Impact of Diabetic Nephropathy on Phosphorus Homeostasis

Authors: Dr. Anjli Pawar, Dr. Suvarna Prasad, Dr. Rajiv Kumar, Dr. Pallav Sharma, Dr. Sunita Manhas, Dr. Karanpreet Bhutani, Dr. Munish Kumar

Conducted at: Department of Biochemistry, MMIMSR, Mullana, 133207

INTRODUCTION

Diabetes Mellitus is the most common metabolic disorder with a high rate of morbidity characterized by impaired metabolism of glucose and other energy-yielding fuels as well as the late development of vascular and neuropathic complications. Diabetes involves a group of disorders having different pathogenic mechanisms in which hyperglycemia is the common denominator. Hyperglycemia has a crucial role in the development of diabetic-related complications. Some of these are atherosclerosis neuropathy, nephropathy, retinopathy, and diabetic foot.[1] There are four potential biochemical pathways linking hyperglycemia to the changes within the kidneys, which can plausibly be linked to the functional, structural changes characterizing diabetic nephropathy. These are polyol pathway, non-enzymatic glycation, glucose auto-oxidation, and de novo synthesis of diacylglycerol leading to protein kinase C and phospholipase A2 activation.[2]

Microalbuminuria (MA) was first described in 1982 in patients having diabetes. In this study, it was found that there was an increased risk of cardiovascular morbidity and mortality in these patients. At the same time, it is accepted as an indicator of the presence of diabetic retinopathy/neuropathy, cardiovascular and peripheral vascular disease, and increased mortality. The presence of MA and overt proteinuria in non-insulin-dependent diabetes mellitus (NIDDM) is an indicator of poor glycemic control. An increase in the prevalence of MA is strongly associated with patients having poor glycemic control, insulin resistance, and low Phosphorus levels.[3]

Direct association of macro elements with Diabetes Mellitus has been observed in many different studies. Insulin action has been potentiated by some trace elements such as chromium, magnesium, zinc, manganese, and phosphate. Phosphorus is a widely distributed element in the human body.[1] It is present in both organic and inorganic form in the serum but the measurable form is inorganic ion. It has an important role in bone mineralization and cellular structural components like phospholipids, nucleotides, and phosphoproteins. It has been also used as an energy store, for oxygen transport as 2,3 DPG, and for its acid-base balance. It has been also used in the formation of creatinine phosphate which is involved in many energy-intensive physiological functions, such as muscle contractility, neurological functions, and electrolyte transport.[4]

Phosphate is absorbed in the small intestine by both paracellular and active transport. Several factors affect tubular phosphate reabsorption through the sodium phosphate cotransporters located in the tubular cell membrane. Reduced phosphate reabsorption can be due to a high intake of dietary phosphate, acidosis, parathyroid hormone, PTH-related peptide, glucocorticoid therapy, calcitonin, and vitamin D.[5] The kidney is the main regulator of phosphorus metabolism.[6]The present study is designed to find any association and correlation of serum phosphorus levels in patients having diabetic nephropathy.

MATERIAL AND METHODS:

This is a cross-sectional study approach on diabetic patients. It was conducted in the Department of Biochemistry, MMIMSR, Mullana, Ambala. Patients were enrolled on the basis of following Inclusion and Exclusion criteria.

Inclusion Criteria: All type 2 diabetic patients; both genders aged 30-55 years.

Exclusion Criteria: Past medical history of hypertension, chronic renal failure, renal replacement therapy, regular hemodialysis, malabsorption, heart failure, bone tumors, hematological disorders, chronic diarrhoea, and on oral supplements of phosphate.

Study area and study population: 150 diabetic patients aged 30 to 55 years which were further equally subdivided into normoalbuminuria, microalbuminuria, and macro albuminuria, and the other 50 healthy subjects were included in the study. All subjects signed informed consent and filled questionnaire. The study was approved by the ethical committee of the University.

METHODOLOGY:

Blood samples were collected after 12 hours fasting period under aseptic conditions. Samples obtained were centrifuged and serum was separated. Serum samples were separated from whole blood collected into the tubes without anticoagulant, after clotting was complete, the tubes were then centrifuged at 2700g for 10 minutes. The serum was removed and assayed for phosphorus by using the UV molybdate method on a fully automated EM 360 ERBA Analyzer. HbA1c was measured using EM 360 ERBA analyzer. This uses the method using the affinity chromatography. For the entire test listed above calibrators used were supplied by the kit manufacturer and the accuracy of the results obtained for all analytes were validated by using Erba Mannheim accuracy control at 2 different levels. Random urine was taken for estimation of microalbuminuria by Pyrogallol red method, and these values were compared with those of normal healthy subjects.

Statistically Analysis:

For statistical analysis of data, software downloaded from the website was used to calculate the correlation coefficient (r), student's distribution (t), and probability (P) between HbA1c and Phosphorus.

RESULTS AND OBSERVATIONS:

The mean age of cases and controls was 46.65(78 males and 72 females) and 44.63 years (with 28 males and 22 females),) respectively which was statistically insignificant (Figure I). The maximum number of patients was in the age group of 46-50 i.e. 32% (Figure II) for both cases and controls. 84% of the control showed a normal serum phosphorus level of 2.5- 4.5 mg/dl. And in diabetic cases, 40.67% had decreased levels than the normal range i.e. < 2.5 mg/dl and 54.67% of them were in the normal range and the remaining 4.67% were above normal levels i.e. > 4.5 mg/dl (Figure III). As shown in Figure IV & Table I, Serum Phosphorus levels of Group IB and IC has a significant association with Group II. Also, a significant association between Group IB and IC was found. As seen in Table II the correlation between Serum HbA1c and Serum Phosphorus of Group IA was moderately negative and statistically significant. However, the correlation between Group IB and Group IC was negative but it is weakly correlated statistically.

