Early apoptotic response in kidney after 177Lu-octreotate administration with or without potential radioprotector α1-microglobulin

Charlotte Ytterbrink; Klara Simonsson; Emman Shubbar; Magnus Gram; Khalil Helou; Eva Forssell-Aronsson

doi:10.1093/rpd/ncaf055

. 2025 Aug 28;201(13-14):877–886. doi: 10.1093/rpd/ncaf055

Early apoptotic response in kidney after ¹⁷⁷Lu-octreotate administration with or without potential radioprotector α₁-microglobulin

Charlotte Ytterbrink ^1,², Klara Simonsson ^3,⁴, Emman Shubbar ^5,⁶, Magnus Gram ^7,⁸, Khalil Helou ^9,¹⁰, Eva Forssell-Aronsson ^11,^12,^13,^✉

¹ Department of Medical Radiation Sciences, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden

² Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden

³ Department of Medical Radiation Sciences, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden

⁴ Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden

⁵ Department of Medical Radiation Sciences, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden

⁶ Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden

⁷ Pediatrics, Department of Clinical Sciences Lund, Lund University, SE-221 85 Lund, Sweden

⁸ Department of Neonatology, Skåne University Hospital, SE-221 84 Lund, Sweden

⁹ Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden

¹⁰ Department of Oncology, Institute of Clinical Sciences, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden

¹¹ Department of Medical Radiation Sciences, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden

¹² Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden

¹³ Department of Medical Physics and Biomedical Engineering, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden

^✉

Corresponding author. Department of Medical Radiation Sciences, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden. E-mail: eva.forssell_aronsson@radfys.gu.se

PMCID: PMC12392902 PMID: 40875262

Abstract

The antioxidant α₁-microglobulin (A1M) has been suggested as kidney protector during ¹⁷⁷Lu-octreotate treatment. The aim of this work was to evaluate apoptotic-related transcript expression in kidney cortex and medulla following injection of ¹⁷⁷Lu-octreotate and/or A1M. Mice were injected with ¹⁷⁷Lu-octreotate, A1M, or ¹⁷⁷Lu-octreotate + A1M. Control groups received PBS or vehicle solution. Animals were killed after 24 hours or 7 d. mRNA was isolated from kidney medulla and cortex. Expression of 84 apoptosis-related genes was assessed by q-PCR. Gene expression profiles in kidney cortex were generally similar in the ¹⁷⁷Lu-octreotate and ¹⁷⁷Lu-octreotate + A1M groups. This was also seen in kidney medulla at 24 hours, but at 7 d anti-apoptotic response of A1M was observed. Altogether, ¹⁷⁷Lu-octreotate exposure induced pro-apoptotic response (e.g. Apaf1, Bax, and Tnfrsf10b genes) in kidney medulla and cortex. A1M co-administration did not inhibit pro-apoptotic response in kidney cortex, while A1M initiated pro-survival mechanisms in kidney medulla.

Introduction

Peptide receptor radionuclide therapy (PRRT) is used to treat patients with neuroendocrine tumours (NETs) that have an overexpression of somatostatin receptor. ¹⁷⁷Lu-octreotate (Lutathera®) was the first conventional drug used for PRRT, approved in 2017 by the European Medicines Agency (EMA) for the treatment of gastroenteropancreatic NETs [1]. Tumour control can be achieved with ¹⁷⁷Lu-octreotate treatment, although relapse of the disease is common since complete remission of the tumours are rare, using the current treatment schedule. There are several strategies that can be used to optimize the treatment [2]. One possible way is to address the limiting factor of potential side effects on kidney and bone marrow. Kidney protective strategies include methods to reduce radiation exposure of the kidney and/or reduce radiobiological effects in kidney tissue [2, 3]. Blocking the kidney uptake of ¹⁷⁷Lu-octreotate is partly achieved in today’s treatment protocol with the co-administration of positively charged amino acids, such as lysine and arginine [1, 4, 5]. However, the amino acids may themselves cause side effects like nausea and vomiting, which limits the dose and makes this method not fully optimal.

Reduction of kidney toxicity after exposure to ¹⁷⁷Lu-octreotate could potentially be achieved by combining the treatment with a radioprotective agent. A promising candidate for this purpose is α₁-microglobulin (A1M), an endogenous antioxidant with radical scavenging properties [6]. Infusion of a recombinant form of A1M has been shown to mitigate radiation induced effects (e.g. DNA double-strand breaks) in mouse kidney when co-infused with ¹⁷⁷Lu-octreotate [7]. The radioprotective function of A1M has also been investigated in mice infused with ¹⁷⁷Lu-PSMA-617, indicating somewhat preserved kidney function [8]. Moreover, our previous study on NET-bearing mice showed that administration of A1M has no effect on the tumour response to ¹⁷⁷Lu-octreotate [9]. Similar findings were reported from the ¹⁷⁷Lu-PSMA-617 study with mice bearing LNCaP xenografts [8]. These results indicate that A1M protects the kidney from ¹⁷⁷Lu induced damage, without interfering with the therapeutic effects of irradiation in tumour [10].

Cells will try to repair cellular damages when exposed to harmful levels of ionizing radiation. Severe cellular damage results in cell death, either in a controlled manner, regulated cell death (RCD) or uncontrolled through accidental cell death (ACD) [11, 12]. Apoptosis, a type of RCD, is one of the major cell death mechanisms in tissues early after radiation exposure [13]. There are two main types of apoptosis; intrinsic apoptosis and extrinsic apoptosis. Intrinsic apoptosis is initiated when the extracellular or intracellular microenvironment is disrupted by harmful events such as DNA damage, reactive oxygen species (ROS) or/and endoplasmic reticulum stress. Mitochondrial outer membrane permeabilization (MOMP) is a key event in intrinsic apoptosis and generally considered to be the point-of-no-return. MOMP is mainly controlled by a family of pro- and anti-apoptotic proteins; the B cell CLL/lymphoma-2 (Bcl-2) family. Extrinsic apoptosis is activated by plasma-membrane receptors (death receptors or dependence receptors) as a consequence of disruptions in the extracellular microenvironment. The extrinsic path to apoptosis can either be mitochondria-dependent (taking the path via MOMP) or mitochondria-independent [12, 13].

