Role of hyperlipidemia in progressive renal disease: Focus on diabetic nephropathy

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1 Kidney International, Vol. 56, Suppl. 71 (1999), pp. S-31 S-36 MECHANISMS OF LIPID-INDUCED RENAL INJURY Role of hyperlipidemia in progressive renal disease: Focus on diabetic nephropathy KARIN JANDELEIT-DAHM, ZEMIN CAO, ALISON J. COX, DARREN J. KELLY, RICHARD E. GILBERT, and MARK E. COOPER Department of Medicine, University of Melbourne, Austin and Repatriation Medical Center (Repatriation Campus), West Heidelberg, Victoria, Australia Role of hyperlipidemia in progressive renal disease: Focus on diabetic nephropathy. Background. It has been suggested that lipids promote renal injury and that 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors confer renoprotection in certain re- nal diseases, including diabetic nephropathy. Methods. Sprague-Dawley rats were randomized to sham, subtotal nephrectomy (STNx) or STNx atorvastatin groups. After 12 weeks, proteinuria, renal function, glomerular injury, renal transforming growth factor- (TGF- ) gene expression and macrophage (ED1-positive cells) accumulation were assessed. In addition, the effects of HMG CoA reductase in human diabetic nephropathy were reviewed. Results. Atorvastatin therapy was associated with a modest reduction in proteinuria and glomerulosclerosis without influencing lipid levels or renal function in STNx rats. These effects were associated with decreased renal TGF- 1 gene expression and less glomerular and tubulointerstitial macrophage accumulation. The renoprotective effects of HMG CoA reductase inhibitors in both insulin- and non insulin-dependent diabetic subjects with either incipient or overt nephropathy appear to be highly variable. Conclusions. HMG CoA reductase inhibition appears to confer renoprotection via effects on prosclerotic cytokines such as TGF- and macrophage accumulation, independent of their lipid-lowering properties. The role of lipid-lowering agents in early or overt diabetic nephropathy remains to be fully ascer- tained. Diabetic nephropathy (DN) remains the major cause of end-stage renal failure in the Western world. This condition is characterized by hypertension, proteinuria, and declining renal function. Before the onset of DN, there is a phase known as microalbuminuria that is associated with a modest increase in blood pressure and subtle changes in lipoproteins. These include minor in- creases in low-density lipoprotein (LDL) cholesterol, in- creases in apolipoprotein B, an increase of the density Key words: diabetes, HMG CoA reductase inhibition, renoprotection, lipid lowering agents, renal injury, atorvastatin therapy by the International Society of Nephrology of LDL, and increases in apoprotein (a) [1]. It has been suggested that hyperlipidemia is an important aggravating factor in the progression of kidney disease. This study explores the role of lipid-lowering treatment in the subtotal nephrectomy (STNx) model, which has many similarities to experimental diabetes, including hyperfil- tration, progressive glomerulosclerosis, and tubulointerstitial injury, as well as changes in growth factor ex- pression, including increased renal transforming growth factor- 1 (TGF- 1) [2]. Furthermore, the possibility that these beneficial effects of lipid-lowering treatment, which have been reported in rodent models of progressive renal injury, are also observed in humans is evalu- ated by reviewing recent clinical data on 3-hydroxy-3- methylglutaryl coenzyme A (HMG CoA) reductase inhibitors in diabetic patients with early and overt DN. METHODS Male Sprague-Dawley rats weighing 200 to 250 g un- derwent 5/6th STNx. As previously described [2], a STNx was performed by right subcapsular nephrectomy and infarction of approximately two thirds of the left kidney by selective ligation of two to three extrarenal branches of the left renal artery. Eight rats underwent sham operation. Following the operation, the rats with STNx were randomly assigned to receive either atorvastatin (20 mg/ kg body wt, N 13) by daily gavage or placebo (N 8) for 12 weeks. After 12 weeks, systolic blood pressure was measured by a plethysmographic tail cuff method. Animals were weighed, and 24-hour urine was collected for the determination of protein excretion. Animals were sacrificed and plasma was collected for urea, creatinine, cholesterol, and triglycerides, which were subsequently measured by an autoanalyzer. The kidneys were removed, weighed, and further processed for in situ hybridization and immunohistochemistry (discussed later in this article). Glomerular filtration rate (GFR) was measured by a single-shot isotopic method using 99m Tc-DTPA [2]. S-31

