Murine models of renal ischemia reperfusion injury: An opportunity for refinement using noninvasive monitoring methods.

BACKGROUND: Renal ischemia reperfusion injury (R-IRI) can cause acute kidney injury (AKI) and chronic kidney disease (CKD), resulting in significant morbidity and mortality. To understand the underlying mechanisms, reproducible small-animal models of AKI and CKD are needed. We describe how innovative technologies for measuring kidney function noninvasively in small rodents allow successful refinement of the R-IRI models, and offer the unique opportunity to monitor longitudinally in individual animals the transition from AKI to CKD. METHODS: Male BALB/c mice underwent bilateral renal pedicle clamping (AKI) or unilateral renal pedicle clamping with delayed contralateral nephrectomy (CKD) under isoflurane anesthetic. Transdermal GFR monitoring and multispectral optoacoustic tomography (MSOT) in combination with statistical analysis were used to identify and standardize variables within these models. RESULTS: Pre-clamping anesthetic time was one of the most important predictors of AKI severity after R-IRI. Standardizing pre-clamping time resulted in a more predictably severe AKI model. In the CKD model, MSOT demonstrated initial improvement in renal function, followed by significant progressive reduction in function between weeks 2 and 4. Performing contralateral nephrectomy on day 14 enabled the development of CKD with minimal mortality. CONCLUSIONS: Noninvasive monitoring of global and individual renal function after R-IRI is feasible and reproducible. These techniques can facilitate refinement of kidney injury models and enable the degree of injury seen in preclinical models to be translated to those seen in the clinical setting. Thus, future therapies can be tested in a clinically relevant, noninvasive manner.

Tubular ß-catenin and FoxO3 interactions protect in chronic kidney disease.

The Wnt/ß-catenin signaling pathway plays an important role in renal development and is reexpressed in the injured kidney and other organs. ß-Catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/ß-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular ß-catenin signaling in CKD remains unclear, we genetically stabilized (i.e., activated) ß-catenin specifically in murine proximal tubules. Mice with increased tubular ß-catenin signaling were protected in 2 murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional T cell factor/lymphoid enhancer factor-dependent ß-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular ß-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine γ-lyase as a potentially novel transcriptional target of ß-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturned the conventional dogma about ß-catenin signaling and CKD by showing a protective effect of proximal tubule ß-catenin in CKD and identified a potentially new transcriptional target of ß-catenin/FoxO3 signaling that has therapeutic potential for CKD.

Multimodal Imaging Techniques Show Differences in Homing Capacity Between Mesenchymal Stromal Cells and Macrophages in Mouse Renal Injury Models.

PURPOSE: The question of whether mesenchymal stromal cells (MSCs) home to injured kidneys remains a contested issue. To try and understand the basis for contradictory findings reported in the literature, our purpose here was to investigate whether MSC homing capacity is influenced by administration route, the type of injury model used, and/or the presence of exogenous macrophages. PROCEDURES: To assess the viability, whole-body biodistribution, and intra-renal biodistribution of MSCs, we used a multimodal imaging strategy comprising bioluminescence and magnetic resonance imaging. The effect of administration route (venous or arterial) on the ability of MSCs to home to injured renal tissue, and persist there, was assessed in a glomerular injury model (induced by the nephrotoxicant, Adriamycin) and a tubular injury model induced by ischaemia-reperfusion injury (IRI). Exogenous macrophages were used as a positive control because these cells are known to home to injured mouse kidneys. To assess whether the homing capacity of MSCs can be influenced by the presence of exogenous macrophages, we used a dual-bioluminescence strategy that allowed the whole-body biodistribution of the two cell types to be monitored simultaneously in individual animals. RESULTS: Following intravenous administration, no MSCs were detected in the kidneys, irrespective of whether the mice had been subjected to renal injury. After arterial administration via the left cardiac ventricle, MSCs transiently populated the kidneys, but no preferential homing or persistence was observed in injured renal tissue after unilateral IRI. An exception was when MSCs were co-administered with exogenous macrophages; here, we observed some homing of MSCs to the injured kidney. CONCLUSIONS: Our findings strongly suggest that MSCs do not home to injured kidneys.

Capillary rarefaction is more closely associated with CKD progression after cisplatin, rhabdomyolysis, and ischemia-reperfusion-induced AKI than renal fibrosis.

