Assessment of Renal Transplant Perfusion by Contrast-Enhanced Ultrasound after Switch from Calcineurin Inhibitor to Belatacept: A Pilot Study.

Calcineurin inhibitors (CNIs) have improved short-term kidney allograft survival but are nephrotoxic and vasoconstrictive. Vasoconstriction is potentially reversible after switching from CNIs to belatacept. The kidney allograft shows optimal requirements for dynamic perfusion imaging using contrast-enhanced ultrasound (CEUS). We performed standardized CEUS in patients after switching from CNIs to belatacept for clinical indication to study the suitability of CEUS, in order to assess the effects of CNI cessation on kidney allograft perfusion. Eleven kidney transplant patients were enrolled from February 2020 until November 2020. Demographic, clinical, and laboratory parameters, as well as perfusion imaging, were assessed at baseline and 6 months after switching immunosuppression. Quantification of perfusion imaging on CEUS was performed using a post-processing software tool on uncompressed DICOM cine loops. After CNI cessation, estimated glomerular filtration rate increased by 4.8 mL/min/1.73 m2 (16%). Despite good quality of fit and comparable regions of interest in baseline and follow-up CEUS examinations, quantification of perfusion imaging showed a slightly improved cortical perfusion without reaching statistical significance after CNI cessation. This is the first study that systematically investigates the suitability of CEUS to detect changes of microvascular perfusion in kidney transplant recipients in vivo. No significant differences could be detected in perfusion measurements before and after CNI cessation.

Drug-induced CYP induction as therapy for tacrolimus intoxication.

Management of calcineurin inhibitor (CNI) therapy in kidney transplant recipients may be complicated due to polypharmacy. As CNI undergo extensive metabolism by cytochrome-P450 enzymes (CYP), drug-mediated CYP inhibition poses a risk for elevated CNI blood concentrations. Here, we report on 2 kidney transplant recipients treated with tacrolimus who presented with signs of tacrolimus intoxication at admission. Patient A was started on antiviral medication ombitasvir, paritaprevir, ritonavir, and dasabuvir for hepatitis C virus treatment 3 days prior to hospitalization. Patient B was treated with clarithromycin for pneumonia. Both therapies cause drug-mediated CYP inhibition, and both patients displayed highly elevated tacrolimus serum concentrations and acute kidney injury (Table 1). After application of the CYP-inducing agents rifampicin and phenytoin, respectively, tacrolimus levels were rapidly reduced, and renal function recovered. Treating severe CNI intoxication is an infrequent yet emergent condition. These results add to the knowledge of therapeutic drug-induced CYP induction as rescue therapy.

"Point of no return" in unilateral renal ischemia reperfusion injury in mice.

BACKGROUND: In the past years evidence has been growing about the interconnection of chronic kidney disease and acute kidney injury. The underlying pathophysiological mechanisms remain unclear. We hypothesized, that a threshold ischemia time in unilateral ischemia/reperfusion injury sets an extent of ischemic tubule necrosis, which as "point of no return" leads to progressive injury. This progress is temporarily associated by increased markers of inflammation and results in fibrosis and atrophy of the ischemic kidney. METHODS: Acute tubule necrosis was induced by unilateral ischemia/reperfusion injury in male C57BL/6 N mice with different ischemia times (15, 25, 35, and 45 min). At multiple time points between 15 min and 5 weeks we assessed gene expression of markers for injury, inflammation, and fibrosis, histologically the injury of tubules, cell death (TUNEL), macrophages, neutrophil influx and kidney atrophy. RESULTS: Unilateral ischemia for 15 and 25 min induced upregulation of markers for injury after reperfusion for 24 h but no upregulation after 5 weeks. None of the markers for inflammation or fibrosis were upregulated after ischemia for 15 and 25 min at 24 h or 5 weeks on a gene expression level, except for Il-6. Ischemia for 35 and 45 min consistently induced upregulation of markers for inflammation, injury, and partially of fibrosis (Tgf-ß1 and Col1a1) at 24 h and 5 weeks. The threshold ischemia time for persistent injury of 35 min induced a temporal association of markers for inflammation and injury with peaks between 6 h and 7 d along the course of 10 d. This ischemia time also induced persistent cell death (TUNEL) throughout observation for 5 weeks with a peak at 6 h and progressing kidney atrophy beginning 7 d after ischemia. CONCLUSIONS: This study confirms the evidence of a threshold extent of ischemic injury in which markers of injury, inflammation and fibrosis do not decline to baseline but remain upregulated assessed in long term outcome (5 weeks). Excess of this threshold as "point of no return" leads to persistent cell death and progressing atrophy and is characterized by a temporal association of markers for inflammation and injury.

