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 renal 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 ascertained.

METHODS

Male Sprague-Dawley rats weighing 200 to 250 g underwent 5/6th STNx. As previously described², 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 ^99mTc-DTPA². Glomerular injury was assessed by examining 70 glomeruli in periodic acid-Schiff–stained sections. Each glomerulus was graded in terms of severity, and a glomerulosclerotic index was calculated as previously described³.

The presence of mononuclear leukocytes was demonstrated immunohistochemically using the indirect avidin-biotinylated peroxidase method. Sections were incubated with a murine monoclonal primary IgG antibody specific for the monocyte/macrophage antigen, ED-1 (Serotec, Oxford, UK). The immunostaining was quantitated by counting the number of positive cells in 20 glomeruli and 20 grid fields (0.1 mm²)⁴. In situ hybridization for TGF-1 was performed on 4 m thick sections of formalin-fixed, paraffin-embedded tissues as previously described². Gene expression for TGF-1 was analyzed on autoradiographs from each animal using a Micro Computer Imaging Device (Imaging Research, Ontario, Canada).

Statistics

Data are shown as mean SEM except for data not normally distributed where medians and interquartile ranges are shown. Analyses were performed by analysis of variance in the case of normally distributed data or using the Kruskal–Wallis test if data were not normally distributed. A P value less than 0.05 was viewed as statistically significant.

RESULTS

After 12 weeks, the body weights of the STNx animals were significantly reduced compared with the sham-operated animals, but there was no significant difference between the body weights of the atorvastatin and placebo-treated rats Table 1. Systolic blood pressure was significantly elevated in both 5/6th nephrectomized groups compared with the sham group, but atorvastatin did not influence blood pressure Table 1. Remnant kidney weights were significantly higher in both STNx groups, but this parameter was not influenced by atorvastatin treatment. All STNx rats had evidence of renal impairment, with elevated plasma urea and creatinine concentrations, as well as reduced GFR. No parameter of renal function was affected by atorvastatin treatment. Total cholesterol was elevated in STNx rats. Atorvastatin did not affect any lipid parameter. STNx rats had increased proteinuria, and this parameter was significantly reduced by atorvastatin after 12 weeks of treatment. STNx was associated with the development of glomerulosclerosis, which was attenuated but not prevented by atorvastatin Table 1. STNx was associated with increased renal TGF-1 gene expression, which was attenuated but not normalized by atorvastatin therapy ( Figure 1 and Table 1). ED-1 staining in both glomeruli and the tubulointerstitium was increased in STNx rats Figure 2. Atorvastatin reduced ED-1 staining in glomeruli with a similar trend in the tubulointerstitium, which did not reach statistical significance ( Figure 2 and Table 1).

Figure 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.

Full figure and legend (238K)

Figure 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.

Full figure and legend (128K)

Table 1 - Functional and structural parameters.

Full table

DISCUSSION

This study has confirmed Kasiske et al's previous study showing that lipid-lowering treatment in the STNx model is associated with attenuated development of proteinuria⁵. In this study, no effect on lipid parameters was observed in these rodents treated with atorvastatin. Therefore, the renoprotective effects observed in this study 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 replication⁶. HMG CoA reductase inhibition was associated with reduced macrophage accumulation in this study, as has been reported in other models of renal disease⁷. Macrophages are a major source of proliferative and prosclerotic cytokines⁸. 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 renin-angiotensin system is associated with reduced macrophage infiltration and local proliferation⁸. 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 protein⁷.

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 damage⁹. 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 experimental DN^10,11,12. 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.

Table 2 - Effect of HMG CoA reductase inhibitors on albuminuria in diabetic subjects with early and overt renal disease.

Full table

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 abnormalities¹³. 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 disease¹³. 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 levels¹⁴. Similar links between plasma cholesterol levels and a decline in renal function have also 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 agents¹³. 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^19,20,21 or type II diabetic subjects²². 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 study²³. 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 patients²⁶. However, these findings must be interpreted with caution in view of the lack of confirmation of similar findings by other groups²⁷.

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 inhibitors will only be considered as additional therapy to other renoprotective agents such as intensified insulin therapy and angiotensin-converting enzyme inhibitors, their major role being the prevention and retardation of diabetes-associated macrovascular disease.

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Acknowledgments

The authors are grateful to Parke-Davis and Pfizer Companies for underwriting the cost of color figures in this article.