In vitro studies have shown that A1M can protect against oxidative stress-induced apoptosis. When cells (mouse CD4⁺ T-cell hybridoma cell line and human primary keratinocytes) were exposed to haem and ROS, A1M was accumulated in apoptotic cells, binding to the mitochondria (complex I subunit) and protecting its structure and function [14]. A1M has also been shown to inhibit haem-induced cell death in human proximal tubule epithelial cells in the kidney [15]. Furthermore, studies in animal models showed that regulation of apoptotic-related genes in mouse kidney was reduced by co-administrating A1M with ¹⁷⁷Lu-octreotate [7]. We have previously shown that the transcriptomic and proteomic response in kidney after ¹⁷⁷Lu-octreotate exposure varies between kidney medulla and kidney cortex [16].

In this study, we evaluated the expression of selected apoptosis-related genes in kidney medulla and kidney cortex separately, with the aim to evaluate differences in apoptotic response in mice 24 hours and 7 d after injection with ¹⁷⁷Lu-octreotate and/or A1M.

Materials and methods

Radiopharmaceutical and recombinant human A1M

LuMark^{® 177}Lu chloride and DOTA-octreotate were obtained from Nuclear Research and Consultancy Group (IDB Holland, the Netherlands). Radiolabelling of octreotate with ¹⁷⁷Lu was performed at Central Radiopharmacy at Sahlgrenska University Hospital, according to the manufacturer’s instructions. Instant thin layer chromatography (ITLC), using Whatman™ Chromatography paper (3 mm, GE Healthcare UK Limited, Amersham, UK) and 0.1 mol sodium citrate (Labservice AB, Sundsvall, Sweden) showed that the amount of peptide bound ¹⁷⁷Lu was higher than 99%. Syringes containing ¹⁷⁷Lu-octreotate were prepared according to previously published method [9]. In short, the syringes were measured before and after administration to correct the injected activity for residual activity.

Human recombinant A1M (modified variant A1M-035 [17]) and vehicle solution containing sterile endotoxin-free 10 mM Na₃PO₄ (pH 7.4), 0.15 mol NaCl, and 12 mM histidine were supplied by A1M Pharma (Lund, Sweden) (new name: Guard Therapeutics International AB, Stockholm, Sweden). The abbreviation A1M will be used for all further description of recombinant human A1M in this paper. A1M was diluted and dosed based on mouse body weight, to a final dose of 5.0 mg kg⁻¹ with maximum injected volume of 0.1 ml.

Animal experiments

Samples of kidney medulla and kidney cortex were collected from female C57BL/6N mice that had received i.v. injection of (a) 150 MBq ¹⁷⁷Lu-octreotate and phosphate buffered saline (PBS) solution, (b) 5 mg kg⁻¹ A1M and PBS, or (c) 150 MBq ¹⁷⁷Lu-octreotate and 5 mg kg⁻¹ A1M. Samples were also collected from control mice that had received either two injections of PBS or PBS and A1M vehicle solution. Collection of the samples was performed at the time of death; three animals in each group were killed 24 hours after injection and another three after 7 d. The animal study protocol was approved by the Ethics Committee for Animal Research in Gothenburg, Sweden (no. 146-2015). We have in a previous study estimated that i.v. injection of 150 MBq ¹⁷⁷Lu-octreotate would result in an absorbed dose to the kidneys of 54 Gy [18].

RNA isolation and gene expression analysis

Expression of 84 apoptosis-related genes were investigated using QIAGEN’s mouse apoptosis RT² PCR profiler array (cat. no. PAMM-012ZA). Total RNA was isolated from individual samples of kidney medulla and kidney cortex using AllPrep® DNA/RNA/Protein Kit (QIAGEN, Hilden, Germany) or RNeasy Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany). The quality and concentration of the RNA were determined using Nanodrop 1000 Spectrometer (Thermo Scientific, Waltham, MA, USA), RNA 6000 Nano LabChip Kit and Agilent 2100 Bioanalyzer (both Agilent Technologies, Santa Clara, CA, USA) (RIN >9) and Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) (RNA concentration > 40 μg ml⁻¹). The RNA was then reverse-transcribed into cDNA and analysed with the RT² PCR profiler array measured in a 7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). Sample amplification was conducted for 40 cycles. Only threshold cycle (C_t) values of <35 were considered for the following gene expression analyses.

The RT² PCR profiler array includes control wells for genomic DNA (1 well), replicate reverse-transcription (3 wells), and replicate positive PCR (3 wells). The RNA from samples with genomic DNA control C_t value of <32 were purified using DNase Max Kit (QIAGEN, Hilden, Germany) before repeating the RT qPCR procedure.

C_t values were normalized against housekeeping genes (Actb, B2m, Gapdh, Gusb, and Hsp90ab1) included in the array. Gene regulations were determined by calculating the fold change (FC) using the 2^−∆∆Ct method [19], where a FC ≥ 1.5 defined upregulation and FC ≤ −1.5 downregulation. Differentially expressed genes (DEGs), were defined as those with statistically significant different normalized C_t values compared to control (Welch’s t-test, P < .05) and |FC| ≥ 1.5. Statistical differences between the DEGs in the groups were analysed using one-way ANOVA and pairwise comparison with Welch’s t-test. The software Perseus (http://www.perseus-framework.org) version 1.6.10.50 was used for the statistical analyses. Gene Ontology (GO) terms for DEGs were obtained from the Mouse Genome Database (http://www.informatics.jax.org/) [20]. Only GO-terms associated with cell death were considered in the analysis.

Results

In total, 82 genes among the 84 apoptotic-related genes included in the array were detected in kidney tissue short time after injection of ¹⁷⁷Lu-octreotate, ¹⁷⁷Lu-octreotate + A1M, or A1M alone. Two genes (Cd70 and Lhx4) were undetected (C_t ≥ 35 or undetermined C_t value) in all groups and at both time-points.

Mean FC of the detected genes are presented in a heat map (Fig. 1). Four genes (Bcl2l10, Casp14, Fasl, and Ll10) were detected on only a few occasions (1–4 occasions considering both time point and tissue type). Most of the detected genes were regulated (|FC| ≥ 1.5) on at least one occasion, and 24 of them were found to be differently expressed (statistically significant from control and |FC| ≥ 1.5). A list of all the observed DEGs with annotated GO terms related to cell death is presented in Supplementary Fig. S1.

