Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron.

Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here, we present a model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death demonstrated pronounced injury propagation and expansion into non-necrotic tissue regions, which predicted tubule atrophy with epithelial VCAM1 expression. In summary, our longitudinal analyses of tubule injury, remodeling, and fate provide important insights into AKI pathology.

Focal segmental glomerulosclerosis in a patient with multiple sclerosis treated with Teriflunomide and Ocrelizumab.

We describe the case of a 24-year-old male patient with multiple sclerosis (MS) who was treated with Teriflunomide for eight months. However, due to MS progression, treatment was switched to Ocrelizumab. After 15 months of therapy with Ocrelizumab the patient developed edema and nephrotic-range albuminuria. Kidney biopsy showed focal segmental glomerulosclerosis (FSGS) and Ocrelizumab treatment was stopped. Teriflunomide is less likely to have caused FSGS due to a three week wash-out period and a timespan of 15 months between the last Teriflunomide dose and development of albuminuria. Treatment with Ocrelizumab has been associated with organ-specific inflammation in MS-patients, thus an association between the development of FSGS and Ocrelizumab therapy is possible, and this case suggests considering this potential association.

Prediction of Renal Function in Living Kidney Donors and Recipients of Living Donor Kidneys Using Quantitative Histology.

BACKGROUND: Living kidney donors (LKDs) are at increased risk of chronic kidney disease, whereas transplant recipients experience progressive reduction of graft function. We examined the predictive value of quantitative stereology on renal function in LKDs and recipients of living donor kidneys, based on perioperative biopsies from the donated kidney. METHODS: Cortex volume of both donor kidneys was determined by contrast-enhanced computed tomography and single-kidney glomerular filtration rate (GFR) by 51 chrome-EDTA clearance together with renography. Glomerular density was used to estimate total glomeruli number in addition to glomerular volume, glomerular sclerosis, kidney fibrosis, and arteriole dimensions. GFR measurements were repeated 1 y after transplantation in both LKDs and recipients. Associations between GFR at follow-up and cortex volume and histomorphometric parameters after adjustment of age, gender, body mass index, smoking status, 24-h blood pressure, and single-kidney GFR were examined. RESULTS: We included 49 LKDs (age, 51 ± 12 y) and 51 recipients (age, 44 ± 13 y). At follow-up, GFR was 71 ± 16 mL/min in LKDs and 61 ± 18 mL/min in recipients with hyperfiltration being more prominent in LKDs (30.4%) as compared to recipients (16.4%; P < 0.05). One-year GFR in donors correlated to cortex volume ( P < 0.001) but not to any histological parameters, whereas GFR in recipients correlated to the amount of interstitial fibrosis ( P < 0.01) but not to other histological parameters or cortex volume. CONCLUSIONS: Kidney cortex volume, but not renal histology parameters, predicts 1-y renal outcome in LKDs. In contrast, the amount of interstitial fibrosis, but not cortex volume, predicts 1-y graft function in recipients.

Increased activity of the metalloproteinase PAPP-A promotes diabetes-induced glomerular hypertrophy.

BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes and a common cause of end stage renal failure. Insulin-like growth factor (IGF)-signaling has been implicated in DN, but is mechanistically poorly understood. Here, we assessed the activity of the metalloproteinase PAPP-A, an activator of IGF activity, and its possible interaction with the endogenous PAPP-A inhibitors stanniocalcin (STC)-1 and -2 in the mammalian kidney under normal and hyperglycemic conditions. METHODS AND RESULTS: Immunohistochemistry demonstrated that PAPP-A, its proteolytic substrate IGF binding protein-4, STC1 and STC2 are present in the human kidney. Endogenous inhibited complexes of PAPP-A (PAPP-A:STC1 and PAPP-A:STC2) were demonstrated in media conditioned by human mesangial cells (HMCs), suggesting that PAPP-A activity is regulated by the STCs in kidney tissue. A method for the selective detection of active PAPP-A in tissue was developed and a significant increase in glomerular active PAPP-A in human diabetic kidney relative to normal was observed. In DN patients, the estimated glomerular filtration rate correlated with PAPP-A activity. In diabetic mice, glomerular growth was reduced when PAPP-A activity was antagonized by adeno-associated virus-mediated overexpression of STC2. CONCLUSION: We propose that PAPP-A activity in renal tissue is precisely balanced by STC1 and STC2. An imbalance in this equilibrium causing increased PAPP-A enzymatic activity potentially contributes to the development of DN, and thus, therapeutic targeting of PAPP-A activity may represent a novel strategy for its treatment.

