The safety and potential efficacy of repeated intravenous administration of allogeneic cryopreserved feline aMSCs to cats with naturally occurring CKD was assessed in this series of pilot studies. MSCs have previously been shown to be effective in suppressing some aspects of renal disease in rodent models of induced CKD. However, MSC therapy has not been evaluated in a naturally occurring animal model of CKD such as the feline CKD model. We modified existing rodent MSC administration protocols to create a clinically feasible trial in cats with CKD with translational potential. The major findings from our preliminary studies of aMSC therapy were that lower doses of allogeneic cryopreserved aMSCs were well tolerated following repeated intravenous administration and appeared to be associated with modest improvement in renal function. The level of improvement in renal function seen in the treated cats is, however, of uncertain clinical significance. The six cats in pilot study 1 that received the 2 × 106 cryopreserved aMSC dose experienced a statistically significant decrease in serum creatinine together with negligible adverse effects from aMSC administration. However, the degree of this decrease (<0.5 mg/dl) is very small and may not translate to any change in clinical disease. In contrast, higher intravenous doses of cryopreserved aMSCs were associated with a high incidence of adverse effects and variable evidence of improvement in renal function as evidenced by GFR estimated by iohexol clearance. Most of the five cats in pilot study 2 that received 4 × 106 cryopreserved aMSCs per injection experienced side effects, including vomiting during infusion and increased respiratory rate and effort including overt dyspnea in one case. Significant changes in serum creatinine, GFR, and urinary cytokine concentrations were not observed in pilot study 2 cats. In pilot study 3, cats that received 4 × 106 aMSCs cultured from cryopreserved adipose experienced no side effects, but little evidence of clinically significant improvement in renal function. Thus, we concluded that although intravenous administration of higher doses of cryopreserved, allogeneic aMSCs was unlikely to be an acceptable treatment option due to the high rate of complications and the lack of clinical or experimental responses; aMSCs expanded from cryopreserved fat appear a safer option although strong indication of efficacy is still lacking.
A recent study by Semedo et al
] in a rat model of CKD provided compelling evidence of the potential efficacy of repeated intravenous administration of bone marrow-derived MSCs for suppression of intrarenal inflammation and fibrosis, and this treatment protocol was the one we adapted for clinical application in CKD cats. Although aMSCs were used instead of bone marrow-derived MSCs, in the field of adipose stem cell research, the two sources are felt to be comparable in characterization and therapeutic potential in inflammatory disease states [23
]. Why then were the results of our preliminary studies of aMSC therapy in cats with CKD so different from the rodent studies, when similar doses of MSCs (based on body weight) were utilized? Firstly, differences in the CKD model should be taken into consideration. Most rodent models involve acute insult to the kidney with subsequent administration of MSCs within a few weeks: a relatively short time post renal insult [4
]. Thus, the induced disease models, with their relatively short time frame, may not be representative of the changes occurring in a truly chronic, naturally occurring disease process [11
]. Cats have an extended lifespan and often have CKD for months to years prior to clinical diagnosis and study enrollment, which is more similar to the disease time frame seen in humans. The possibility that this potential therapy may not be as effective in patients with more long-standing disease should be considered.
Secondly, in our study, we utilized allogeneic aMSCs, whereas autologous MSCs were used in the Semedo et al
. study [4
]. The relative efficacy of autologous vs. allogeneic cells is an area of controversy. Although allogeneic MSCs are immune privileged and are not expected to incite an immune response, according to some authors they may not be as effective as autologous cells [35
]. It is argued that autologous MSCs may survive longer in the body in comparison to allogeneic cells, which could reduce efficacy of the latter. Decreased efficacy of allogeneic MSCs in comparison to autologous MSCs has been observed in one acute renal failure rodent study [35
]. However, allogeneic MSCs have been widely used in experimental stem cell transfer investigations, including clinical trials in humans, with positive results [35
A third major difference was the use of freshly-thawed, cryopreserved aMSCs in pilot studies 1 and 2. This decision was made based on the logistical ease afforded by use of cryopreserved rather than freshly-cultured MSCs. Successful cryopreservation of cells has been previously described [37
]. However, it is unknown if cryopreservation affects the functional properties of aMSCs necessary for successful use in this model. Previous studies assessing the effects of cryopreservation on MSCs did not examine effects on potential immunogenicity and further work is needed to fully assess this subject [37
Based on the results of the three pilot studies, it appears that use of higher doses of cryopreserved aMSCs was the source of the treatment-related adverse effects in pilot study 2 as similar doses of aMSCs cultured from cryopreserved adipose tissue did not result in any adverse effects. Although the specific reasons for the increased incidence of side effects is not known, it is likely related to the increase in dose of cryopreserved cells. The most likely explanation for this reaction is an instant blood mediated inflammatory reaction (IBMIR) which results in clumping of the cells as they contact the blood and potential subsequent micro pulmonary thromboembolism [39
]. The IBMIR phenomenon has been described previously in cryopreserved cells and increases in severity with dose and passage number [39
]. It can result in lysis of the administered MSCs and subsequent poor efficacy. Although all cells given in pilot study 2 were of the same passage (P3) as those used in the other two pilot studies, the reaction was only seen in the pilot study group where cells were taken directly from cryopreservation and used at the higher dose. In pilot study 3 no complications during or after administration of aMSCs cultured from cryopreserved fat were appreciated. Thus, we have concluded that the administration of a higher dose of aMSCs taken directly from cryopreservation, despite careful washing, was the source of the toxic reactions observed, and this form of administration is not recommended.
Although several cats in the pilot study 1 group experienced a statistically significant improvement in serum creatinine concentrations during the study, the degree of increase is unlikely to be clinically significant. Creatinine is not the most sensitive measurement of renal function even though it is the routinely measured clinical marker of renal function. Several factors that are not directly related to renal function can influence serum creatinine values, including muscle mass and hydration status [28
]. Decreases in weight and/or muscle mass can potentially decrease serum creatinine, while a worsening in hydration status can increase serum creatinine. In addition, changes to renal function may occur without any concomitant alteration in serum creatinine, although this is thought to be less true once an animal reaches the stage of moderate renal dysfunction [28
]. Although there were no significant changes in body weight noted in these pilot studies, individual changes in body weight in the study cats could potentially have confounded serum creatinine measurements, while changes in bladder fullness at the time of weighing could potentially have compromised weight values. Therefore, in cats in pilot studies 2 and 3 we also measured estimated GFR, as a more objective way to monitor changes in renal function. Quantitating GFR allowed us to assess potential overall kidney function changes induced by the paracrine and/or autocrine effects of MSC administration. GFR results were variable in cats in pilot studies 2 and 3; two of five animals in pilot study 1 and two of five cats in pilot study 3 did experience a marked increase from pretreatment baseline; however the change in GFR was not significant overall for the group. Overall, definitive evidence of significant efficacy of intravenous administration of allogeneic aMSCs for treatment of CKD in cats is lacking at this time. Closer evaluation of the mechanism of MSC action in CKD, effect of disease stage, necessary cell dosage (for example, a cell/kg dose rather than a per cat dose as used in these pilot studies), timing of injections, and other variables remain to be determined.