A human tissue-based model of renal inflammation.

Inflammation plays a key role in both the onset and progression of various kidney diseases. However, the specific molecular and cellular mechanisms by which inflammation drives kidney diseases from different etiologies remain to be elucidated. To enhance our understanding of these mechanisms, a reliable and translational human model of renal inflammation is needed. Here, we aim to establish such a model using human precision-cut kidney slices (PCKS). The PCKS were prepared from fresh, macroscopically healthy kidney tissue and cultured for 3h-48h with or without tumor necrosis factor-α (TNFα), or its inhibitor Etanercept. The ensuing inflammatory response in the slices was evaluated using both qPCR and a cytokine array. Furthermore, the presence of immune cells was visualized using immunofluorescent staining, and the activation potential of tissue-resident macrophages was examined with ELISA. We observed a culture-induced inflammatory response, reflected by increased expression of pro-inflammatory genes TNF, IL1B, CCL2, and IL6. This response could be partially inhibited by Etanercept, indicating that TNFα plays a role in the observed response. Moreover, we found that TNFα stimulation further increased the gene expression of TNF, IL1B, CCL2, and IL6, as well as the production of several chemokines and cytokines, including CXCL5, MCP1, MCP3, and IL-6. Lastly, we observed the presence of CD14- and HLA-DR-positive cells, as well as proliferating (CD68- and PCNA-positive) and activated macrophages in the slices during incubation. In conclusion, this study presents a novel human model for investigating renal inflammation.

Metabolic MRI With Hyperpolarized 13 C-Pyruvate for Early Detection of Fibrogenic Kidney Metabolism.

OBJECTIVES: Fibrosis is the final common pathway for chronic kidney disease and the best predictor for disease progression. Besides invasive biopsies, biomarkers for its detection are lacking. To address this, we used hyperpolarized 13 C-pyruvate MRI to detect the metabolic changes associated with fibrogenic activity of myofibroblasts. MATERIALS AND METHODS: Hyperpolarized 13 C-pyruvate MRI was performed in 2 pig models of kidney fibrosis (unilateral ureteral obstruction and ischemia-reperfusion injury). The imaging data were correlated with histology, biochemical, and genetic measures of metabolism and fibrosis. The porcine experiments were supplemented with cell-line experiments to inform the origins of metabolic changes in fibrogenesis. Lastly, healthy and fibrotic human kidneys were analyzed for the metabolic alterations accessible with hyperpolarized 13 C-pyruvate MRI. RESULTS: In the 2 large animal models of kidney fibrosis, metabolic imaging revealed alterations in amino acid metabolism and glycolysis. Conversion from hyperpolarized 13 C-pyruvate to 13 C-alanine decreased, whereas conversion to 13 C-lactate increased. These changes were shown to reflect profibrotic activity in cultured epithelial cells, macrophages, and fibroblasts, which are important precursors of myofibroblasts. Importantly, metabolic MRI using hyperpolarized 13 C-pyruvate was able to detect these changes earlier than fibrosis-sensitive structural imaging. Lastly, we found that the same metabolic profile is present in fibrotic tissue from human kidneys. This affirms the translational potential of metabolic MRI as an early indicator of fibrogenesis associated metabolism. CONCLUSIONS: Our findings demonstrate the promise of hyperpolarized 13 C-pyruvate MRI for noninvasive detection of fibrosis development, which could enable earlier diagnosis and intervention for patients at risk of kidney fibrosis.

Magnetic resonance imaging as a noninvasive adjunct to conventional assessment of functional differences between kidneys in vivo and during ex vivo normothermic machine perfusion.

Normothermic machine perfusion (NMP) is increasingly considered for pretransplant kidney quality assessment. However, fundamental questions about differences between in vivo and ex vivo renal function, as well as the impact of ischemic injury on ex vivo physiology, remain unanswered. This study utilized magnetic resonance imaging (MRI), alongside conventional parameters to explore differences between in vivo and ex vivo renal function and the impact of warm ischemia on a kidney's behavior ex vivo. Renal MRI scans and samples were obtained from living pigs (n = 30) in vivo. Next, kidney pairs were procured and exposed to minimal, or 75 minutes of warm ischemia, followed by 6 hours of hypothermic machine perfusion. Both kidneys simultaneously underwent 6-hour ex vivo perfusion in MRI-compatible NMP circuits to obtain multiparametric MRI data. Ischemically injured ex vivo kidneys showed a significantly altered regional blood flow distribution compared to in vivo and minimally damaged organs. Both ex vivo groups showed diffusion restriction relative to in vivo. Our findings underscore the differences between in vivo and ex vivo MRI-based renal characteristics. Therefore, when assessing organ viability during NMP, it should be considered to incorporate parameters beyond the conventional functional markers that are common in vivo.

