Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chronic kidney disease DOI: https://doi.org/10.31351/vol32iss1pp59-66 59 Clinical Significance of Osteoprotegerin, Vitamin D, Obestatin and some Biochemical Variables in Kidney Failure Patients Hazhar M. Balaky*,1, Akam Jasim Mustafa** and Parween Abdulsamad Ismail*** *Mergasor Technical Institute, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq. **Department of Chemistry, Faculty of Science, Soran University, Kurdistan Region. Iraq. ***Department of Chemistry, College of Education, University of Salahaddin, Iraq Abstract Chronic kidney disease (CKD) is described as an abnormality of renal function existing for a long period of time. Because the initial stages of CKD can be asymptomatic, its early diagnosis is difficult. The earlier stage of CKD can lead to several complications, such as anemia and bone mineral metabolism disorders. The study was conducted to assess the effect of the chronic renal disease stage on osteoprotegerin, 1,25 dihydroxyvitamin D, obestatin levels and some biochemical parameters in patients who have not undertaken dialysis therapy. In this case-control study, fifty-five patients with kidney failure and forty healthy people were examined. Circulating concentrations of osteoprotegerin, 1,25 dihydroxyvitamin D and obestatin were estimated by ELISA technique, serum urea, uric acid, total protein and total Calcium were estimated using spectrophotometry technique. The serum concentration of osteoprotegerin, obestatin, and renal function markers (Urea, Creatinine and Uric Acid) in patient groups were higher (328.3±41.68 pg/mL; 15.52±4.28 pg/mL; 183.2±10.35 mg/dL; 8.88±0.54 mg/dL; 6.57±0.22 mg/dL) respectively in comparison with the control group (172.6±55.48 pg/mL; 11.64±3.26 pg/mL; 33.45±1.08 mg/dL; 0.95±0.03 mg/dL; 4.35±0.22 mg/dL) respectively. Vitamin D, total Calcium and total Protein level in the patient group were lower (24.49±2.53 ng/mL; 8.06±0.18 mg/dL; 6.49±0.12 g/dL), respectively, as compared to controls (57.11±12.39 ng/mL; 10.15±0.23 mg/dL; 6.82±0.10 g/dL) respectively. The current study reveals that circulating levels of osteoprotegerin and obestatin are remarkably linked with renal failure; the current data identify a high prevalence deficiency and insufficiency of 1,25 dihydroxyvitamin D in patients with moderate and severe chronic nephropathy. Keywords: Osteoprotegerin, 1,25 dihydroxyvitamin D, Obestatin, Kidney failure. Introduction Chronic Kidney Disease (CKD) is one of the leading causes of death and disability. CKD is associated with a wide range of life-threatening diseases (1). It is a disease condition associated with premature mortality, increased healthcare expenditures, and decreased quality of life. The terminal stage of the disease, End-Stage Renal Disease (ESRD), necessitates dialysis or kidney transplantation (2). Osteoprotegerin (OPG) is a glycoprotein that plays an important regulatory role in the skeletal, vascular, and immune systems. It has been shown that OPG predicts chronic kidney disease (CKD). PG is increased in patients with non- diabetic and diabetic chronic kidney disease (CKD), where it predicts deterioration of kidney function, vascular events, and cardiovascular and all-cause mortality. Consistent with it, it has been recently reported that elevated OPG is associated with increased 5- and 10-year risk of rapid renal decline, renal disease hospitalization, and/or deaths in elderly women (3). Vitamin D deficiency is very frequent in CKD, affecting more than 80% of patients in pre- dialysis. Vitamin D insufficiency arises at an early stage of the disease and tends to worsen with the progressive loss of renal function (4). Moreover, decline in glomerular filtration in CKD patients causes vitamin D deficiency, derangements in calcium and phosphate homeostasis, and secondary hyperparathyroidism resulting in bone destruction and vascular calcification (5). However, the role of native vitamin D supplementation (ergocalciferol, cholecalciferol or calcifediol) remains unclear in chronic kidney disease (CKD), particularly in the pre-dialytic phase (4). Obestatin, a peptide hormone, might be participated in the loss of appetite in end- stage renal disease patients. The obestatin hormone is encoded by the same gene as ghrelin and has the opposite effects compared with ghrelin; it blocks nourishment and causes weight gain, whereas