DISCUSSION:

Out of the 200 subjects studied, 150 were diabetic and 78 (52%) were males and 72 (48%) were females. Raul et al reported 52.73% males and 47.27% females in their study.[4] Willer et al and Rao et al, in their studies also show male preponderance which is statistically insignificant.[7,8]In our study, the maximum number of patients was in the age group of 46-50 years. The mean age of diabetic patients was 46.45±6.55. In our study, the mean phosphorus levels in diabetic patients were 2.84±1.1 which was lower as compared to controls (3.84±0.66). A significant association between Group IA, IB with Group II, and Group IB with Group IC was found for the serum phosphorous(Table I). Many studies have also found decreased concentration of phosphate in poorly regulated diabetic patients and the level increases when blood glucose is controlled.[9]According to Gartner et al study in juvenile-onset diabetic patients found that as plasma glucose decreased from 221 mg/dl to 95.5 mg/dl, serum inorganic phosphorus was rose from 4.09 to 5 mg/dl.[10] In our study, there is decreased phosphorus concentration in the diabetics, and as the disease progresses, the level of phosphorus further deteriorates. This is in concordance with a study done by Revathi [1] and Ugwuja.[11] The findings of the earlier studies are in concordance with our work which can be due to osmotic diuresis and intracellular shift.[9]As osmotic diuresis is the most common factor for enhanced urinary secretion and decreased electrolytes, an intracellular shift can also be responsible.[12] Osmotic diuresis in hyperglycaemic and acidemic states may cause competition between phosphate and glucose for excretion in the proximal tubular system. Uncontrolled diabetics have glucose accompanied by phosphate in the cells, resulting in low blood phosphorous levels.[13] Dietzel et al found that the maximal capacity of renal tubular reabsorption of phosphate was significantly suppressed in diabetic patients and also reported that urinary phosphate excretion was three times higher in diabetic patients when compared to healthy controls [6,14]Sultan et al revealed that there is decreased serum calcium which is due to hyperglycemia that enhances calcium and phosphorus excretion in urine.[15] Hyperglycemia causes excess urinary phosphate in patients with type 2 diabetes mellitus. Nagasaka et al suggested that hyperglycemia caused excess urinary calcium and phosphorus excretion in patients with NIDDM.[16] Serum phosphorus levels were decreased in type 2 diabetic patients indicating that there is an alteration in the phosphorus metabolism, represented by the study done by Zhong et al.[17] Kalaitzidis et al observed that patients with metabolic syndrome showed significantly lower phosphate and magnesium levels compared with controls.[18] Raskin and Pak studied 21 diabetic patients in whom treatment results ranged from “suboptimal” to “optimal” control and found that, as the mean plasma glucose decreased from 17.1 mmol/L to 5.2 mmol/L over 4 to 10 days, serum phosphate level raised from 1.12 to 1.26 mmol/L.[19]

A conflicting metabolic variance in phosphate occurs from the early onset of diabetes and may lead to reduced high-energy phosphate and hypoxia of tissues. These variations take place in the cell and tissues in which access of glucose is not regulated by insulin and specifically in poorly controlled diabetic patients in which chronic vascular complications are more likely to occur. Decreased serum phosphate levels are related to the severity of diabetes mellitus.[14]

A disturbance in phosphate regulation occurs in kidney nephrons, where the increased sodium-dependent glucose entry in diabetics impairs phosphate reabsorption. Glucose is more potent than inorganic phosphate in stimulating the uptake of sodium in the renal microvillus vesicles. The elevated glucose concentration depolarizes the transmembrane electrochemical sodium gradient of the brush border membrane for inorganic phosphate entry into the tubular cells and decreases intracellular phosphate leading to hyperphosphaturia.[6,20] This can cause a decreased level of phosphate in the diabetics and normalization of blood glucose levels to improve the capacity of kidney tubules to reabsorb phosphate and ultimately increase phosphate concentration. It is also suggested by the DCCT and UKPDS studies that improved glucose regulation definitely influences or also prevents long-term diabetic complications. 21,22] This decreased urinary phosphorus loss can be due to reduced glycosuria and is affected by the direct action of insulin on renal tubules or suppression of glucagon and parathyroid hormone secretion.[13]

Haglin et al[23] in 2001, and Park W et al[24] in 2009 showed a negative correlation between serum phosphate levels and fasting blood sugar levels. In our study, a significant negative correlation between phosphorus and HbA1c was found in T2DM patients.

CONCLUSION:

The inorganic phosphate is essential for the resynthesis of 2,3 diphosphoglycerate and ATP, therefore phosphate depletion results in tissue hypoxia and a decrease in energy-rich phosphate with disturbances of various organ systems. Our findings suggest that there is a low phosphorus level in type 2 diabetes mellitus. Increased microalbuminuria was seen with reduced glucose tolerance hence early estimation of both the parameters should be done while monitoring the cases of type 2 diabetes and thus will help to decrease the incidence of renal failure. Phosphorus depletion may increase the risk of secondary complications; preventing low phosphorus in diabetics can be beneficial in the management of the disease. Regular control of plasma phosphate levels and prophylactic substitution of phosphate is recommended.[25]

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