Multiplex immunofluorescence staining of coverslip-mounted paraffin-embedded tissue sections.

Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

H2S-Enriched Flush out Does Not Increase Donor Organ Quality in a Porcine Kidney Perfusion Model.

Kidney extraction time has a detrimental effect on post-transplantation outcome. This study aims to improve the flush-out and potentially decrease ischemic injury by the addition of hydrogen sulphide (H2S) to the flush medium. Porcine kidneys (n = 22) were extracted during organ recovery surgery. Pigs underwent brain death induction or a Sham operation, resulting in four groups: donation after brain death (DBD) control, DBD H2S, non-DBD control, and non-DBD H2S. Directly after the abdominal flush, kidneys were extracted and flushed with or without H2S and stored for 13 h via static cold storage (SCS) +/- H2S before reperfusion on normothermic machine perfusion. Pro-inflammatory cytokines IL-1b and IL-8 were significantly lower in H2S treated DBD kidneys during NMP (p = 0.03). The non-DBD kidneys show superiority in renal function (creatinine clearance and FENa) compared to the DBD control group (p = 0.03 and p = 0.004). No differences were seen in perfusion parameters, injury markers and histological appearance. We found an overall trend of better renal function in the non-DBD kidneys compared to the DBD kidneys. The addition of H2S during the flush out and SCS resulted in a reduction in pro-inflammatory cytokines without affecting renal function or injury markers.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14 days posttransplant. After 75 min of kidney warm ischemia, four experimental groups of n = 7 underwent 14 h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240 min of NMP with infusion of vehicle, 10 million porcine, or 10 million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14 days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Goal-Directed Fluid Therapy Does Not Improve Early Glomerular Filtration Rate in a Porcine Renal Transplantation Model.

BACKGROUND: Insufficient fluid administration intra- and postoperatively may lead to delayed renal graft function (DGF), while fluid overload increases the risk of heart failure, infection, and obstipation. Several different fluid protocols have been suggested to ensure optimal fluid state. However, there is a lack of evidence of the clinical impact of these regimens. This study aimed to determine whether individualized goal-directed fluid therapy (IGDT) positively affects the initial renal function compared to a high-volume fluid therapy (HVFT) and to examine the effects on renal endothelial glycocalyx, inflammatory and oxidative stress markers, and medullary tissue oxygenation. The hypothesis was that IGDT improves early glomerular filtration rate (GFR) in pigs subjected to renal transplantation. METHODS: This was an experimental randomized study. Using a porcine renal transplantation model, animals were randomly assigned to receive IGDT or HVFT during and until 1 hour after transplantation from brain-dead donors. The kidneys were exposed to 18 hours of cold ischemia. The recipients were observed until 10 hours after reperfusion, which included GFR measured as clearance of chrom-51-ethylendiamintetraacetat (Cr-EDTA), animal weight, and renal tissue oxygenation by fiber optic probes. The renal expression of inflammatory and oxidative stress markers as well as glomerular endothelial glycocalyx were analyzed in the graft using polymerase chain reaction (PCR) technique and immunofluorescence. RESULTS: Twenty-eight recipient pigs were included for analysis. We found no evidence that IGDT improved early GFR compared to HVFT (P = .45), while animal weight increased more in the HVFT group (a mean difference of 3.4 kg [1.96-4.90]; P < .0001). A better, however nonsignificant, preservation of glomerular glycocalyx (P = .098) and significantly lower levels of the inflammatory marker cyclooxygenase 2 (COX-2) was observed in the IGDT group when compared to HVFT. COX-2 was 1.94 (1.50-2.39; P = .012) times greater in the HVFT group when compared to the IGDT group. No differences were observed in outer medullary tissue oxygenation or oxidative stress markers. CONCLUSIONS: IGDT did not improve early GFR; however, it may reduce tissue inflammation and could possibly lead to preservation of the glycocalyx compared to HVFT.

Mesenchymal Stromal Cells Are Retained in the Porcine Renal Cortex Independently of Their Metabolic State After Renal Intra-Arterial Infusion.

