The aim of our study was to establish the importance of the SDF-1/CXCR4-axis in the migration of HSC to the ischemic injured kidney, in order to eventually manipulate this axis for therapeutic purposes. The contribution of HSC in post-ischemic renal tissue repair is controversial. First of all, the capacity of HSC to engraft tubules after ischemic injury is much lower than originally reported. Initial studies in chimeric mice stated that 21% [6
] or even 80% [7
] of the tubules contained BMC. However, some of these numbers are thought to be overestimated due to false-positive results as a consequence of β-gal leaking by the BMC [23
]. Sex-mismatched and GFP-chimeric mice studies published in 2005 showed only a few BMC in the tubules after I/R injury [8
], whereas Duffield and colleagues could not observe any BMC in post-ischemic tubules [24,25
]. Secondly, the biological significance of HSC in repair after I/R injury is challenged, since low numbers of BMC in the tubules do not make a significant contribution to renal functional recovery [8
]. Therefore, mechanisms to increase the migration of HSC may result in a significant contribution of these cells to tubular repair and hence have therapeutic potential.
To determine whether the SDF-1/CXCR4-axis is the driving force for the recruitment of HSC to the injured kidney, we followed the migration of exogenous HSC in a unilateral renal I/R model. This model gives the opportunity to compare the migration of HSC in injured and normal tissue within one mouse, in contrast to experimental models such as cisplatin-induced renal injury and bilateral renal I/R injury. Here, we demonstrate that HSC migrate to the injured kidney and are even able to migrate into damaged tubules. In line with our data, Lin et al
] and Tögel et al
] have shown that the homing process of BMC is selective for the ischemic injured kidney. Our study, however, extends these findings as we have studied migration of purified HSC instead of whole bone marrow that is a heterogeneous population of cells consisting, amongst others, of inflammatory cells, mesenchymal stem cells and only a small percentage of HSC. Surprisingly, manipulating the SDF-1/CXCR4-axis in three different and independent manners did not result in a significantly altered migration of exogenous HSC.
Previous studies have shown the importance of the SDF-1/CXCR4-axis in the migration of BMC. This was done either by blocking endogenous SDF-1, blocking BMC-associated CXCR4 or increasing locally SDF-1 [11,12
]. Our results are at variance with the current opinion that SDF-1 is the mediator of HSC trafficking to injured organs. The neutralizing antibodies were chosen based on their proven functionality in previous studies. Neutralization with the same function-blocking anti-SDF-1 monoclonal antibody and dose as used in the present study significantly attenuated HSC accumulation within the growing platelet-rich thrombus in an arterial thrombosis mouse model [21
]. In order to neutralize HSC-associated CXCR4, the same protocol was used as reported previously to be effective in blocking the recruitment of BMC [18
] or HSC [11
]. In addition, our in vitro
and in vivo
HSC migration assays demonstrated a decreased migration towards high recSDF-1 levels and bone marrow, respectively, after neutralizing SDF-1 or CXCR4. Therefore, we believe that our experimental set-up is highly reliable to study the role of this axis. Despite this, we could not observe a significant effect of neutralizing SDF-1 or its receptor CXCR4 on the migration of HSC to the injured kidney. Since these results do not support the current reports on SDF-1-involvement in HSC-migration, we conducted a third independent experiment to determine the effect of local SDF-1 administration on the migration of exogenous HSC. Despite the previously reported preferred migration of HSC towards high SDF-1 gradient in liver, bone marrow and spleen [11,12
], we could not observe this in both the unilateral and bilateral renal I/R injury model. However, in accordance with our results, it has been reported that BMC migration to the lung could not be influenced significantly by manipulating the SDF-1/CXCR4-axis in both a non-injured [12
] and a bleomycine-injured [27
] mouse model. This indicates that other mechanisms may be involved in HSC migration to the kidney and lung.
We also did not find an effect of the local administration of recSDF-1 in the non-injured (contralateral) kidney on renal engraftment of HSC compared to the non-treated contralateral kidney. These results suggest that injury is necessary for HSC migration and that SDF-1 alone is not sufficient for migration. This was also observed in the heart, where overexpression of SDF-1 without injury could not induce the homing of BMC [16
]. In addition, the results from the bilateral I/R model, where both the SDF-1 and saline-injected kidney had the same percentage of migration, indicate that the exogenous administered HSC cells did not migrate preferentially towards high SDF-1 gradient.
Our results indicate that I/R injury induces the homing of HSC in the injured kidney and that this preferred migration is not exclusively dependent on the SDF-1/CXCR4-axis. Homing of HSC towards the ischemic damaged kidney could be much more complex. Several other factors are described to be involved in stem cell migration and in addition are known to be induced after I/R injury. These factors include the chemokine GROβ [28
], the glycosaminoglycan hyaluronic acid [29
], the nuclear factor HMGB1 [30
] and the growth factor HGF [11
]. Interestingly, in a recently published report, a flexible hierarchy of homing molecules was proposed, providing an explanation for some of the contradictory findings in the literature [31
]. Herein, the authors show that in a mouse model of HSC transplantation, SDF-1 was not required for stem cell homing due to compensatory action via the VLA-4/VCAM-1 interaction [31
In conclusion, our initial hypothesis that SDF-1/CXCR4 interactions are implicated in the migration of HSC to the injured kidney is not supported by the results in this report. In sharp contrast to studies in other injured organs, our results indicate that migration of HSC to the ischemic damaged kidney is not solely dependent on the SDF-1/CXCR4 signalling axis.