Protection of Kidney Function with Human Antioxidation Protein α1-Microglobulin in a Mouse 177Lu-DOTATATE Radiation Therapy Model

Abstract

Aims: Peptide receptor radionuclide therapy (PRRT) is in clinical use today to treat metastatic neuroendocrine tumors. Infused, radiolabeled, somatostatin analog peptides target tumors that are killed by irradiation damage. The peptides, however, are also retained in kidneys due to glomerular filtration, and the administered doses must be limited to avoid kidney damage. The human radical scavenger and antioxidant, α₁-microglobulin (A1M), has previously been shown to protect bystander tissue against irradiation damage and has pharmacokinetic and biodistribution properties similar to somatostatin analogs. In this study, we have investigated if A1M can be used as a renal protective agent in PRRT.

Results: We describe nephroprotective effects of human recombinant A1M on the short- and long-term renal damage observed following lutetium 177 (¹⁷⁷Lu)-DOTATATE (150 MBq) exposure in BALB/c mice. After 1, 4, and 8 days (short term), ¹⁷⁷Lu-DOTATATE injections resulted in increased formation of DNA double-strand breaks in the renal cortex, upregulated expression of apoptosis and stress response-related genes, and proteinuria (albumin in urine), all of which were significantly suppressed by coadministration of A1M (7 mg/kg). After 6, 12, and 24 weeks (long term), ¹⁷⁷Lu-DOTATATE injections resulted in increased animal death, kidney lesions, glomerular loss, upregulation of stress genes, proteinuria, and plasma markers of reduced kidney function, all of which were suppressed by coadministration of A1M. Innovation and Conclusion: This study demonstrates that A1M effectively inhibits radiation-induced renal damage. The findings suggest that A1M may be used as a radioprotector during clinical PRRT, potentially facilitating improved tumor control and enabling more patients to receive treatment.

Keywords: Lu-DOTATATE; PRRT; cancer; radionuclide therapy; renal protection; α-microglobulin.

FIG. 1.

Overview of design, groups, and results of the experiment presented in this article. Four groups of nude female BALB/c mice received different injections: (i) control (PBS), (ii) ¹⁷⁷Lu-DOTATATE, (iii) ¹⁷⁷Lu-DOTATATE+A1M, and (iv) A1M. Animals from each group were terminated and sampled at six various time points after injection: 1 day, 4 days, 8 days, 6 weeks, 12 weeks, and 24 weeks. Injection of ¹⁷⁷Lu-DOTATATE resulted in significant formation of DNA breaks, kidney damage (structural lesions and compromised renal function), and lower survival rate. Coadministration with A1M resulted in less DNA damage, renal function impairment, and fewer radiation-related deaths. A1M, α1-microglobulin; ¹⁷⁷Lu, lutetium 177; PBS, phosphate-buffered saline. Color images are available online.

FIG. 2.

Coadministration of A1M with ¹⁷⁷Lu-DOTATATE increased survival. Table presents total number of animals in all groups at all time points (A). Number of animal deaths and animals removed from study (censored) are also presented. (B, C) Kaplan–Meier survival curve of control, ¹⁷⁷Lu-DOTATATE, ¹⁷⁷Lu-DOTATATE+A1M, and A1M groups. The experiment for the different groups ran 12 (B), respectively, 24 (C) weeks postinjections. Censored animals are marked in the graph at the time point of the censoring. The causes of death of censored mice were weight loss, skin lesions, and swollen joints. Deaths were classified as radiation related when any of the following were found: swollen liver, swollen kidneys, swollen spleen, pathological spleen, and pathological liver. p = 0.0397; control mice compared with ¹⁷⁷Lu-DOTATATE mice and p = 0.2731 (n.s.); ¹⁷⁷Lu-DOTATATE mice compared with ¹⁷⁷Lu-DOTATATE+A1M mice in the 24-week groups. (Mantel–Cox log-rank test).

FIG. 3.

A1M decreases double-strand DNA breaks in the kidneys and reduces kidney damage after injection with ¹⁷⁷Lu-DOTATATE. (A–H) Immunofluorescence labeling of the DNA damage marker γH2AX in kidney sections from the four groups. (A–D) Representative images of γH2AX immunofluorescence-labeled foci (green) and cell nuclei (blue) in kidney glomeruli and surrounding tubuli of kidney sections, 4 days after injections of ¹⁷⁷Lu-DOTATATE (A, C) or ¹⁷⁷Lu-DOTATATE+A1M (B, D). Scale bar in (A) indicates 20 μm, applicable in (A, B), and in–(C) indicates 10 μm, applicable in (C, D). (E–H) Percentage of γH2AX-positive cells in the four groups in cortex (E), medulla (F), cortical tubules (G), and glomeruli (H) 4 days postinjections. (I–R) Histopathological findings in the kidney from a ¹⁷⁷Lu-DOTATATE-injected nude female BALB/c mouse (24 weeks) showing advanced stage of nephropathy (J) compared with the corresponding cortical region in a normal kidney from a control animal (I). (K–N) depict higher magnification fields from (J) showing areas of fibrosis (white arrows), glomerular changes, including glomerular degeneration and atrophy, and glomerulosclerosis (black arrows). Inflammation (white arrowhead) and tubular dilation (asterisk) are also depicted. The pale pink areas depict tubular necrosis and degeneration found in ¹⁷⁷Lu-DOTATATE-injected animals (M). Scale bar in (I) represents 500 μm in (I, J), 125 μm in (K, M, N), and 250 μm in (L). (O–R) Total scores of kidney lesions found in the four mouse groups. Eight different lesions were separately scored (0–4 severity; shown in Supplementary Fig. S1) and totaled for each animal. The mean of each group is presented at (O) 6 weeks, (P) 12 weeks, and (Q) 24 weeks postinjections. The glomerular count of morphologically defined normal glomerular units is shown as 24 weeks postinjections (R). Assessments were performed in hematoxylin–eosin-stained kidney sections (10 μm). Statistical comparison was made between control and ¹⁷⁷Lu-DOTATATE, control and ¹⁷⁷Lu-DOTATATE+A1M, respectively, and ¹⁷⁷Lu-DOTATATE and ¹⁷⁷Lu-DOTATATE+A1M (E, F, O–R). Only significant differences are presented in the figure. No statistical calculations are presented in (G, H) due to the low number of samples. Values are presented as mean ± SEM. Differences in groups were analyzed using one-way ANOVA with post hoc Tukey's test (E, F, R) and Kruskal–Wallis test with post hoc Dunn's test for histological scoring (O–Q). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. γH2AX, H2AX phosphorylated on serine 139; ANOVA, analysis of variance. Color images are available online.