The regenerative capacities of mesenchymal stromal cells (MSCs) make them suitable for renal regenerative therapy. The most common delivery route of MSC is through intravenous infusion, which is associated with off-target distribution. Renal intra-arterial delivery offers a targeted therapy, but limited knowledge is available regarding the fate of MSCs delivered through this route. Therefore, we studied the efficiency and tissue distribution of MSCs after renal intra-arterial delivery to a porcine renal ischemia-reperfusion model. MSCs were isolated from adipose tissue of healthy male pigs, fluorescently labeled and infused into the renal artery of female pigs. Flow cytometry allowed MSC detection and quantification in tissue and blood. In addition, quantitative polymerase chain reaction was used to trace MSCs by their Y-chromosome. During infusion, a minor number of MSCs left the kidney through the renal vein, and no MSCs were identified in arterial blood. Ischemic and healthy renal tissues were analyzed 30 min and 8 h after infusion, and 1-4 × 104 MSCs per gram of tissue were detected, predominantly, in the renal cortex, with a viability >70%. Confocal microscopy demonstrated mainly glomerular localization of MSCs, but they were also observed in the capillary network around tubuli. The infusion of heat-inactivated (HI) MSCs, which are metabolically inactive, through the renal artery showed that HI-MSCs were distributed in the kidney in a similar manner to regular MSCs, suggesting a passive retention mechanism. Long-term MSC survival was analyzed by Y-chromosome tracing, and demonstrated that a low percentage of the infused MSCs were present in the kidney 14 days after administration, while HI-MSCs were completely undetectable. In conclusion, renal intra-arterial MSC infusion limited off-target engraftment, leading to efficient MSC delivery to the kidney, most of them being cleared within 14 days. MSC retention was independent of the metabolic state of MSC, indicating a passive mechanism.

The unsuitability of implantable Doppler probes for the early detection of renal vascular complications - a porcine model for prevention of renal transplant loss.

BACKGROUND: Vascular occlusion is a rare, but serious complication after kidney transplantation often resulting in graft loss. We therefore aimed to develop an experimental porcine model for stepwise reduction of the renal venous blood flow and to compare an implantable Doppler probe and microdialysis for fast detection of vascular occlusion. METHODS: In 20 pigs, implantable Doppler probes were placed on the renal artery and vein and a microdialysis catheter was placed in the renal cortex. An arterial flowprobe served as gold standard. Following two-hour baseline measurements, the pigs were randomised to stepwise venous occlusion, complete venous occlusion, complete arterial occlusion or controls. RESULTS: All parameters were stable through baseline measurements. Glutamate and lactate measured by microdialysis increased significantly (p = 0.02 and p = 0.03 respectively) 30 minutes after a 2/3 (66%) reduction in renal blood flow. The implantable Doppler probe was not able to detect flow changes until there was total venous occlusion. Microdialysis detected changes in local metabolism after both arterial and venous occlusion; the implantable Doppler probe could only detect vascular occlusions on the vessel it was placed. CONCLUSIONS: We developed a new model for stepwise renal venous blood flow occlusion. Furthermore, the first comparison of the implantable Doppler probe and microdialysis for detection of renal vascular occlusions was made. The implantable Doppler probe could only detect flow changes after a complete occlusion, whereas microdialysis detected changes earlier, and could detect both arterial and venous occlusion. Based on these results, the implantable Doppler probe for early detection of vascular occlusions cannot be recommended.

Testing Danegaptide Effects on Kidney Function after Ischemia/Reperfusion Injury in a New Porcine Two Week Model.

INTRODUCTION: Ischemia/reperfusion injury (I/R-I) is a leading cause of acute kidney injury (AKI) and is associated with increased mortality. Danegaptide is a selective modifier of the gap junction protein connexion 43. It has cytoprotective as well as anti-arrhythmic properties and has been shown to reduce the size of myocardial infarct in pigs. The aim of this study was to investigate the ischemia-protective effect of Danegaptide in a porcine renal I/R-I model with two weeks follow up. METHODS: Unilateral renal I/R-I was induced in pigs by clamping the left renal artery over a two hour period. The model allowed examination of renal blood flow by magnetic resonance imaging (MRI) and the measurement of single kidney GFR two weeks after injury. Eleven animals were randomized to Danegaptide-infusion while nine animals received placebo. Kidney histology and urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion were included as markers of AKI. RESULTS: Unilateral kidney I/R-I resulted in an immediate ~50% GFR reduction, associated with a four-fold increase in urinary NGAL-excretion. Fourteen days after I/R-I, the total GFR was ~75% of baseline with a significantly lower GFR in the injured left kidney compared to the right kidney. No differences in GFR were observed between the treated and non-treated animals immediately after I/R-I or at Day 14. Furthermore, no differences were observed in the urinary excretion of NGAL, renal blood flow or other markers of renal function. CONCLUSIONS: As expected this porcine renal I/R-I model was associated with reduced GFR two weeks after injury. Danegaptide did not improve renal function after I/R-I.

