dig>
§
<dig>  ±  <dig> °
<dig>  ±  <dig>
↑
§
p <  <dig>  vs b, c; °p <  <dig>  vs c
il-6
<dig>  ±  <dig> °
<dig>  ±  <dig>
<dig>  ±  <dig>
ns
°p <  <dig>  vs b, c
il-2
<dig>  ±  <dig> °
227 ±  <dig>
262 ±  <dig>
ns
°p <  <dig>  vs b, c
il-1
<dig>  ±  <dig> °
<dig>  ±  <dig>
493 ± 142
ns
°p <  <dig>  vs b, c
il-4
<dig>  ±  <dig>
<dig>  ± 6
<dig>  ±  <dig>
ns
ns
groups are defined in figure  <dig>  a indicates cytokine levels in kidneys of healthy rats, b and c indicate cytokine levels in renal tissue of recipient rats 7 days after transplantation.
data represent means ± sd.
the symbols § and ° indicate the different statistical power.
serum cytokine levels
pg/ml
a
b
c
b vs c
p
inf-γ
14 ±  <dig> °
<dig>  ±  <dig>
§
<dig>  ±  <dig>
↓
°p <  <dig>  vs b, c;
§
p <  <dig>  vs c
il-10
0°
<dig>  ±  <dig> *
<dig>  ±  <dig>
↑
°p <  <dig>  vs b, c; *p <  <dig>  vs c
il-6
0°
183 ±  <dig>
#
<dig>  ±  <dig>
↓
°p <  <dig>  vs b, c;
#
p <  <dig>  vs c
il-2
0°
61 ±  <dig>
<dig>  ± 61
ns
°p <  <dig>  vs b, c
il-1
0°
1515 ± 613
1843 ± 670
ns
°p <  <dig>  vs b, c;
il-4
0
0
0
ns
groups are defined in figure  <dig>  a indicates serum cytokine levels in healthy rats.
b and c indicate serum cytokine levels in recipient rats 7 days after transplantation.
data are means ± sd.
the symbols §, °, *, # indicate the different statistical power.
tubular necrosis and tubular cell proliferation
tubules with necrotic cells were significantly less in grafts injected with msc compared to untreated grafts .
the expression of pcna was suppressed in grafts not injected with msc, but it was spared by msc treatment .figure  <dig>
tubular necrosis and tubular cell proliferation.
groups are defined as in figure  <dig>  panel a: columns represent the ratio between necrotic tubules number and total counted tubules .
°p <  <dig>  vs c; *p <  <dig>  vs b and c. panel b: boxes represent 25-75% percentile of tubular pcna-positive cell number/hpf .
*p <  <dig>  vs a and b.
foxp <dig> cells infiltrate
in the grafted kidneys not injected with msc we found few foxp <dig> positive cells  and the number increased in msc treated rats .
in the spleen we detected 15 ±  <dig> cells/section .figure  <dig>
foxp <dig> positive cells.
foxp <dig> staining of representative kidney and spleen sections.
panel a shows foxp <dig> positive cells in cortical tubules after 7 days from transplantation in rats of group c .
panel b shows foxp <dig> positive cells in medullary tubules after 7 days from transplantation in rats of group c .
panel c shows foxp <dig> positive cells in spleen after 7 days from transplantation in rats of group c .
hgf/met system
serum and renal tissue hgf levels, hgf mrna expression in kidneys
hgf serum levels significantly decreased in grafted rats that were not injected with msc compared to control normal rats.
msc treatment returned serum hgf to normal levels .figure  <dig>
serum hgf levels, hgf mrna and hgf protein in kidney of control and allografted rats on day  <dig>  groups are defined as in figure  <dig>  upper panel: serum hgf levels in a, b, c groups of rats.
columns are means, bars are sd.
*p <  <dig>  vs a and c. middle panel: western blot performed with anti-hgf antibody in renal tissue of groups a, b, c. the p69-kda band represents the α sub of active heterodimer hgf.
lower panel: hgf mrna expression in renal tissue analyzed by rt pcr in all groups of rats.
columns indicate hgf mrna expression normalized to the beta-actin expression and converted into fold change.