Gene expression of apoptotic-related genes in kidney medulla and kidney cortex at 24 h and 7 d after injection of ¹⁷⁷Lu-octreotate (Lu-oct), ¹⁷⁷Lu-octreotate and A1M (Lu-oct + A1M), or A1M. The expression is presented as fold change relative to control (n = 1–3) with standard error of the mean (SEM) in italic.

Apoptosis-related DEGs in kidney cortex

Regulation of the totally 11 apoptosis-related DEGs in kidney cortex (compared to control), detected in at least one of the groups, are shown in Fig. 2: Apaf1, Bax, Bcl2a1a, Bcl2l1, Bcl2l11, Casp1, Casp3, Casp4, Cd40lg, Tnfrsf10b, and Traf1. Nine DEGS were detected at 24 hours and six after 7 d.

Regulation of apoptotic-related genes in kidney cortex. Differentially expressed genes compared to control (^*) in at least one of the groups are shown at (a) 24 h and (b) 7 d after injection of ¹⁷⁷Lu-octreotate, ¹⁷⁷Lu-octreotate + A1M, or A1M. Positive fold change indicates upregulation and negative fold change indicates downregulation of the gene. Error bars show SEM of the fold change values, and statistically significant difference between the groups is represented with ‘X’.

After injection of ¹⁷⁷Lu-octreotate, the gene expression analyses detected eight DEGs at 24 hours (Apaf1, Bax, Bcl2a1a, Bcl2l1, Casp1, Casp3, Cd40lg, and Tnfrsf10b) and four DEGs at 7 d (Apaf1, Bax, Casp 1, and Tnfrsf10b). Apaf1, Bax, and Tnfrsf10b were upregulated and Casp1 was downregulated at both time-points in the ¹⁷⁷Lu-octreotate group. Highest difference in expression (¹⁷⁷Lu-octreotate versus control) was observed for Cd40lg (FC = −6.9, SEM = 0.0) at 24 hours.

After injection of ¹⁷⁷Lu-octreotate + A1M, a similar pattern of gene expression was observed: six DEGs were detected at 24 hours (Apaf1, Bax, Bcl2l1, Casp3, Casp4, and Tnfrsf10b) and four DEGs at 7 d (Apaf1, Bax, Bcl2l11, and Tnfrsf10b). The Apaf1, Bax, and Tnfrsf10b genes were upregulated at both time-points. None of the DEGs were found to be downregulated at any of the time-points in the ¹⁷⁷Lu-octreotate + A1M group.

In the A1M groups, only the Casp1 (upregulated at 24 hours) and Traf1 (upregulated at 7 d) genes were differentially expressed.

Statistically significant differences between the gene regulation in the ¹⁷⁷Lu-octreotate and the ¹⁷⁷Lu-octreotate + A1M groups were only found for Bax and Casp1, both at 24 hours.

Apoptosis-related DEGs in kidney medulla

Expression of the 19 detected DEGs (observed in at least one of the groups) in kidney medulla is shown in Fig. 3: Abl1, Apaf1, Bad, Bak1, Bax, Bcl2a1a, Bcl2l1, Bnip3l, Bok, Casp4, Cidea, Dad1, Fas, Polb, Prdxk, Ripk1, Tnfrsf10b, Trp53, and Trp73. The Bcl2a1a gene was observed to be highly upregulated in the ¹⁷⁷Lu-octreotate (FC = 31.8, SEM = 19.1) and in the ¹⁷⁷Lu-octreotate + A1M group (FC = −6.5, SEM = 6.2), although the expressions were not statistically significant different from control.

Regulation of apoptotic-related genes in kidney medulla. Differentially expressed genes compared to control (^*) in at least one of the groups are shown at (a) 24 h and (b) 7 d after injection of ¹⁷⁷Lu-octreotate, ¹⁷⁷Lu-octreotate + A1M, or A1M. Positive fold change indicates upregulation and negative fold change indicates downregulation of the gene. Error bars show SEM of the fold change values, and statistically significant difference between the groups is represented with ‘X’.

After injection of ¹⁷⁷Lu-octreotate group, four DEGs were observed at 24 hours (Bax, Bcl2l1, Cidea, and Tnfrsf10b) and five DEGs (Bad, Bax, Bnip3l, Dad1, and Polb) at 7 d. Of these, the Cidea was the only downregulated DEG (FC = −3.8, SEM = 0.0) and the Tnfrsf10b was the highest upregulated DEG (FC = 5.0, SEM = 0.4). Bax was upregulated at both time-points.

After injection of ¹⁷⁷Lu-octreotate + A1M, three DEGs were detected at 24 hours (Bax, Casp4, and Tnfrsf10b) and seven DEGs at 7 d (Abl1, Apaf1, Bok, Fas, Prdx2, Tnfrsf10b, and Trp53). The Tnfrsf10b gene was upregulated at both time-points. Bok, Fas, and Prdx2 were downregulated at 7 d. Tnfrsf10b was the highest upregulated DEG (FC = 5.9, SEM = 0.5) and Bok the highest downregulated DEG (FC = −6.0, SEM = 0.1).

Unlike the result in kidney cortex, most of the DEGs in kidney medulla were also observed in the A1M group. In the A1M groups, no DEG was found at 24 hours, while 13 were detected at 7 d (Abl1, Bad, Bak1, Bax, Bcl2a1a, Casp4, Dad1, Fas, Polb, Prdxk, Ripk1, Trp53, and Trp73), all downregulated except Abl1 and Trp53.

Statistically significant differences between the gene regulation in the ¹⁷⁷Lu-octreotate and the ¹⁷⁷Lu-octreotate + A1M groups were only found for Bcl2l1 and Casp4 at 24 hours, and Apaf1, Bad, Bak1, Bax, Bnip3l, Dad 1, Polb, and Trp53 at 7 d. Interestingly, after 7 d five genes (Bad, Bak1, Bnip3l, Dad 1, and Polb) were upregulated after ¹⁷⁷Lu-octreotate exposure, while downregulated after co-exposure with ¹⁷⁷Lu-octreotate and A1M. Bax and Tnfrsf10b were the only common DEGs for the ¹⁷⁷Lu-octreotate and ¹⁷⁷Lu-octreotate + A1M groups and then only at 24 hours.

