INTRODUCTION:

Glomerulonephritis describes an acute inflammatory disease on the kidney functions especially in the glomerular.⁽¹⁾. The clinical presentation are varies from asymptomatic microscopic hematuria to acute kidney injury. Proteinuria, edema as well as high blood pressure was generally noticed particularly GN types that required to limit clinical symptoms, while others patients with necessitate urgent of therapeutic treatment⁽²⁾.

podocyte is diverse interacting proteins which maintain cell to cell and cell to glomerular basement membrane (GBM) contacts⁽³⁾. Podocytes cells is found in the surface area of the glomerular basement membranes (GBM)⁽⁴⁾.

It is decreased in Patients with early diagnoses of albumin excretion and decline in podocyte number^(5,6) which includes focal segmental glomerulosclerosis, glomerular nephritis, diabetic nephropathy, lupus nephritis and proliferative membranous nephropathy⁽⁷⁾. While, despite the kidney injury complication but losses of podocyte numbers is the main contributor for glomerulosclerosis development⁽⁸⁾. Cystatin-C have a low molecular mass therefore freely filtred through the glomeruli of the kidney.⁽¹⁰⁾. It was used as endogenous marker for CVD as well as in individuals with chronic kidney disease (CKD), dialysis and kidney transplante ⁽¹¹⁾. Cystatin-C is important role for predicting of severe cardiovascular disease in patients with renal proplems which direct related with various other factors associate with CVD including pulse wave rate and accountable for arterial tightness, so increased levels of serum Cystatin-C indicate renal impairment and arterial stiffness^(12,13). Fibroblasts cells product a large amount of interstitial matrix elements, consisting of fibronectin as well as type I and III collagens⁽¹⁴⁾Nephrosis is induced by doxorubicin, an experimental model of glomerulosclerosis in relation to stable proteinuria.⁽¹⁵⁾ Doxorubicin is administrated in vivo related to several metabolites besides generated of reactive oxygen species ⁽¹⁶⁾. The incretin hormones; glucagon-like peptide-1(GLP-1)) are produced from intestinal cells in response to food intake which stimulate secretion of insulin hormone from beta cells of pancreas as well as decreased glucagon levels from alpha cells in the way of glucose dependent, these endogenous peptide hormones was reduced plasma glucose and controlled postprandial sugar homeostasis⁽¹⁷⁾. In the pancreas GLP-1R was controlled glycaemia by stimulate insulin secretion while glucagon was suppression with delayed gastric empty. These actions was confirmed by clinical examination of fasting and postprandial glucose⁽¹⁸⁾. The mechanism of GLP-1 is antioxidant⁽¹⁹⁾. It is reported that GLP-1 inhibited of the anti-inflammatory on the pancreatic islands , adipose tissue besides reduced levels of glucose in the diabetic patient⁽²⁰⁾

MATERIALS AND METHODS:

Thirty six of adult male wister rats with weight of (250 – 300) mg from animal house of biotechnology research center/Nahrain university have been used in the experiment after seeking approval ethical committee of the college of pharmacy/Al Mustansiriyah University. It was used plastic cages with diameters of (20x25x35cm) to keep four animals in each of cage. Food and Water was commercially provided in addition to marked colored tail for discrimination. For optimal environment air vacuum was supplied in the animal house. Before starting of the study protocol, the animals were placed for seven days under controlled condition at room temperature (21 to 22)⁰C and dark light about twelve hours cycles. In addition to that; Laboratory tests must be checked of of all animals. Rats were divided into two groups; First groups consist of twelve rats were received normal saline as group A(control). Second group consist of twenty four of rats were received single dose (7.5mg/kg) of doxorubicin (Adriamycin) intraperitoneally depended on the data of literature and pilot experiment for glomerulonephritis induction, after injection waiting of 2^th weeks for complicated to proteinuria then administered 0.5ml/kg normal saline intraperitoneal for 28 days as group B (induction)⁽²¹⁾. Third groups consist of twelve rats are received 200μg/kg/day intraperitoneal liraglutide (victoza^®) intraperitoneally (200μg/kg daily dose) dissolved freshly in normal saline 0.5ml/kg. after 2^th weeks of induction as group C (treatment)⁽²²⁾. At the end of the 6^th weeks, the animals anesthetized by intramuscular administration of 100 mg/kg ketamine and 10 mg/kg xylazine⁽²³⁾. Blood was collected by cardiac puncture (~6 ml) then animals euthanized by decapitation. The kidney was removed and washed with tap water which placed in the neutral buffered formalin 10%. Serum sample preparation which thirty six of blood samples were collected in the gel tube. Serum was obtained by centrifuged blood sample for 10 minute (~ 1000 x g) or (3000rpm) then serum was kept in Eppendorf tubes at twenty centigrade beside marked numbers for diagnosis. Biochemical tests include; creatinine, Urea, Total. protein, Albumin were determined by Chemistry analyzer AU480 while Fibronectin and Cystatin-C by Enzyme Linked Immunosorbent Assay technique.

