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INTRODUCTION

The pathology of diabetic nephropathy manifests histologically as diabetic


glomerulosclerosis, and is characterized by glomerular basement membrane thickness and mesangial expension with increased extracellular matrix deposition . in Type 1 diabetics, there is a direct relationship between the extent of mesangial expansion and clinical severity of disease. In this regard ,Mauers seminal paper demonstrated a direct correlation between the degree of mesangial expansion, and magnitude of proteinuria, severity of hypertension,and degree og renal impairment.Taken together, these. Cardiovascular disease is recognized is as the predominant cause of death in the patient with diabetic nephropathy.In addition to traditional risk factors, uraemia related risk factors including endothelial dysfunction inflammation hyperhomocytenemia hypoalbuminemia oxidative stress abnormal divalent ion metabolism and advanced glycation end products also contribute to increased incidence of cardiovascular disease. Endothelial dysfunction is recognized to occur in diabetic nephropathy of any cause and is associated with increased acute phase reactant,plasma cytokines and homocystneie levels.Modulation of endothelial dysfunction may prove a novel meachanism in reducing cardiovascular disease. CRP is an acute phase reactant and a marker for inflammation . It has become recognized as an important link between development of atheroma in DN.

Salacia chinansis is native to region of India and Sri Lanka it is a herbal alpha glycoside inhibitor, effective in lowering post-Prandial glycemia and insulinemia.Hippophae rhomboids it grows on hill and hill sides in vellyes and river bed. It has anti-oxidant and immunomodulatory property. Coccinia indica, it grows in wild states abundantly in Bengal and other parts of India. Beta-sitosterol and taraxerol is located, an orally effective hypoglycaemic active principle comparable to tolbutamid. With this background we are prompted to undertake this pilot study to evaluate renoprotective effect of Salacia chinansis,Hippophae rhamnoides and COccinia indica(RF3).

MATERIAL AND METHODS

Present

study was conducted in Department of Nephrology, Institute of

Medical Sciences, Banaras Hindu University. A total of 60 cases of diabetic nephropathy attending nephrology OPD were included in the study .All patients had given informed consent for clinical examination, investigations renal biopsy and drug administration for the purpose of this study .Patients after including in the study were randomly assigned into two groups. Each group consisting of 25 patients. Group 1:25 patients of diabetic nrphtopathy who received herbal compounds(Salacia chinesis,Coccinia indica and Hippophar rambnoidis) and calcium channel blocker,central acting antihypertensive(conventional drug). Group 2: 25 patients of diabetic nephropathy who received calcium channel blocker, central acting antihypertensive. Section of cases Inclusion criteria Patients of diabetic nephropathy who fulfilled following criteria were included in the study. 1. Age Adult patients with age more than 18 years.
2.

Sex both sexes.

3.

Serum creatinine of >1.5 mg/dl but

3 mg/dl.

Exclusion criteria Patients were excluded from study in presence of following:

Acute Renal failure

Obstructive uropathy Diabetic nephropathy patients with acute deterioration History of allergy to trial drug

Patient receiving Angiotensin converting enzyme inhibitor or Angiotensin receprot blocker

Terminal criteria

Rapid decline in renal function requiring renal replacement therapy. Death of the patient during to any cause.

Sudden detenioration of kidney functions during study. Creatinine >3mg/dl at any time during study period. Acute myocardial infarction. Congestive heart failure. Study design and follow up

50 patients fulfilling the selection criteria were included in the study into two groups. Group 1 consisting of 25 patients of diabetic nephropathy channel who received central herbal compounds (Salacia chinesis,Coccinia indica and Hippophae rambnoidis) and calcium blocker, acting antihypertensive(conventional drug).group 2 consisting of 25 patients of diabetic nephropathy who received calcium channel blocker ,central acting antihypertensive. Antidiabetic drugs Apart from trial drug other drugs which were used included antihypertensive drugs (calcium channel blockers ,centrally acting drugs), dirtetics,phosphate binders,calcium vitamin D# supplements and iron. Detailed history, examination investigations were performed at baseline. Patients were followed at monthly intervals and any new symptom adverse drug reaction was noted. Physical examination estimation of creatinine levels,creatinine clearance,24 hour protein excretion routine bio-chemical investigations were performed at each visit. Estimation of c-reactive protein homocysteine interleukin-6 and detailed lipid estimation including cholesterol LDL,HDL and triglyceride was done at 0,3 and 6 months. Electron microscopy was done at 6 months in 2 patients who received trial drug. Collection of blood samples

