Rongjun Chen

Kidney Transplantation: Multiparametric Functional Magnetic Resonance Imaging for Assessment of Renal Allograft Pathophysiology in Mice.

ObjectivesThe aims of this experimental study were to investigate renal allograft pathophysiology by multiparametric functional magnetic resonance imaging (MRI) and to directly correlate MRI parameters with renal histopathology in mouse models of allogenic and isogenic kidney transplantation (ktx). Materials and MethodsAllograft rejection was induced by transplantation of C57BL/6 (B6) donor kidneys into BALB/c recipients (allogenic ktx). B6 mice that received B6 kidneys served as controls (isogenic ktx). Three weeks after ktx, MRI was performed using a 7-T small-animal scanner. Flow sensitive alternating inversion recovery echoplanar imaging arterial spin labeling, multiecho turbo spin echo, and diffusion-weighted imaging sequences were acquired. Maps of renal perfusion, T2 and T1 relaxation times, and apparent diffusion coefficients were calculated. Histological changes in the kidney were evaluated according to Banff criteria. Renal cell infiltrates and fibrosis were quantified by immunohistochemistry. Differences between groups were assessed using the Mann-Whitney U test, and the correlation of MRI parameters with renal histopathology was determined by Spearman correlation analysis. ResultsAfter allogenic, but not isogenic, ktx, animals developed acute allograft rejection. Allogenic grafts were infiltrated by macrophages and T-lymphocytes and exhibited marked renal fibrosis. Magnetic resonance imaging revealed stronger impairment of renal perfusion (56 ± 7 vs 293 ± 44 mL/[min × 100 g]; P < 0.01) and more pronounced increases in T2 (60.1 ± 2.0 vs 45.7 ± 1.2 milliseconds, P < 0.01) and T1 relaxation times (1938 ± 53 vs 1350 ± 27 milliseconds, P < 0.01) in allogenic than in isogenic kidneys. Apparent diffusion coefficient was reduced to 1.39 ± 0.14 × 10−3 mm2/s in kidneys with an acute rejection and was 1.83 ± 0.05 × 10−3 mm2/s in isogenic kidneys without rejection (P < 0.05). Magnetic resonance imaging parameters significantly correlated with the amount of cellular infiltration and renal fibrosis observed histologically. ConclusionsFunctional MRI allows detection of acute renal allograft rejection after allogenic ktx in mice. Functional MRI parameters correlate with cell infiltrates and fibrosis. Thus, MRI may be used noninvasively and longitudinally to investigate mechanisms of renal allograft rejection and evaluate novel therapeutic strategies in experimental studies.

A Novel Therapy to Attenuate Acute Kidney Injury and Ischemic Allograft Damage after Allogenic Kidney Transplantation in Mice

Ischemia followed by reperfusion contributes to the initial damage to allografts after kidney transplantation (ktx). In this study we tested the hypothesis that a tetrapeptide EA-230 (AQGV), might improve survival and attenuate loss of kidney function in a mouse model of renal ischemia/reperfusion injury (IRI) and ischemia-induced delayed graft function after allogenic kidney transplantation. IRI was induced in male C57Bl/6N mice by transient bilateral renal pedicle clamping for 35 min. Treatment with EA-230 (20–50mg/kg twice daily i.p. for four consecutive days) was initiated 24 hours after IRI when acute kidney injury (AKI) was already established. The treatment resulted in markedly improved survival in a dose dependent manner. Acute tubular injury two days after IRI was diminished and tubular epithelial cell proliferation was significantly enhanced by EA-230 treatment. Furthermore, CTGF up-regulation, a marker of post-ischemic fibrosis, at four weeks after IRI was significantly less in EA-230 treated renal tissue. To learn more about these effects, we measured renal blood flow (RBF) and glomerular filtration rate (GFR) at 28 hours after IRI. EA-230 improved both GFR and RBF significantly. Next, EA-230 treatment was tested in a model of ischemia-induced delayed graft function after allogenic kidney transplantation. The recipients were treated with EA-230 (50 mg/kg) twice daily i.p. which improved renal function and allograft survival by attenuating ischemic allograft damage. In conclusion, EA-230 is a novel and promising therapeutic agent for treating acute kidney injury and preventing IRI-induced post-transplant ischemic allograft injury. Its beneficial effect is associated with improved renal perfusion after IRI and enhanced regeneration of tubular epithelial cells.

Oral treatment of rodents with soluble epoxide hydrolase inhibitor 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea (TPPU): Resulting drug levels and modulation of oxylipin pattern.