Discussion

Co-infusion of A1M is a promising option for the improvement of ¹⁷⁷Lu-octreotate treatment of patients with NETs. Enhanced kidney protection by A1M could result in better tumour control by allowing higher total amount of administered ¹⁷⁷Lu-octreotate. A better understanding of the apoptosis protective abilities of A1M could benefit optimization of kidney protection. In this study, transcriptional regulation of 84 apoptosis-related genes was investigated short time after injection of ¹⁷⁷Lu-octreotate, ¹⁷⁷Lu-octreotate + A1M, or A1M. The gene expression was investigated separately in mouse kidney tissue: cortex and medulla. The activity level (150 MBq) was chosen based on data from a previous long-term study in the same mouse strain, demonstrating reduced kidney function and increased kidney toxicity biomarkers versus controls [21].

In agreement with previously published data, a tissue specific response was observed in regulated transcripts [16, 22]. Out of the 84 investigated genes, 53 were found to be regulated in any of the tissue types, groups, and time-points. For most of these genes the |FC| values compared to controls were minor (below 2), while some had higher |FC| levels. Low |FC| values are sometimes thought to imply low biological impact, especially if the regulation lasts a short time and the biological halt-time of the corresponding protein is limited. However, it has been shown that regulation of many genes with low |FC| values (between 1.5 and 2) have a great impact on biological effects in tissue and should not be omitted during analysis [23]. Statistically significant differences compared with control were found for 24 of these genes, defined as DEGs. Only six of the DEGs were found in both kidney medulla and cortex: apoptotic peptidase activating factor 1 (Apaf1), effector proteins BCL2-associated X protein (Bax), BCL2-like 1 (Bcl2l1), B cell leukaemia/lymphoma 2 related protein A1a (Bcl2a1a), caspase 4, apoptosis-related cysteine peptidase (Casp4), and tumour necrosis factor receptor superfamily, member 10b (Tnfrsf10b).

Most of the identified DEGs have pro-apoptotic GO annotations like ‘apoptotic process’ and ‘positive regulation of apoptotic process’. Many of them are also related to anti-apoptotic processes like ‘negative regulation of apoptotic process’ as well as regulatory processes (not specified as positive or negative) like ‘regulation of apoptotic process’. DEGs with pro-apoptotic annotations were in general upregulated in both tissue types after injection of ¹⁷⁷Lu-octreotate with or without A1M, indicating that radiation exposure induced apoptosis in both kidney tissue types. Only a small set of genes were differently expressed after injection of A1M only, except for kidney medulla at 7 d, where 13 genes were identified as DEGs. These DEGs are associated with pro-cell death processes or both pro and anti-cell death processes and were all found to be downregulated, indicating that A1M may induce anti-apoptotic response in kidney medulla and that the response increases with time.

Seven of the identified DEGs are genes encoding proteins belonging to the BCL-2 gene family: BCL2-associated agonist of cell death (Bad), Bcl2-antagonist/killer 1 (Bak1), Bax, Bcl2a1a, Bcl2l1, Bcl2-like 11 (Bcl2l11), and BCL2-related ovarian killer (Bok). BAX and BAK1 are known as effector proteins in the BCL-2 family and are key-players in induction of MOMP [24]. In this study, the Bax gene was found to be upregulated in both irradiated groups in kidney cortex at both time-points, and co-injection of A1M did not seem to inhibit the regulation of the Bax gene. On the other hand, in kidney medulla at 7 d, the expression of the Bax gene seemed to be inhibited by co-injection of A1M, demonstrated by statistically significant higher expression in ¹⁷⁷Lu-octreotate group compared with ¹⁷⁷Lu-octreotate + A1M group. In support of this, negative regulation of both the Bax and Bak1 gene was observed in the A1M group at 7 d. The Bad gene, which has an indirect role in the activation of BAX/BAK1 [24], was also found to be downregulated in kidney medulla 7 d after injection of A1M. The Bok gene encodes an effector protein that can mediate MOMP independently of BAX/BAK1 [12, 25–27]. In this study, Bok was downregulated in kidney medulla in the ¹⁷⁷Lu-octreotate + A1M group at 7 d. Although not statistically significant, the Bok gene was downregulated in almost all groups at both time-points in kidney medulla. High upregulation (not statistically significant) of the Bok gene was observed in kidney cortex at 24 h after injection of ¹⁷⁷Lu-octreotate, although the large spread in FC values (FC = 140, SEM = 140) makes this data point difficult to interpret. The Bok gene was also upregulated (not statistically significant) 24 h after injection of ¹⁷⁷Lu-octreotate + A1M in kidney medulla. The regulation pattern of the Bok gene can be interpreted as an anti-apoptotic response in kidney medulla at 7 d and that this pro survival response is strengthened by A1M.

The receptor interacting protein kinase (RIPK) 1 gene was upregulated in kidney medulla 7 d after injection of ¹⁷⁷Lu-exposure and downregulated in the combination group, both values, however, not statistically significant from controls. Furthermore, A1M alone exposure resulted in statistically significant downregulation. RIPK1 is expressed in kidney tubules and to some extent also in glomeruli, and takes part in activation of the extrinsic apoptotic pathway [28].

The proteins BCL2A1A and BCL2L1 are members of the BCL-2 family and inhibit apoptosis by binding to pro-apoptotic BCL-2 proteins [24]. In the present study, the Bcl2l1 gene was upregulated at 24 h after injection of ¹⁷⁷Lu-octreotate in both kidney tissues. In kidney medulla, the expression of the Bcl2l1 gene was higher with a statistical significance than the combination group, while no such difference was found in cortex.

The expression pattern of the Bcl2a1a gene differed substantially between the kidney tissue types; it was in general downregulated in kidney cortex and upregulated in kidney medulla. However, no statistically significant difference was found between any of the treatment groups in expression of the Bcl2a1a gene.