5 ml of blood was collected aseptically from median cubital veins of patients using a disposable syringe with a 2 gauge needle. Blood was immediately transferred to a sterile test tube/vial and left on the laboratory bench to clot at room temperature. After the clot retracted serum was transferred by a sterile Pasteur pipette to sterile Kahn tube, centrifuged kept 40C in a refrigerator. Special test Interleukin Assay The interleukin immune assay kit is a 4-5 hour solid phase ELISA designed to measure IL-6 in serum, plasma and cultured supernatant. Manufactured distributed by R&D system maineneapolin, MN55413 United States of America. c-Reactive Proteins (CRP) Kit:For quantitative nephelometric determination CRP human serum or plasma=Turbox/Turbox analyzer. Manufactured by orion Dignostica Supplied By:B.M. System,New Delhi. Measurement of Homocysteine Homocysteine measured by high performance liquid chromatographic method based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole4-sulphonate) pre-coloum derivatization. TINF-

TINF- done by ELISA kit with the help of standardized solution and experimental material. ELISA kit was brought from Diaclone Research.

Endothelin-1 Endothelin-1 done with the of quantogo chemilumineescent Sanduich ELISA kit with standardized solution and experimental material. Electron microscopy: The tissue were cut into pieces of 1-2 mm 2 and fixed in 3%

gluteralaldehyde prepared in sodium cacodylate buffer overnight at 4 degree Celsius and pH 7.4. after fixation the tissue was washed on 0.1 M sodium cacodylate buffer for 45 minutes thrice and then post fixed in 1% OsO4 in 0.2 M sodium cacodylate for 1-2 degree celcius and pH7.4. Section were made of 1m thickness from the blocks and stained with toludene blue for light microscopy.After the careful examination of the section the area of interest was processed for ultrathin sectioning.section of 60 m thickness was cut and stained with uranyl acetate and lead citrate. Section obtained were subjected to electron microscopy for observation. Statical analysis: Statical analysis was done using S.P.S.S. Software (version 12.0) for window. Studentt test, Pairedt test and Chi- square test were used. P-value< 0.05 as considered as statically significant.

OBSERVATIONS Patients of diabetic nephropathy attending Nephrology OPD between May, 2008 to june,2009 who met the inclusion criteria were enrolled in the study. Patients were broadly divided into two groups. Group 1 consisting of patients of diabetic nephropathy with conventional drug and herbal compound(Salacia chinensis,Hippophae rhamnoides and Coccinia indiaca) at the dose of 1000 mg twice daily and Group 2 consisting of patients of diabetic nephropathy with conventional drug(calcium channel blocker, central acting antihypertensive).Both the group is consist of diabetic nephropathy which have protenuria>300 mg/day. 60 patients were initially enrolled for the study. 10 patients dropped out of the study, 6 patients were lost to follow up in the both groups. 2 patient die in group 1 due to acute complication (myocardial infarction) and 1 patient in group was die because of intracranial hemorrhage and 1 patient die due to infection (sepsis). 50 patients, 25 in each group who complete the 6 months were included in final analysis. In group 1 mean age was 57.369.03 with range from 38 to 75 years whereas in group 2, the mean age was 53.0010.94 years with age range 30 to 72 years. Although, the mean age in group 1 was higher compared to group 2, the difference was not statistically significant.

There was male predominance in our patients. Overall 78% of our patients were males and 28% of patients were females. In group 1, 80.0 % were males and in group 2, 76.0% of the patients were males. In group 2 females constituted 20.0% of patients where as In group 2, females accounted for 24.0%. Most common clinical symptoms at the time of 1st visit were generalized weakness and edema in both the groups. Other symptoms included anorexia, nausea and vomiting. Patients were observed for any deterioration or improvement in symptoms and also for appearance of any new symptom at monthly intervals for a period of 6 months. In both the groups there was consistent and comparable improvement in symptoms particularly edema, anorexia vomiting whereas there was no significant improvement in feeling of generalized weakness.