°p <  <dig>  vs a.
both hgf protein and hgf mrna were reduced in grafted kidneys compared to native organs.
msc injection prevented the loss of hgf protein , although hgf mrna levels were lower probably for a feedback mechanism .
renal expression of met
the widespread expression of met protein in tubules of native kidneys  was almost completely abrogated in untreated grafted kidneys .
msc infusion prevented met loss .
the result was confirmed by western blot for met performed on kidney tissue .figure  <dig>
met expression in kidney of control and allografted rats on day  <dig>  groups are defined as in figure  <dig>  upper panel: met expression in representative renal sections of control and allografted rats on day  <dig>  panel a: normal kidney, panel b: untreated allograft, panel c: msc treated allograft .
middle panel: bars represent the medians of met positive tubules number/hpf in all groups of rats, boxes represent the 25-75% percentile, whiskers: min to max.
°p <  <dig>  vs b, § p <  <dig>  vs a, * p <  <dig>  vs c. bottom panel: western blot performed with anti-met antibody in renal tissue of all groups of rats.
the p170-kda band represents pro-met, the p140-kda band represents mature met β subunit.
msp/ron system
renal expression of msp/ron
immunohistochemistry showed that msp was expressed in tubular cells of native kidneys  and it was undetectable in untreated grafts except for some tubules in which it was distributed with luminal pattern .
msc restored tubular msp in transplanted rats, but in a different pattern from normal, i.e.
luminal instead of cytoplasmic .
as msp also its receptor, ron, was diffusely expressed with cytoplasmic pattern in tubules of healthy rats , while in transplanted rats with acute rejection tubular ron expression was significantly decreased  and reappeared in tubules of allografts treated with msc .
in untreated grafts ron was expressed also by some inflammatory cells in the interstitium, but it was absent in infiltrating cells of msc treated rats .figure  <dig>
msp and ron expression in kidney of control and allografted rats on day  <dig>  groups are defined as in figure  <dig>  panel a: msp expression in representative renal section of normal kidney, note the strong cytoplasmic pattern of staining, panel b: msp expression in representative renal section of untreated allograft, note the almost complete absence of msp expression, panel c: msp expression in representative renal section of msc treated allograft, note the luminal pattern of msp staining .
panel d: ron expression in representative renal sections of normal kidney, panel e: ron expression in representative renal sections of untreated allograft, note the presence of ron positive inflammatory cells and the significant reduction of ron tubular staining, panel f: ron expression in representative renal sections of msc treated allograft, note the reappearance of ron tubular staining and absence of ron positive inflammatory cells .
panel g: bars represent the medians of msp positive tubules number/hpf in all groups of rats; boxes represent 25-75% percentile of msp positive tubules, whiskers: min to max) i: °p <  <dig>  vs b; *p <  <dig>  vs c, § p <  <dig>  vs a. panel h: bars represent the medians of ron positive tubule number/hpf in all groups, boxes represent the 25-75% percentile, whiskers: min to max.
*p <  <dig>  vs c, °p <  <dig>  vs b and c.
msp mrna expression in kidneys
rt-pcr showed a significant decrease of msp mrna in the untreated graft.
msc injection was associated with recovery of msp mrna expression .figure  <dig>
msp mrna expression of control and allografted rats.
groups are defined as in figure  <dig>  msp mrna expression in renal tissue analyzed by rt pcr in all groups of rats.
columns indicate msp mrna expression normalized to the beta-actin expression and converted into fold change.
*p <  <dig>  vs a and c.
msc express msp mrna
we performed in vitro experiments aimed to understand whether msc are possible producers of msp.
pbmc known to be constitutive msp producers  were used as controls.
the expression of mspmrna was investigated by quantitative pcr in pbmc and in msc.
the expression of msp mrna in pbmc was held as the arbitrary unit.
msc expressed a greater amount of constitutive msp compared with pbmc .figure  <dig>
semiquantitative mspmrna expression in pbmc, msc in basal condition evaluated by reverse transcription –pcr that used 1 mg of rna as the template.
pbmc known for their capacity to produce msp were used as controls.
columns indicate msp mrna expression normalized to the beta-actin expression and converted into fold change.
*p <  <dig>  vs.
ron mrna and ron protein expression in monocytes
ron mrna expression normalized to the beta-actin significantly increased in monocytes stimulated in vitro for 6 h with lps and inf-γ than in monocytes cultured in basal condition  respectively   .
ron positive monocytes percentage in basal condition  was  <dig>  ± 2%, but it increased significantly after in vitro monocytes stimulation for 24 h with respectively lps and inf-γ  .