Circulating tumor DNA for prognosis assessment and postoperative management after curative-intent resection of colorectal liver metastases.

The recurrence rate of colorectal liver metastases (CRLM) patients treated with curative intent is above 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging as well as carcinoembryonic antigen (CEA) measurements. Nonetheless, relapse detection often happens too late to resume curative treatment. This longitudinal cohort study enrolled 115 patients with plasma samples (N = 439) prospectively collected before surgery, postoperatively at day 30 and every third month for up to 3 years. Droplet digital PCR (ddPCR) was used to monitor serial plasma samples for somatic mutations. Assessment of ctDNA status either immediately after surgery, or serially during surveillance, stratified the patients into groups of high and low recurrence risk (hazard ratio [HR], 7.6; 95% CI, 3.0-19.7; P < .0001; and HR, 4.3; 95% CI, 2.3-8.1; P < .0001, respectively). The positive predictive value (PPV) of ctDNA was 100% in all postoperative analyses. In multivariable analyses, postoperative ctDNA status was the only consistently significant risk marker associated with relapse (P < .0001). Indeterminate CT findings were observed for 30.8% (21/68) of patients. All patients (9/21) that were ctDNA positive at the time of the indeterminate CT scan later relapsed, contrasting 42.6% (5/12) of those ctDNA negative (P = .0046). Recurrence diagnoses in patients with indeterminate CT findings were delayed (median 2.8 months, P < .0001). ctDNA status is strongly associated with detection of minimal residual disease and early detection of relapse. Furthermore, ctDNA status can potentially contribute to clinical decision-making in case of indeterminate CT findings, reducing time-to-intervention.

The Clinical Value of Measuring Circulating HPV DNA during Chemo-Radiotherapy in Squamous Cell Carcinoma of the Anus.

BACKGROUND AND PURPOSE: Circulating tumor DNA (ctDNA) is investigated in various cancers. In squamous cell carcinoma of the anus (SCCA) infection with human papilloma virus (HPV) is found in around 90% of cases and here, plasma HPV (pHPV) can be used as ctDNA. Preliminary data have proved the ability to detect pHPV16 and -18 in SCCA. We have developed a highly sensitive method for measurement of six relevant pHPV subtypes, to investigate the elimination pattern of pHPV during chemo-radiotherapy (CRT) for SCCA and its clinical value. MATERIAL AND METHODS: Patients treated at Aarhus University Hospital from 2016-2020 were included. P16 status in the primary biopsy was measured and 82% of patients had P16 positive tumor. Blood samples were collected prior to treatment (PT), mid treatment (MT), end of therapy (EOT), and during follow-up (FU). An in-house multiplex digital droplet PCR method measured pHPV subtypes 16, 18, 31, 33, 51, 58. RESULTS: Samples from 88 patients were drawn PT (n = 73), MT (n = 72), EOT (n = 64) and during FU (n = 41). Plasma HPV was detectable in 52 patients and PT pHPV levels correlated to tumor stages. Three elimination patterns were observed during CRT with correlation to outcome: fast responders with no local or distant failures (0/12); slow responders with high risk of local failures (4/20), no distant failures; persistent molecular responders with high risk of distant failures (4/13), but no local failures, p < 0.01. CONCLUSION: During CRT, pHPV can divide patients with SCCA into three groups with significantly different risk of failure. The use of pHPV can potentially assist in clinical treatment decision.

Improved Normothermic Machine Perfusion After Short Oxygenated Hypothermic Machine Perfusion of Ischemically Injured Porcine Kidneys.

