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Revisiting the Role of Chronic Kidney Disease and Its Association With Anemia in Diabetics and Non-Diabetics: A Cross Sectional Audit of 450 Cases

Background: Anemia is twice common in diabetics compared with non-diabetics. The etiopathogenesis of anemia in diabetes is multifactorial [Chronic Kidney Disease (CKD), Iron Deficiency Anemia (IDA), B12 and folate deficiency, etc]. Anemia in diabetic patients prior to the evidence of renal impairment is rarely reported. Objective: To determine the role of CKD and other etiological factors of anemia in diabetics and non-diabetics. To correlate the degree of anemia with severity of CKD and glycemic status. Methods: A cross-sectional study carried out in SRM Hospital and Research Centre, Chennai. Total 450 subjects (150 controlled & 150 poorly controlled diabetics, 150 non-diabetics) with equal number of anemic and not-anemic patients in each group were included. Further categorized into those with CKD and without CKD. Results: In our study, only 33% anemic patients with diabetes had low eGFR and CKD (p<0.05). Among the rest 67% majority of anemia in diabetics was due to IDA which became evident from low iron profile and microcytic blood picture. As CKD progressed, anemia worsened. Moreover poorly controlled diabetics with CKD showed statistical significant difference between anemic and not-anemic patients. Anemia was highly prevalent in females and CKD in males in both diabetics and non-diabetics. Conclusion: In this study we insist on the utter importance of evaluating other etiological factors apart from CKD such as IDA for accurate diagnosis, treatment of anemia in diabetics and thereby preventing the prognosis of complications.


Introduction
Anemia is twice common in diabetics compared with non-diabetics. [1] The etiopathogenesis of anemia in diabetes is multifactorial which includes deficiency in erythropoietin (EPO) synthesis and release due to Chronic Kidney Disease (CKD), nutritional deficiency (iron, vitamin B 12 ), systemic inflammation, concomitant autoimmune diseases, drugs (ACE inhibitors) and hormonal changes. [2] Anemia potentially contributes to the development and progression of diabetic micro and macro vascular complications such as diabetic kidney disease, retinopathy, neuropathy etc. Despite these facts, anemia is still unrecognized in 25% of diabetic patients. [3] Anemia occurs earlier and more severe in CKD in Diabetes Mellitus (DM) than in patients with renal impairment from other causesand is associated with more rapid decline in Glomerular Filtration Rate (GFR) [4] .Probably this may be due to EPO deficiency and hypo responsiveness to the action of EPO. [4,5] Al Khowry et aldemonstrated that for each CKD stage, hemoglobin is 1g/dl lower in diabetics than non-diabetics. [6] But some recent studies have linked anemia with relatively low serum EPO in persons with either type 1 or type 2 diabetes, even without advanced kidney disease or overt uremia. [7][8][9][10] While limited studies have reported the incidence of anemia in diabetic patients prior to the evidence of renal impairment. [9] To shed additional light on this dispute, we aimed in this study to determine the role of chronic kidney disease and its association with anemia in diabetics and non-diabetics. Additionally we correlated the degree of anemia with severity of CKD and glycemic status in these patients. Based on the data observed among our study subjects we categorized them into those with CKD (78 diabetics and 37 non-diabetics) and without CKD (222 diabetics and 113 non-diabetics). Among 78 diabetic CKD patients 50 had anemia and 28 without anemia. Out of 222diabetic without CKD, 100 had anemia and 122 were without anemia. Among 37 non-diabetic CKD patients, 23 had anemia and 14were without anemia. Out of 113nondiabetic non-CKD patients, 52 had anemia and 61 were without anemia. [Fig-1] Anemia: The anemic patients were selected based on their hemoglobin levels (Hb<13 gm% in males and <12 gm% in females) based on definition of World Health Organization (WHO). [11] Anemic patients were further categorized as mild anemia (male 12-12.9 gm/dl and female 11-11.9gm/ dl), moderate anemia (male 9-11.9 gm/dl and female 8-10.9 gm/dl) and severe anemia (male <9 gm/dl and female <8 gm/dl). Anemia was defined as normocytic with a mean corpuscular volume (MCV) of 80 to 100 fl, microcytic with the MCV <80 fl and macrocytic with the MCV >100 fl. [12] Chronic  [12][13][14] Measurements: Fasting plasma glucose (FPG) estimated by glucose oxidase/peroxidase method. Hemoglobin (Hb), Mean corpuscular volume (MCV) was estimated by sysmex XT-1800 analyzer. Serum creatinine was estimated by Jaffe's kinetic method.HbA 1 C was measured by HPLC method using Bio-Rad analyzer.

Statistical analysis:
The data were analyzed using SPSS version 21.All the categorical variables were analyzed using chi square test. Continuous variables were presented as mean ± S.D. Numerical variables werecompared using independent sample t-test. P value <0.05 was considered statistically significant.

Results
Anemia: Our study result showsa high incidence of anemia in females (62%) when compared with males (38%) in both diabetics and non-diabetics and was statistically significant (p <0.05) [ Chronic kidney disease: In our study, CKD was more prevalent in males (62%) than females (38%) [ Table-3/ Fig-2].In poorly controlled diabetic patients with CKD, statistical significant difference was observed between anemic and not-anemic patients (p < 0.05) but however in case of controlled diabetics with CKD there was no significant difference between anemic and not-anemic patients(p >0.05).
In case of non-diabetics with CKD, no statistical significant difference was observed between anemic and not-anemic patients (p > 0.05). Mean HbA 1 C between CKD (7.98 ± 0.21) and non-CKD (7.58 ± 0.13) patients was not statistically significant in both diabetics and non-diabetics (p > 0.05) .
Correlation between stages of CKD with severity of anemia: Majority (54.3 %) of non-anemic patients were found in CKD stage 1. Also majority of patients with severe anemia (57.1%) were found in CKD stage 5. This implies that as CKD progresses, severity of anemia worsens. [