The Tnfrsf10b gene was one of the most recurrently observed DEGs in this study. It was upregulated in the ¹⁷⁷Lu-octreotate and ¹⁷⁷Lu-octreotate + A1M group at both time-points and tissue types. TNFRSF10B gene (also known as TRAILR2 or DR5) is a member of the tumour necrosis factor receptor superfamily (TNFRSF) and is involved in activation of the extrinsic apoptosis pathway. Hence, upregulation of the Tnfrsf10b gene can be considered as a pro-apoptotic response. The Fas gene is another pro-apoptotic DEG belonging to the TNFRSF family and was found to be downregulated in kidney medulla 7 d after injection of A1M. The regulation of the Tnfrsf10b gene did not indicate any difference between the ¹⁷⁷Lu-octreotate and ¹⁷⁷Lu-octreotate + A1M group, but the regulation of the Fas gene could potentially mean that A1M alone has a mitigating effect of the extrinsic apoptosis pathway [29].

Once MOMP is initiated, several mitochondrial proteins, such as cytochrome C, are leaked from the mitochondria intermembrane space into the cytoplasm. Cytochrome C then activates and binds to APAF1 and pro-caspase 9 (CASP9), creating a complex known as apoptosome. This process is followed by activation of the effector caspases CASP3, CASP6, and CASP7, which in turn initiate the final steps needed to carry out the apoptosis process. In this study, the Apaf1 gene was found to be differently expressed in both irradiated groups at both time-points in kidney cortex and in kidney medulla at 7 d in the combination group. The Casp3 gene was found to be differently expressed from control in kidney cortex 24 hours after injection of ¹⁷⁷Lu-octreotate, but not in the combination group. The upregulation of the Apaf1 and Casp3 genes can be interpreted as a radiation induced pro-apoptotic response late in the apoptotic process [12, 13].

Previously, Kristiansson et al. showed that ¹⁷⁷Lu-octreotate-induced regulation of apoptosis-related genes in kidney was reduced by co-infusion of A1M [7]. That study was performed using the same mouse apoptosis RT² PCR profiler array as in the present study, but did not distinguish medulla from cortex. Our results present a less pronounced difference in regulation of the genes between the ¹⁷⁷Lu-octreotate and ¹⁷⁷Lu-octreotate + A1M group. In agreement with Kristiansson et al., the Bax, Bcl2l1, and Tnfrsf10b genes were among the genes most affected by the irradiation. Their study also demonstrated a high regulation of growth arrest and DNA-damage-inducible 45 alpha (Gadd45a), which was not differently expressed in the present study. The differences in results between the studies could partly be explained by difference in mouse strains: C57BL/6N in the present study and immune-deficient BALB/c nude mice in the other study. The two strains differ in radiosensitivity and the BALB/c strain has consistently been found to be more radiosensitive than C57BL/6 [30–32]. Furthermore, based on our previous results we do not expect the same transcriptional response when analysing the entire kidney tissue compared with separate analyses of kidney medulla and cortex [16].

An important criterion for an agent to function as radioprotector is that its effects reach the irradiated tissue it is supposed to protect. A1M has shown to have similar biodistribution as ¹¹¹In-labeled octreotide, a somatostatin analogue with similar biokinetic properties as octreotate, in mouse kidney short time after injection [33]. The distribution of the two molecules was primarily found in proximal tubules in kidney cortex, similar to ¹⁷⁷Lu [34]. These findings strongly advocate that the protective effect of A1M will reach the irradiated areas following co-administration of ¹⁷⁷Lu-octreotate and A1M. Interestingly, our results show an overall more pronounced effect of A1M in kidney medulla compared with kidney cortex. The reason for this is unknown and needs to be further investigated. It should be noted that a higher γH2AX signal was found in cortex but not in medulla after ¹⁷⁷Lu-octreotate exposure, with lower frequency after co-exposure with A1M in cortex in the study on nude mice 4 d after exposure [7]. The γH2AX signal then consisted of both single foci, representing DNA strand breaks, and pan-nuclear staining, representing apoptosis. The majority of the signal was pan-nuclear staining, and the effect of A1M seemed to primarily reduce the frequency of apoptosis-signalling cells in cortex. This finding may be in contrast to the results from the present study, where no clear protection was found in cortex, but the discrepancy could also be due to difference in time after exposure.

In the present study, the apoptosis-related gene regulation following ¹⁷⁷Lu-octreotate and/or A1M exposure was examined, demonstrating tissue specific response in mouse kidney. It should be noted that this study did not include kidney toxicity measurements and the observed pro-apoptotic response needs to be validated on the protein level. Furthermore, the kidney is a late responding organ and the impact of the observed gene regulation on kidney damage and loss of function at later time-points is not known. However, 150 MBq ¹⁷⁷Lu-octreotate administration should result in kidney damage, since a previous long-term study revealed dose-dependent regulation of several kidney toxicity biomarkers and late occurring functional effects after 8 and 12 months after administration of up to 150 MBq ¹⁷⁷Lu-octreotate in mice of the same animal strain [20]. As a next step in the investigation of the potential renal protective abilities of A1M, we suggest protein validation of the most DEGs, together with further long-term studies focusing on potential protection of renal function with co-administration of A1M.

Conclusion

In summary, this study demonstrated a transcriptional pro-apoptotic response in mouse kidney early after injection of ¹⁷⁷Lu-octreotate. The radiation induced effects depended on kidney tissue type (medulla versus cortex) and to some extent also time after injection. The proposed kidney protector A1M seems to have a pro-survival effect in kidney medulla, but not in kidney cortex. Long-term nephrotoxicity after administration of ¹⁷⁷Lu-octreotate with or without A1M should be studied to further assess the potential clinical application of A1M as radioprotector of the kidneys.

Supplementary Material

Supplemental_figure_1_ncaf055

supplemental_figure_1_ncaf055.docx^{(31.9KB, docx)}

Acknowledgements

The authors are most grateful to Britta Langen, PhD, for conceiving the study, contributing to planning of experiment design and supervision of RNA isolation. The authors thank the staff at Central Radiopharmacy at Sahlgrenska University Hospital, Annika Bergentall, MSc, Hana Hameed Bakr, MSc, Petra Bergström, PhLic, and Ylva Surac, MSc, for preparation of ¹⁷⁷Lu-octreotate. Thanks also to Guard Therapeutics International AB (Stockholm, Sweden) for providing the A1M.

Contributor Information

Charlotte Ytterbrink, Department of Medical Radiation Sciences, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden; Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden.

Klara Simonsson, Department of Medical Radiation Sciences, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden; Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden.

Emman Shubbar, Department of Medical Radiation Sciences, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden; Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden.