2 S-32 Jandeleit-Dahm et al: Lipids and diabetic nephropathy Table 1. Functional and structural parameters Sham STNx STNx atorvastatin N SBP mm Hg a a Body weight g a a Kidney weight g a a Urea mmol/liter a a Creatinine mol/liter a 87 4 a Cholesterol mmol/liter a a Triglycerides mmol/liter GFR ml/min a a Proteinuria mg/24 hr a a,b Glomerulosclerotic index a a,b TGF- 1 dpm/mm a a,b Glomerular ED-1 staining positive cells/glomeruli 0.10 ( ) 0.55 ( ) a 0.13 (0 0.48) b Tubulointerstitial ED-1 staining positive cells/field 1.4 ( ) 8.0 ( ) a 2.9 ( ) c Data are shown as mean sem except for ED-1 staining data, which are shown as median (interquartile range). Abbreviations are: STNx, subtotal nephrectomy; N, number; SBP, systolic blood pressure; Body wt, body weight; GFR, glomerular filtration rate; TGF- 1, transforming growth factor- 1. a P 0.01 vs. sham b P c P 0.07 vs. STNx Glomerular injury was assessed by examining 70 glome- placebo-treated rats (Table 1). Systolic blood pressure ruli in periodic acid-schiff stained sections. Each glome- was significantly elevated in both 5/6th nephrectomized rulus was graded in terms of severity, and a glomerulo- groups compared with the sham group, but atorvastatin sclerotic index was calculated as previously described [3]. did not influence blood pressure (Table 1). Remnant The presence of mononuclear leukocytes was demon- kidney weights were significantly higher in both STNx strated immunohistochemically using the indirect avidin- groups, but this parameter was not influenced by atorvasbiotinylated peroxidase method. Sections were incubated tatin treatment. All STNx rats had evidence of renal with a murine monoclonal primary IgG antibody specific impairment, with elevated plasma urea and creatinine for the monocyte/macrophage antigen, ED-1 (Serotec, concentrations, as well as reduced GFR. No parameter Oxford, UK). The immunostaining was quantitated by of renal function was affected by atorvastatin treatment. counting the number of positive cells in 20 glomeruli Total cholesterol was elevated in STNx rats. Atorvastatin and 20 grid fields (0.1 mm 2 ) [4]. In situ hybridization did not affect any lipid parameter. STNx rats had infor TGF- 1 was performed on 4 m thick sections of formalin-fixed, paraffin-embedded tissues as previously creased proteinuria, and this parameter was significantly described [2]. Gene expression for TGF- 1 was analyzed reduced by atorvastatin after 12 weeks of treatment. on autoradiographs from each animal using a Micro STNx was associated with the development of glomeru- Computer Imaging Device (Imaging Research, Ontario, losclerosis, which was attenuated but not prevented by Canada). atorvastatin (Table 1). STNx was associated with increased renal TGF- 1 gene expression, which was atten- Statistics uated but not normalized by atorvastatin therapy (Fig. Data are shown as mean sem except for data not 1 and Table 1). ED-1 staining in both glomeruli and the normally distributed where medians and interquartile tubulointerstitium was increased in STNx rats (Fig. 2). ranges are shown. Analyses were performed by analysis Atorvastatin reduced ED-1 staining in glomeruli with a of variance in the case of normally distributed data or similar trend in the tubulointerstitium, which did not using the Kruskal Wallis test if data were not normally reach statistical significance (Fig. 2 and Table 1). distributed. A P value less than 0.05 was viewed as statistically significant. DISCUSSION This study has confirmed Kasiske et al s previous study RESULTS showing that lipid-lowering treatment in the STNx model After 12 weeks, the body weights of the STNx animals is associated with attenuated development of proteinuria were significantly reduced compared with the shamserved [5]. In this study, no effect on lipid parameters was oboperated animals, but there was no significant difference in these rodents treated with atorvastatin. There- between the body weights of the atorvastatin and fore, the renoprotective effects observed in this study