No effect of remote ischaemic conditioning on inflammation in a porcine kidney transplantation model.

MAIN PROBLEM: Delayed graft function after kidney transplantation is associated with decreased graft survival and increased patient mortality but the pathogenesis is poorly understood. Remote ischaemic conditioning (rIC) may prevent delayed graft function by an anti-inflammatory effect. In a porcine model of transplantation from adults to children, we investigated the inflammatory response in the transplanted kidney and the effect of rIC. METHODS: Kidneys were recovered from brain dead donor pigs(63kg) and transplanted into two groups of recipient pigs(15kg) after 22h of cold ischaemia. Recipients were randomised to either: rIC (n=8) performed before the 10-h reperfusion period or no-rIC (n=8). Non-transplanted kidneys from eight brain dead pigs served as controls. RESULTS: Compared to controls, transplantation increased the number of apoptotic cells, macrophages and neutrophils in the kidney. After transplantation, IL-10 levels increased and IL-6 levels decreased in the kidney, whereas levels of TNF-α and IL-8 were not affected. A significant rise in plasma IL-1ß and IL-6 was observed in the recipients after transplantation. Plasma IL-10 was not affected by transplantation and TNF-α and IL-8 were below detection limit. No effect of rIC was found with regards to cell infiltration or cytokine production. CONCLUSION: Renal transplantation elicits an inflammatory response in the kidney manifested as apoptotic cell death, macrophage and neutrophil infiltration, and an anti-inflammatory cytokine response 10h after transplantation. This response was not modified by rIC.

α-Melanocyte stimulating hormone treatment in pigs does not improve early graft function in kidney transplants from brain dead donors.

Delayed graft function and primary non-function are serious complications following transplantation of kidneys derived from deceased brain dead (DBD) donors. α-melanocyte stimulating hormone (α-MSH) is a pleiotropic neuropeptide and its renoprotective effects have been demonstrated in models of acute kidney injury. We hypothesized that α-MSH treatment of the recipient improves early graft function and reduces inflammation following DBD kidney transplantation. Eight Danish landrace pigs served as DBD donors. After four hours of brain death both kidneys were removed and stored for 18 hours at 4°C in Custodiol preservation solution. Sixteen recipients were randomized in a paired design into two treatment groups, transplanted simultaneously. α-MSH or a vehicle was administered at start of surgery, during reperfusion and two hours post-reperfusion. The recipients were observed for ten hours following reperfusion. Blood, urine and kidney tissue samples were collected during and at the end of follow-up. α-MSH treatment reduced urine flow and impaired recovery of glomerular filtration rate (GFR) compared to controls. After each dose of α-MSH, a trend towards reduced mean arterial blood pressure and increased heart rate was observed. α-MSH did not affect expression of inflammatory markers. Surprisingly, α-MSH impaired recovery of renal function in the first ten hours following DBD kidney transplantation possibly due to hemodynamic changes. Thus, in a porcine experimental model α-MSH did not reduce renal inflammation and did not improve short-term graft function following DBD kidney transplantation.

Cell injury after ischemia and reperfusion in the porcine kidney evaluated by radiolabelled microspheres, sestamibi, and lactadherin.

BACKGROUND: The purpose of the present study was to quantify renal cell injury after ischemia and reperfusion in a pig model using (99m)Tc-lactadherin as a marker of apoptosis and (99m)Tc-sestamibi as a marker of mitochondrial dysfunction. METHODS: Thirty-four pigs were randomized into unilateral renal warm ischemia of 120 (WI120) or 240 min (WI240). The glomerular filtration rate (GFR) was calculated by renal clearance of (51)Cr-ethylenediaminetetraacetic acid, and apoptosis was quantified by immunohistochemical detection of caspase-3. After 240 min of reperfusion, intravenous (99m)Tc-lactadherin or (99m)Tc-sestamibi was injected simultaneously with (153)Gd microspheres into the aorta. Ex-vivo static planar images of the kidneys were acquired for determination of the differential renal function of tracer distribution using a gamma camera. RESULTS: In WI120, there was no significant difference in the uptake of microspheres in the ischemic and contralateral normal kidney indicating adequate perfusion (uptake in ischemic kidney relative to the sum of uptake in both kidneys; 46% ± 12% and 51% ± 5%). In WI240, the uptake of microspheres was severely reduced in both groups (17% ± 11% and 27% ± 17%). GFR was severely reduced in the post ischemic kidney in both groups. In both groups, the uptake of lactadherin was reduced (41% ± 8%, 17% ± 13%) but not different from the uptake of (153)Gd microspheres. Caspase-3-positive cell profiles were increased in the post-ischemic kidneys (p < 0.001) and increased as the length of ischemia increased (p = 0.003). In both WI120 and WI240, the amount of (99m)Tc-sestamibi in the ischemic kidney was significantly lower than the amount of (153)Gd microspheres (40 ± 5 versus 51 ± 5 and 20 ± 11 versus 27 ± 17; p < 0.05). CONCLUSIONS: In an established pig model with unilateral renal warm ischemia, we found significantly reduced (99m)Tc-sestamibi uptake relative to perfusion in the kidneys exposed to ischemia indicating a potential ability to detect renal ischemic and reperfusion injuries. However, apoptosis was not detected using (99m)Tc-lactadherin in the post-ischemic kidneys despite increased number of caspase-3-positive cell profiles. TRIAL REGISTRATION: This study is approved by the Danish Inspectorate of Animal Experiments (2010/561-1837).