Gene expression profiling of the Notch-AhR-IL22 axis at homeostasis and in response to tissue injury.

Notch and interleukin-22 (IL-22) signaling are known to regulate tissue homeostasis and respond to injury in humans and mice, and the induction of endogenous aryl hydrocarbon receptor (Ahr) ligands through Notch links the two pathways in a hierarchical fashion. However in adults, the species-, organ- and injury-specific gene expression of the Notch-AhR-IL22 axis components is unknown. We therefore performed gene expression profiling of DLL1, DLL3, DLL4, DLK1, DLK2, JAG1, JAG2, Notch1, Notch2, Notch3, Notch4, ADAM17/TNF-α ADAM metalloprotease converting enzyme (TACE), PSEN1, basigin (BSG)/CD147, RBP-J, HES1, HES5, HEY1, HEYL, AHR, ARNT, ARNT2, CYP1A1, CYP24A1, IL-22, IL22RA1, IL22RA2, IL10RB, and STAT3 under homeostatic conditions in ten mature murine and human organs. Additionally, the expression of these genes was assessed in murine models of acute sterile inflammation and progressive fibrosis. We show that there are organ-specific gene expression profiles of the Notch-AhR-IL22 axis in humans and mice. Although there is an overall interspecies congruency, specific differences between human and murine expression signatures do exist. In murine tissues with AHR/ARNT expression CYP1A1 and IL-22 were correlated with HES5 and HEYL expression, while in human tissues no such correlation was found. Notch and AhR signaling are involved in renal inflammation and fibrosis with specific gene expression changes in each model. Despite the presence of all Notch pathway molecules in the kidney and a model-specific induction of Notch ligands, IL-22 was only up-regulated in acute inflammation, but rapidly down-regulated during regeneration. This implies that for targeting injury responses, e.g. via IL-22, species-specific differences, injury type and time points have to be considered.

Histones and Neutrophil Extracellular Traps Enhance Tubular Necrosis and Remote Organ Injury in Ischemic AKI.

Severe AKI is often associated with multiorgan dysfunction, but the mechanisms of this remote tissue injury are unknown. We hypothesized that renal necroinflammation releases cytotoxic molecules that may cause remote organ damage. In hypoxia-induced tubular epithelial cell necrosis in vitro, histone secretion from ischemic tubular cells primed neutrophils to form neutrophil extracellular traps. These traps induced tubular epithelial cell death and stimulated neutrophil extracellular trap formation in fresh neutrophils. In vivo, ischemia-reperfusion injury in the mouse kidney induced tubular necrosis, which preceded the expansion of localized and circulating neutrophil extracellular traps and the increased expression of inflammatory and injury-related genes. Pretreatment with inhibitors of neutrophil extracellular trap formation reduced kidney injury. Dual inhibition of neutrophil trap formation and tubular cell necrosis had an additive protective effect. Moreover, pretreatment with antihistone IgG suppressed ischemia-induced neutrophil extracellular trap formation and renal injury. Renal ischemic injury also increased the levels of circulating histones, and we detected neutrophil infiltration and TUNEL-positive cells in the lungs, liver, brain, and heart along with neutrophil extracellular trap accumulation in the lungs. Inhibition of neutrophil extracellular trap formation or of circulating histones reduced these effects as well. These data suggest that tubular necrosis and neutrophil extracellular trap formation accelerate kidney damage and remote organ dysfunction through cytokine and histone release and identify novel molecular targets to limit renal necroinflammation and multiorgan failure.

Targeting Inflammation in So-Called Acute Kidney Injury.