CONCLUSIONS
we formerly showed that msc injected in a rat model of renal transplantation attenuated the severity of rejection and we found also that such effect was independent of msc endowing in the transplanted kidney.
the latter findings indicate that msc operate through intermediate effectors, as it was elegantly demonstrated by bi et al.
<cit>  who reproduced msc effects by substituting msc for their culture medium.
aim of the present study was to identify possible effectors of the protection provided by msc in the renal transplant model.
actually, the study consisted of a straightforward continuation of our former ones in which we demonstrated that msc prevent the rejection  <cit> .
we have pointed out two systems:  the cytokine network that regulates the immune response in acute rejection, and  the scatter factors systems that have been shown to promote repair and to modulate immune cell traffic in renal tissue in different models of kidney disease  <cit> .
in addition to confirming that msc significantly decrease cd <dig>  cd <dig> cells and monocytes infiltration  <cit> , here we report that on day  <dig> of grafting in msc untreated rats, e.g.
rats with acute severe rejection ifn-γ and il- <dig> levels rose impressively both in serum and in graft tissue, while msc injection prevented the rise in ifn-γ and simultaneously it caused a further rise in il- <dig> levels.
since ifn-γ is a nominal marker of th <dig> lymphocyte activity, while il- <dig> is th <dig> cells product  <cit> , these results suggest that msc reset the balance between the two t helper subpopulations, contrasting the prevalence of th <dig> over th <dig>  msc effects on t cell subsets included also a rise of foxp3+ cells number, i.e.
treg lymphocytes in the transplanted kidney.
this finding confirms that msc induce tregs, as shown in patients with sle  <cit>  and renal graft  <cit>  in which circulating tregs rose after msc infusion and in a mouse model of renal transplantation in which pre-transplant msc infusion was associated with a significant prolongation of graft survival by treg-dependent mechanism  <cit> .
altogether the results of our study suggest that msc reset t cell subpopulations, decreasing the prevalence of th <dig> cells that are main effectors of rejection, increasing the activity of the immunosuppressive th <dig> subset and recruiting tolerogenic tregs.
in addition to changing the t cell phenotypic distribution, msc blocked il- <dig> overproduction, a major inflammatory product of monocyte/macrophage cell and effector of acute rejection  <cit> .
therefore, msc suppressed the cytokines that drive the graft assault by the two major cell effectors of acute rejection, th <dig> lymphocytes and monocytes.
a new information given by our study is that msc have relevant effects on the scatter factor systems.
in fact, in msc untreated rats the hgf/met couple was suppressed in serum  and in renal tissue .
msc prevented such abatement of hgf/met, thus saving a system that has been proved to protect the kidney in diverse experimental models of renal disease.
actually, hgf has several features that facilitate renal healing: it stimulates proliferation and blocks apoptosis of injured tubular cells, it induces formation of new tubular structures in renal epithelial cells and generates new capillary vessels  <cit> , it downregulates the inflammatory and immune response, e.g.
by inactivating dendritic cells  <cit> , resetting cytokine network and addressing transformation of t cells to the th <dig> phenotype  <cit> .
hgf was shown also to interfere with the expression of immune co-stimulatory molecules, and to prolong survival of cardiac allograft by preventing acute rejection in a rodent model  <cit> .
furthermore, in a model of acute renal rejection, administration of recombinant hgf expanded treg cell subset  <cit> .
indeed, there is an impressive overlap between the activities that are attributed to hgf and the changes induced by msc in grafted rats, e.g.
the shift of t cells to a tolerogenic phenotype, the rescue of tubular cells from death and the increase in tubular cell proliferation, that are the most distinctive features of hgf activity on renal tubular cells.
therefore, we believe that hgf/met is a system that mediates the protective msc effect on the kidney graft.
interestingly, msc induced a rise in hgf levels also in another rat model of renal diseases, i.e.
anti-thy  <dig> nephritis  <cit>  and ischemia-reperfusion injury  <cit>  and in both models hgf overexpression was associated with prevention of renal damage.
in contrast to the abundant literature available on the pleiotropic activities of hgf/met, the homologous factor msp and its receptor ron are less known and their role in renal physiology and disease has been scarcely investigated.
we have shown that tubular cells produce msp and that msp and ron are diffusely expressed in the normal kidney  <cit> .
studies in vitro have demonstrated that msp induces in tubular cells proliferation, resistance to apoptosis, migration and branching morphogenesis, i.e.