Fast detection of renal ischemia in transplanted kidneys with delayed graft function-an experimental study.

BACKGROUND: The newly transplanted kidney is difficult to monitor with regard to postoperative vascular thrombosis, especially when there is delayed graft function. We evaluated microdialysis as a tool for early ischemia detection in porcine kidneys with delayed graft function early after transplantation. METHODS: Sixteen pigs were transplanted with 26-hr cold ischemia kidneys. A microdialysis catheter was placed in the lateral renal cortex. Five hours after graft reperfusion, the pigs were randomized to renal arterial clamping or open artery, n=8 in each group, and further observed for 2 hr. RESULTS: The diuresis and glomerular filtration rate were low and decreasing throughout the study, with no significant differences between groups. Until arterial clamping, there were no significant differences in the development of local renal metabolites between the two groups. Renal artery clamping immediately caused significantly different development of all metabolites (P<0.02 for all) compared to the open artery group. After clamping, levels of glutamate and glycerol were significantly increased within 30 min (P=0.0049 and P=0.0061, respectively). CONCLUSIONS: Microdialysis provided an early warning of arterial occlusion in transplanted grafts with delayed graft function. It may become a valuable tool for postoperative monitoring and detection of thrombosis after renal transplantation.

Improved GFR and renal plasma perfusion following remote ischaemic conditioning in a porcine kidney transplantation model.

Delayed graft function (DGF) complicates approximately 25% of kidney allografts donated after brain death (DBD). Remote ischaemic conditioning (rIC) involves brief, repetitive, ischaemia in a distant tissue in connection with ischaemia/reperfusion in the target organ. rIC has been shown to induce systemic protection against ischaemic injuries. Using a porcine kidney transplantation model with donor (63 kg) recipient (15 kg) size mismatch, we investigated the effects of recipient rIC on early renal plasma perfusion and GFR. Brain death was induced in donor pigs (n = 8) and kidneys were removed and kept in cold storage until transplantation. Nephrectomized recipient pigs were randomized to rIC (n = 8) or non-rIC (n = 8) with one kidney from the same donor in each group. rIC consisted of 4 × 5 min clamping of the abdominal aorta. GFR was significantly higher in the rIC group compared with non-rIC (7.2 ml/min vs. 3.4 ml/min; ΔGFR = 3.7 ml/min, 95%-CI: 0.3-7.2 ml/min, P = 0.038). Renal plasma perfusion in both cortex and medulla measured by dynamic contrast-enhanced magnetic resonance imaging (MRI) was significantly higher over time in the rIC group compared with non-rIC. This experimental study demonstrated a positive effect of rIC on early graft perfusion and function in a large animal transplantation model.

Early outcome in renal transplantation from large donors to small and size-matched recipients - a porcine experimental model.

Kidney transplantation from a large donor to a small recipient, as in pediatric transplantation, is associated with an increased risk of thrombosis and DGF. We established a porcine model for renal transplantation from an adult donor to a small or size-matched recipient with a high risk of DGF and studied GFR, RPP using MRI, and markers of kidney injury within 10 h after transplantation. After induction of BD, kidneys were removed from ∼63-kg donors and kept in cold storage for ∼22 h until transplanted into small (∼15 kg, n = 8) or size-matched (n = 8) recipients. A reduction in GFR was observed in small recipients within 60 min after reperfusion. Interestingly, this was associated with a significant reduction in medullary RPP, while there was no significant change in the size-matched recipients. No difference was observed in urinary NGAL excretion between the groups. A significant higher level of HO-1 mRNA was observed in small recipients than in donors and size-matched recipients indicating cortical injury. Improvement in early graft perfusion may be a goal to improve short- and long-term GFR and avoid graft thrombosis in pediatric recipients.