FIG. 4.

¹⁷⁷Lu-DOTATATE injections in nude BALB/c mice change gene expression of genes associated with apoptosis. (A) Messenger RNA expression of an apoptosis-related gene array in pooled renal tissue from all individuals (n = 4 control and ¹⁷⁷Lu-DOTATATE+A1M and n = 5 ¹⁷⁷Lu-DOTATATE) terminated 1 day postinjection, normalized to expression of the β2-microglobulin gene, and expressed as fold change relative to the control group, where fold change 1 equals the control group. Green indicates a fold change of 0–1.5, orange 1.5–2.0, red 2.0–5.0, and black above 5. (B–E) Analysis of mRNA expression of Bax (B), Gadd45a (C), Tnfrsf10b (D), and Bcl2l1 (E) at the individual level, 1 day postinjections, normalized to expression of GAPDH. Statistical comparison was made between control and ¹⁷⁷Lu-DOTATATE, control and ¹⁷⁷Lu-DOTATATE+A1M, respectively, and ¹⁷⁷Lu-DOTATATE and ¹⁷⁷Lu-DOTATATE+A1M. Only significant differences are presented in the figure. Values, normalized against the control group, are presented as mean fold change with whiskers indicating 5 to 95 percentiles. Outliers were removed using the ROUT method (Q = 1%) (B–E). Differences in groups were analyzed using one-way ANOVA with post hoc Tukey's test (B–E). *p < 0.05, **p < 0.01, ****p < 0.0001. Bax, bcl-2-like protein 4; Bcl211, Bcl-2-like 1; Gadd45a, growth arrest and DNA damage-inducible protein alpha; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA; Tnfrsf10b, tumor necrosis factor receptor superfamily member 10B. Color images are available online.

FIG. 5.

¹⁷⁷Lu-DOTATATE injections in nude BALB/c mice change gene expression of genes associated with inflammation and oxidative stress. (A–D) Analysis of mRNA expression of NGAL (A), Hsp70 (B) in kidney tissue, and A1M (C) and HO-1 (D) in the liver at the individual level, 6 weeks postinjection. Statistical comparison was made between control and ¹⁷⁷Lu-DOTATATE, control and ¹⁷⁷Lu-DOTATATE+A1M, respectively, and ¹⁷⁷Lu-DOTATATE and ¹⁷⁷Lu-DOTATATE+A1M. Only significant differences are presented in the figure. Values, normalized against the control group, are presented as mean fold change normalized to GAPDH expression values, with whiskers indicating 5 to 95 percentiles. Outliers were removed using the ROUT method (Q = 1%). Differences in groups were analyzed using one-way ANOVA with post hoc Tukey's test. *p < 0.05, ***p < 0.001. HO-1, heme oxygenase 1; Hsp70, heat shock protein 70; NGAL, neutrophil gelatinase-associated lipocalin.

FIG. 6.

¹⁷⁷Lu-DOTATATE injections in nude BALB/c mice affect serum levels of markers associated with kidney function. Scatter plots depict (A) serum cystatin C, (B) A1M, (C) BUN, (D) Na⁺, and (E) K⁺ levels 24 weeks postinjections measured by (A) ELISA, (B) radioimmunoassay, or (C–E) the VetScan VS2 chemistry analyzer in the four mouse groups. Statistical comparison was made between control and ¹⁷⁷Lu-DOTATATE, control and ¹⁷⁷Lu-DOTATATE+A1M, respectively, and ¹⁷⁷Lu-DOTATATE and ¹⁷⁷Lu-DOTATATE+A1M. Only significant differences are presented in the figure. Values are presented as mean with whiskers indicating 5 to 95 percentiles. Outliers were removed using the ROUT method (Q = 1%). Differences in groups were analyzed using one-way ANOVA with post hoc Tukey's test. *p < 0.05. K⁺, potassium ion; Na⁺, sodium ion.

FIG. 7.

¹⁷⁷Lu-DOTATATE injections in nude BALB/c mice affect urine albumin levels, a marker of proteinuria. Urine level of the functional marker albumin in BALB/c nude mice (A) 6 weeks, (B) 12 weeks, and (C) 24 weeks postinjections, measured by ELISA. Statistical comparison was made between control and ¹⁷⁷Lu-DOTATATE, control and ¹⁷⁷Lu-DOTATATE+A1M, respectively, and ¹⁷⁷Lu-DOTATATE and ¹⁷⁷Lu-DOTATATE+A1M. Only significant differences are presented in the figure. No statistical comparison is presented in the 24-week animals due to low number of samples from the ¹⁷⁷Lu-DOTATATE animals (n = 2). Values are presented as mean ± SEM. Outliers were removed using the ROUT method (Q = 1%). Differences in groups were analyzed using one-way ANOVA with post hoc Tukey's test. *p < 0.05, **p < 0.01.