DISCUSSION The present study conducted in the Department of Nephrology was aimed at exploring the potential nephroprotective effect of Salacia chinensis, Hippophae rhamnoides and Coccinia indica herb in patients nephropathy. In experimental studies it has been demonstrated that Salacia chinensis, Hippophae rhamnoides and Coccinia indica, has anti-inflammatory, antiprotinuric and hypolipidemic action and improvement in endothelial dysfunction so with this background in mind anti-inflammatory, antiprotinuric and hypolipidemic action, and improvement in endothelial dysfunction were also explored and we do the kidney biopsy to evaluate the ultra structural changes in diabetic nephropathy. During study period 6 patients were lost to follow up, 2 patients die in group due to acute complication (mi) and in group 2,1 patient die to intracranial hemorrhage and 1 patient due to sepsis. 50 patient completed follow up of 6 month and 25 in each of the following group. Group 1: 25 patients of diabetic nephropathy who received herbal compound and calcium channel blocker, central acting antihypertensive (conventional drug). Group2 :25 patients of diabetic nephropathy who received calcium channel blocker, central acting antihypertensive. Hypertension in all groups was treated by calcium channel blockers (5 mg) and centrally acting agents (clonidine) either alone or in combination with of diabetic

target to achieve blood pressure of 140/90 nm Hg and herbal compound 1000mg twice daily in group 2 patients only calcium channel blocker(5 mg O.D) central acting drug 0.1g TDS. In both the group there was significant decline of BP was study period and comparing the two group at 6 month BP was controlled equally .Antihypertensive drug used were calcium channel blocker, central acting antihypertencive. No use of ACRE inhibitor or ARB blocker. The blood pressure reduction the concern of renoprotective intervention of associated with a more favorable course of long term renal function in diabetic nephropathy. In our study aimed at exploring the potential renoprotective role of herbal compound, strict blood pressure control was obtained in group receiving the trial drug. The effect of herbal compound on diabetic nephropathy was evaluated by measuring blood urea and serum creatinine at the start of trial and monthly for 6 month and comparing it with well matched control receiving the conventional drug. In diabetic nephropathy patient receiving herbal compound (group1) the mean blood urea at baseline was78.9636.15mg/dl at 6 month was not statically significant in group 2 patient received conventional drug and blood urea at start of study was 58.3625.32 and at 6 month 55.1825.12 mg/dl show no statically significant difference either in two group. So suggested that herbal drug does not stabilizing the urea level. In group 1 serum creatinine at 0 and 6 month was 2.400.7 and 2.140.69 mg/dl respectively. This decline in serum creatinine did not assume statistically significant. but there was stabilization of serum creatinine level

during the treatment with herbal compound . in group 2 baseline creatinine 2.060.61 at 0 month and 2.00.49 at 6 month but both both group comparable . so it suggest that herbal compound are stabilization of serum ceratinine level. These observations on blood urea and serum creatinine suggest that herbal compound may have renoprotective role by retarding the progression of diabetic nephropathy.Further studies are needed to elucidate the mechanism of renoprotection. There is previous study assressing the issue of renoprotective effect offered by salacia chinensis(Dr.Ranjit,IMS BHU).but not on COccinia indica and Hipopophae rhamnoides. Since the follow up was short. Long term studies was needed to conform renoprotective role of herbal compound. 24 hour urinary protein excretion 1 patient with diabetic neprhropathy the mean proteinuria decrease from 1.420.95 to 1.170.67 gm/day at 6 month. This decrease was statically significant. Hence the herbal compound have antiproteinuria effect. In group 2 decrease of protein excretion 2.281.3 to 2.311.28 but statistically not significant. Observations suggest that the decrease in 24 hour urinary protein. In group 1 and group 2 the base line FBS level were 19.9636.47 and 169.6849.44 mg/dl respectively. During 6 months follow up patient received oral hupoglycemic agent with the attempt to control blood glucose level. Three was significant reduction of blood glucose level drug study period and at 6 month. The FBS level in two groups was 130.6824.43 and 145.3218.83 mg/dl respectively (p=0.033) which suggest herbal compound