BACKGROUND: In an era where global kidney shortage has pushed the field of transplantation towards using more marginal donors, modified kidney preservation techniques are currently being reviewed. Some techniques require further optimization before implementation in full scale transplantation studies. Using a porcine donation after circulatory death kidney model, we investigated whether initial kidney hemodynamics improved during normothermic machine perfusion if this was preceded by a short period of oxygenated hypothermic machine perfusion (oxHMP) rather than static cold storage (SCS). METHODS: Kidneys subjected to 75 minutes of warm ischemia were randomly assigned to either SCS (n = 4) or SCS + oxHMP (n = 4), with a total cold storage time of 240 minutes. Cold preservation was followed by 120 minutes of normothermic machine perfusion with continuous measurement of hemodynamic parameters and renal function. RESULTS: oxHMP preserved kidneys maintained significantly lower renal resistance throughout the normothermic machine perfusion period compared to SCS kidneys (P < 0.001), reaching lowest levels at 60 minutes with means of 0.71 ± 0.35 mm Hg/mL/min/100 g (SCS) and 0.45 ± 0.15 mm Hg/mL/min/100 g (oxHMP). Accordingly, the oxHMP group had a higher mean renal blood flow versus SCS kidneys (P < 0.001). oxHMP kidneys had higher oxygen consumption during normothermic machine perfusion compared to SCS preserved kidneys (P < 0.001). Creatinine clearance remained similar between groups (P = 0.665). CONCLUSIONS: Preceding oxHMP significantly improved initial normothermic machine perfusion hemodynamics and increased total oxygen consumption. With the long period of warm ischemia, immediate kidney function was not observed, reflected by the findings of low creatinine clearance in both groups.

Tumour blood flow for prediction of human prostate cancer aggressiveness: a study with Rubidium-82 PET, MRI and Na+/K+-ATPase-density.

PURPOSE: Tumour blood flow (TBF) is a crucial determinant of cancer growth. Recently, we validated Rubidium-82 (82Rb) positron emission tomography (PET) for TBF measurement in prostate cancer (PCa) and found TBF and cancer aggressiveness positively correlated. The aims of the present study were to determine the ability of TBF for separating significant from insignificant PCa and to examine the relation to underlying Na+/K+-ATPase density, which is relevant as 82Rb is transported intracellularly via the Na+/K+-ATPase. METHODS: One hundred and two patients were included for pelvic 82Rb PET scan prior to magnetic resonance imaging (MRI)-guided prostate biopsy. Findings constituted 100 PCa lesions (86 patients) and 25 benign lesions (16 patients). Tumours were defined on MRI and transferred to 82Rb PET for TBF measurement. Immunohistochemical Na+/K+-ATPase staining was subsequently performed on biopsies. RESULTS: TBF was the superior predictor (rho = 0.68, p < 0.0001, inflammatory lesions excluded) of MRI-guided biopsy grade group (GG) over lowest apparent diffusion coefficient (ADC) value (rho = -0.23, p = 0.01), independent of ADC value and tumour volume (p < 0.0001). PET could separate GG-2-5 from GG-1 and benign lesions with an area under the curve (AUC), sensitivity, and specificity of 0.79, 96%, and 59%, respectively. For separating GG-3-5 from GG-1-2 and benign lesions the AUC, sensitivity, and specificity were 0.82, 95%, and 63%, respectively. Na+/K+-ATPase density per PCa cell profile was 38% lower compared with that of the benign prostate cell profiles. Neither cell density nor Na+/K+-ATPase density determined tumour 82Rb uptake. CONCLUSION: TBF is an independent predictor of PCa aggressiveness and deserves more attention, as it may be valuable in separating clinically significant from insignificant PCa.

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.

Measurement of circulating free DNA in squamous cell carcinoma of the anus and relation to risk factors and recurrence.

BACKGROUND: Measuring circulating-free-deoxyribonucleic-acid (cfDNA) has created a new framework for personalized treatment in oncology. The aim of this study was to analyze the relation between cfDNA and risk factors and outcome in squamous cell carcinoma of the anus (SCCA). METHODS: Patients treated with radiotherapy for localized SCCA were included in Aarhus, Denmark from 2016 to 2019. Serum samples from baseline, during and after therapy, were measured for the level of cfDNA in copies per mL by a direct fluorescent assay. RESULTS: Eighty patients were included. Samples were available at baseline (n = 73) mid-therapy (n = 74), end-therapy (n = 67) and one-year follow-up (1Y) (n = 29). P16-positivity was found in 89% (n = 55). The median level of cfDNA was higher for P16 negative tumors (1.48) compared with the P16 positive tumors (0.90, P = 0.04). Data showed a correlation between baseline cfDNA levels and Gross Tumor Volume (R2 = 0.13, P < 0.01), and increasing levels with increasing T-stage (T1 = 0.80, T2 = 0.94, T3 = 1.11, T4 = 1.3). Higher cfDNA levels were observed in patients with poor performance status (P < 0.01). The cfDNA level decreased from baseline to mid-therapy (0.92-0.78, P < 0.01) and from baseline to 1Y (0.92-0.71, P < 0.01). Baseline levels for patients with treatment failure (n = 8) were above the 25th percentile (p = 0.05) which translates into difference in disease free survival. CONCLUSION: Results indicate an association between baseline cfDNA levels and risk factors in SCCA and a low baseline level correlates to lower risk of treatment failure. Findings contribute with new knowledge of the biological role of cfDNA in SCCA and holds potential knowledge for personalized treatment of SCCA.