Acute kidney injury (AKI) is a strong independent predictor of mortality and often results in incomplete recovery of renal function, leading to progressive chronic kidney disease (CKD). Many clinical trials have been conducted on the basis of promising preclinical data, but no therapeutic interventions have been shown to improve long-term outcomes after AKI. This is partly due to the failure of preclinical studies to accurately model clinically relevant injury and long-term outcomes on CKD progression. Here, we evaluated the long-term effects of AKI on CKD progression in three animal models reflecting diverse etiologies of AKI: repeat-dose cisplatin, rhabdomyolysis, and ischemia-reperfusion injury. Using transdermal measurement of glomerular filtration rate as a clinically relevant measure of kidney function and quantification of peritubular capillary density to measure capillary rarefaction, we showed that repeat-dose cisplatin caused capillary rarefaction and decreased renal function in mice without a significant increase in interstitial fibrosis, whereas rhabdomyolysis-induced AKI led to severe interstitial fibrosis, but renal function and peritubular capillary density were preserved. Furthermore, long-term experiments in mice with unilateral ischemia-reperfusion injury showed that restoration of renal function 12 wk after a contralateral nephrectomy was associated with increasing fibrosis, but a reversal of capillary rarefaction was seen at 4 wk. These data demonstrate that clear dissociation between kidney function and fibrosis in these models of AKI to CKD progression and suggest that peritubular capillary rarefaction is more strongly associated with CKD progression than renal fibrosis.

Cell-free hemoglobin augments acute kidney injury during experimental sepsis.

Acute kidney injury is a common complication of severe sepsis and contributes to high mortality. The molecular mechanisms of acute kidney injury during sepsis are not fully understood. Because hemoproteins, including myoglobin and hemoglobin, are known to mediate kidney injury during rhabdomyolysis, we hypothesized that cell-free hemoglobin (CFH) would exacerbate acute kidney injury during sepsis. Sepsis was induced in mice by intraperitoneal injection of cecal slurry (CS). To mimic elevated levels of CFH observed during human sepsis, mice also received a retroorbital injection of CFH or dextrose control. Four groups of mice were analyzed: sham treated (sham), CFH alone, CS alone, and CS + CFH. The addition of CFH to CS reduced 48-h survival compared with CS alone (67% vs. 97%, P = 0.001) and increased the severity of illness. After 24 and 48 h, CS + CFH mice had a reduced glomerular filtration rate from baseline, whereas sham, CFH, and CS mice maintained baseline glomerular filtration rate. Biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), were markedly elevated in CS+CFH compared with CS (8-fold for NGAL and 2.4-fold for KIM-1, P < 0.002 for each) after 48 h. Histological examination showed a trend toward increased tubular injury in CS + CFH-exposed kidneys compared with CS-exposed kidneys. However, there were similar levels of renal oxidative injury and apoptosis in the CS + CFH group compared with the CS group. Kidney levels of multiple proinflammatory cytokines were similar between CS and CS + CFH groups. Human renal tubule cells (HK-2) exposed to CFH demonstrated increased cytotoxicity. Together, these results show that CFH exacerbates acute kidney injury in a mouse model of experimental sepsis, potentially through increased renal tubular injury.

Long-term outcomes in mouse models of ischemia-reperfusion-induced acute kidney injury.

Severe acute kidney injury has a high mortality and is a risk factor for progressive chronic kidney disease. None of the potential therapies that have been identified in preclinical studies have successfully improved clinical outcomes. This failure is partly because animal models rarely reflect the complexity of human disease: most preclinical studies are short term and are commonly performed in healthy, young, male mice. Therapies that are effective in preclinical models that share common clinical features seen in patients with acute kidney injury, including genetic diversity, different sexes, and comorbidities, and evaluate long-term outcomes are more likely to predict success in the clinic. Here, we evaluated susceptibility to chronic kidney disease after ischemia-reperfusion injury with delayed nephrectomy by monitoring long-term functional and histological responses to injury. We defined conditions required to induce long-term postinjury renal dysfunction and fibrosis without increased mortality in a reproducible way and evaluate effect of mouse strains, sexes, and preexisting diabetes on these responses.

A Noninvasive Imaging Toolbox Indicates Limited Therapeutic Potential of Conditionally Activated Macrophages in a Mouse Model of Multiple Organ Dysfunction.

Cell-based regenerative medicine therapies require robust preclinical safety, efficacy, biodistribution, and engraftment data prior to clinical testing. To address these challenges, we have developed an imaging toolbox comprising multispectral optoacoustic tomography and ultrasonography, which allows the degree of kidney, liver, and cardiac injury and the extent of functional recovery to be assessed noninvasively in a mouse model of multiorgan dysfunction. This toolbox allowed us to determine the therapeutic effects of adoptively transferred macrophages. Using bioluminescence imaging, we could then investigate the association between amelioration and biodistribution. Macrophage therapy provided limited improvement of kidney and liver function, although not significantly so, without amelioration of histological damage. No improvement in cardiac function was observed. Biodistribution analysis showed that macrophages homed and persisted in the injured kidneys and liver but did not populate the heart. Our data suggest that the limited improvement observed in kidney and liver function could be mediated by M2 macrophages. More importantly, we demonstrate here the utility of the imaging toolbox for assessing the efficacy of potential regenerative medicine therapies in multiple organs.

Transdermal Measurement of Glomerular Filtration Rate in Mice.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Non-invasive imaging reveals conditions that impact distribution and persistence of cells after in vivo administration.