Epoxides from polyunsaturated fatty acids (PUFAs) are potent lipid mediators. In vivo stabilization of these epoxides by blockade of the soluble epoxide hydrolase (sEH) leads to anti-inflammatory, analgesic and normotensive effects. Therefore, sEH inhibitors (sEHi) are a promising new class of drugs. Herein, we characterized pharmacokinetic (PK) and pharmacodynamic properties of a commercially available potent sEHi 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea (TPPU). Cell culture studies suggest its high absorption and metabolic stability. Following administration in drinking water to rats (0.2, 1, and 5mg TPPU/L with 0.2% PEG400), TPPUs blood concentration increased dose dependently within the treatment period to reach an almost steady state after 8 days. TPPU was found in all the tissues tested. The linoleic epoxide/diol ratios in most tissues were dose dependently increased, indicating significant sEH inhibition. Overall, administration of TPPU with the drinking water led to systemic distribution as well as high drug levels and thus makes chronic sEH inhibition studies possible.

Multiparametric Functional MRI: Non-Invasive Imaging of Inflammation and Edema Formation after Kidney Transplantation in Mice.

Background Kidney transplantation (ktx) in mice is used to learn about rejection and to develop new treatment strategies. Past studies have mainly been based on histological or molecular biological methods. Imaging techniques to monitor allograft pathology have rarely been used. Methods Here we investigated mice after isogenic and allogenic ktx over time with functional MRI with diffusion-weighted imaging (DWI) and mapping of T2-relaxation time (T2-mapping) to assess graft inflammation and edema formation. To characterize graft pathology, we used PAS-staining, counted CD3-positive T-lymphocytes, analyzed leukocytes by means flow cytometry. Results DWI revealed progressive restriction of diffusion of water molecules in allogenic kidney grafts. This was paralleled by enhanced infiltration of the kidney by inflammatory cells. Changes in tissue diffusion were not seen following isogenic ktx. T2-times in renal cortex were increased after both isogenic and allogenic transplantation, consistent with tissue edema due to ischemic injury following prolonged cold ischemia time of 60 minutes. Lack of T2 increase in the inner stripe of the inner medulla in allogenic kidney grafts matched loss of tubular autofluorescence and may result from rejection-driven reductions in tubular water content due to tubular dysfunction and renal functional impairment. Conclusions Functional MRI is a valuable non-invasive technique for monitoring inflammation, tissue edema and tubular function. It permits on to differentiate between acute rejection and ischemic renal injury in a mouse model of ktx.

Functional MRI for characterization of renal perfusion impairment and edema formation due to acute kidney injury in different mouse strains

Purpose The purpose was to characterize acute kidney injury (AKI) in C57BL/6 (B6)- and 129/Sv (Sv)-mice by noninvasive measurement of renal perfusion and tissue edema using functional MRI. Methods Different severities of AKI were induced in B6- and Sv-mice by renal ischemia reperfusion injury (IRI). Unilateral clamping of the renal pedicle for 35 min (moderate AKI) or 45 min (severe AKI) was done. MRI (7-Tesla) was performed 1, 7 and 28 days after surgery using a flow alternating inversion recovery (FAIR) arterial spin labeling (ASL) sequence. Maps of perfusion and T1-relaxation time were calculated. Relative MRI-parameters of the IRI kidney compared to the contralateral not-clipped kidney were compared between AKI severities and between mouse strains using unpaired t-tests. In addition, fibrosis was assessed by Masson Trichrome and collagen IV staining. Results After moderate AKI relative perfusion impairment was significantly higher in B6- than in Sv-mice at d7 (55±7% vs. 82±8%, p<0.05) and d28 (76±7% vs. 102±3%, p<0.01). T1-values increased in the early phase after AKI in both mouse strains. T1-increase was more severe after prolonged ischemia times of 45 min compared to 35 min in both mouse strains, measured in the renal cortex and outer stripe of outer medulla. Kidney volume loss (compared to the contralateral kidney) occurred already after 7 days but proceeded markedly towards 4 weeks in severe AKI. Early renal perfusion impairment was predictive for later kidney volume loss. The progression to chronic kidney disease (CKD) in the severe AKI model was similar in both mouse strains as revealed by histology. Conclusion Quantification of renal perfusion and tissue edema by functional MRI allows characterization of strain differences upon AKI. Renal perfusion impairment was stronger in B6- compared to Sv-animals following moderate AKI. Prolonged ischemia times were associated with more severe perfusion impairment and edema formation in the early phase and progression to CKD within 4 weeks of observation.

