Resistance to Recombinant Human Erythropoietin Therapy in a Rat Model of Chronic Kidney Disease Associated Anemia

Abstract

This study aimed to elucidate the mechanisms explaining the persistence of anemia and resistance to recombinant human erythropoietin (rHuEPO) therapy in a rat model of chronic kidney disease (CKD)-associated anemia with formation of anti-rHuEPO antibodies. The remnant kidney rat model of CKD induced by 5/6 nephrectomy was used to test a long-term (nine weeks) high dose of rHuEPO (200 UI/kg bw/week) treatment. Hematological and biochemical parameters were evaluated as well as serum and tissue (kidney, liver and/or duodenum) protein and/or gene expression of mediators of erythropoiesis, iron metabolism and tissue hypoxia, inflammation, and fibrosis. Long-term treatment with a high rHuEPO dose is associated with development of resistance to therapy as a result of antibodies formation. In this condition, serum EPO levels are not deficient and iron availability is recovered by increased duodenal absorption. However, erythropoiesis is not stimulated, and the resistance to endogenous EPO effect and to rHuEPO therapy results from the development of a hypoxic, inflammatory and fibrotic milieu in the kidney tissue. This study provides new insights that could be important to ameliorate the current therapeutic strategies used to treat patients with CKD-associated anemia, in particular those that become resistant to rHuEPO therapy.

Keywords: anemia; chronic kidney disease; erythropoiesis; inflammation and fibrosis; iron metabolism; kidney hypoxia; remnant kidney rat model; resistance to rHuEPO therapy.

Figure 1

Hematological and renal data throughout the follow-up period of 12 weeks: hemoglobin concentration (A); red blood cell count (B); hematocrit (C); reticulocyte count (D); creatinine (E); and BUN concentrations (F). Results are presented as mean ± SEM (seven rats per group): a: p < 0.05 and aaa: p < 0.001 vs. Sham; b: p < 0.05 and and bbb: p < 0.001 vs. CRF.

Figure 2

Erythropoietin (EPO) and erythropoietin receptor (EPOR): EPO in serum and kidney and liver mRNA levels (A); and EPOR kidney and liver mRNA levels (B), at the end of the study (12 weeks). Results are presented as mean ± SEM (seven rats per group): a: p < 0.05; aa: p < 0.01; and aaa: p < 0.001 vs. Sham; bb: p < 0.01; and bbb: p < 0.001 vs. CRF.

Figure 3

Iron metabolism: Serum iron, ferritin and transferrin at final time (A); Relative gene expression mRNA levels/18S of DMT1 and SLC40A1 in the duodenum (B); and of iron regulatory proteins in the liver (C) at the end of protocol (12 weeks). Results are presented as mean ± SEM (7 rats per group): a: p < 0.05; aa: p < 0.01; and aaa: p < 0.001 vs. Sham; b: p < 0.05; and bbb: p < 0.001 vs. CRF. Ferroportin (SLC40A1), hemojuvelin (HJV), soluble transferrin receptor (STFR), hemochromatosis (Hfe), divalent metal transporter 1 (DMT1), transferrin receptor 1 (TfR1), matriptase-2 (TMPRSS6), interleukin-6 (IL-6) and bone morphogenic protein 6 (BMP6).

Figure 4

Liver hepcidin (A); and HIF2α (B) expression of mRNA and protein (immunohistochemical staining). Original magnification ×400. Results are presented as mean ± SEM (seven rats per group): a: p < 0.05; and aa: p < 0.01 vs. Sham.

Figure 5

Kidney histopathology. Representative glomerular and tubulointerstitial lesions observed in kidneys of rat groups under study, at the final time (PAS staining, original magnification ×400). (A1) glomerular hypercellularity; (A2) dilatation of the Bowman’s space and glomerular atrophy; (A3) total score of mild glomerular lesions for each rat group; (A4) nodular sclerosis; (A5) global glomerulosclerosis; (A6) total score of advanced glomerular lesions for each rat group; (B1) interstitial inflammatory infiltration; (B2) tubular basements membrane irregularity; (B3) total score of mild tubulointerstitial lesions in lesions for each rat group; (B4) Interstitial fibrosis and tubular atrophy (IFTA); (B5) tubular calcification; and (B6) total score of advanced tubulointerstitial lesions for each rat group. Results are presented as mean ± SEM (seven rats per group): aaa: p < 0.001 vs. Sham; b: p < 0.05, and bbb: p < 0.001 vs. CRF.

Figure 5

Kidney histopathology. Representative glomerular and tubulointerstitial lesions observed in kidneys of rat groups under study, at the final time (PAS staining, original magnification ×400). (A1) glomerular hypercellularity; (A2) dilatation of the Bowman’s space and glomerular atrophy; (A3) total score of mild glomerular lesions for each rat group; (A4) nodular sclerosis; (A5) global glomerulosclerosis; (A6) total score of advanced glomerular lesions for each rat group; (B1) interstitial inflammatory infiltration; (B2) tubular basements membrane irregularity; (B3) total score of mild tubulointerstitial lesions in lesions for each rat group; (B4) Interstitial fibrosis and tubular atrophy (IFTA); (B5) tubular calcification; and (B6) total score of advanced tubulointerstitial lesions for each rat group. Results are presented as mean ± SEM (seven rats per group): aaa: p < 0.001 vs. Sham; b: p < 0.05, and bbb: p < 0.001 vs. CRF.

Figure 6

Kidney expression of mediators of inflammation and fibrosis. Gene (mRNA) expression of thrombospondin-1 (TSP-1), pro-(III) collagen, cytochrome c (Cyt C) and interleukin 1β (IL-1β) (A); Kidney expression of nuclear factor kappa B (NF-κB) gene and protein (immunohistochemical staining) (B) and connective tissue growth factor (CTGF) gene and protein (C). Original magnification × 400. Results are presented as mean ± SEM (seven rats per group): a: p < 0.05, aa: p < 0.01, and aaa: p < 0.001 vs. Sham; bb: p < 0.01, and bbb: p < 0.001 vs. CRF.

Figure 6

Kidney expression of mediators of inflammation and fibrosis. Gene (mRNA) expression of thrombospondin-1 (TSP-1), pro-(III) collagen, cytochrome c (Cyt C) and interleukin 1β (IL-1β) (A); Kidney expression of nuclear factor kappa B (NF-κB) gene and protein (immunohistochemical staining) (B) and connective tissue growth factor (CTGF) gene and protein (C). Original magnification × 400. Results are presented as mean ± SEM (seven rats per group): a: p < 0.05, aa: p < 0.01, and aaa: p < 0.001 vs. Sham; bb: p < 0.01, and bbb: p < 0.001 vs. CRF.

Figure 7

Kidney hypoxia-inducible factor 2α (A); and 2β (B). mRNA and protein (immunohistochemical staining) expression. Original magnification ×400. Results are presented as mean ± SEM (7 rats per group): aa: p < 0.01, and aaa: p < 0.001 vs. Sham; b: p < 0.05, and bbb: p < 0.001 vs. CRF.

Figure 7

Kidney hypoxia-inducible factor 2α (A); and 2β (B). mRNA and protein (immunohistochemical staining) expression. Original magnification ×400. Results are presented as mean ± SEM (7 rats per group): aa: p < 0.01, and aaa: p < 0.001 vs. Sham; b: p < 0.05, and bbb: p < 0.001 vs. CRF.

Figure 8

Proposed mechanisms for the impact of resistance to rHuEPO therapy due to anti-EPO antibodies formation in a rat model of CKD-associated anemia.