ghrelin promotes nourishment. The balance between concentration of obestatin and ghrelin in final-stage nephropathic patients influences balance of energy and appetite, which involves malnourishment in final-stage nephropathic patients. Thus, obestatin is a nourishment parameter identifying the fattening of the body and insulin resistance (6). Urea is the primary metabolite derived from dietary protein and tissue protein turnover. It is freely filtered at the glomerulus but not secreted, and it is reabsorbed by the renal tubules. In addition, as urine flow rates decrease, more urea is reabsorbed. Blood urea nitrogen (BUN) measures the nitrogen component of serum urea levels are inversely 1Corresponding author E-mail: hazharbalaky86@yahoo.com Received: 27/12 /2021 Accepted:6 /4 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol32iss1pp59-66 Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chronic kidney disease 60 correlated with the decline of kidney function and are also affected by extrarenal factors such as protein intake, gastrointestinal bleeding, catabolic states, malnutrition, heart failure, dehydration, use of glucocorticoids, and hepatic urea synthesis (7). The role of uric acid in chronic kidney disease (CKD) has been a controversial topic for decades. Studies supporting the role of uric acid as an important mediator of CKD are extensive. Experimentally raising uric acid could cause low-grade kidney damage and accelerate established CKD (8). Creatinine is the most commonly used marker to measure GFR. GFR is estimated from measurements of creatinine concentrations in blood, using various prediction equations. In adults, the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) and the Modification of Diet in Renal Disease (MDRD) study equations are the most widely used (9). Several pieces of research have demonstrated that nourishment factors are essential in averting the development of persistent nephropathy. According to prior research, a very low protein food source as a section of nourishment treatment has serviceable impacts in decelerating the development of chronic kidney disease (10). Nevertheless, restricted research has investigated the effect of continuous protein consumption and its crucial sources on the beginning of chronic kidney disease, comprising the protein of both plant and animal sources. In an investigation, it was recorded that a higher intake of animal protein (red meat) contributes to elevated chronic kidney disease health risk, and replacing animal protein with plant protein diminished the health risk of chronic kidney disease (11). Methods Study population and design Ninety-five participants with age range (29-67) years, have been subjected to the present study, and these selected participants were divided into two groups. Group (1) includes subjects with chronic renal failure (n=55). Group (2) consisted of apparently healthy individuals (n=40). Patients were clinically diagnosed with kidney failure. The samples were collected from Ashty Hospital in Soran City. Patients were assessed by full medical history to exclude any existing systemic disease. The two groups were matched in age and gender. Collection of blood samples Five to six mL of venous blood were taken from each participant, and collected in gold-top serum separator tubes (SST), to get the serum, the samples were centrifuged at (3000 rpm) for fifteen minutes. The obtained serum samples were transferred immediately to pre-labelled and coded Eppendorf tubes. These samples were frozen at – 20°C for subsequent analysis. Biochemical assays Circulating concentrations of osteoprotegerin (OPG), 1,25- dihydroxycholecalciferol and obestatin in serum samples were estimated by sandwich enzyme-linked immunosorbent assay (ELISA) technique using BioVision (USA) research-purpose kits. The concentration of urea, creatinine, uric acid, total protein and total Calcium were estimated using the enzymatic colorimetric method using BIOLABO kits (France). Statistical analysis Statistical data analysis was performed using SPSS version 21 and GraphPad Prism version 8 computer programs. Comparing the study parameter means between patients and control groups was performed by using Unpaired T-test (Man-Whitney U) test. Bar graphs and Statistical test results were expressed as Mean±SE. Results and Discussion As shown in Table 1, osteoprotegerin, urea, creatinine, and