3 Jandeleit-Dahm et al: Lipids and diabetic nephropathy S-33 Table 2. Effect of HMG CoA reductase inhibitors on albuminuria in diabetic subjects with early and overt renal disease Type of Agent Type of study Duration N diabetes Stage of renal disease Proteinuria Reference Simvastatin/placebo Placebo 12 weeks 14 1 Proteinuria Hommel et al Controlled 12 (1992) [19] Simvastatin/placebo Placebo months 11 1 Microalbuminuria Barnes et al Controlled 12 (1998) [27) Pravastatin/placebo Cross-over 12 weeks active/ 20 1 Microalbuminuria Zhang et al 12 weeks placebo (1995) [20] Lovastatin Uncontrolled 12 weeks 10 1 Proteinuria Biesenbach et al (1992) [21] Simvastatin/placebo Placebo 36 weeks 8 2 Microalbuminuria Nielsen et al Controlled 10 (1993) [22] Pravastatin Uncontrolled 12 weeks 9 2 Proteinuria Sasaki et al (1990) [17] Pravastatin Uncontrolled 12 weeks 12 2 Predominantly ( 50%) Shoji et al proteinuric (1991) [18] Lovastatin/placebo Placebo 2 years 16 2 Proteinuria Lam et al Controlled 18 (1995) [23] ( GFR in placebo group) Simvastatin/placebo Cross-over 1 year active/ 19 2 Microalbuminuria/ ( 25%) Tonolo et al 1 year placebo Normal BP (1997) [26] appear to be independent of the drug s hypolipidemic action. The beneficial effect on proteinuria was associated with reduced renal TGF- 1 expression and less macrophage infiltration in both glomeruli and the tubulointerstitium. Multiple mechanisms for these effects of HMG CoA reductase inhibitors have been described, including those caused by reduction in nonsterol products such as isoprenoids, which are intimately involved in DNA replication [6]. HMG CoA reductase inhibition was associated with reduced macrophage accumulation in this study, as has been reported in other models of renal disease [7]. Macrophages are a major source of proliferative and prosclerotic cytokines [8]. Our own group has documented localization of TGF- 1 within macrophages in the model of STNx and has shown that reduction in TGF- 1 gene expression with inhibition of the reninangiotensin system is associated with reduced macrophage infiltration and local proliferation [8]. Furthermore, the chemokine, monocyte chemoattractant protein-1 (MCP-1), has been suggested to play a pivotal role in mediating lipid-induced nephrotoxicity, and lovastatin reduces gene expression of this protein [7]. Although atorvastatin retarded the development of proteinuria, there was no effect on renal function. This would imply that in the STNx model, the major determinants of progressive decline in GFR are other factors such as systemic hypertension and the status of the local renin-angiotensin system. Atorvastatin did not affect blood pressure and is unlikely to influence the intrarenal renin-angiotensin system directly. The importance of MCP-1 is further suggested from studies by Remuzzi, Ruggenenti, and Benigni, which emphasize the role of this protein in mediating tubular protein-induced renal damage [9]. Although speculative, it is possible that the concomitant effect of atorvastatin on proteinuria and macrophage accumulation may be linked to the capacity of these agents to inhibit MCP-1 gene expression. Although lipid-lowering agents have been shown in a range of experimental models to confer variable degrees of renoprotection, their role in both experimental and clinical diabetes remains uncertain. Indeed, the role of lipids per se in aggravating experimental DN remains controversial. There are reports of cholesterol feeding attenuating, aggravating, or having no effect on experi- mental DN [10 12]. Nevertheless, the potential renoprotective role of HMG CoA reductase inhibitors in human DN has been investigated over the last decade in a range of studies that have, in general, been of relatively short duration and only in a limited number of subjects (Table 2). These lipid-lowering agents are already widely used in diabetes, as this is a state of accelerated atherosclerosis with increased prevalence of a range of lipid abnormalities [13]. Furthermore, lipid-lowering therapy has been shown to be particularly useful in reducing cardiovascular morbidity and mortality in diabetic subjects with moderate hypercholesterolemia and established vascular disease [13]. Hypercholesterolemia has been shown in a number of epidemiological studies to be linked to the progression of nephropathy. For example, in an analysis of 439 type 1 diabetic patients with nephropathy who participated in the Diabetic Retinopathy Study, a rapid decline in renal function was associated not only with elevated blood pressure, but also with increased plasma cholesterol levels [14]. Similar links between plasma cholesterol levels and a decline in renal function have also

4 S-34 Jandeleit-Dahm et al: Lipids and diabetic nephropathy Fig. 1. Autoradiographs are shown for renal transforming growth factor- 1 (TGF- 1) gene expression in sham (A), subtotal nephrectomy (STNx) (B) and STNx atorvastatin groups (C). Publication of this figure in color was made possible by a grant from Parke-Davis and Pfizer.