Statistical analysis:

Statistical packages of social sciences ver-16 was used in the statistical analysis of this research⁽²⁴⁾data descriptive expressed as mean ± st. error and One way Anova with Tukey test which used to differentiate between three groups (control, induction and treatment groups)^(25). Significant difference between parameters depended on P. value less or equal than 0.05 while high significance expressed as P. value less or equal than 0.01. ROC curve was utilized for comparison between biomarker as best indicator of kidney deterioration with highest possible level of sensitivity as well as highest specificity. The correlation coefficient (r) between two tails is taken depending on the significant value of its. If (r) is positive value means direct correlation while negative value is inverse correlation, strong correlation should be more than 0.7.⁽²⁶⁾

RESULTS:

In this study mean value of urea showed significant increased (P< 0.05) in the group B as compared with other groups while group C showed significantly decreased (P< 0.05) than group B and approach to group A (P= 0.147). serum creatinine level revealed significant increased (P< 0.05 ) in the group B as compared with group A and group C meanwhile, also study was appeared improvement of creatinine (P< 0.05) in the group C than group B, but still significantly higher (P< 0.05) as comparison with normal value of group A, in the other hand, total protein in the group B showed significant decreased ( P< 0.05 ) as compared with other groups while group C appeared significant increased (P< 0.05) than group B however mean value of group C still significantly lower(P< 0.05 )as comparison with group A, Otherwise, serum albumin in the group B was noticed significant decreased (P< 0.05) as compared with group A, while group C revealed no significant difference (P=0.11) than group B but group C still significant decreased (P< 0.05) as comparison with group A.Table1.

Table 1: Clinical characteristics the effect of Liraglutide on Glomerulonephritis

Parameters	Groups	Mean± SEM	Minimum	Maximum
Urea mg/ dl	Group A	34.5±1.143 ^b	28.00	40.50
	Group B	70.7±7.107 ^a,c	44.00	121.00
	Group C	47.13±3.542 ^b	33.70	76.80
Creatinine mg/dl	Group A	0.54±0.009 ^b,c	0.50	0.60
	Group B	1.65±0.0882 ^a,c	1.30	2.30
	Group C	1.20±0.074 ^a,b	0.80	1.70
Total protein g/dl	Group A	6.63±0.125 ^b,c	6.12	7.45
	Group B	2.10±0.154 ^a,c	1.24	2.92
	Group C	2.70±0.107 ^b,c	2.23	3.14
Albumin g/dl	Group A	2.78±0.096 ^b,c	2.31	3.51
	Group B	1.47±0.136 ^a	0.76	2.05
	Group C	1.83±0.132^a	1.22	2.50

Data expressed as Mean± SEM, Group A = control , Group B = induction , Group C = treatment Number of animal for each group = 12, ^a Significantly different when compared with group A at P < 0.05.^b Significantly different when compared with group B at P < 0.05.^c Significantly different when compared with group C at P < 0.05.

Statistical analysis of Cystatin-C expressed as significant increased (P<0.05) in the group B in relation to kidney deterioration as compared with group A and group C. In addition, the study was appeared significant decreased (P<0.05) in the group C as comparison with group B however, group C still significantly higher (P<0.05) than normal value of group A. The statistical study of fibronectin was revealed significant increased (P<0.05) in the group B as compared with group A and group C. Furthermore, it was showed significant decreased (P<0.05) in the group C than group B and approach of group A (p =0.129), Table (2).

Table 2: Variation of serum Cystatin-C and Fibronectin of all groups.

