Effect of histidine and carnosine on haemoglobin recovery in anaemia induced-kidney damage and iron-loading mouse models

Abstract

Histidine and carnosine can form complexes with divalent metal ions such as Fe²⁺, potentially providing stability to intracellular labile iron. Anaemia is a common comorbidity in the late stages of kidney disease, and patients are treated with erythropoiesis-stimulating agents (ESAs) and iron supplementation. However, iron supplementation is also associated with worse long-term outcomes. The purpose of this study is to investigate how histidine and carnosine supplementation can reduce symptoms of anaemia of chronic kidney disease (CKD) and the effects associated with iron-overloaded conditions. Adenine-induced chronic kidney disease mice were treated with histidine and carnosine by oral gavage for 10 days. Additionally, a model involving iron overload in mice was established, and these mice received concurrent treatment with histidine and carnosine. Haemoglobin, non-haem iron, malondialdehyde (MDA) and iron parameters were measured. Carnosine increased erythropoietin (EPO) levels (35.62 µg/ml ± 11.43) and resulted in haemoglobin repletion (16.7 g/dL ± 3.4). When iron was supplemented alongside with histidine or carnosine, there were better effects on haemoglobin repletion (14.22 ± 1.7 and 13.82 ± 2.15 g/ dL respectively), ferritin (59.5 ± 16.4, 52 ± 29.5 µg/ml) and non-haem iron (0.8 ± 0.21, 0.7 ± 0.38 nmol/mg), than the group receiving iron alone (p < 0.05). Furthermore, histidine and carnosine reduced non-haem iron and MDA, in iron-loaded conditions (p < 0.05). These positive effects observed in histidine and carnosine could be associated with reactive oxygen species (ROS) scavenging. EPO restoring levels in CKD model and the increment in haemoglobin and ferritin in carnosine treatments suggested the potential formation of a ternary complex with iron-glutathione. In conclusion, our results indicate the beneficial effect of histidine and carnosine in the context of iron supplementation for the correction of haemoglobin and protection against iron-loaded conditions.

Keywords: Adenine; Anaemia; Carnosine; Disease; Histidine; Kidney.

Fig. 1

Haemoglobin levels pre and post-treatment with histidine or carnosine. All the groups were compared at the baseline to observe differences with the control group. * significantly different (p ˂ 0.05) from the control group. * (p ˂ 0.05) **(p ˂ 0.005) ***(p ˂ 0.001) significant difference between the adenine group and the experimental treatments. Two-way ANOVA. Sample size n = 8. Ferric carboxymaltose (FCM)

Fig. 2

Erythropoietin (EPO) levels in kidney tissue. Carnosine treatment ((A) + Carnosine) presented levels similar to the control untreated group. * treatment significantly different from the control group (p ˂ 0.05). ANOVA one-way test. sample size n = 8. Ferric carboxymaltose (FCM)

Fig. 3

Blood urea nitrogen (BUN) levels in plasma. Urea nitrogen levels were measured pre and post-treatment. All the groups treated with adenine presented similar levels of BUN at the baseline and were significantly different from the control untreated group (# p ˂ 0.05). After treatment with histidine or carnosine, BUN levels were decreased significantly. # significant difference (p ˂ 0.05) between the control and treatment groups. * significant difference (p ˂ 0.05) between the adenine group and the other treatments. ANOVA two-way test. Sample size n = 8. Ferric carboximaltose (FCM)

Fig. 4

Haematoxylin and eosin (H & E) staining of kidney tissues. Black arrows show alteration in tubular morphology, and thinner arrows indicate crystal accumulation and necrotic cellular debris. (n = 2 slides)

Fig. 5

Non-haem iron levels in the liver (A) and spleen (B). Significant differences were found in the liver of the group supplemented with Ferric carboxymaltose (FCM) in combination with carnosine (* p ˂ 0.05). ANOVA two-way test. Sample size n = 8

Fig. 6

Ferritin levels in plasma. The groups with Ferric carboxymaltose (FCM) presented higher values than the control and the adenine group (# p ˂ 0.05). The groups with iron supplementation with Ferric FCM combined with histidine and carnosine showed higher values than those that received FCM alone (* p ˂ 0.05). ANOVA two-way test. Sample size n = 8

Fig. 7

Hepcidin levels in plasma. Values presented in the bars are means ± SEM (n =8). The asterisk represents a significant difference between the control group (**p ˂ 0.005) and the treatments. Hepcidin levels were higher in the iron-supplemented groups with Ferric carboxymaltose (FCM) than in the control and adenine groups. ANOVA one-way test. Sample size n = 8

Fig. 8

Non-haem iron levels in tissues. Figure (A) shows the non-haem levels in the liver, (B) spleen and (C) kidney. In the liver (A), all the groups presented higher levels of iron (p< 0.05) compared with the control group. Carnosine reduced non-haem levels in the spleen (B) and kidney (C).iron-loaded group with iron dextran (ID). # significantly different from the control group (p ˂ 0.05). * Significantly different from the iron dextran group (p ˂ 0.05) for ANOVA one-way test. Sample size n = 5

Fig. 9

Glutathione levels in the liver. Histidine and carnosine groups presented higher (p< 0.05) levels of glutathione levels (GSH) in the liver compared with the control. Iron-loaded group with iron dextran (ID). *** Significantly different from the Fenton substrate (FS) group (p ˂ 0.001) for ANOVA one-way test. n = 5

Fig. 10

Malondialdehyde (MDA) assay in the liver (A) and kidney (B) tissue. In the liver (A) iron dextran (ID) and histidine groups showed higher levels of MDA (p< 0.05) compared with the control group in liver samples. In contrast, the carnosine group has lower levels of MDA, similar to the control group. In the kidney (B), histidine and carnosine groups presented lower levels of MDA, similar to the control group (p< 0.05). iron-loaded group with iron dextran (ID). Histidine and carnosine were supplemented at 1 g/L. ANOVA one-way, n = 5