effects that altogether suggest a role of msp expressed in the kidney as an autocrine/paracrine factor that protects survival and stimulates proliferation of tubular cells.
in fact, msp was shown to attenuate renal injury in the glycerol-induced model of acute renal failure  <cit> .
in the present study we found that msp and ron were suppressed in renal grafts not injected with msc i.e.
in the setting of unopposed acute rejection.
msc injection had significant effects on msp/ron system that consisted of  recovery of msp mrna levels in tubular cells,  recovery of msp and ron expressed in tubular cells with msp shifted in a different cell location, i.e.
on the cell surface rather than inside the cytoplasm,  suppression of ron in infiltrating monocyte/macrophage cells.
we interpret this combination as a series of actions that benefit the kidney by  restarting the translation of msp and the production of msp protein in an amount that makes it detectable in the tubular cell  shifting the msp molecule to the cell surface, i.e.
in a site where it can meet its receptor and possibly activate it in an autocrine fashion  suppressing ron in monocytes thus restraining their ability to address tubular cells.
as for the last effects, in order to understand the underlying mechanism we designed an in vitro experiment that demonstrated for the first time that msc express constitutively mrna of msp in amount greater than that expressed by pbmc, i.e.
a reference constitutive msp producer  <cit> .
ron expression in monocytes is induced by ifn-γ.
since msc suppress ifn-γ both in serum and in renal tissue, it seems reasonable that this mechanism accounts for the absence of ron in monocytes infiltrating the renal graft injected with msc.
recent studies in a mouse model of renal transplantation have shown that msc effects on graft function depend on the time of their injection, i.e.
msc worsen kidney function when injected after grafting, while they prevent rejection and ameliorate renal outcome when administered  <dig> or 7 days before the grafting  <cit> .
these observations have a relevant impact on deciding the mode of msc administration that should be used in clinical transplantation.
we have chosen to inject msc into the renal artery soon after reperfusion of the graft for two reasons:  to avoid changes in msc activity caused by their transit in the lung, and  to administer msc in a setting that is easily reproducible in the circumstances of transplantation from a cadaver donor, in which the identity of the recipient is known just at the last minute.
our demonstration that this mode is feasible offers a potential advantage, mainly because, once our model is proven safe in humans, it allows to deliver msc to recipients of kidney from cadaver donors that represent the majority of patients crowding the waiting lists.
however, the two models are hardly comparable not only because of the different mode of administration, but also because the studies were performed in different species.
nonetheless it is interesting that both of them give useful information to programme use of msc in humans, and both identify tregs as effectors of msc immunosuppressive action.
in summary, we have demonstrated that the prevention of rejection provided by msc is associated with a shift of t cells to an immune suppressive phenotype and with the recruitment of the scatter factor systems.
these observations identify candidate mediators of msc activity.

 mesenchymal stromal cells are pluripotent cells that differentiate into various mature cell types. a distinctive property of msc is that they are not immunogenic, and inhibit cell and antibody-mediated immunity in several ways, including the induction of t regulatory cell differentiation. in a rat model of kidney transplantation we found that msc injected in the graft improved its function and attenuated renal injury, reducing significantly tubulitis, vasculitis, glomerulitis and immune cell infiltration. furthermore, we traced msc in the recipient tissues and found an irrelevant number of them in the kidney. the last finding suggests that mediators account for the protection provided by msc to the renal graft. a renal protective effect of msc was shown also in a mouse model of renal transplantation, in which msc suppressed rejection when they were infused before transplantation, while post-transplant infusion worsened graft outcome. interestingly, msc injected before transplantation were found in lymphoid organs and did not localize in the graft, confirming the need for some mediation of msc effects. indeed a mediated action of msc has been proved in diverse experimental disease models, and cytokines have been proposed to play the role of effectors. however, as yet no evidence has been given that msc modify the cytokine network in the setting of renal graft, so that we have felt it interesting to test the hypothesis in the present study. in addition, we have thought of the scatter factors as a system that could be reset by msc and provide protection in the renal graft model. the scatter factor system consists of hepatocyte growth factor and macrophage stimulating protein and their receptors met and ron respectively. there are several reasons to think that msc operate through the sf system. in fact, the sfs are expressed in normal kidney and participate in the regulation of cell growth and inflammation in various renal diseases including autoimmune forms, e.g. they stimulate renal cell growth and modulate monocyte traffic in the kidney. furthermore, we demonstrated that in experimental anti thy- nephritis msc improve renal injury by modulating sfs. in summary, we investigated whether msc injected into the renal graft modify the cytokine network and sfs in a way that fits with the concept that these systems are the effectors of msc-induced graft protection.  