The clinical category of acute kidney injury includes a wide range of completely different disorders, many with their own pathomechanisms and treatment targets. In this review we focus on the role of inflammation in the pathogenesis of acute tubular necrosis (ATN). We approach this topic by first discussing the role of the immune system in the different phases of ATN (ie, early and late injury phase, recovery phase, and the long-term outcome phase of an ATN episode). A more detailed discussion focuses on putative therapeutic targets among the following mechanisms and mediators: oxidative stress and reactive oxygen species-related necroinflammation, regulated cell death-related necroinflammation, immunoregulatory lipid mediators, cytokines and cytokine signaling, chemokines and chemokine signaling, neutrophils and neutrophils extracellular traps (NETs) associated neutrophil cell death, called NETosis, extracellular histones, proinflammatory mononuclear phagocytes, humoral mediators such as complement, pentraxins, and natural antibodies. Any prioritization of these targets has to take into account the intrinsic differences between rodent models and human ATN, the current acute kidney injury definitions, and the timing of clinical decision making. Several conceptual problems need to be solved before anti-inflammatory drugs that are efficacious in rodent ATN may become useful therapeutics for human ATN.

Optimizing Mouse Surgery with Online Rectal Temperature Monitoring and Preoperative Heat Supply. Effects on Post-Ischemic Acute Kidney Injury.

Body temperature affects outcomes of tissue injury. We hypothesized that online body core temperature recording and selective interventions help to standardize peri-interventional temperature control and the reliability of outcomes in experimental renal ischemia reperfusion injury (IRI). We recorded core temperature in up to seven mice in parallel using a Thermes USB recorder and ret-3-iso rectal probes with three different protocols. Setup A: Heating pad during ischemia time; Setup B: Heating pad from incision to wound closure; Setup C: A ventilated heating chamber before surgery and during ischemia time with surgeries performed on a heating pad. Temperature profile recording displayed significant declines upon installing anesthesia. The profile of the baseline experimental setup A revealed that <1% of the temperature readings were within the target range of 36.5 to 38.5°C. Setup B and C increased the target range readings to 34.6 ± 28.0% and 99.3 ± 1.5%, respectively. Setup C significantly increased S3 tubular necrosis, neutrophil influx, and mRNA expression of kidney injury markers. In addition, using setup C different ischemia times generated a linear correlation with acute tubular necrosis parameters at a low variability, which further correlated with the degree of kidney atrophy 5 weeks after surgery. Changing temperature control setup A to C was equivalent to 10 minutes more ischemia time. We conclude that body temperature drops quickly in mice upon initiating anesthesia. Immediate heat supply, e.g. in a ventilated heating chamber, and online core temperature monitoring can help to standardize and optimize experimental outcomes.

Glomerular parietal epithelial cell activation induces collagen secretion and thickening of Bowman's capsule in diabetes.

The metabolic and hemodynamic alterations in diabetes activate podocytes to increase extracellular matrix (ECM) production, leading to thickening of the glomerular basement membrane (GBM). We hypothesized that diabetes would activate parietal epithelial cells (PECs) in a similar manner and cause thickening of Bowman's capsules. Periodic acid Schiff staining of human kidney biopsies of 30 patients with diabetic nephropathy (DN) revealed a significantly thicker Bowman's capsule as compared with 20 non-diabetic controls. The average thickness was 4.55±0.21 µm in the group of patients with DN compared with 2.92±0.21 µm in the group of non-diabetic controls (P<0.001). Transmission electron microscopy confirmed this finding. In vitro, short-term exposure of human PECs to hyperglycemic conditions (30 mM glucose) advanced glycation end products (100 µg/ml) or transforming growth factor-ß1 (TGF-ß1; 5 ng/ml) increased the mRNA expression of collagen type I α-1, collagen type IV (all six α-chains), bamacan, nidogen 1, laminin α-1, and perlecan. Western blot and colorimetric collagen assays confirmed these results for collagen type IV at the protein level. The production and secretion of TGF-ß1 as a possible positive feedback loop was excluded as a mechanism for the autocrine activation of human PECs. To validate these findings in vivo, activation of the PECs was assessed by immunohistochemical staining for CD44 of 12 human biopsy cases with DN. Thickening of the Bowman's capsule showed strong association with CD44-positive PECs. In summary, metabolic alterations in diabetes activate PECs to increase the expression and secretion of Bowman's capsule proteins. This process may contribute to the thickening of the Bowman's capsule, similar to the thickening of the GBM that is driven by activated podocytes. These data may also imply that activated PECs contribute to ECM production once they migrate to the glomerular tuft, a process resulting in glomerular scaring, for example, in diabetic glomerulosclerosis.