Introduction
Regenerative medicine refers to the process of using living function tissues to repair or replace organs that are functionally damaged. Stem cell therapy in particular is an innovative new field of scientific investigation and clinical application that holds promise for a variety of diseases in veterinary medicine as well as human medicine. Recent years have brought increased interest in the potential for adult stem cells to help in the treatment of many diseases through both their regenerative properties as well as their apparent ability to alter the environment in injured and diseased tissues. In particular, adult stem cells called mesenchymal stem cells can migrate to affected areas and may be able to support the growth of other stem cells as well as moderate the response of the immune system.
Stem Cells
A stem cell is a generic term referring to any unspecialized cell that is capable of long-term self-renewal through cell division but that can be induced to differentiate into a specialized, functional cell. Stem cells are generally divided into two groups, embryonic stem cells and adult stem cells. Adult stem cells can be obtained from many differentiated tissues including but not limited to bone marrow, bone, fat, and muscle. Obtaining adult stem cells also does not raise ethical concerns. For most studies, the adult stem cell in question is actually a mesenchymal stem cell or mesenchymal stromal cell. Mesenchymal stem cells are multipotent but not pluripotent, which means they can differentiate into some, or “multiple,” but not all tissue types.1
Characterization
Mesenchymal stem cells or mesenchymal stromal cells (MSC) are plastic adherent and assume a fibroblast-like morphology during culture. They proliferate easily in culture and can be cryopreserved without loss of phenotype or differentiation potential. Additionally cell surface marker characterization via flow cytometry differentiates them from hematopoetic cells, though no truly unique MSC molecule has been identified. In part, the lack of definitive markers probably reflects the diverse lineage of MSC and the fact that each MSC population reflects to some degree the characteristics of tissues from which they were derived. Most importantly stem cells possess the ability to differentiate into cell types of multiple lineages including adipocytes, chrondrocytes, and osteocytes.1
Immunologic Properties
Mesenchymal stem cells clearly modulate immune responses, as demonstrated by both in vitro and in vivo studies. For example, MSC are poor antigen presenting cells and do not express MHC class II or co-stimulatory molecules and only low levels of MHC class I molecules.1 Thus, MSC are very non-immunogenic and can be transferred to fully allogeneic recipients and still mediate their immunologic effects. Among their other immunological properties, MSC inhibit lymphocyte proliferation and cytokine production, suppress dendritic cell function and alter DC cytokine production, and decrease IFN-g production by NK cells. 1 These properties of MSC can be harnessed therapeutically (as discussed below). In addition, it is also important to keep the immune modulatory properties of MSC in mind when interpreting the results of many stem cell therapy studies.
Stem Cells and Kidney Disease
There are numerous studies of MSC therapy in rodent models of renal failure, though most studies have focused on models of short-term protection from acute renal disease.2-5 The majority of these studies provide evidence that systemic administration of bone marrow-derived or adipose-tissue derived MSC can help preserve renal function in the face of acute insults and can also help reduce tubular injury and fibrosis. 2-5 Several studies have also demonstrated incorporation of small numbers of MSC into the renal parenchyma.4,6,7 It has been proposed that some of these MSC may actually differentiate into functional renal tubular epithelial cells, though this theory remains controversial. Other investigators propose that paracrine effects from the injected MSC are more important than the effects of direct cellular incorporation into the kidney.8,9 Thus, the available data indicate that systemically administered MSC can help improve or stabilize renal function in acute renal disease by a variety of mechanisms.
Fewer studies have investigated the effects of MSC therapy in chronic renal failure models in rodents.10-15 In the CKD rodent models that have been investigated, administration of MSC has been beneficial, especially with respect to reducing intra-renal inflammation and suppressing fibrosis and glomerulosclerosis.10,11,13,14,16 Results from a recent study, which utilized a rat remnant kidney model of CKD, are particularly noteworthy.10 In this study, the authors found that 3 i.v. injections of relatively low numbers of MSC led to significant and sustained improvement in renal function (eg, reduced serum creatinine concentrations and proteinuria) and markedly suppressed intra-renal fibrosis and inflammation. Repeated injections of MSC were found to be more effective than a single injection, and MSC additionally MSC were identified in the kidney after systemic injection. Thus, there is compelling evidence from rodent models of CKD that MSC injections can lead to significant improvement in renal function. MSC secrete a variety of biologically active factors and release of these factors, either systemically or locally into the kidney parenchyma, could affect renal function both directly and indirectly. In vitro studies have demonstrated that MSC can produce growth factors, cytokines, and anti-inflammatory mediators, all of which could help maintain or improve renal function and suppress intra-renal inflammation.8,17,18 The ability of MSC to suppress inflammation appears to be mediated both by secreted factors and by direct contact with inflammatory cells.17,18 Thus, MSC have the potential to strongly suppress intra-renal inflammation.
Chronic Kidney Disease
Prevalence of CKD
CKD remains a leading cause of illness and death in cats in the United States. This is a progressive disease, and currently no treatment short of renal transplantation has been shown to reverse or halt declining renal function for any significant period of time. This condition is characterized by tubulointerstitial damage, fibrosis and progressive loss of renal function, and is commonly described as the final common pathway after any one of multiple types of renal insults. Regardless of the initial insult, once a threshold of renal damage has been reached, progression is irreversible and appears consistent in character.19 Stem cell therapy has the potential to improve or stabilize renal function in animals with renal failure, based on evidence from rodent model studies. An appreciation of the pathophysiology of kidney disease is helpful in understanding why this might be the case.