Discussion
Even thoughit's a well-known fact that anemia is a common accompaniment to diabetes leading to its various complications, diabetics are rarely evaluated and treated for anemia. [3] While several reports have indicated that anemia mostly occurs in diabetic patients with renal insufficiency,some studies have also reported anemia without renal dysfunction. [7,9] But in our study, only 33% of diabetic patients with anemia had CKD having low eGFR (<90 mL/min/1.73 m 2 ). This contrasts with the commonly presumed fact that CKD is the usual cause of anemia in diabetes. Hence some other factors could have influenced the occurrence of anemia in these diabetic patients.
The main cause of anemia in CKD may be EPO deficiency due to reduction in the number of specific EPO synthesizing interstitial cells, disruption of interstitial anatomy or vascular architecture of kidney. [16,17] Few other studies also proposed that hypo responsiveness to the action of EPO may be due to renal denervation in diabetic autonomic neuropathy reducing splanchnic sympathetic stimulation of EPO production. [8,9,18] Apart from CKD, anemia in diabetes may be due to other cause such as nutritional deficiency (iron, vitamin B12 and folate), systemic inflammation, concomitant autoimmune disease, drugs(ACE inhibitors) and hormonal changes. We observed in both diabetics and non-diabetics that anemia was more prevalent in females62% thanmales38% and was statistically significant (p<0.05). This implies that anemia is influenced by gender in both these groups which correlates with the study results of Cawood et al. [19] Ourresult contradicts with the study result of Craig et al who reported that anemia is common in males. [20] In this study with reference to age, in subjects <60 years, anemia was more prevalent in females and in subjects >60 years anemia was common in males. [21] Data from our study show CKD with anemia was more common in males (55%) than females (45%) in both diabetics and non-diabetics and was statistically significant (p<0.05). The possible pathogenesis for this male preponderance of anemia in kidney dysfunction is due to decreased lutenizing hormone production and decreased prolactin clearance lowering the androgen levels. Androgens stimulate erythropoiesis by increasing erythropoietin production and by direct augmentation of marrow stem cells. [22] Finally, CKD has been associated with reduced testosterone levels in men. [23] On the other hand, premenopausal and postmenopausal women with diabetes have higher bioavailable testosterone level. [24] Regardless of the positive and significant relationship between HbA 1 C and CKD observed in other studies of

Inaba M et al, Chujo K et al, Riveline JP et al, our study result doesn't show a significant relationship between
HbA 1 C and CKD in both diabetics and non-diabetics [25,26,27] . This result of ours goes in hand with the study results of Vos FE et al. [28] Comparison of CKD with Anemia: CKD contributed to only 33% of anemia in diabetics and 31% in non-diabetics, hence causes other than CKD would have contributed to anemia in these patients. This figure is almost same as compared to the prevalence of 25% documented by Janmohamed et al. [17] Out of 115 patients with CKD, majority of patients(64%) were anemic and rest (36%)were not-anemic. This implies that anemia is not always due to CKD but other factors like nutritional deficiency (iron, B12, folic acid), hormones etc may play a role. Among patients having CKD with anemia in our study, we observed normocytic blood picture in only 34% of diabetics and 26% of non-diabetics. Majority showed microcytic blood picture with iron deficiency(56% in diabetics and 56.5% in non-diabetics) and few showed macrocytic blood picture with vitamin B12 deficiency (10% in diabetics and 17.3% in non-diabetics).
Iron deficiency was found in among 56% of diabetics with anemia, which is higher than the figure of 15.5% reported by Sharif et al. [18] However, Katherine et al found iron deficiency in none of their diabetic patients in a study conducted in UK. [19] Higher prevalence of iron deficiency in these diabetics may be due to lower socioeconomic class who frequently has nutritional deficiencies particularly iron deficiency which is common in Asian countries, lack of awareness and inability to access appropriate healthcare due to financial constraints. [20] Hyperglycemia has a direct relationship with the development of an inflammatory condition showed by the increased expression of pro-inflammatory cytokines such as IL-6, TNF-α, and NFκB. Thus, diabetes, as well as hyperglycemia due to its nature, is also an inflammatory disease character. Studies show that the longer the duration of the disease and/or loss of glycemic control, the higher the inflammatory process. [7,8] The elevation of pro-inflammatory cytokines plays an essential role in insulin resistance and induces the appearance of diabetic micro and macrovascular complications like, kidney disease, cardiovascular disease and anemia. By increasing especially IL-6, antierythropoietic effect occurs, since this cytokine changes the sensitivity of progenitors to erythropoietin (erythroid growth factor) and also promotes apoptosis of immature erythrocytes causing a decrease, further, in the number of circulating erythrocytes and consequently causing a reduction of circulating hemoglobin. [7,9]

Conclusion
To summarize, a high incidence of anemia was observed in diabetics even without renal insufficiency. Furthermore majority of these anemic patients had microcytic blood picture with low iron profile which in turn indicates iron deficiency. This implies that causes other than CKD such as nutritional deficiency have a significant role in the development of anemia in diabetes. This contrast with the commonly presumed fact that the predominant risk factor for the development of anemia in a diabetic population has been found to be the presence of CKD. We also observed that severity of anemia worsens as CKD progresses. The prevalence of anemia was increased at depressed levels of eGFR in patients with poor controlled diabetics compared to controlled diabetics and non-diabetics. Hence in this study we insist on the utter importance of evaluating the causes of anemia other than CKD like nutritional (iron) deficiencies for accurate diagnosis, treatment and thereby preventing the prognosis of diabetic complications.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964Helsinki declaration and its later amendments or comparable ethical standards.