Magnus Gram, Pediatrics, Department of Clinical Sciences Lund, Lund University, SE-221 85 Lund, Sweden; Department of Neonatology, Skåne University Hospital, SE-221 84 Lund, Sweden.

Khalil Helou, Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden; Department of Oncology, Institute of Clinical Sciences, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden.

Eva Forssell-Aronsson, Department of Medical Radiation Sciences, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden; Sahlgrenska Center for Cancer Research, Sahlgrenska Academy at the University of Gothenburg, Box 425, SE-405 30 Gothenburg, Sweden; Department of Medical Physics and Biomedical Engineering, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden.

Conflict of interest

None declared.

Funding

This study was supported by grants from the Swedish Research Council (grants no. 2021-02636), the Swedish Cancer Society (grants no. 23 2975), the Swedish state under the agreement between the Swedish government and the county councils—the ALF-agreement (ALFGBG-966074), the King Gustaf V Jubilee Clinic Cancer Research Foundation, the Sahlgrenska University Hospital Research Funds, the Wilhelm and Martina Lundgren Research Foundation, the Assar Gabrielsson Cancer Research Foundation and the Herbert & Karin Jacobsson Foundation, and Adlerbertska Research Foundation.

References

1.Authorization details for Lutathera® in Europe. https://www.ema.europa.eu/en/medicines/human/EPAR/lutathera#authorisation-details-section (21 February 2020, date last accessed).
2. Forssell-Aronsson E, Spetz J, Ahlman H. Radionuclide therapy via SSTR: future aspects from experimental animal studies. Neuroendocrinology 2013;97:86–98. 10.1159/000336086 [DOI] [PubMed] [Google Scholar]
3. Geenen L, Nonnekens J, Konijnenberg M. et al. Overcoming nephrotoxicity in peptide receptor radionuclide therapy using [(177)Lu]Lu-DOTA-TATE for the treatment of neuroendocrine tumours. Nucl Med Biol 2021;102-103:1–11. 10.1016/j.nucmedbio.2021.06.006 [DOI] [PubMed] [Google Scholar]
4. Rolleman EJ, Bernard BF, Breeman WA. et al. Molecular imaging of reduced renal uptake of radiolabelled [DOTA0,Tyr3]octreotate by the combination of lysine and Gelofusine in rats. Nuklearmedizin 2008;47:110–5. 10.3413/nukmed-0069 [DOI] [PubMed] [Google Scholar]
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6. Åkerstrom B, Gram M. A1M, an extravascular tissue cleaning and housekeeping protein. Free Radic Biol Med 2014;74:274–82. 10.1016/j.freeradbiomed.2014.06.025 [DOI] [PubMed] [Google Scholar]
7. Kristiansson A, Ahlstedt J, Holmqvist B. et al. Protection of kidney function with human antioxidation protein alpha1-microglobulin in a mouse (177)Lu-DOTATATE radiation therapy model. Antioxid Redox Signal 2019;30:1746–59. 10.1089/ars.2018.7517 [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Kristiansson A, Örbom A, Ahlstedt J. et al. (177)Lu-PSMA-617 therapy in mice, with or without the antioxidant α(1)-microglobulin (A1M), including kidney damage assessment using (99m)Tc-MAG3 imaging. Biomolecules 2021;11:263. 10.3390/biom11020263 [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Andersson CK, Shubbar E, Schüler E. et al. Recombinant α(1)-microglobulin is a potential kidney protector in (177)Lu-octreotate treatment of neuroendocrine tumors. J Nucl Med 2019;60:1600–4. 10.2967/jnumed.118.225243 [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Kristiansson A, Örbom A, Vilhelmsson TO. et al. Kidney protection with the radical scavenger α(1)-microglobulin (A1M) during peptide receptor radionuclide and radioligand therapy. Antioxidants (Basel) 2021;10:1271. 10.3390/antiox10081271 [DOI] [PMC free article] [PubMed] [Google Scholar]
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12. Galluzzi L, Vitale I, Aaronson SA. et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ 2018;25:486–541. 10.1038/s41418-017-0012-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Eriksson D, Stigbrand T. Radiation-induced cell death mechanisms. Tumour Biol 2010;31:363–72. [DOI] [PubMed] [Google Scholar]
14. Olsson MG, Rosenlöf LW, Kotarsky H. et al. The radical-binding lipocalin A1M binds to a complex I subunit and protects mitochondrial structure and function. Antioxid Redox Signal 2013;18:2017–28. 10.1089/ars.2012.4658 [DOI] [PubMed] [Google Scholar]
15. Kristiansson A, Davidsson S, Johansson ME. et al. α(1)-Microglobulin (A1M) protects human proximal tubule epithelial cells from heme-induced damage in vitro. Int J Mol Sci 2020;21:21. 10.3390/ijms21165825 [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Schüler E, Rudqvist N, Parris TZ. et al. Transcriptional response of kidney tissue after ¹⁷⁷Lu-octreotate administration in mice. Nucl Med Biol 2014;41:238–47. 10.1016/j.nucmedbio.2013.12.001 [DOI] [PubMed] [Google Scholar]
17. Åkerström B, Rosenlöf L, Hägerwall A. et al. rA1M-035, a physicochemically improved human recombinant α(1)-microglobulin, has therapeutic effects in rhabdomyolysis-induced acute kidney injury. Antioxid Redox Signal 2019;30:489–504. 10.1089/ars.2017.7181 [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Schüler E, Österlund A, Forssell-Aronsson E. The amount of injected ¹⁷⁷Lu-octreotate strongly influences biodistribution and dosimetry in C57BL/6N mice. Acta Oncologica 2016;55:68–76. 10.3109/0284186x.2015.1027001 [DOI] [PubMed] [Google Scholar]
19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402–8. 10.1006/meth.2001.1262 [DOI] [PubMed] [Google Scholar]
20.Mouse Genome Database (MGD). https://www.informatics.jax.org/ (6 December 2021, date last accessed).
21. Schüler E, Larsson M, Parris TZ. et al. Potential biomarkers for radiation-induced renal toxicity following ¹⁷⁷Lu-octreotate administration in mice. PLoS One 2015;10:e0136204. 10.1371/journal.pone.0136204 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Schüler E, Rudqvist N, Parris TZ. et al. Time- and dose rate-related effects of internal (177)Lu exposure on gene expression in mouse kidney tissue. Nucl Med Biol 2014;41:825–32. 10.1016/j.nucmedbio.2014.07.010 [DOI] [PubMed] [Google Scholar]
23. Dalman MR, Deeter A, Nimishakavi G. et al. Fold change and p-value cutoffs significantly alter microarray interpretations. BMC Bioinformatics 2012;13:S11. 10.1186/1471-2105-13-S2-S11 [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Chipuk JE, Moldoveanu T, Llambi F. et al. The BCL-2 family Reunion. Mol Cell 2010;37:299–310. 10.1016/j.molcel.2010.01.025 [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Llambi F, Wang YM, Victor B. et al. BOK is a non-canonical BCL-2 family effector of apoptosis regulated by ER-associated degradation. Cell 2016;165:421–33. 10.1016/j.cell.2016.02.026 [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Fernández-Marrero Y, Bleicken S, Das KK. et al. The membrane activity of BOK involves formation of large, stable toroidal pores and is promoted by cBID. FEBS J 2017;284:711–24. 10.1111/febs.14008 [DOI] [PubMed] [Google Scholar]
27. Einsele-Scholz S, Malmsheimer S, Bertram K. et al. Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak. J Cell Sci 2016;129:2213–23. 10.1242/jcs.181727 [DOI] [PubMed] [Google Scholar]
28. Green DR, Oberst A, Dillon CP. et al. RIPK-dependent necrosis and its regulation by caspases: a mystery in five acts. Mol Cell 2011;44:9–16. 10.1016/j.molcel.2011.09.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Gaur U, Aggarwal BB. Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol 2003;66:1403–8. 10.1016/S0006-2952(03)00490-8 [DOI] [PubMed] [Google Scholar]
30. Okayasu R, Suetomi K, Yu Y. et al. A deficiency in DNA repair and DNA-PKcs expression in the radiosensitive BALB/c mouse. Cancer Res 2000;60:4342–5. [PubMed] [Google Scholar]
31. Grahn D, Hamilton KF. Genetic variation in the acute lethal response of four inbred mouse strains to whole body X-irradiation. Genetics 1957;42:189–98. 10.1093/genetics/42.3.189 [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Kallman RF. The effect of dose rate on mode of acute radiation death of C57BL and BALB/c mice. Radiat Res 1962;16:796–810. 10.2307/3571279 [DOI] [PubMed] [Google Scholar]
33. Ahlstedt J, Tran TA, Strand F. et al. Biodistribution and pharmacokinetics of recombinant alpha1-microglobulin and its potential use in radioprotection of kidneys. Am J Nucl Med Mol Imaging 2015;5:333–47. [PMC free article] [PubMed] [Google Scholar]
34. Melis M, Krenning EP, Bernard BF. et al. Localisation and mechanism of renal retention of radiolabelled somatostatin analogues. Eur J Nucl Med Mol Imaging 2005;32:1136–43. 10.1007/s00259-005-1793-0 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental_figure_1_ncaf055

