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Pathological deposition of collagen is a hallmark of renal fibrosis. We employed multimodal optical imaging to visualize and quantify collagen deposition in murine models of kidney fibrosis using the collagen-binding agent CNA35. For in vivo imaging, we used hybrid computed tomography-fluorescence molecular tomography (CT-FMT) and CNA35 labeled with the near-infrared fluorophore Cy7. Upon i.v. injection, CNA35-Cy7 accumulation was significantly higher in fibrotic kidneys compared to non-fibrotic kidneys. This difference was not detected for a non-specific scrambled version of CNA35-Cy7. Ex vivo, on kidney sections of animals and patients with renal fibrosis, CNA35-FITC co-localized with scar collagen type I and III, but not with the basement membrane collagen type IV. Upon i.v. injection, CNA35-FITC bound to both interstitial and perivascular fibrotic areas. In line with this perivascular accumulation, we observed significant perivascular fibrosis in animal models and patients using computer-based morphometry quantification. In conclusion, molecular imaging of collagen using CNA35 enables specific non-invasive quantification of renal fibrosis. Collagen imaging revealed significant perivascular fibrosis as a consistent component next to the more commonly assessed interstitial fibrosis. Our results lay the basis for further probe and protocol optimization towards the clinical translation of molecular imaging of kidney fibrosis.

In the last decade, the role of apoptosis in the pathophysiology of acute kidney injury (AKI) and AKI to chronic kidney disease (CKD) progression has been revisited as our understanding of ferroptosis and necroptosis has emerged. A growing body of evidence, reviewed here, ascribes a central pathophysiological role for ferroptosis and necroptosis to AKI, nephron loss, and acute tubular necrosis. We will introduce concepts to the non-cell-autonomous manner of kidney tubular injury during ferroptosis, a phenomenon that we refer to as a "wave of death." We hypothesize that necroptosis might initiate cell death propagation through ferroptosis. The remaining necrotic debris requires effective removal processes to prevent a secondary inflammatory response, referred to as necroinflammation. Open questions include the differences in the immunogenicity of ferroptosis and necroptosis, and the specificity of necrostatins and ferrostatins to therapeutically target these processes to prevent AKI-to-CKD progression and end-stage renal disease.

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

Immunofluorescent staining is a widespread tool in basic science to understand organ morphology and (patho-) physiology. The analysis of imaging data is often performed manually, limiting throughput and introducing human bias. Quantitative analysis is particularly challenging for organs with complex structure such as the kidney. In this study we present an approach for automatic quantification of fluorescent markers and histochemical stainings in whole organ sections using open source software. We validate our novel method in multiple typical challenges of basic kidney research and demonstrate its general relevance and applicability to other complex solid organs for a variety of different markers and stainings. Our newly developed software tool “AQUISTO”, applied as a standard in primary data analysis, facilitates efficient large scale evaluation of cellular populations in various types of histological samples. Thereby it contributes to the characterization and understanding of (patho-) physiological processes.

The prognosis of patients with de novo myelodysplastic syndrome (MDS) who are red blood cell transfusion–dependent (TD) and receive supportive care is inferior to that of patients who do not require transfusions. Whether TD also affects outcome after allogeneic transplantation is unknown. Consequently, in 172 de novo MDS patients (median age, 51 years), we analyzed the impact of TD on outcome after high-dose conditioning and allogeneic peripheral blood stem cell transplantation (PBSCT). With a median follow-up of 37 months, the probability of 3-year overall survival (OS) did not differ significantly between patients who were TD and those who were not TD before PBSCT (P = .1); however, transfusion burden, as reflected by ferritin levels, was correlated with a greater probability of severe acute graft-versus-host disease (aGVHD; P = .03) and a higher comorbidity index (P = .01), and OS was inferior in those patients with a ferritin level >1000 μg/L before PBSCT (P = .03). In multivariate analysis, only marrow myeloblast count (P = .01) and comorbidity index (P = .001) had a significant impact on OS. Our data do not identify TD as an independent negative prognostic factor for outcome after allogeneic PBSCT' however, iron overload (presumably transfusion-related) may contribute to poor transplantation success by adding to the overall comorbidities. Whether clinical intervention in the form of iron chelation can improve the outcome of allogeneic PBSCT in TD patients with MDS remains to be determined.