characterization of rat msc as detailed elsewhere the msc used in the present study were isolated from sprague dawley egfp rats and differentiated into osteogenic and adipogenic cells. flow fluorocytometry analysis showed that msc were positive for cd and cd and were negative for cd, cd11b, cd, cd. tissue and serum levels of cytokines the expression in renal tissue of ifn-γ, il- il- il- il- was significantly increased in grafted kidneys not injected with msc compared with native kidneys. msc injection attenuated significantly the rise of ifn-γ and caused a further increase of il- msc did not modify tissue levels of any else cytokine. il- tissue levels were similar in all groups. in msc untreated rats grafting was associated with a rise in serum levels of all tested cytokines, except for il- msc treatment reduced serum ifn-γ and il- and increased il- compared to untreated rats.table cytokine levels in renal tissue pg/mg a b c b vs c p inf-γ  ±  §  ±  °  ±  ↓ § p <  vs b, c; °p <  vs c il-10  ±  §  ±  °  ±  ↑ § p <  vs b, c; °p <  vs c il-6  ±  °  ±   ±  ns °p <  vs b, c il-2  ±  ° 227 ±  262 ±  ns °p <  vs b, c il-1  ±  °  ±  493 ± 142 ns °p <  vs b, c il-4  ±   ± 6  ±  ns ns groups are defined in figure  a indicates cytokine levels in kidneys of healthy rats, b and c indicate cytokine levels in renal tissue of recipient rats 7 days after transplantation. data represent means ± sd. the symbols § and ° indicate the different statistical power. serum cytokine levels pg/ml a b c b vs c p inf-γ 14 ±  °  ±  §  ±  ↓ °p <  vs b, c; § p <  vs c il-10 0°  ±  *  ±  ↑ °p <  vs b, c; *p <  vs c il-6 0° 183 ±  #  ±  ↓ °p <  vs b, c; # p <  vs c il-2 0° 61 ±   ± 61 ns °p <  vs b, c il-1 0° 1515 ± 613 1843 ± 670 ns °p <  vs b, c; il-4 0 0 0 ns groups are defined in figure  a indicates serum cytokine levels in healthy rats. b and c indicate serum cytokine levels in recipient rats 7 days after transplantation. data are means ± sd. the symbols §, °, *, # indicate the different statistical power. tubular necrosis and tubular cell proliferation tubules with necrotic cells were significantly less in grafts injected with msc compared to untreated grafts. the expression of pcna was suppressed in grafts not injected with msc, but it was spared by msc treatment.figure tubular necrosis and tubular cell proliferation. groups are defined as in figure  panel a: columns represent the ratio between necrotic tubules number and total counted tubules. °p <  vs c; *p <  vs b and c. panel b: boxes represent 25-75% percentile of tubular pcna-positive cell number/hpf. *p <  vs a and b. foxp cells infiltrate in the grafted kidneys not injected with msc we found few foxp positive cells and the number increased in msc treated rats. in the spleen we detected 15 ±  cells/section.figure foxp positive cells. foxp staining of representative kidney and spleen sections. panel a shows foxp positive cells in cortical tubules after 7 days from transplantation in rats of group c. panel b shows foxp positive cells in medullary tubules after 7 days from transplantation in rats of group c. panel c shows foxp positive cells in spleen after 7 days from transplantation in rats of group c. hgf/met system serum and renal tissue hgf levels, hgf mrna expression in kidneys hgf serum levels significantly decreased in grafted rats that were not injected with msc compared to control normal rats. msc treatment returned serum hgf to normal levels.figure serum hgf levels, hgf mrna and hgf protein in kidney of control and allografted rats on day groups are defined as in figure  upper panel: serum hgf levels in a, b, c groups of rats. columns are means, bars are sd. *p <  vs a and c. middle panel: western blot performed with anti-hgf antibody in renal tissue of groups a, b, c. the p69-kda band represents the α sub of active heterodimer hgf. lower panel: hgf mrna expression in renal tissue analyzed by rt pcr in all groups of rats. columns indicate hgf mrna expression normalized to the beta-actin expression and converted into fold change. °p <  vs a. both hgf protein and hgf mrna were reduced in grafted kidneys compared to native organs. msc injection prevented the loss of hgf protein, although hgf mrna levels were lower probably for a feedback mechanism. renal expression of met the widespread expression of met protein in tubules of native kidneys was almost completely abrogated in untreated grafted kidneys. msc infusion prevented met loss. the result was