uric acid levels were significantly higher in renal failure patients (328.3±41.68pg/mL; 183.2±10.35 mg/dL; 8.88±0.54 mg/dL; 6.57±0.22 mg/dL) respectively. Similarly, the levels of Obestatin were higher in Kidney failure patients (15.52±4.28 pg/mL), but the variation was not statistically remarkable. The levels of vitamin D, total proteins and total Calcium were significantly lower in kidney failure patients (24.49±2.53 ng/mL; 6.49±0.12; g/dL 8.06±0.18 mg/dL) respectively than that of the control group (57.11±12.39 ng/mL; 6.82±0.10 g/dL; 10.15±0.23 mg/dL) respectively. Table 1. Comparison between biochemical variable levels in KF and control group Parameters Controls Patients P-Value Osteoprotegerin (pg/mL) 172.6±55.48 328.3±41.68* 0.0001 Obestatin (pg/mL) 11.64±3.26 15.52±4.28 0.241 Vitamin D (ng/mL) 57.11±12.39 24.49±2.53* 0.030 Urea (mg/dL) 33.45±1.08 183.2±10.35** <0.0001 Creatinine (mg/dL) 0.95±0.03 8.88±0.54** <0.0001 Uric Acid (mg/dL) 4.35±0.22 6.57±0.22** <0.0001 Total Proteins (g/dL) 6.82±0.10 6.49±0.12* 0.0503 Total Calcium (mg/dL) 10.15±0.23 8.06±0.18** <0.0001 Value of * indicates significant, ** indicates highly significant Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chronic kidney disease 61 Serum level of OPG Table (1) and Figure (1) showed significant elevation (P=0.0001) in circulating concentration of OPG in patients (328.3±41.68 pg/mL) as compared to controls (172.6±55.48 pg/mL). Figure 1. Serum level of OPG Possible explanations for increased serum concentration of osteoprotegerin (OPG) with CKD may include decreased clearance of OPG or increased inflammatory activity. Inflammatory mediators are known to promote the production of OPG. As renal failure is associated with increased inflammatory activity, likely, inflammatory responses may partly explain increased serum OPG in CKD. Several mechanisms underlying OPG increase in CKD might include low-grade inflammation, fibroblast growth factor 23 (FGF-23) elevation, and kidney function. Inflammation might elevate OPG levels as several proinflammatory cytokines, such as TNF-α, regulate OPG production in vascular smooth muscle cells (12). Secondly, increased concentrations of Fibroblast growth factor 23 found in persistent nephropathic patients might also promote osteoprotegerin expression (13). Thirdly, given that renal secretion is assumed to control the clearance of osteoprotegerin, the reservation of osteoprotegerin attributed to kidney destruction might supply another section to explain the mechanisms whereby osteoprotegerin levels are elevated in chronic kidney disease (14). As for inflammation, it has been revealed that osteoprotegerin promoted the expression of endothelial cells of cell surface proteins mediating the interaction between cells. It elevated white blood cell adhesion to the single layer of squamous endothelial cells, as for scarring of the tissue. It has been revealed that osteoprotegerin could begin Transforming growth factor beta 1 -dependent alters in stromal cells of the vascular wall, promoting multiplication, fibrosis and inflammation. Furthermore, osteoprotegerin expression is elevated in response to transforming growth factor beta 1, which could contribute to a vicious cycle that causes the self-elicitation of both Osteoprotegerin and Transforming growth factor beta 1 (3). Osteoprotegerin transportation remarkably elevated the gene expression of interleukin six and transforming growth factor beta 1, as well as the quantity of protein cysteine nitrosylation, which all participated in the progression of renal damage (15). In this process, not only Transforming growth factor beta 1 but also various cytokines and growth factors stimulate the abnormal aggregation of collagen protein, fibronectin glycoprotein, and other constituents accountable for kidney scarring (16). Consequently, our finding supposes that osteoprotegerin has the possibility to directly instigate renal damage (17). Serum levels of obestatin Table (1) and Figure (2) showed a non- significant increase (0.241) in the circulating level of obestatin in patients (15.52±4.28 ng/mL) as compared to controls (11.64±3.26 ng/mL). Figure 2. Serum level of obestatin Obestatin, originally identified as an anorectic peptide, plays a vital role in several pathological activities besides physiological activities, such as substance and generation of energy from the metabolic pathway, motility