Jandeleit-Dahm et al: Lipids and diabetic nephropathy S-35 been reported in post hoc analyses of several studies exploring the role of angiotensin-converting enzyme inhibition in type I and type II

5 Jandeleit-Dahm et al: Lipids and diabetic nephropathy S-35 been reported in post hoc analyses of several studies exploring the role of angiotensin-converting enzyme inhibition in type I and type II diabetic patients with early or overt renal disease [15, 16]. However, these studies only describe association, and therefore, intervention studies with lipid-lowering agents are required to test directly the hypothesis that lipid-lowering agents confer renoprotection in the diabetic population. HMG CoA reductase inhibitors have been shown to be effective lipid-lowering agents in both type I and type II diabetic patients, with no evidence that diabetic patients are resistant to the LDL-lowering effects of these agents [13]. However, their role as renoprotective agents remains controversial. Most studies suggest no or only a minimal effect on proteinuria over weeks to months (Table 2). Furthermore, the effects on renal function have not been generally performed or have used insensitive markers such a serum creatinine. The initial studies were uncontrolled and involved pretreatment and posttreatment assessment of the HMG CoA reductase inhibitor pravastatin for 12 weeks [17, 18]. These pilot studies were suggestive of an effect of pravastatin on albuminuria. This was followed by a series of placebo-controlled and cross-over studies with a range of HMG CoA reductase inhibitors, most of these studies showing minimal evidence of a reduction in albuminuria with these agents in type I [19 21] or type II diabetic subjects [22]. In a placebo-controlled study performed in Hong Kong over two years, it was suggested that despite no significant effect on proteinuria, a decline in renal function was attenuated by lovastatin, particularly over the second year of the study [23]. However, these findings have been disputed by several investigators who suggested that the statistical analysis used was inappropriate [24, 25]. Tonolo et al reported in a cross-over study, which included a 12-month active treatment period with simvastatin, an approximate 25% decrease in proteinuria in a group of normotensive, microalbuminuric type II diabetic patients [26]. However, these findings must be interpreted with caution in view of the lack of confirmation of similar findings by other groups [27]. Despite generally positive findings for lipid-lowering agents in experimental models of renal disease, these agents cannot be considered first-line renoprotective agents. Even if these agents are potentially beneficial in human DN, it appears likely that HMG CoA reductase Fig. 2. ED-1 staining is shown in glomeruli and the tubulointerstitium in sham (A), STNx (B), and STNx atorvastatin groups (C) (magnification 400). Publication of this figure in color was made possible by a grant from Parke-Davis and Pfizer.