BACKGROUND: Cell-based regenerative medicine therapies are now frequently tested in clinical trials. In many conditions, cell therapies are administered systemically, but there is little understanding of their fate, and adverse events are often under-reported. Currently, it is only possible to assess safety and fate of cell therapies in preclinical studies, specifically by monitoring animals longitudinally using multi-modal imaging approaches. Here, using a suite of in vivo imaging modalities to explore the fate of a range of human and murine cells, we investigate how route of administration, cell type and host immune status affect the fate of administered cells. METHODS: We applied a unique imaging platform combining bioluminescence, optoacoustic and magnetic resonance imaging modalities to assess the safety of different human and murine cell types by following their biodistribution and persistence in mice following administration into the venous or arterial system. RESULTS: Longitudinal imaging analyses (i) suggested that the intra-arterial route may be more hazardous than intravenous administration for certain cell types, (ii) revealed that the potential of a mouse mesenchymal stem/stromal cell (MSC) line to form tumours depended on administration route and mouse strain and (iii) indicated that clinically tested human umbilical cord (hUC)-derived MSCs can transiently and unexpectedly proliferate when administered intravenously to mice. CONCLUSIONS: In order to perform an adequate safety assessment of potential cell-based therapies, a thorough understanding of cell biodistribution and fate post administration is required. The non-invasive imaging platform used here can expose not only the general organ distribution of these therapies, but also a detailed view of their presence within different organs and, importantly, tumourigenic potential. Our observation that the hUC-MSCs but not the human bone marrow (hBM)-derived MSCs persisted for a period in some animals suggests that therapies with these cells should proceed with caution.

Preclinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies.

Regenerative medicine therapies hold enormous potential for a variety of currently incurable conditions with high unmet clinical need. Most progress in this field to date has been achieved with cell-based regenerative medicine therapies, with over a thousand clinical trials performed up to 2015. However, lack of adequate safety and efficacy data is currently limiting wider uptake of these therapies. To facilitate clinical translation, non-invasive in vivo imaging technologies that enable careful evaluation and characterisation of the administered cells and their effects on host tissues are critically required to evaluate their safety and efficacy in relevant preclinical models. This article reviews the most common imaging technologies available and how they can be applied to regenerative medicine research. We cover details of how each technology works, which cell labels are most appropriate for different applications, and the value of multi-modal imaging approaches to gain a comprehensive understanding of the responses to cell therapy in vivo.

Imaging technologies for monitoring the safety, efficacy and mechanisms of action of cell-based regenerative medicine therapies in models of kidney disease.

The incidence of end stage kidney disease is rising annually and it is now a global public health problem. Current treatment options are dialysis or renal transplantation, which apart from their significant drawbacks in terms of increased morbidity and mortality, are placing an increasing economic burden on society. Cell-based Regenerative Medicine Therapies (RMTs) have shown great promise in rodent models of kidney disease, but clinical translation is hampered due to the lack of adequate safety and efficacy data. Furthermore, the mechanisms whereby the cell-based RMTs ameliorate injury are ill-defined. For instance, it is not always clear if the cells directly replace damaged renal tissue, or whether paracrine effects are more important. Knowledge of the mechanisms responsible for the beneficial effects of cell therapies is crucial because it could lead to the development of safer and more effective RMTs in the future. To address these questions, novel in vivo imaging strategies are needed to monitor the biodistribution of cell-based RMTs and evaluate their beneficial effects on host tissues and organs, as well as any potential adverse effects. In this review we will discuss how state-of-the-art imaging modalities, including bioluminescence, magnetic resonance, nuclear imaging, ultrasound and an emerging imaging technology called multispectral optoacoustic tomography, can be used in combination with various imaging probes to track the fate and biodistribution of cell-based RMTs in rodent models of kidney disease, and evaluate their effect on renal function.

Measures of kidney function by minimally invasive techniques correlate with histological glomerular damage in SCID mice with adriamycin-induced nephropathy.

Maximising the use of preclinical murine models of progressive kidney disease as test beds for therapies ideally requires kidney function to be measured repeatedly in a safe, minimally invasive manner. To date, most studies of murine nephropathy depend on unreliable markers of renal physiological function, exemplified by measuring blood levels of creatinine and urea, and on various end points necessitating sacrifice of experimental animals to assess histological damage, thus counteracting the principles of Replacement, Refinement and Reduction. Here, we applied two novel minimally invasive techniques to measure kidney function in SCID mice with adriamycin-induced nephropathy. We employed i) a transcutaneous device that measures the half-life of intravenously administered FITC-sinistrin, a molecule cleared by glomerular filtration; and ii) multispectral optoacoustic tomography, a photoacoustic imaging device that directly visualises the clearance of the near infrared dye, IRDye 800CW carboxylate. Measurements with either technique showed a significant impairment of renal function in experimental animals versus controls, with significant correlations with the proportion of scarred glomeruli five weeks after induction of injury. These technologies provide clinically relevant functional data and should be widely adopted for testing the efficacies of novel therapies. Moreover, their use will also lead to a reduction in experimental animal numbers.