of gastrointestinal tract, pancreatic secretion, cell multiplication, memory and sleep, etc. (18). Obestatin-arginine vasopressin (AVP) contributes in the regulation of metabolic pathway of body water. The secretion of arginine vasopressin (AVP) in basic status is elevated after the management of obestatin antiserum; the secretion of arginine vasopressin is additionally elevated after water impoverishment. Earlier investigations have examined that the release of obestatin is abnormally elevated in patients with persistent heart failure and nephropathy. Compared to other groups, The current study found that patients with complicated renal failure have remarkably increased concentrations of obestatin (19,20). Circulating level of 1,25-dihydroxycholecalciferol The results in Table (1) and Figure (3) showed that there was a remarkable decline P=0.030) in circulating concentration of vitamin D in patients (24.49±2.53 ng/mL) as compared to controls (57.11 ± 12.39 ng/mL). Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chronic kidney disease 62 Figure 3. Serum level of Vitamin D. Our results accompany previous findings proposing that 1,25 dihydroxyvitamin D insufficiency is vigorously linked with significant advancement in chronic kidney diseases among adults. Among 14,679 united states adult participants, the mean circulating concentration of 1,25- dihydroxycholecalciferol was lower in patients with stage four-five persistent nephropathy in comparison with normal renal function (21). Likewise, another research estimated the circulating concentration of 1,25-dihydroxy cholecalciferol in patients with persistent nephropathy. The mean serum concentration of 1,25- dihydroxycholecalciferol was lower in patients with stages three-four (22). Furthermore, participants with a serum concentration of 1,25- dihydroxycholecalciferol less than 15 ng/mL had a 2fold greater occurrence of final stage nephropathy than those with more than 15 ng/mL during a long- lasting, Follow-up (23). Additionally, few researches revealed that low 1,25 dihydroxyvitamin D concentrations were independently linked with albumin-urea in chronic kidney diseases and T1DM (24,25). The present result illustrates that 1,25 dihydroxyvitamin D mild decrease / greater decrease is a persistent state in patients with chronic kidney diseases, mainly those with an estimated glomerular filtration rate of less than 15 mL/min/1.73 m2. Various mechanisms might describe the deficiency of 1,25- dihydroxycholecalciferol in persistent nephropathic patients. First, most patients with chronic kidney diseases have limited protein sources besides caloric consumption, so 1,25 dihydroxyvitamin D is fairly few. Second, many chronic kidney disease patients have restricted environmental activities with declined sunlight exposure (26). Finally, a considerable lack of urinary 1,25 dihydroxyvitamin D metabolites takes place in patients with urinary protein-creatinine ratio ≥1.0 g protein/g creatinine. Investigators reported an interrelationship between parathyroid hormone, 1,25- dihydroxycholecalciferol and persistent nephropathy (27). In chronic kidney diseases, the kidney 1α-hydroxylase expression is blocked to remunerate for phosphate reservation, which results in an elevated expression of 24‐hydroxylase for the breakdown of 1,25-dihydroxycholecalciferol. The concentration of 1,25 dihydroxyvitamin D in dialysis patients is lower than in healthy individuals (28). Renal malfunctioning, usually found among patients with renal diseases, is involved in the generation of 1,25-dihydroxycholecalciferol insufficiency that contributes to lowering serum calcium levels and secondary hyperparathyroidism, which results in secondary osteoporosis (29). It has been recorded that 1,25 dihydroxyvitamin D insufficiency in renal disorders is elevated in severe stages of chronic kidney disease (28). Serum level of kidney function markers Table (1) and Figure (4) revealed a significant (P<0.0001) increase in serum level of urea, creatinine and uric acid in patients (183.2±10.35 mg/dL), (8.88±0.54 mg/dL) and (6.57±0.22 mg/dL) respectively as compared to controls (33.45±1.08 mg/dL), (0.95±0.03 mg/dL) and (04.35±0.22 mg/dL), respectively. Figure 4. Serum levels of (a) urea, (b) creatinine (c) uric acid. Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chronic kidney disease 63 The current research revealed that higher blood urea nitrogen concentrations were linked with negative kidney consequences unconstrained of estimated glomerular filtration rate in patients with moderate to severe kidney damage. Previous cohort study recorded that both blood urea nitrogen and serum osmolality were unconstrained health risks for persistent nephropathy in patients with conserved renal action (30). Urea formation directly corresponds to dietary protein consumption, and curtailment of daily protein consumption contributes to decrease urea formation. Various research concerning the potency of daily protein curtailment on renal disease development have been performed; however, findings have been incompatible. The wide-ranging irregular trial, the improvement of diet in kidney disorder study, revealed that a diet with very low- protein provided with oxoacids contributes to the reduction of the degree of development of chronic kidney disease compared with a diet that contains a limiting amount of high protein foods, but this was not statistically remarkable (7). Serum creatinine descriptive of glomerular filtration rate has acquired its restriction since a decline in glomerular filtration only contributes to an imperceptible elevate in serum creatinine since its tubular release elevated; so, an imperceptible elevate in serum creatinine does not definitely means that glomerular filtration rate is normal, but a serum creatinine elevate more than 2 mg/dL result in saturation of excretion begins to consider glomerular filtration rate (31). The current study supports that hyperuricemia is associated with a greater decline in renal function and a higher risk of progressing to kidney failure. The influences of hyperuricemia on renal function decline and the risk of kidney failure are more significant in patients without proteinuria than those with proteinuria (32). Uric acid is the final oxidation product of purine metabolism and is renally excreted. Therefore, elevated serum uric acid levels are seen in patients with reduced glomerular filtration rate (GFR). However, it has been proposed that uric acid itself plays a causal role in the pathophysiology of chronic kidney disease and possibly in acute kidney injury. A review of the literature demonstrates uric acid- related cellular changes that contribute to renal disease. Studies performed on rats have demonstrated that there are fundamental changes in the renal vasculature in the presence of hyperuricemia. It was found that uric acid decreased the expression of E-cadherin in epithelial cells resulting in a loss of cell-to-cell contact in the renal tubular cells of rats. Without cell-to-cell contact, epithelial cells are unable to coordinate efforts to secrete substances needed to increase renal blood flow, such as nitric oxide (33). In addition, a recent study utilizing immortalized proximal tubular epithelial cells from normal adult human male kidneys has demonstrated that increased uric acid levels cause NAPDH-dependent oxidative changes that promote apoptosis (34). This finding shed light on the connection between hyperuricemia and tubulointerstitial renal damage. Serum level of total protein Table (1) & Figure (5) revealed a non- significant decrease (P=0.0503) in serum level of total protein in patients (6.49±0.12 mg/dL) as compared to controls (6.82±0.10 mg/dL). Figure 5. Serum level of total protein It has been reported that serum total protein concentrations are strongly associated with estimated glomerular filtration rate decline, rapid eGFR decline and incident CKD. These findings were independent of albuminuria, self-reported health, biomarkers of inflammation, as well as demographic and clinical risk factors for kidney disease progression (35). Serum total protein concentration is a well-known predictor of mortality in patients with CKD. Only a few studies have examined serum albumin as a risk factor for the development of ESRD (36,37). Few studies have examined the associations of serum albumin with kidney function decline. However, in one study of eight inflammatory markers, including serum albumin, serum albumin was the only laboratory measure associated with kidney function decline. Few studies have examined the associations of serum albumin with kidney function decline. However, in one study of eight inflammatory markers, including serum albumin, serum albumin was the only laboratory measure associated with kidney function