Kidney structural characteristics based on a kidney biopsy and contrast-enhanced computed tomography in healthy living kidney donors.

The demands for kidney transplantations are increasing, and so is the number of live kidney donors (LKDs). Recent studies show that LKDs have an increased risk of developing end-stage renal disease compared with healthy non-donors. However, the knowledge about factors predicting renal disease in kidney donors is sparse. Some evidence points to increased glomerular sclerosis and kidney fibrosis, as well as a low number of glomeruli as associated with a worse renal outcome. This methodological study investigated that which estimates are obtainable with a standard kidney biopsy taken from the donated kidney during the transplantation, and a standard contrast-enhanced computed tomography (CT) in kidney donors. CT-scans were used to obtain total volume of the kidney and kidney cortex using the Cavalieri estimator and 2D-nucleator. Glomerular number density in the biopsies was estimated by a model-based method, and was multiplied by total cortex volume in order to estimate the total number of glomeruli in the kidney. Glomerular volume was estimated by the 2D-nucleator and a model-based stereological technique. Kidney fibrosis (point-counting), glomerular sclerosis (evaluation of glomerular profiles), and arteriole dimensions (2D-nucleator) were also estimated in the biopsy sections from the donated kidney. Various studies have attempted to identify predictors of renal outcome in LKDs. There is no consensus yet, and further studies are needed to elucidate if and how the estimates described in this study are associated with renal outcome in LKDs.

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.

Correlation between early dynamics in circulating tumour DNA and outcome from FOLFIRI treatment in metastatic colorectal cancer.

Chemotherapy resistance remains a challenge in the clinical management of metastatic colorectal cancer (mCRC). Here, early changes in cell-free circulating tumour DNA (ctDNA) levels were explored as a marker of therapeutic efficacy. Twenty-four mCRC patients were enrolled and treated with FOLFIRI based first-line therapy. Blood samples collected pre-treatment, at day 7, 14, 21, 60 and at progression were analysed for cell-free DNA (cfDNA) and ctDNA levels using digital droplet PCR. A subset of samples were additionally analysed by targeted sequencing. Patients with high pre-treatment ctDNA or cfDNA levels (≥75th centile) had significantly shorter progression free survival (PFS) than patients with lower levels. Despite an overall decline in ctDNA levels from pre-treatment to first CT-scan, serial analysis identified seven patients with temporary increases in ctDNA consistent with growth of resistant cells. These patients had shorter PFS and shorter overall survival. Targeted sequencing analyses of cfDNA revealed dramatic changes in the clonal composition in response to treatment. Our study suggests that increasing ctDNA levels during the first cycles of first-line FOLFIRI treatment is a predictor of incipient progressive disease and poorer survival. Thus, we demonstrate the importance of monitoring ctDNA levels as early as one week after treatment onset to enable early detection of treatment failure.

Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients With Stages I to III Colorectal Cancer.