6 S-36 Jandeleit-Dahm et al: Lipids and diabetic nephropathy inhibitors will only be considered as additional therapy progression of lipid-induced nephrotoxicity in diabetic nephropa- thy. J Diabetes Complications 9: , 1995 to other renoprotective agents such as intensified insulin 12. Cooper ME, Vranes D, Vranes DA, Allen TJ, Panagiotopoulos therapy and angiotensin-converting enzyme inhibitors, S, Komers R, Jerums G: The effects of dietary cholesterol on experimental diabetic nephropathy. Diabetes Res 22: , 1993 their major role being the prevention and retardation of 13. Pyorala K, Pedersen TR, Kjekshus J, Faergeman O, Olsson diabetes-associated macrovascular disease. AG, Thorgeirsson G: Cholesterol lowering with simvastatin improves prognosis of diabetic patients with coronary heart disease: A Reprint requests to Dr. Mark E. Cooper, Department of Medicine, subgroup analysis of the Scandinavian Simvastatin Survival Study Austin and Repatriation Medical Center, Repatriation Campus, W Hei- (4S). Diabetes Care 20: , 1997 delberg, Vic, 3081, Australia. 14. Krolewski AS, Warram JH, Christlieb AR: Hypercholesterol- cooper@austin.unimelb.edu.au emia: A determinant of renal function loss and deaths in IDDM patients with nephropathy. Kidney Int 45(Suppl 45):S125 S131, ACKNOWLEDGMENTS Ravid M, Brosh D, Ravidsafran S, Levy Z, Rachmani R: Main risk factors for nephropathy in type 2 diabetes mellitus are plasma The authors are grateful to Parke-Davis and Pfizer Companies for cholesterol levels, mean blood pressure, and hyperglycemia. Arch underwriting the cost of color figures in this article. Intern Med 158: , Mulec H, Johnsen SA, Wiklund O, Bjorck S: Cholesterol: A renal risk factor in diabetic nephropathy? Am J Kidney Dis 22:196 REFERENCES 201, Jerums G, Allen TJ, Tsalamandris C, Akdeniz A, Sinha A, 17. Sasaki T, Kurata H, Nomura K, Utsunomiya K, Ikeda Y: Amelio- Gilbert R, Cooper ME: Relationship of progressively increasing ration of proteinuria with pravastatin in hypercholesterolemic paalbuminuria to apoprotein(a) and blood pressure in type-2 (non- tients with diabetes mellitus. Jpn J Med 29: , 1990 insulin-dependent) and type-1 (insulin-dependent) diabetic pa- 18. Shoji T, Nishizawa Y, Toyokawa A, Kawagishi T, Okuno Y, tients. Diabetologia 36: , 1993 Morii H: Decreased albuminuria by pravastatin in hyperlipidemic diabetics. (letter) Nephron 59: , Wu L, Cox A, Roe C, Dziadek M, Cooper ME, Gilbert RE: 19. Hommel E, Andersen P, Gall MA, Nielsen F, Jensen B, Rossing Transforming growth factor 1 and renal injury following subtotal P, Dyerberg J, Parving HH: Plasma lipoproteins and renal funcnephrectomy in the rat: Role of the renin-angiotensin system. Kidtion during simvastatin treatment in diabetic nephropathy. 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Kasiske BL, O Donnell MP, Garvis WJ, Keane WF: Pharmacoduring KG, Mogensen CE: Renal function and insulin sensitivity logic treatment of hyperlipidemia reduces glomerular injury in rat simvastatin treatment in type 2 (non-insulin-dependent) 5/6 nephrectomy model of chronic renal failure. Circ Res 62:367 diabetic patients with microalbuminuria. Diabetologia 36: , , O Donnell MP, Kasiske BL, Kim Y, Atluru D, Keane WF: 23. Lam KS, Cheng IK, Janus ED, Pang RW: Cholesterol-lowering Lovastatin inhibits proliferation of rat mesangial cells. J Clin Invest therapy may retard the progression of diabetic nephropathy. Dia- 91:83 87, 1993 betologia 38: , Park YS, Guijarro C, Kim Y, Massy ZA, Kasiske BL, Keane 24. Bender R: The effect of cholesterol-lowering therapy on the pro- WF, O Donnell MP: Lovastatin reduces glomerular macrophage gression of diabetic nephropathy is unproved. 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