decline (38). The underlying mechanisms for these associations are unclear. To our knowledge, there is no direct physiological basis for low serum albumin to cause the development of CKD. Serum albumin levels may be reduced for several reasons: liver damage and disease may decrease production; they may decrease as a negative acute phase reactant or in response to inflammation; and substantial and sustained albuminuria can lead to lower serum concentrations. Uncommonly low serum albumin can also reflect malnutrition, but the only malnourished state that consistently produces lower Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chronic kidney disease 64 serum albumin is kwashiorkor, a rare disease in the developing world (39). Previous studies hypothesized the strong relationship between lower serum protein and kidney function decline in their research must be multi-factorial, representing general ill health. Lower protein concentrations may reflect much broader abnormalities in inflammation, thrombosis and microvascular function processes than can be captured by the markers available in this cohort. Lower serum protein and kidney function decline may also be related to multiple factors that are currently poorly understood. These findings are consistent with the results from our previous study of HIV-infected women and other studies of serum protein in ESRD (35). In a 2011 retrospective cohort analysis of 1800 men and women from the UK, Wagner et al. found that serum albumin levels were an essential component of a multivariable risk model for mortality in patients with ESRD (36). With some types of kidney disease, protein may be lost in the urine (proteinuria). With peritoneal dialysis, some protein crosses the peritoneal membrane and exits the body in the effluent dialysate (the solution drained from the peritoneal cavity). This loss increases in a person with peritonitis, an infection of the peritoneum. Serum level of total calcium Table (1) and Figure (6) revealed a remarkable reduction (P<0.0001) in circulating concentration of total calcium in patients (8.06±0.18 mg/dL) as compared to controls (10.15±0.23 mg/dL). Figure 6. Serum level of total Calcium Throughout nephropathy, the renal may no more filter out additional phosphorus and eliminate it from the body. With time, phosphorus may elevate in the blood. Ca and PO-34 in the body interact in opposite directions: as blood Ca levels rise, PO-34 levels fall. With renal disorder development, elevated phosphorus concentrations may cause low circulating calcium concentration by accumulating onto the osseous matter and other tissues. Disturbances in the metabolic pathway of calcium metabolism disorders are characteristic features of chronic kidney disease (40). In various investigations, the circulating concentration of phosphorus was confirmed as a principal health risk for heart failure and all-cause death in kidney failure patients. In an American group of 35114 patients undertaken hemodialysis with an average follow-up of one year, among the patients with excessive remaining kidney urea authorization, there was an increased mortality health risk in patients with an elevated circulating concentration of phosphorus and decreased serum calcium (41). Conclusion The study showed disturbances in the circulating concentration of osteoprotegerin, obestatin, and 1,25 dihydroxyvitamin D in patients with kidney failure. The serum osteoprotegerin level and obestatin were considered contributors to Kidney failure. These findings indicated that OPG, a bone-modulating protein, might have a role in developing KF patients. Decreased concentrations levels of 1,25 dihydroxyvitamin D are frequently observed in patients suffering from chronic kidney failure; these serve as a principal element in the progression of KF. More investigations are needed to explain the importance of these findings in the etiopathogenesis of Kidney failure. Acknowledgement We would like to express our gratitude to the technical and support staff of the Dialysis Center - Ashty Hospital in Soran City for providing specimens. Conflict of Interest The authors declare no conflict of interest during this study. References 1. Hasan M, Sutradhar I, Gupta RD, Sarker M. Prevalence of chronic kidney disease in South Asia: a systematic review. BMC nephrology. 2018;19(1):1-2. 