confirmed by western blot for met performed on kidney tissue.figure met expression in kidney of control and allografted rats on day groups are defined as in figure  upper panel: met expression in representative renal sections of control and allografted rats on day panel a: normal kidney, panel b: untreated allograft, panel c: msc treated allograft. middle panel: bars represent the medians of met positive tubules number/hpf in all groups of rats, boxes represent the 25-75% percentile, whiskers: min to max. °p <  vs b, § p <  vs a, * p <  vs c. bottom panel: western blot performed with anti-met antibody in renal tissue of all groups of rats. the p170-kda band represents pro-met, the p140-kda band represents mature met β subunit. msp/ron system renal expression of msp/ron immunohistochemistry showed that msp was expressed in tubular cells of native kidneys and it was undetectable in untreated grafts except for some tubules in which it was distributed with luminal pattern. msc restored tubular msp in transplanted rats, but in a different pattern from normal, i.e. luminal instead of cytoplasmic. as msp also its receptor, ron, was diffusely expressed with cytoplasmic pattern in tubules of healthy rats, while in transplanted rats with acute rejection tubular ron expression was significantly decreased and reappeared in tubules of allografts treated with msc. in untreated grafts ron was expressed also by some inflammatory cells in the interstitium, but it was absent in infiltrating cells of msc treated rats.figure msp and ron expression in kidney of control and allografted rats on day groups are defined as in figure  panel a: msp expression in representative renal section of normal kidney, note the strong cytoplasmic pattern of staining, panel b: msp expression in representative renal section of untreated allograft, note the almost complete absence of msp expression, panel c: msp expression in representative renal section of msc treated allograft, note the luminal pattern of msp staining. panel d: ron expression in representative renal sections of normal kidney, panel e: ron expression in representative renal sections of untreated allograft, note the presence of ron positive inflammatory cells and the significant reduction of ron tubular staining, panel f: ron expression in representative renal sections of msc treated allograft, note the reappearance of ron tubular staining and absence of ron positive inflammatory cells. panel g: bars represent the medians of msp positive tubules number/hpf in all groups of rats; boxes represent 25-75% percentile of msp positive tubules, whiskers: min to max) i: °p <  vs b; *p <  vs c, § p <  vs a. panel h: bars represent the medians of ron positive tubule number/hpf in all groups, boxes represent the 25-75% percentile, whiskers: min to max. *p <  vs c, °p <  vs b and c. msp mrna expression in kidneys rt-pcr showed a significant decrease of msp mrna in the untreated graft. msc injection was associated with recovery of msp mrna expression.figure msp mrna expression of control and allografted rats. groups are defined as in figure  msp mrna expression in renal tissue analyzed by rt pcr in all groups of rats. columns indicate msp mrna expression normalized to the beta-actin expression and converted into fold change. *p <  vs a and c. msc express msp mrna we performed in vitro experiments aimed to understand whether msc are possible producers of msp. pbmc known to be constitutive msp producers were used as controls. the expression of mspmrna was investigated by quantitative pcr in pbmc and in msc. the expression of msp mrna in pbmc was held as the arbitrary unit. msc expressed a greater amount of constitutive msp compared with pbmc.figure semiquantitative mspmrna expression in pbmc, msc in basal condition evaluated by reverse transcription –pcr that used 1 mg of rna as the template. pbmc known for their capacity to produce msp were used as controls. columns indicate msp mrna expression normalized to the beta-actin expression and converted into fold change. *p <  vs. ron mrna and ron protein expression in monocytes ron mrna expression normalized to the beta-actin significantly increased in monocytes stimulated in vitro for 6 h with lps and inf-γ than in monocytes cultured in basal condition respectively. ron positive monocytes percentage in basal condition was  ± 2%, but it increased significantly after in vitro monocytes stimulation for 24 h with respectively lps and inf-γ.  we formerly showed that msc injected in a rat model of renal transplantation attenuated the severity of rejection and we found also that such effect was independent of msc endowing in the transplanted kidney. the latter findings indicate that msc operate through intermediate effectors, as it was elegantly demonstrated by bi et al. who reproduced msc effects by substituting msc for their culture medium. aim of the present study was to identify possible effectors of the protection provided by msc in the renal transplant model. actually, the study consisted of a straightforward continuation of our former ones in which we demonstrated that msc prevent the rejection. we have pointed out