also effect on FBS level. But study was short need long term follow up for further evolution. Postprandial blood sugar (PPBS) level was not controlled by the trial drug. Baseline PPBS in both the group are at start of therapy and 6 month interval was 207.6687.48 and 245.0852.54 at 0 month and at 6 month was 189.9234.42 and 214.7669.48 which was statistically not significant. Mean serum albumin in group 1,baseline mean serum albumin was 3.750.66 mg/dl with no significant change at 6 month 3.220.43 mg/dl. In group 2 mean serum albumin concentration at baseline and 6 month was 3.390.72 and 3.290.72 and 3.290.49 mg/dl respectively. So herbal compound did not show any effect on serum albumin level in both the group. Effect of herbal compound on lipid was studied by measuring serum cholesterol,triglyseride, HDL and LDL at 3 month interval. In group 1 patient receiving herbal compound, the mean total cholesterol at baseline In group 1 the mean homocystine level at 0 month was 31.3210.12 and at 6 month 23.849.13 ml/l which was not statistically significant.IN group 1 versus group 2 comparison at 0 month the main homocystine level was 31.3210.13 respectively which was statistically significant(p<0.001).|It show that patient receiving the herbal compound where decrease the level of homocystine significantly. It indicate that trial drug have effect on indothelial dysfunction immunomodulation. C-reactive protein is an acute phase reactant and marker for

inflammation.CRP has been recognized as an important link in the development of the artheroma in diabetic nephropathy patient. Experimental

study have shown at herbal compound has anti-inflammatory activity with this background in the mind. The present study, effect of herbal compound on CRP was studied by measuring the level at the start of the study an at 3 monthly interval in diabetic nephropathy patient which on trial drug (herbal compound). In group 1,the mean CRP was level decrease from 4.091.18 at start to 3.070.87mg/dl at 6 month which show statistically significant(p<0.001).In group 2 the mean CRP level decrease from 4.161.00 at start to 3.891.16 mg/dl at 6 month which show not statistically significant difference. In group 1 versus group 2 comparison at 0 month the main CRP level was 4.091.18mg/dl and 4.161.00 which was not statistically significant and at 6 month the maion CRP level was 3.070.86mg/dl and 3.891.16mg/dl respectively which was statistically significant|(p=0.007).So trial drug so that have effect on inflammation (*decrease inflammatory activity). In group 1 the mean endothelin-1 level at 0 month was 1131.96183.65 and at 6 month `1024.94164.78pg/ml which was statistically significant (p<0.001). In group 2 the main endothelin -1 level at 0 month was 1084.14212.98and at 6 month 1134.94211.06pg/ml which was statistically significant(p<0.001).In group 1 versus group 2 comparison at 0 month the mean endothelia -1 level was 1131.96183.65 and 1084.14212.98pg/ml which was not statistically significant and at 6 month the mean endothelin-1 level was 1024.94164.78 and 1134.9211.06pg/ml respectively which was statistically significant(p=0.045) difference i.e. the trail drug also reduced the endothelin1 level, it indicate that improvement in endothelial dysfunction.

In group 1 the mean TNF- level at 0 month was 819.90174.70 and at 6 month 764.92170.63pg/ml which was statically significant(p<0.001).In group 2 the mean TNF- level at 0 month was 785.93190.77 and at 6 month 752.81229.68pg/ml which was statistically not significant. In group 1 versus group 2 comparison at 0 month the mean TNF- level was 819.90174.70 and 785.93190.77pg/ml which was not statistically significant and at 6 month the mean TNF- level a level was 764.92170.63 and 752.81229.68pg/ml respectively which was not statistically significant. Electron microscopic study Electron microscopic study (at 6 month)was done in 2 patients in which shows resin section two glomeruli with thick basement membrane. Electron microscopy shows expansion of mesangial matrix basement membrane thickness in various are varies from 260-1094nm with a mean of 600nm.No electron dense deposition scene. Podocytes and endothelial cells are normal.These findings are suggestive of diabetic glomerulopathy so biopsy is favor of diabetic nephropathy.