2. Chukwuonye II, Ohagwu KA, Adelowo OO, Chuku A, Obi EC, Onwuchekwa U, Anyabolu EN, Oviasu E. Prevalence and predictors of chronic kidney disease in a Semiurban Community in Lagos. International journal of nephrology. 2019 ; 2. 3. Bernardi S, Toffoli B, Bossi F, Candido R, Stenner E, Carretta R, Barbone F, Fabris B. Circulating osteoprotegerin is associated with chronic kidney disease in hypertensive patients. BMC nephrology. 2017;18(1):1-9. 4. Cardoso MP, Pereira LA. Native vitamin D in pre-dialysis chronic kidney disease. Nefrología (English Edition). 2019;39(1):18-28. 5. Kamińska J, Stopiński M, Mucha K, Pac M, Gołębiowski M, Niewczas MA, Pączek L, Foroncewicz B. Circulating osteoprotegerin in chronic kidney disease and all-cause mortality. International Journal of General Medicine. 2021;14:2413. 6. Banaei S, Rezagholizadeh L. The role of hormones in renal disease and ischemia- Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chronic kidney disease 65 reperfusion injury. Iranian journal of basic medical sciences. 2019;22(5):469. 7. Seki M, Nakayama M, Sakoh T, Yoshitomi R, Fukui A, Katafuchi E, Tsuda S, Nakano T, Tsuruya K, Kitazono T. Blood urea nitrogen is independently associated with renal outcomes in Japanese patients with stage 3–5 chronic kidney disease: a prospective observational study. BMC nephrology. 2019;20(1):1-0. 8. Goldberg A, Garcia-Arroyo F, Sasai F, Rodriguez-Iturbe B, Sanchez-Lozada LG, Lanaspa MA, Johnson RJ. Mini Review: Reappraisal of uric acid in chronic kidney disease. American Journal of Nephrology. 2021 ;21:1-8. 9. Temilola DO, Bezuidenhout K, Erasmus RT, Stephen L, Davids MR, Holmes H. Salivary creatinine as a diagnostic tool for evaluating patients with chronic kidney disease. BMC nephrology. 2019;20(1):1-6. 10. Di Micco L, Di Lullo L, Bellasi A, Di Iorio BR. Very low protein diet for patients with chronic kidney disease: Recent Insights. Journal of clinical medicine. 2019 ;8(5):718. 11. Mirmiran P, Yuzbashian E, Aghayan M, Mahdavi M, Asghari G, Azizi F. A prospective study of dietary meat intake and risk of incident chronic kidney disease. Journal of Renal Nutrition. 2020;30(2):111-8. 12. Bernardi S, Fabris B, Thomas M, Toffoli B, Tikellis C, Candido R, Catena C, Mulatero P, Barbone F, Radillo O, Zauli G. Osteoprotegerin increases in metabolic syndrome and promotes adipose tissue proinflammatory changes. Molecular and cellular endocrinology. 2014;394(1-2):13-20. 13. Nakahara T, Kawai-Kowase K, Matsui H, Sunaga H, Utsugi T, Iso T, Arai M, Tomono S, Kurabayashi M. Fibroblast growth factor 23 inhibits osteoblastic gene expression and induces osteoprotegerin in vascular smooth muscle cells. Atherosclerosis. 2016 Oct 1;253:102-10. 14. Gordin D, Soro-Paavonen A, Thomas MC, Harjutsalo V, Saraheimo M, Bjerre M, Forsblom C, Flyvbjerg A, Groop PH, FinnDiane Study Group. Osteoprotegerin is an independent predictor of vascular events in Finnish adults with type 1 diabetes. Diabetes care. 2013 Jul 1;36(7):1827-33. 15. Cortinovis M, Ruggenenti P, Remuzzi G. Progression, remission and regression of chronic renal diseases. Nephron. 2016;134(1):20-4. 16. Eddy AA. Overview of the cellular and molecular basis of kidney fibrosis. Kidney international supplements. 2014;4(1):2-8. 17. Meng XM, Nikolic-Paterson DJ, Lan HY. TGF- β: the master regulator of fibrosis. Nature Reviews Nephrology. 2016;12(6):325-38. 18. Gargantini E, Lazzari L, Settanni F, Taliano M, Trovato L, Gesmundo I, Ghigo E, Granata R. Obestatin promotes proliferation and survival of adult hippocampal progenitors and reduces amyloid-β-induced toxicity. Molecular and cellular endocrinology. 2016;422:18-30. 19. Catak Z, Aydin S, Sahin İ, Kuloglu T, Aksoy A, Dagli AF. Regulatory neuropeptides (ghrelin, obestatin and nesfatin-1) levels in serum and reproductive tissues of female and male rats with fructose-induced metabolic syndrome. Neuropeptides. 2014;48(3):167-77. 20. Moraes C, Borges NA, Barboza J, Barros AF, Mafra D. Effects of acute resistance exercise on acyl-ghrelin and obestatin levels in hemodialysis patients: a pilot study. Renal failure. 2015;37(10):338-42. 21. Chonchol M, Scragg R. 25-Hydroxyvitamin D, insulin resistance, and kidney function in the Third National Health and Nutrition Examination Survey. Kidney international. 2007;71(2):134-9. 22. LaClair RE, Hellman RN, Karp SL, Kraus M, Ofner S, Li Q, Graves KL, Moe SM. Prevalence of calcidiol deficiency in CKD: a cross- sectional study across latitudes in the United States. American Journal of Kidney Diseases. 