Sodium Channel Nav1.3 Is Expressed by Polymorphonuclear Neutrophils during Mouse Heart and Kidney Ischemia In Vivo and Regulates Adhesion, Transmigration, and Chemotaxis of Human and Mouse Neutrophils In Vitro

Background: Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed noncanonical roles in nonexcitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils. Methods: Expression of voltage-gated sodium channels was analyzed by polymerase chain reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration, and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with a whole cell patch clamp. Results: Mouse and human neutrophils express multiple voltage-gated sodium channels. Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7%, n = 14) and kidney (19 ± 2%, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9%, unselective Nav-inhibitor), ICA121431 (53 ± 10%), and Pterinotoxin-2 (55 ± 9%; preferential inhibitors of Nav1.3, n = 10). Tetrodotoxin (56 ± 19%), ICA121431 (62 ± 22%), and Pterinotoxin-2 (59 ± 22%) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3 × 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9% (n = 10) and transmigration to 54 ± 8% (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2. Conclusions: Nav1.3 is expressed in neutrophils in vivo; regulates attachment, transmigration, and chemotaxis in vitro; and may serve as a relevant target for antiinflammatory effects of lidocaine.

IL-17A blockade or deficiency does not affect progressive renal fibrosis following renal ischaemia reperfusion injury in mice

IL‐17A contributes to acute kidney injury and fibrosis. Therefore, we asked whether IL‐17A deficiency or treatment with a IL‐17A blocking antibody impacts severe renal ischaemia reperfusion injury (IRI) and the progression to chronic kidney disease (CKD).

Longitudinal evaluation of perfusion changes in acute and chronic renal allograft rejection using arterial spin labeling in translational mouse models

To examine the longitudinal changes of renal perfusion due to acute and chronic renal allograft rejection by using arterial spin labeling (ASL) MRI in translational mouse models of isogenic and allogenic kidney transplantation (ktx).

Alpha1‐antitrypsin binds hemin and prevents oxidative activation of human neutrophils: putative pathophysiological significance

Heme is a ubiquitous compound of human tissues, and it is involved in cellular physiology and metabolism. Once released from the cell, free heme oxidizes to the ferric state (hemin). High levels of hemin can cause oxidative stress and inflammation if not neutralized immediately by specialized scavenger proteins. Human alpha1‐antitrypsin (A1AT), an acute‐phase glycoprotein and important inhibitor of neutrophil proteases, is also a hemin‐binding protein. A short‐term exposure of freshly isolated human blood neutrophils to 4 µM hemin results in cell spreading, surface expression of filament protein, vimentin, free radical production, expression of heme oxygenase‐1 (HO‐1), release of IL‐8, and enhanced neutrophil adhesion to human endothelial cells. Consequently, the phosphorylation of protein kinase C (PKC) occurs after 25 min. Under the same experimental conditions, addition of 1 mg/ml A1AT markedly reduces or abolishes neutrophil‐activating effects of hemin and prevents PKC phosphorylation. In a mouse model of acute kidney injury (AKI) plus injection of hemin, monotherapy with 4 mg/mouse A1AT significantly lowered serum levels of free hemin at 2 h after surgery. Moreover, a tendency toward lower AKI scores, reduced infiltration of neutrophils, and lower levels of serum chemokine [CXCL1/keratinocyte‐derived chemokine (KC)] was observed. Our findings highlight A1AT as a potential serum scavenger of hemin and suggest that the commercial preparations of human plasma A1AT might prove to be useful therapeutics in conditions associated with hemolysis.

Ischemia Reperfusion Injury (IRI) causes Local Release of Free Heme which Aggravates Inflammation and Contributes to Delayed Graft Function

Ischemia reperfusion injury (IRI) is relevant in solid organ transplantation and contributes to delayed graft function (DGF). In this study, release of free heme after renal IRI and the consecutive inflammatory response were studied in mice. Methods Renal IRI was induced by 15, 35 and 45 min unilateral renal pedicle clamping in mice. Sham surgery served as control. Mice were sacrificed at 2 and 4 and 24 hours after IRI. Free heme was measured in the kidney and systemic complement activation was measured in blood samples. qPCR for pro-inflammatory cytokine expression, histology and immunohistochemistry for acute kidney injury were done. Results In correlation with increased duration of ischemia time the free heme generation in the tissue increased and enhanced local pro-inflammatory cytokine release (TNF-alpha, MCP-1, IL-6) was measured. AKI score and inflammatory cell infiltration into the tissue increased as well. Complement activation was higher in correlation with longer ischemia time Conclusion Free heme release in ischemic organs aggravates local inflammation. Strategies to reduce free heme production prior to solid organ transplantation would be promising therapeutic approaches to reduce the risk of DGF.