supplemental_figure_1_ncaf055.docx^{(31.9KB, docx)}

[ref1] 1.Authorization details for Lutathera® in Europe. https://www.ema.europa.eu/en/medicines/human/EPAR/lutathera#authorisation-details-section (21 February 2020, date last accessed).

[ref2] 2. Forssell-Aronsson E, Spetz J, Ahlman H. Radionuclide therapy via SSTR: future aspects from experimental animal studies. Neuroendocrinology 2013;97:86–98. 10.1159/000336086 [DOI] [PubMed] [Google Scholar]

[ref3] 3. Geenen L, Nonnekens J, Konijnenberg M. et al. Overcoming nephrotoxicity in peptide receptor radionuclide therapy using [(177)Lu]Lu-DOTA-TATE for the treatment of neuroendocrine tumours. Nucl Med Biol 2021;102-103:1–11. 10.1016/j.nucmedbio.2021.06.006 [DOI] [PubMed] [Google Scholar]

[ref4] 4. Rolleman EJ, Bernard BF, Breeman WA. et al. Molecular imaging of reduced renal uptake of radiolabelled [DOTA0,Tyr3]octreotate by the combination of lysine and Gelofusine in rats. Nuklearmedizin 2008;47:110–5. 10.3413/nukmed-0069 [DOI] [PubMed] [Google Scholar]

[ref5] 5. Rolleman EJ, Valkema R, de Jong M. et al. Safe and effective inhibition of renal uptake of radiolabelled octreotide by a combination of lysine and arginine. Eur J Nucl Med Mol Imaging 2003;30:9–15. 10.1007/s00259-002-0982-3 [DOI] [PubMed] [Google Scholar]

[ref6] 6. Åkerstrom B, Gram M. A1M, an extravascular tissue cleaning and housekeeping protein. Free Radic Biol Med 2014;74:274–82. 10.1016/j.freeradbiomed.2014.06.025 [DOI] [PubMed] [Google Scholar]

[ref7] 7. Kristiansson A, Ahlstedt J, Holmqvist B. et al. Protection of kidney function with human antioxidation protein alpha1-microglobulin in a mouse (177)Lu-DOTATATE radiation therapy model. Antioxid Redox Signal 2019;30:1746–59. 10.1089/ars.2018.7517 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref8] 8. Kristiansson A, Örbom A, Ahlstedt J. et al. (177)Lu-PSMA-617 therapy in mice, with or without the antioxidant α(1)-microglobulin (A1M), including kidney damage assessment using (99m)Tc-MAG3 imaging. Biomolecules 2021;11:263. 10.3390/biom11020263 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref9] 9. Andersson CK, Shubbar E, Schüler E. et al. Recombinant α(1)-microglobulin is a potential kidney protector in (177)Lu-octreotate treatment of neuroendocrine tumors. J Nucl Med 2019;60:1600–4. 10.2967/jnumed.118.225243 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref10] 10. Kristiansson A, Örbom A, Vilhelmsson TO. et al. Kidney protection with the radical scavenger α(1)-microglobulin (A1M) during peptide receptor radionuclide and radioligand therapy. Antioxidants (Basel) 2021;10:1271. 10.3390/antiox10081271 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref11] 11. Mishra KN, Moftah BA, Alsbeih GA. Appraisal of mechanisms of radioprotection and therapeutic approaches of radiation countermeasures. Biomed Pharmacother 2018;106:610–7. 10.1016/j.biopha.2018.06.150 [DOI] [PubMed] [Google Scholar]