2005;45(6):1026-33. 23. Melamed ML, Astor B, Michos ED, Hostetter TH, Powe NR, Muntner P. 25-hydroxyvitamin D levels, race, and the progression of kidney disease. Journal of the American Society of Nephrology. 2009;20(12):2631-9. 24. Isakova T, Gutiérrez OM, Patel NM, Andress DL, Wolf M, Levin A. Vitamin D deficiency, inflammation, and albuminuria in chronic kidney disease: complex interactions. Journal of Renal Nutrition. 2011 Jul 1;21(4):295-302. 25. De Boer IH, Sachs MC, Cleary PA, Hoofnagle AN, Lachin JM, Molitch ME, Steffes MW, Sun W, Zinman B, Brunzell JD, Diabetes control and complication trial/epidemiology of diabetes interventions and complications study research group. circulating vitamin D metabolites and kidney disease in type 1 diabetes. The Journal of Clinical Endocrinology & Metabolism. 2012 Dec 1;97(12):4780-8. 26. Tonelli M. Vitamin D in patients with chronic kidney disease: nothing new under the sun. Annals of Internal Medicine. 2007; 147(12) :880-1. 27. Wang WH, Chen LW, Lee CC, Sun CY, Shyu YC, Hsu HR, Chien RN, Wu IW. Association between parathyroid hormone, 25 (OH) vitamin D, and chronic kidney disease: a population- based study. BioMed Research International. 2017;2017. 28. Vahdat S. Vitamin D and kidney diseases: A narrative review. International Journal of Preventive Medicine. 2020;11. Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Chronic kidney disease 66 29. Jean G, Souberbielle JC, Chazot C. Vitamin D in chronic kidney disease and dialysis patients. Nutrients. 2017;9(4):328. 30. Kuwabara M, Hisatome I, Roncal-Jimenez CA, Niwa K, Andres-Hernando A, Jensen T, Bjornstad P, Milagres T, Cicerchi C, Song Z, Garcia G. Increased serum sodium and serum osmolarity are independent risk factors for developing chronic kidney disease; 5 year cohort study. PLoS One. 2017;12(1):e0169137. 31. Shemesh O, Golbetz H, Kriss JP, Myers BD. Limitations of creatinine as a filtration marker in glomerulopathic patients. Kidney international. 1985;28(5):830-8. 32. Tsai CW, Lin SY, Kuo CC, Huang CC. Serum uric acid and progression of kidney disease: a longitudinal analysis and mini-review. PloS one. 2017;12(1):e0170393 33. Ryu ES, Kim MJ, Shin HS, Jang YH, Choi HS, Jo I, Johnson RJ, Kang DH. Uric acid-induced phenotypic transition of renal tubular cells as a novel mechanism of chronic kidney disease. American Journal of Physiology-Renal Physiology. 2013;304(5):F471-80 34. Verzola D, Ratto E, Villaggio B, Parodi EL, Pontremoli R, Garibotto G, Viazzi F. Uric acid promotes apoptosis in human proximal tubule cells by oxidative stress and the activation of NADPH oxidase NOX 4. PloS one. 2014;9(12):e115210 35. Lang J, Katz R, Ix JH, Gutierrez OM, Peralta CA, Parikh CR, Satterfield S, Petrovic S, Devarajan P, Bennett M, Fried LF. Association of serum albumin levels with kidney function decline and incident chronic kidney disease in elders. Nephrology Dialysis Transplantation. 2018;33(6):986-92 36. Wagner M, Ansell D, Kent DM, Griffith JL, Naimark D, Wanner C, Tangri N. Predicting mortality in incident dialysis patients: an analysis of the United Kingdom Renal Registry. American Journal of Kidney Diseases. 2011;57(6):894-902. 37. Phelan PJ, O'Kelly P, Walshe JJ, Conlon PJ. The importance of serum albumin and phosphorous as predictors of mortality in ESRD patients. Renal failure. 2008;30(4):423-9. 38. Keller C, Katz R, Sarnak MJ, Fried LF, Kestenbaum B, Cushman M, Shlipak MG, CHS study. Inflammatory biomarkers and decline in kidney function in the elderly: the Cardiovascular Health Study. Nephrology Dialysis Transplantation. 2010 ;25(1):119-24. 39. Friedman, A.N. and Fadem, S.Z., 2010. Reassessment of albumin as a nutritional marker in kidney disease. Journal of the American Society of Nephrology, 21(2), pp.223-230 40. Yu G, Cheng J, Jiang Y, Li H, Li X, Chen J. Serum phosphorus and calcium levels and kidney disease progression in immunoglobulin A nephropathy. Clinical Kidney Journal. 2021 Jan 25. 41. Wang M, Obi Y, Streja E, Rhee CM, Lau WL, Chen J, Hao C, Hamano T, Kovesdy CP, Kalantar-Zadeh K. Association of parameters of mineral bone disorder with mortality in patients on hemodialysis according to level of residual kidney function. Clinical Journal of the American Society of Nephrology. 2017;12(7):1118-27 This work is licensed under a Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/