IMPORTANCE: Novel sensitive methods for detection and monitoring of residual disease can improve postoperative risk stratification with implications for patient selection for adjuvant chemotherapy (ACT), ACT duration, intensity of radiologic surveillance, and, ultimately, outcome for patients with colorectal cancer (CRC). OBJECTIVE: To investigate the association of circulating tumor DNA (ctDNA) with recurrence using longitudinal data from ultradeep sequencing of plasma cell-free DNA in patients with CRC before and after surgery, during and after ACT, and during surveillance. DESIGN, SETTING, AND PARTICIPANTS: In this prospective, multicenter cohort study, ctDNA was quantified in the preoperative and postoperative settings of stages I to III CRC by personalized multiplex, polymerase chain reaction-based, next-generation sequencing. The study enrolled 130 patients at the surgical departments of Aarhus University Hospital, Randers Hospital, and Herning Hospital in Denmark from May 1, 2014, to January 31, 2017. Plasma samples (n = 829) were collected before surgery, postoperatively at day 30, and every third month for up to 3 years. MAIN OUTCOMES AND MEASURES: Outcomes were ctDNA measurement, clinical recurrence, and recurrence-free survival. RESULTS: A total of 130 patients with stages I to III CRC (mean [SD] age, 67.9 [10.1] years; 74 [56.9%] male) were enrolled in the study; 5 patients discontinued participation, leaving 125 patients for analysis. Preoperatively, ctDNA was detectable in 108 of 122 patients (88.5%). After definitive treatment, longitudinal ctDNA analysis identified 14 of 16 relapses (87.5%). At postoperative day 30, ctDNA-positive patients were 7 times more likely to relapse than ctDNA-negative patients (hazard ratio [HR], 7.2; 95% CI, 2.7-19.0; P < .001). Similarly, shortly after ACT ctDNA-positive patients were 17 times (HR, 17.5; 95% CI, 5.4-56.5; P < .001) more likely to relapse. All 7 patients who were ctDNA positive after ACT experienced relapse. Monitoring during and after ACT indicated that 3 of the 10 ctDNA-positive patients (30.0%) were cleared by ACT. During surveillance after definitive therapy, ctDNA-positive patients were more than 40 times more likely to experience disease recurrence than ctDNA-negative patients (HR, 43.5; 95% CI, 9.8-193.5 P < .001). In all multivariate analyses, ctDNA status was independently associated with relapse after adjusting for known clinicopathologic risk factors. Serial ctDNA analyses revealed disease recurrence up to 16.5 months ahead of standard-of-care radiologic imaging (mean, 8.7 months; range, 0.8-16.5 months). Actionable mutations were identified in 81.8% of the ctDNA-positive relapse samples. CONCLUSIONS AND RELEVANCE: Circulating tumor DNA analysis can potentially change the postoperative management of CRC by enabling risk stratification, ACT monitoring, and early relapse detection.

Metformin attenuates renal medullary hypoxia in diabetic nephropathy through inhibition uncoupling protein-2.

BACKGROUND: The purpose of the study is to examine the effect of metformin on oxygen metabolism and mitochondrial function in the kidney of an animal model of insulinopenic diabetes in order to isolate any renoprotective effect from any concomitant effect on blood glucose homeostasis. METHODS: Sprague-Dawley rats were injected with streptozotocin (STZ) (50 mg kg-1 ) and when stable started on metformin treatment (250 mg kg-1 ) in the drinking water. Rats were prepared for in vivo measurements 25 to 30 days after STZ injection, where renal function, including glomerular filtration rate and sodium transport, was estimated in anesthetized rats. Intrarenal oxygen tension was measured using oxygen sensors. Furthermore, mitochondrial function was assessed in mitochondria isolated from kidney cortex and medulla analysed by high-resolution respirometry, and superoxide production was evaluated using electron paramagnetic resonance. RESULTS: Insulinopenic rats chronically treated with metformin for 4 weeks displayed improved medullary tissue oxygen tension despite of no effect of metformin on blood glucose homeostasis. Metformin reduced UCP2-dependent LEAK and differentially affected medullary mitochondrial superoxide radical production in control and diabetic rats. CONCLUSIONS: Metformin attenuates diabetes-induced renal medullary tissue hypoxia in an animal model of insulinopenic type 1 diabetes. The results suggest that the mechanistic pathway to attenuate the diabetes-induced medullary hypoxia is independent of blood glucose homeostasis and includes reduced UCP2-mediated mitochondrial proton LEAK.

Unilateral nephrectomy diminishes ischemic acute kidney injury through enhanced perfusion and reduced pro-inflammatory and pro-fibrotic responses.