[ref12] 12. Galluzzi L, Vitale I, Aaronson SA. et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ 2018;25:486–541. 10.1038/s41418-017-0012-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref13] 13. Eriksson D, Stigbrand T. Radiation-induced cell death mechanisms. Tumour Biol 2010;31:363–72. [DOI] [PubMed] [Google Scholar]

[ref14] 14. Olsson MG, Rosenlöf LW, Kotarsky H. et al. The radical-binding lipocalin A1M binds to a complex I subunit and protects mitochondrial structure and function. Antioxid Redox Signal 2013;18:2017–28. 10.1089/ars.2012.4658 [DOI] [PubMed] [Google Scholar]

[ref15] 15. Kristiansson A, Davidsson S, Johansson ME. et al. α(1)-Microglobulin (A1M) protects human proximal tubule epithelial cells from heme-induced damage in vitro. Int J Mol Sci 2020;21:21. 10.3390/ijms21165825 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref16] 16. Schüler E, Rudqvist N, Parris TZ. et al. Transcriptional response of kidney tissue after ¹⁷⁷Lu-octreotate administration in mice. Nucl Med Biol 2014;41:238–47. 10.1016/j.nucmedbio.2013.12.001 [DOI] [PubMed] [Google Scholar]

[ref17] 17. Åkerström B, Rosenlöf L, Hägerwall A. et al. rA1M-035, a physicochemically improved human recombinant α(1)-microglobulin, has therapeutic effects in rhabdomyolysis-induced acute kidney injury. Antioxid Redox Signal 2019;30:489–504. 10.1089/ars.2017.7181 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref18] 18. Schüler E, Österlund A, Forssell-Aronsson E. The amount of injected ¹⁷⁷Lu-octreotate strongly influences biodistribution and dosimetry in C57BL/6N mice. Acta Oncologica 2016;55:68–76. 10.3109/0284186x.2015.1027001 [DOI] [PubMed] [Google Scholar]

[ref19] 19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402–8. 10.1006/meth.2001.1262 [DOI] [PubMed] [Google Scholar]

[ref20] 20.Mouse Genome Database (MGD). https://www.informatics.jax.org/ (6 December 2021, date last accessed).

[ref21] 21. Schüler E, Larsson M, Parris TZ. et al. Potential biomarkers for radiation-induced renal toxicity following ¹⁷⁷Lu-octreotate administration in mice. PLoS One 2015;10:e0136204. 10.1371/journal.pone.0136204 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref22] 22. Schüler E, Rudqvist N, Parris TZ. et al. Time- and dose rate-related effects of internal (177)Lu exposure on gene expression in mouse kidney tissue. Nucl Med Biol 2014;41:825–32. 10.1016/j.nucmedbio.2014.07.010 [DOI] [PubMed] [Google Scholar]

[ref23] 23. Dalman MR, Deeter A, Nimishakavi G. et al. Fold change and p-value cutoffs significantly alter microarray interpretations. BMC Bioinformatics 2012;13:S11. 10.1186/1471-2105-13-S2-S11 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref24] 24. Chipuk JE, Moldoveanu T, Llambi F. et al. The BCL-2 family Reunion. Mol Cell 2010;37:299–310. 10.1016/j.molcel.2010.01.025 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref25] 25. Llambi F, Wang YM, Victor B. et al. BOK is a non-canonical BCL-2 family effector of apoptosis regulated by ER-associated degradation. Cell 2016;165:421–33. 10.1016/j.cell.2016.02.026 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref26] 26. Fernández-Marrero Y, Bleicken S, Das KK. et al. The membrane activity of BOK involves formation of large, stable toroidal pores and is promoted by cBID. FEBS J 2017;284:711–24. 10.1111/febs.14008 [DOI] [PubMed] [Google Scholar]

[ref27] 27. Einsele-Scholz S, Malmsheimer S, Bertram K. et al. Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak. J Cell Sci 2016;129:2213–23. 10.1242/jcs.181727 [DOI] [PubMed] [Google Scholar]

[ref28] 28. Green DR, Oberst A, Dillon CP. et al. RIPK-dependent necrosis and its regulation by caspases: a mystery in five acts. Mol Cell 2011;44:9–16. 10.1016/j.molcel.2011.09.003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref29] 29. Gaur U, Aggarwal BB. Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol 2003;66:1403–8. 10.1016/S0006-2952(03)00490-8 [DOI] [PubMed] [Google Scholar]

[ref30] 30. Okayasu R, Suetomi K, Yu Y. et al. A deficiency in DNA repair and DNA-PKcs expression in the radiosensitive BALB/c mouse. Cancer Res 2000;60:4342–5. [PubMed] [Google Scholar]

[ref31] 31. Grahn D, Hamilton KF. Genetic variation in the acute lethal response of four inbred mouse strains to whole body X-irradiation. Genetics 1957;42:189–98. 10.1093/genetics/42.3.189 [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref32] 32. Kallman RF. The effect of dose rate on mode of acute radiation death of C57BL and BALB/c mice. Radiat Res 1962;16:796–810. 10.2307/3571279 [DOI] [PubMed] [Google Scholar]

[ref33] 33. Ahlstedt J, Tran TA, Strand F. et al. Biodistribution and pharmacokinetics of recombinant alpha1-microglobulin and its potential use in radioprotection of kidneys. Am J Nucl Med Mol Imaging 2015;5:333–47. [PMC free article] [PubMed] [Google Scholar]

[ref34] 34. Melis M, Krenning EP, Bernard BF. et al. Localisation and mechanism of renal retention of radiolabelled somatostatin analogues. Eur J Nucl Med Mol Imaging 2005;32:1136–43. 10.1007/s00259-005-1793-0 [DOI] [PubMed] [Google Scholar]

PERMALINK

Early apoptotic response in kidney after ¹⁷⁷Lu-octreotate administration with or without potential radioprotector α₁-microglobulin

Charlotte Ytterbrink

Klara Simonsson

Emman Shubbar

Magnus Gram

Khalil Helou

Eva Forssell-Aronsson

Abstract

Introduction

Materials and methods