two systems: the cytokine network that regulates the immune response in acute rejection, and the scatter factors systems that have been shown to promote repair and to modulate immune cell traffic in renal tissue in different models of kidney disease. in addition to confirming that msc significantly decrease cd cd cells and monocytes infiltration, here we report that on day of grafting in msc untreated rats, e.g. rats with acute severe rejection ifn-γ and il- levels rose impressively both in serum and in graft tissue, while msc injection prevented the rise in ifn-γ and simultaneously it caused a further rise in il- levels. since ifn-γ is a nominal marker of th lymphocyte activity, while il- is th cells product, these results suggest that msc reset the balance between the two t helper subpopulations, contrasting the prevalence of th over th msc effects on t cell subsets included also a rise of foxp3+ cells number, i.e. treg lymphocytes in the transplanted kidney. this finding confirms that msc induce tregs, as shown in patients with sle and renal graft in which circulating tregs rose after msc infusion and in a mouse model of renal transplantation in which pre-transplant msc infusion was associated with a significant prolongation of graft survival by treg-dependent mechanism. altogether the results of our study suggest that msc reset t cell subpopulations, decreasing the prevalence of th cells that are main effectors of rejection, increasing the activity of the immunosuppressive th subset and recruiting tolerogenic tregs. in addition to changing the t cell phenotypic distribution, msc blocked il- overproduction, a major inflammatory product of monocyte/macrophage cell and effector of acute rejection. therefore, msc suppressed the cytokines that drive the graft assault by the two major cell effectors of acute rejection, th lymphocytes and monocytes. a new information given by our study is that msc have relevant effects on the scatter factor systems. in fact, in msc untreated rats the hgf/met couple was suppressed in serum and in renal tissue. msc prevented such abatement of hgf/met, thus saving a system that has been proved to protect the kidney in diverse experimental models of renal disease. actually, hgf has several features that facilitate renal healing: it stimulates proliferation and blocks apoptosis of injured tubular cells, it induces formation of new tubular structures in renal epithelial cells and generates new capillary vessels, it downregulates the inflammatory and immune response, e.g. by inactivating dendritic cells, resetting cytokine network and addressing transformation of t cells to the th phenotype. hgf was shown also to interfere with the expression of immune co-stimulatory molecules, and to prolong survival of cardiac allograft by preventing acute rejection in a rodent model. furthermore, in a model of acute renal rejection, administration of recombinant hgf expanded treg cell subset. indeed, there is an impressive overlap between the activities that are attributed to hgf and the changes induced by msc in grafted rats, e.g. the shift of t cells to a tolerogenic phenotype, the rescue of tubular cells from death and the increase in tubular cell proliferation, that are the most distinctive features of hgf activity on renal tubular cells. therefore, we believe that hgf/met is a system that mediates the protective msc effect on the kidney graft. interestingly, msc induced a rise in hgf levels also in another rat model of renal diseases, i.e. anti-thy nephritis and ischemia-reperfusion injury and in both models hgf overexpression was associated with prevention of renal damage. in contrast to the abundant literature available on the pleiotropic activities of hgf/met, the homologous factor msp and its receptor ron are less known and their role in renal physiology and disease has been scarcely investigated. we have shown that tubular cells produce msp and that msp and ron are diffusely expressed in the normal kidney. studies in vitro have demonstrated that msp induces in tubular cells proliferation, resistance to apoptosis, migration and branching morphogenesis, i.e. effects that altogether suggest a role of msp expressed in the kidney as an autocrine/paracrine factor that protects survival and stimulates proliferation of tubular cells. in fact, msp was shown to attenuate renal injury in the glycerol-induced model of acute renal failure. in the present study we found that msp and ron were suppressed in renal grafts not injected with msc i.e. in the setting of unopposed acute rejection. msc injection had significant effects on msp/ron system that consisted of recovery of msp mrna levels in tubular cells, recovery of msp and ron expressed in tubular cells with msp shifted in a different cell location, i.e. on the cell surface rather than inside the cytoplasm, suppression of ron in infiltrating monocyte/macrophage cells. we interpret this combination as a series of actions that benefit the kidney by restarting the translation of msp and the production of msp protein in an amount that makes it detectable in the tubular cell shifting the msp molecule to the cell