Anaemia is a common complication in CKD patients, who have a mortality rate 20 - 100 times higher than the normal population. Several studies have revealed the association of hyperparathyroidism with CKD. Parathyroid hormone (PTH) is a major uremic toxin which may be responsible for long term consequences in CKD such as renal osteodystrophy, vascular calcification, altered cardiovascular function, immune dysfunction and anaemia [1]. One of the lesser known functions of the kidneys is the production of erythropoietin, a signaling molecule that stimulates red blood cell production, in response to decreased oxygen levels in the blood. Any disruption of this process, e.g., secondary to a functional abnormality due to CKD, has the potential to produce anemia, a condition in which the number of circulating red blood cells, and therefore the level of hemoglobin, is lower than normal [2].

Secondary hyperparathyroidism is characterized by elevated serum parathyroid hormone (PTH) levels. Excess PTH may impair erythropoiesis by exerting a direct toxic effect on erythroid progenitor cells and an indirect effect through bone marrow fibrosis induction. In addition, low hemoglobin concentration in uremic patients may have resulted from an increase in erythrocyte osmotic fragility due to high PTH concentration [3]. In advanced CKD, high PTH levels may cause “resistance” to erythropoietin by increasing bone marrow fibrosis, increase the osmotic fragility of red blood cells, leading to a shortened lifespan, and inhibit the proliferation of erythroid precursors [4]. According to the various studies, an increase in serum level of iPTH leads to a decrease in serum level of hemoglobin among hemodialysis patients [5].

This work is aimed to study the relationship between red blood cells osmotic fragility and level of parathyroid hormone in patients with different stages of chronic kidney disease including end stage renal disease.

This cross-sectional study was conducted on 111 patients with CKD during the period of one year (from March 2019 till February 2020), patients were divided into three groups according to GFR into Group I: ESRD (GFR from <15 min/ml/1.73 m²) patient on regular hemodialysis more than 3 months. Group 2: CKD stage 4 (GFR from 15 - 29 min/ml/1.73 m²). Group 3: CKD stage 3 (GFR from 30 - 59 min/ml/1.73 m²). We included patients older than 18 years with different stages of chronic kidney disease and we excluded patients with cardiovascular diseases, inflammatory diseases, patients with hemolysis and active bleeding. The study followed the ethical standards of Faculty of Medicine, Menofia University and was approved by ethical committee of the faculty. The following data were analyzed for all patients: Age, Gender and routine laboratory investigations: CBC, Ca, Phos, Na, K, Iron profile, kidney functions, liver functions, iPTH (Serum intact Parathyroid Hormone (iPTH) was measured using chemiluminiscence immuno assay by Beckmann Coulte and by using Serolia tm Human Intact PTH ELISA Kit) and MOF (Red cell osmotic fragility was evaluated by incubating 50 μl of the RBC of the patients’ sample with gradually increasing concentrations of NaCl ranging from 0.1% to 0.9%. After one hour, the incubated solutions were centrifuged and the absorbance of the supernatant was taken at 540 nm. A curve was plotted based on the concentration of NaCl and absorbance reading. The concentration of NaCl causing 50% hemolysis is taken as the Median Osmotic Fragility (MOF).

The collected data were coded, processed and analyzed using the SPSS (Statistical Package for Social Sciences) version 22 for Windows^® (IBM SPSS Inc, Chicago, IL, USA). Data were tested for normal distribution using the Shapiro Walk test. Qualitative data were represented as frequencies and relative percentages. Chi square test (χ²) was used to calculate difference between qualitative variables as indicated. Quantitative data were expressed as mean ± SD (Standard deviation). One-way analysis of the variance one-way ANOVA was used to compare between three independent groups of normally distributed variables (parametric data) while Kruskal wallis test was used for non-normally distributed Data (non-parametric data). Significance test results are quoted as two-tailed probabilities. For all the above-mentioned tests, the level of significance was tested, expressed as the probability of (p-value) and the results were explained as following:

Non-significant if the p value is >0.05, Significant if the p value is ≤0.05, highly significant if the p value < 0.001. There is a statistically significant negative correlation between MOF with HGB, RBCs, MCV, platelets count, calcium and GFR (Figure 1). There is a statistically significant difference between the cases within the three study groups in Calcium, Phosphorous, Ca*PH, Na, K, TIBC, Total saturation, Serum albumin, iPTH and MOF. iPTH and MOF were significantly lower in cases with stage 3 as compared with cases in stage 5. Also, iPTH and MOF were significantly lower in cases in stage 4 as compared with cases in stage 5. (Figure 2) There is a statistically significant positive correlation between MOF with Creatinine, Urea, Phosphorous, Ca*phosphorous product, K, KTV and PTH.

There are various causes of renal anemia such as decreased production of erythropoietin (EPO), resistance to EPO, shortened survival of red blood cells (RBCs), and bone marrow fibrosis. Secondary hyperparathyroidism (SHPT) is a less recognized, but potentially significant cause of renal anemia in CKD patients.

Parathyroid hormone (PTH) has been regarded as a uremic toxin that potentially inhibits EPO synthesis, shortens the survival of RBCs, and causes myelofibrosis and thereby decreases hematopoiesis [6]. This current study was conducted to study the relationship between red blood cells osmotic fragility and

level of parathyroid hormone in patients with different stages of chronic kidney disease including end Stage Renal disease. To the best of our knowledge, there is gust one study available in the literature that studies the effect of CKD on the osmotic fragility of RBCs. This study included 111 patients with chronic kidney disease, stage 3 - 5. There were 37 patients in each stage. In this study, the mean age of the cases didn’t reveal a statistically significant difference in the study groups (p = 0.586). There was no statistically significant difference in the sex distribution between the cases within the three study groups (Males represent 56.8%, 48.7% and 54.1% in the cases in Stage III, IV and V CKD respectively) (Table 1). Similar results were reported by Janmaat et al. who showed that participants with CKD stage 5 were younger and more often men than the patients with CKD stages 3 to 4 [7]. A gender-different prevalence of CKD was discovered in most related studies. Higher prevalence of CKD in women compared with men as reported in many studies [8]. Also, the serum hemoglobin level was 9.65 ± 1.28 g/dl in cases with stage V CKD, 11.18 ± 1.16 g/dl in cases with stage IV CKD and 11.89 ± 1.12 g/dl in cases with stage III with high statistically significant difference between the three groups. This agreed with Janmaat et al. who showed that the mean serum of hemoglobin level was 134.7  ±  15.2 g/dl, 129.2  ±  15.7 g/dl, 123.8  ±  15.7 g/dl and 119.1  ±  16.5 g/dl in cases with stage 3a, 3b, 4 and 5 CKD respectively, with high statistically significant difference between the four groups (Table 2, Table 3) [7]. Similar results were shown by Finkelstein et al. where the mean Hgb levels were 12.8 in stage 3 patients, 12.0 in stage 4 patients, and 11.4 in stage 5 patients (P = 0.023) [9], Anemia in CKD patients is recognized to be multifactorial. Anemia prevalence in CKD patients increases from 26% to 75% when the renal function decreases from >60 ml/min to <15 ml/min most probably because of EPO deficiency [10], EPO deficiency is sometimes not only attributed to the kidney disease but also to another concomitant chronic disease [11]. Interestingly also, Mercadal et al. have shown that anemia in CKD patients with eGFR > 30 ml/min should be explained by other factors than EPO deficiency [12]. Treating patients with ESA while they suffer from other causes of anemia will lead to EPO resistance. Therefore, anemia in CKD patients when secondary to iron deficiency does not need ESA treatment unless it persists after iron replenishing [13]. In the current study, the serum level of iPTH was 916.49 ± 142.34 ng/L in cass with stage V CKD, 530.27 ± 86.25