	Groups	Mean± SEM	Minimum	Maximum
Cystatin-C	Group A	30 ± 3.097 ^b,c	16.50	47.25
	Group B	187.81 ± 7.290 ^a,c	144.63	230.75
	Group C	146.87 ± 6.361 ^a,b	118.50	197.00
Fibronectin	Group A	0.53±0.042 ^b	0.23	0.79
	Group B	1.93±0.257^a,c	0.69	3.32
	Group C	0.97±0.081 ^b	0.55	1.35

Data expressed as Mean± SEM, Group A = control, Group B = induction, Group C = treatment, Number of animal for each group = 12, ^a Significantly different when compared with group A at P < 0.05.^b Significantly different when compared with group B at P < 0.05.^c Significantly different when compared with group C at P < 0.05.

Receiver operating characteristic (Roc) curve was used to identify the ability of the Cystatin-C to diagnose renal injury among ordinary biomarkers (creatinine and urea). Cystatin C was observed at cut-off (149 pg/ml), the sensitivity is 92 % and specificity 87% while in the urea at (49.7 mg/dl) the sensitivity is 91% and specificity 56% and finally creatinine at (1.3 mg/ml) the sensitivity is 83% and specificity 83%Table (3) and Figure (1)

Table 3: Comparsion study of Receiver operator characteristic by AUC of Cystatin-C, creatinine and urea

Biomarker	AUC	95% CI	P value
Cystatin-C	0.946	0.874-1	< 0.001
Creatinine	0.941	0.870-1	< 0.001
Urea	0.901	0.803-0.99	< 0.001

Roc: Receiver Operating Characteristic, AUC: Area Under Curve, CI: Confidence Interval

Cystatin C was observed at cut-off (149pg/ml), the sensitivity is 92 % and specificity 87% while in the urea at (49.7mg/dl) the sensitivity is 91% and specificity 56% and finally creatinine at (1.3mg/ml) the sensitivity is 83% and specificity 83% as showed in Table (4)

Table 4: Table (3-2C): Cut- off value of some biomarker to predict renal progression

parameter	Cut-off	Sensitivity (%)	Specificity (%)
Cystatin C	159	92	87
Urea	49.7	91	83
Creatinine	1.3	83	83

Figure 1: Receiver operating characteristic (ROC) curves explain the diagnostic efficacy of kidney injury by comparing their areas of serum Cystatin- C, creatinine and urea under Roc curves.

Correlation coefficient between Cystatin-C, Fibronectin and other biomarker was demonstrated in the Table (5):

DISCUSSION:

In the past decade more biomarkers are specific than serum creatinine to determine kidney function. Cystatin-C is one of the important biomarkers to predict early diagnoses within 24 hour of acute kidney injury⁽²⁷⁾ it may be better than creatinine in glomerular filtration rate which observer at a late stage of ICU clients also Patients with paralysis as well as ICU catabolism were lose muscle mass, thus a gradual decreased levels of creatinine⁽²⁸⁾. In this study , serum Cystatin-C levels was significantly reduced after 28 day of liraglutide treatment when compared with induction groups these results combined with another studies which appeared the sensivity of Cystatin-C and NGAL is predict early stage of diadetes and re-treatment with liraglutide as a restorative therapy^(29,30). In the narrawing glomerular of AKI, Cystatin-C is freely filtered by renal glomeruli and completely reabsorbed by proximal tubule and has molecular size about 100 times than creatinine therefore Cystatin-C is more specific than creatinine that freely excreted in the urine⁽³¹⁾. The area under the ROC determines the ability of the statistics to discriminate appropriately between some study analyzes that have the best prediction of renal injury. Overall, the ROC curve ranges from 0.5 to 1.0 as 0.5 (weak) and 1.0 (best) to test. Moreover, the most effective test for medical diagnosis will certainly be sensitivity and specificity 100%. ROC analysis is the best test for a medical diagnostic disease using a biomarker. it has been used to compare the highest level of sensitivity as well as the highest specificity⁽³²⁾. In this study, serum Cystatin-C was showed more active diagnostic test in comparsion with creatinine and urea. These findings relites with Al-Saedy AAK et al. 2017 to sugest that Cystatin-C is more specific marker in diadetic iraqi patient with early microalbuminurea⁽³³⁾ also the study of Gandhi S et al. 2012 in renal injury induced by streptozocin in rats confirmed that Cystatin-C as specific biomarker of others disgnosis⁽³⁴⁾^. The current study showed a significant correlation between serum Cystatin-C and other biomarker (urea, creatinine, total protein and albumin) also positive correlation of Cystatin-C with urea and creatinine these results corespond with other studies⁽³¹⁾^. In adition to negative correlation of Cystatin-C with albumin and total protein which in line with another study showing this association with albumin in urine for primary hypertension patient ⁽³⁵⁾^. Fibronectin is a high molecular weight of glycoprotein in the body fluid and is widely distributed through extracellular matrices⁽³⁶⁾. Extracellular fibronectin on the surface of cell is related with ECM regulation and accumulation, the first step of renal fibrosis which confirmed by many studies^(37,38). immune function related with Plasma fibronectin and other body fluid such as cerebral and amniotic fluid more than fibronectin on the surface. It was revealed that plasma fibronectin increased in the animal induced by toxic or immunolgical kidney damage by increased synthetic rate of glycoprotein⁽³⁹⁾ recently appeared circulating fibronectin is responsible of mesangial expansion and albuminurea⁽⁴⁰⁾^. From the above data, fibronectin is related with renal function by associated with albuminurea therefore in this study , group B appeared increased significant in serum fibronectin which glomerulonephritus induction by doxorubicin compared to group A as control group while liraglutide treatment group C showed significantly improvement in serum fibronectin corresponds to several studies conducted on liraglutide and other analog GLP-1 that show an improvement of fibronectin in renal tissue by reducing albumin^(41,42). It has been used as renoprotective by controlled fibronectin in addition to the effect on cellular fibronectin that responsible of ECM accumulation. Curent study appeared asignificant correlation between serum fibrnectin and other renal biomarker ( urea , creatinine, total protein, albumin ). It was positive correlation of fibronectin with urea and creatinine but negative correlation with albumin and total proetien showing liver reduces fibronectin and other glycoprotein as improvement level of albumin^(43).

CONCLUSION:

1. Cystatin-C and Fibronectin are more useful than creatinine to determine chronic kidney disease especially in the individuals with low muscular mass because Cystatin-C have a low molecular mass which freely filtred through the glomeruli therefore used for early diagnosis of renal failure.

2. In ROC analysis, Cystatin-C is more sensitive to diagnose renal injury among ordinary biomarkers. It was found more specific and sensitive in prediction kidney disease progression at cut-off value (149 pg/ml) when compared with other biomarkers.

3. Significant correlation either positive or negative was found between Cystatin-C, fibronectin and other biomarkers which upregulation and down regulation of the pro and anti-inflammatory of creatine and albumin respectively.

RECOMMENDATION:

1. Measured of Cystatin-C levels with glomerular filtration rate as a reliable and sensitive marker for identified the filtration changes in the glomerular and tubular.

2. Study the correlated between creatinine clearance and Cystatin-C as prognosis assessment of renal function.

3. Study the combinative of Fibronectin with currently available markers that related with tissue glycation to diagnose structural alternation of kidney.

ACKNOWLEDGEMENT:

The researcher introduced thankful for the College of Pharmacy, Mustansiriyah University for its support to complete this research.

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Received on 12.06.2019 Modified on 04.07.2019

Research J. Pharm. and Tech. 2020; 13(1): 147-151.

DOI: 10.5958/0974-360X.2020.00029.3

		Urea mg/dl	Creatinine mg/dl	Total protein g/dl	Albumin g/dl
Cystatin-C g/ml	Correlation	0.623^**	0.884^**	-0.936^**	-0.780^**
	Sig. (2-tailed)	0.0001	0.0001	0.0001	0.0001
	95% C. I of r	0.36 _ 0.78	0.78 _ 0.93	-0.96- -0.87	-0.88- -0.60
	N	36	36	36	36
Fibronectin ug/ml	Correlation	0.732^**	0.609^**	-0.592^**	-0.506^**
	Sig. (2-tailed)	0.000	0.000	0.000	0.002
	95% C.I of r	0.53_ 0.83	0.35 _ 0.78	-0.77 _ -0.32	-0.71 _ -0.213
	N	36	36	36	36