surface, i.e. in a site where it can meet its receptor and possibly activate it in an autocrine fashion suppressing ron in monocytes thus restraining their ability to address tubular cells. as for the last effects, in order to understand the underlying mechanism we designed an in vitro experiment that demonstrated for the first time that msc express constitutively mrna of msp in amount greater than that expressed by pbmc, i.e. a reference constitutive msp producer. ron expression in monocytes is induced by ifn-γ. since msc suppress ifn-γ both in serum and in renal tissue, it seems reasonable that this mechanism accounts for the absence of ron in monocytes infiltrating the renal graft injected with msc. recent studies in a mouse model of renal transplantation have shown that msc effects on graft function depend on the time of their injection, i.e. msc worsen kidney function when injected after grafting, while they prevent rejection and ameliorate renal outcome when administered or 7 days before the grafting. these observations have a relevant impact on deciding the mode of msc administration that should be used in clinical transplantation. we have chosen to inject msc into the renal artery soon after reperfusion of the graft for two reasons: to avoid changes in msc activity caused by their transit in the lung, and to administer msc in a setting that is easily reproducible in the circumstances of transplantation from a cadaver donor, in which the identity of the recipient is known just at the last minute. our demonstration that this mode is feasible offers a potential advantage, mainly because, once our model is proven safe in humans, it allows to deliver msc to recipients of kidney from cadaver donors that represent the majority of patients crowding the waiting lists. however, the two models are hardly comparable not only because of the different mode of administration, but also because the studies were performed in different species. nonetheless it is interesting that both of them give useful information to programme use of msc in humans, and both identify tregs as effectors of msc immunosuppressive action. in summary, we have demonstrated that the prevention of rejection provided by msc is associated with a shift of t cells to an immune suppressive phenotype and with the recruitment of the scatter factor systems. these observations identify candidate mediators of msc activity. 
systems that are committed to defend survival and stimulate regeneration of tubular cells. in this study we investigated whether msc operate by resetting cytokine network and scatter factor systems, i.e. in addition, msc sustain the expression of the scatter factor systems expression, i.e. our results demonstrate that msc modify cytokine network to a tolerogenic setting, they suppress th cells, inactivate monocytes/macrophage, recruit tregs. in former studies we showed in a rat model of renal transplantation that mesenchymal stromal cells prevent acute rejection in an independent way of their endowing in the graft. mesenchymal stromal cellsacute kidney rejectionexperimental modelhepatocyte growth factormacrophage stimulating proteinscatter factorsissue-copyright-statement© the author 2014 hepatocyte growth factor, macrophage stimulating protein and their receptors met and ron, respectively. serum and renal tissue levels of ifn-γ, il- il- il- il- il- msp/ron, hgf/met systems, treg lymphocytes were investigated. controls were grafted untreated and normal rats. in grafted untreated rats ifn-γ increased in serum and renal tissue and il- rose in serum. msc were injected into the renal artery soon after reperfusion.
 in former studies we showed in a rat model of renal transplantation that mesenchymal stromal cells prevent acute rejection in an independent way of their endowing in the graft. in this study we investigated whether msc operate by resetting cytokine network and scatter factor systems, i.e. hepatocyte growth factor, macrophage stimulating protein and their receptors met and ron, respectively. methods msc were injected into the renal artery soon after reperfusion. controls were grafted untreated and normal rats. rats were sacrificed 7 days after grafting. serum and renal tissue levels of ifn-γ, il- il- il- il- il- msp/ron, hgf/met systems, treg lymphocytes were investigated.  in grafted untreated rats ifn-γ increased in serum and renal tissue and il- rose in serum. msc prevented both the phenomena, increased il- serum levels and treg number in the graft. furthermore msc increased serum and tissue hgf levels, met tubular expression and prevented the suppression of tubular msp/ron expression.  our results demonstrate that msc modify cytokine network to a tolerogenic setting, they suppress th cells, inactivate monocytes/macrophage, recruit tregs. in addition, msc sustain the expression of the scatter factor systems expression, i.e. systems that are committed to defend survival and stimulate regeneration of tubular cells. keywords mesenchymal stromal cellsacute kidney rejectionexperimental modelhepatocyte growth factormacrophage stimulating proteinscatter factorsissue-copyright-statement© the author 2014 