While unilateral nephrectomy (UNx) is suggested to protect against ischemia-reperfusion injury (IRI) in the remaining kidney, the mechanisms underlying this protection remain to be elucidated. In this study, functional MRI was employed in a renal IRI rat model to reveal global and regional changes in renal filtration, perfusion, oxygenation and sodium handling, and microarray and pathway analyses were conducted to identify protective molecular mechanisms. Wistar rats were randomized to either UNx or sham UNx immediately prior to 37 minutes of unilateral renal artery clamping or sham operation under sevoflurane anesthesia. MRI was performed 24 hours after reperfusion. Blood and renal tissue were harvested. RNA was isolated for microarray analysis and QPCR validation of gene expression results. The perfusion (T1 value) was significantly enhanced in the medulla of the post-ischemic kidney following UNx. UNx decreased the expression of fibrogenic genes, i.a. Col1a1, Fn1 and Tgfb1 in the post-ischemic kidney. This was associated with a marked decrease in markers of activated myofibroblasts (Acta2/α-Sma and Cdh11) and macrophages (Ccr2). This was most likely facilitated by down-regulation of Pdgfra, thus inhibiting pericyte-myofibroblast differentiation, chemokine production (Ccl2/Mcp1) and macrophage infiltration. UNx reduced ischemic histopathologic injury. UNx may exert renoprotective effects against IRI through increased perfusion in the renal medulla and alleviation of the acute pro-inflammatory and pro-fibrotic responses possibly through decreased myofibroblast activation. The identified pathways involved may serve as potential therapeutic targets and should be taken into account in experimental models of IRI.

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.

Significant Individual Variation Between Pathologists in the Evaluation of Colon Cancer Specimens After Complete Mesocolic Excision.

BACKGROUND: After the introduction of complete mesocolic excision, a new pathological evaluation of the resected colon cancer specimen was introduced. This concept has quickly gained acceptance and is often used to compare surgical quality. The grading of colon cancer specimens is likely to depend on both surgical quality and the training of the pathologist. OBJECTIVE: The purpose of this study was to validate the principles of the pathological evaluation of colon cancer specimens. DESIGN: This was an exploratory study. SETTINGS: The study was conducted in Aarhus, Denmark, and Leeds, United Kingdom. PATIENTS: Colon cancers specimens were used. MAIN OUTCOME MEASURES: The agreement of gradings between participants was of interest. Four specialist GI pathologists and 2 abdominal surgeons evaluated 2 rounds of colon cancer specimens, each at 2 separate time points. Each round contained 50 specimens. After the first round, a protocol of detailed principles for the grading procedure was agreed on. Results from an experienced pathologist were considered as the reference results. RESULTS: In the first round, the distribution of gradings between participants showed substantial variation. In the second round, the variation was reduced. Intraobserver agreement was mostly fair to good, whereas interobserver agreement was frequently poor. This did not significantly change from round 1 to round 2. LIMITATIONS: The small sample size of 100 specimens provided a very small number of specimens resected in the muscularis propria plane, which renders the evaluation of this group potentially unreliable. The evaluations were made on photos and not on fresh specimens. CONCLUSIONS: This study demonstrates significant variation in the pathological evaluation of colon cancer specimens. It demonstrates that it cannot be used in clinical studies, and care should be taken when comparing results between different hospitals.

Remote ischemic perconditioning attenuates ischemia/reperfusion-induced downregulation of AQP2 in rat kidney.

Renal ischemia/reperfusion (I/R) can lead to impaired urine concentration ability and increased fractional excretion of sodium (FeNa). Local ischemic preconditioning improves renal water and sodium handling after I/R injury. Here, we investigate whether remote ischemic perconditioning (rIPeC) prevents dysregulation of renal water and salt handling in response to I/R injury and mechanisms that may be involved. Rats were subjected to right nephrectomy and randomized into a sham group or an I/R group. In the I/R group, rats were subjected to 37 min of renal ischemia and 3 days of reperfusion. rIPeC was applied to the abdominal aorta. Blood and urine were collected on day 3 postoperatively for clearance studies. The expression of aquaporins (AQPs) and the sodium transporter Na-K-ATPase were analyzed using immunoblotting and immunohistochemistry. I/R injury resulted in polyuria, increased FeNa, and decreased urine osmolality compared to sham rats. rIPeC attenuated the increase in FeNa and the decrease in urine osmolality. Expression of AQP1, AQP2, phosphorylated AQP2 (pAQP2), and Na-K-ATPase was downregulated in I/R rats. rIPeC attenuated the reductions in AQP2 and pAQP2 expression. Immunohistochemistry revealed decreased labeling of Na-K-ATPase in the outer medulla in I/R kidneys compared to kidneys from sham and I/R + rIPeC rats. After renal ischemia, the expression of Na-K-ATPase was substantially reduced in the outer medullary thick ascending limb. In conclusion, our data suggest that rIPeC might prevent dysregulation of renal water and salt handling via regulation of AQP2 expression and phosphorylation as well as via regulation of Na-K-ATPase expression in I/R rat kidneys.