Progression of CKD
Impairment of renal function is correlated with the degree of tubulointerstitial injury, including tubular atrophy, atubular glomeruli and tubulointerstitial fibrosis. Hyperfiltration, proteinuria, tubulointerstitial inflammation, oxidative damage and induction of the renin-angiotensin-aldosterone system (RAAS) are major factors that are thought to contribute to the process of tubulointerstitial injury.19-21
Loss of functional nephrons results in hyperfiltration due to an increase in remaining single nephron GFR. Combined with a loss of the ability to autoregulate blood flow to the kidney, hyperfiltration results in intraglomerular hypertension, which can be further exacerbated by systemic hypertension. Mechanical and sheer stress on the glomerular apparatus causes damage resulting in stretching of the membrane and leakage of proteins. Although the kidney filters a large amount of protein on a daily basis, urinary protein levels probably underestimate the filtered protein load due to the enormous capacity of the proximal tubule for protein reabsorption. However, proximal tubule cells are therefore prone to damage as a result. Proteins are reabsorbed through megalin- cubulin mediated endocytosis and then processed lysosomally. Protein excess results in overload of the lysosomal system and resultant rupture is damaging to tubular cells and instigates recruitment of inflammatory cells to the area. In addition, substances carried by the proteins such as, LPS, FFA, PG, heavy metals and hormones result in oxidative stress as well as direct damage to the cells.20
Tubulointerstitial inflammation is characterized by an inflammatory infiltrate in the interstitium that usually consists of mononuclear leucocytes including T-cells, monocytes and macrophages. These cells are thought to play a pivotal role in progression and fibrosis. Nephritogenic T-cells are thought to exacerbate the conditions present in the kidney through direct cytotoxic effects as well as non-cytotoxic mechanisms such as cytokine release, altered tubular function and proliferation of interstitial fibroblasts and fibrosis.3 Once fibrosis occurs, conditions become increasingly suboptimal as loss of peritubular capillaries and increased resistance to oxygen diffusion result in hypoxia, which in turn stimulates fibrosis.
Oxidative damage to tubular cells can occur as a result of direct harm from reabsorbed substances. However, increased filtration of substances such as glucose or protein also greatly increases the metabolism of tubule cells and this leads to increased production of free radicals. It has been shown that anti-oxidant defense systems are decreased in ESRD and thus the kidney is little prepared to address this increase and oxidative stress results. In addition, increase metabolic demand results in hypoxia as cells receive the same amount of blood flow, but have a higher demand. This condition is further exacerbated if anemia is present.20
The RAAS system is also a critical component in the pathophysiology of renal progression.21 Although normally protective in emergency situations such as shock or hypotension, the RAAS system becomes maladaptive in ESRD. Combined with a loss of autoregulation in the diseased state, Angiotensin II plays a major role in this pathologic process as it vasoconstricts the efferent arteriole to maintain GFR. Although GFR is maintained, the downsides of this mechanism are hypoperfusion of post-glomerular capillaries, including the interstitium, and glomerular capillary hypertension. Angiotensin II also has detrimental non-vascular effects including activating tubular cells, oxidative stress, stimulation of inflammatory cell accumulation and promotion of fibrosis. The importance of the RAAS system is further supported by clinical evidence demonstrating a distinct clinical benefit in RAAS blockade.21
All of the discussed factors; hyperfiltration, proteinuria, tubulointerstitial inflammation, oxidative damage and induction of the RAAS contribute to disruption of tubuloglomerular continuity and chronic hypoxia, which leads to further inflammation and fibrosis. The process of inflammation appears to be integral to the progressive, irreversible nature of CKD. It is based on this premise that mesenchymal stem cells have potential to ameliorate this disease process.
Current Clinical Stem Cell Research for Feline Chronic Kidney Disease
At present, there is little published work regarding the use of MSC for treatment of CKD. At the Center for Immune and Regenerative Medicine at Colorado State University, we are currently conducting research into the immunological properties of feline MSC, as well as the potential use of MSC for treatment of CKD in cats. Over the past 6 years the feline stem cell program at Colorado State has investigated basic feline stem cell biology and therapeutic applications. Other feline diseases under investigation for MSC therapy include feline asthma and inflammatory bowel disease. We previously determined that the most non-invasive and rich source for stem cells in cats is collection from fat.22 We initially completed a pilot study investigating the safety and potential effectiveness of unilateral intra-renal autologous MSC injections for cats with CKD.23 In that study, we found that the MSC injections were well-tolerated and may have improved renal function in some animals. However, the number of sedations required for the procedure limited its clinical applicability. In a recent series of pilot studies, we investigated the effectiveness of repeated intravenously delivered MSC for treatment of feline CKD, using allogeneic MSC derived from healthy young donor animals.24 The effect of MSC administration on kidney function in this clinical trial was variable. We continue to develop clinical trials to explore and optimize this potentially powerful treatment modality.
Summary
The fields of stem cell therapy and regenerative medicine are expanding rapidly. Veterinary medicine is poised to take a leading role in these fields, as there are a number of diseases in companion animals that would be amenable to stem cell therapy. Among the challenges facing these emerging fields are standardization of treatment protocols and adherence to strict principles of evidence-based medicine in reporting study results and conclusions. Nonetheless, it is likely that stem cell therapy will make significant progress in changing treatment paradigms for a number of important diseases of dogs and cats in the relatively near future.
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