*3.2.2. Delayed in vivo catabolism of LDL and IDL in HD patients as potential cause of premature atherosclerosis*

For better understanding the atherogeneity of apoB-containing lipoproteins in HD patients and to resolve the apparent discrepancy between their obviously impaired lipoprotein metabolism and e.g. normal LDL plasma concentrations, we studied the in vivo kinetics of VLDL, IDL and LDL by stable isotope technology in HD patients and compared them to those of healthy controls [12].

This study demonstrated for the first time severely decreased FCRs of IDL- and LDL-apoB in HD patients as compared to controls (Figure 4), whereas the in vivo kinetics of VLDL did not change significantly. A decreased FCR of IDL- and LDL-apoB is identical to a prolonged RT of these highly atherogenic particles. The longer RT of these lipoproteins results in an extended exposure to oxidation for IDL and LDL in a highly oxidative environment. This is in line with experimental data showing a highly significant correlation of 5-hydroxy-2 aminovaleric acid (HAVA) in LDL, an oxidation product of apoB, with LDL RT in normolipidemic controls [99]. In accordance with these results, two previously conducted randomized placebo-controlled studies revealed a significant reduction in composite cardiovascular disease endpoints when HD patients were treated for two years with supplementation of antioxidants such as vitamin E [100] or acetylcysteine [101].

Most remarkably, the observed impaired metabolism of apoB-containing lipoproteins is accompanied by normal concentrations of LDL-apoB and elevated levels of IDL-apoB (Figure 4), in line with previous reports which found increased concentrations of IDL as an independent risk factor for atherosclerosis in HD patients [102]. A closer look at the kinetic data reveals that the normal concentrations of LDL are the result of a combination of decreased FCR and PR. This pattern therefore demonstrates convincingly the strength of kinetic studies in contrast to simply quantifying blood concentrations of a target marker such as LDL concentrations. Its normal concentrations are masked by two metabolic disorders which neutralise each other and result in normal values such as observed in the

general population. The altered lipoprotein metabolism therefore puts HD patients at high risk for developing atherosclerotic disease despite their normal total and LDL cholesterol concentrations. Since most lipid-lowering drugs act by "normalising" the RT of the major atherogenic lipoproteins IDL and LDL [103], these drugs are expected to correct some of the basic defects of the severely disturbed lipoprotein metabolism in HD patients. Therefore, kinetic studies on the impact of lipid-lowering medication on the lipoprotein metabolism in CKD patients were a logic consequence of the observed, above-described findings (see chapter 3.2.5.).

**Figure 4.** Kinetic parameters of apoB in LDL, IDL and Lp(a) and apo(a) in Lp(a). Plasma concentrations, production rates (PR), fractional catabolic rates (FCR) and residence times (RT) are given for healthy controls (green columns) and HD patients (red columns). Columns represent mean values ± SD. Results for LDL and IDL are taken from Ikewaki et al. [12], those for Lp(a) from Frischmann et al. [44].

Due to the laborious nature of these studies and the complexity of the metabolic modeling, only few studies have been performed so far in CKD patients either by radiotracer or stable isotope technology. Our kinetic data seem to contrast with a previously published turnover study in Finnish HD patients performed with conventional radiotracer techniques. While the authors found decreased LDL clearance rates in predialysis CKD patients [104] they could not find a significant difference in LDL-apoB FCR between HD patients and controls [105]. More recently, Prinsen et al., by using stable isotopes, found unchanged FCRs for LDL-apoB in CKD patients treated with peritoneal dialysis [106]. Chan et al. injected radiolabeled VLDL into HD patients with or without hyperlipidemia and found decreased FCRs of VLDL-apoB and IDL-apoB (the latter only in hyperlipidemic patients) [107]. LDL kinetics were not investigated in this study. The reason for these discrepancies is not clear. There might be ethnic differences in the lipoprotein metabolism between the investigated patient populations of different ethnic origin. One major difference between our and the Finnish study is an age difference between patients and controls in our but not in the Finnish study. Our control subjects were considerably younger than the HD patients (35 vs. 51 years). At first glance, this age difference might explain to some extent the dramatic differences found in our study, since LDL clearance rates have been repeatedly described to decrease with age presumably due to down-regulated hepatic LDL receptor expression in the elderly [108, 109]. Based on the results of these studies, an age difference of 15 years (as observed in our work) would result in an approximately 10% change in FCR values and could therefore not explain the more than two-fold difference in our study. The observed differences in kinetic parameters can therefore not be explained by age differences between study groups.

Several mechanisms may contribute to our observations. First, the diminished LDL catabolism in HD patients might be explained by a possible contribution of LDL uptake by the healthy human kidney which does not function appropriately (or at all) in chronic kidney failure. In fact, glomerular cells like mesangial or epithelial cells have been shown *in vitro* to express lipoprotein receptors and to take up LDL comparably to fibroblasts and hepatocytes [110]. It is, however, completely unclear whether the kidney plays a significant role in LDL catabolism *in vivo*. Perfusion studies in rat kidneys indicate that virtually no intact LDL is cleared from the circulation by the kidney [111]. Second, an impaired lipolytic cascade in HD patients most likely also contributes to our results. The relatively normal VLDL concentrations and kinetic parameters and the correspondingly impaired IDL parameters are in good accordance with previous findings of normal lipoprotein lipase (LPL) but significantly decreased activities of hepatic triglyceride lipase (HL) in HD patients [59]. Since HL promotes the conversion of IDL to LDL, a decrease in HL activity might contribute to the accumulation of IDL and reduced production rates of LDL (without accumulating small, dense LDL) in HD patients.

#### *3.2.3. Kinetics of Lp(a) in hemodialysis patients*

32 Lipoproteins – Role in Health and Diseases

chapter 3.2.5.).

general population. The altered lipoprotein metabolism therefore puts HD patients at high risk for developing atherosclerotic disease despite their normal total and LDL cholesterol concentrations. Since most lipid-lowering drugs act by "normalising" the RT of the major atherogenic lipoproteins IDL and LDL [103], these drugs are expected to correct some of the basic defects of the severely disturbed lipoprotein metabolism in HD patients. Therefore, kinetic studies on the impact of lipid-lowering medication on the lipoprotein metabolism in CKD patients were a logic consequence of the observed, above-described findings (see

**Figure 4.** Kinetic parameters of apoB in LDL, IDL and Lp(a) and apo(a) in Lp(a). Plasma concentrations, production rates (PR), fractional catabolic rates (FCR) and residence times (RT) are given for healthy controls (green columns) and HD patients (red columns). Columns represent mean values ± SD. Results

Due to the laborious nature of these studies and the complexity of the metabolic modeling, only few studies have been performed so far in CKD patients either by radiotracer or stable isotope technology. Our kinetic data seem to contrast with a previously published turnover study in Finnish HD patients performed with conventional radiotracer techniques. While

for LDL and IDL are taken from Ikewaki et al. [12], those for Lp(a) from Frischmann et al. [44].

We previously performed *in vivo* kinetic studies using stable-isotope techniques to elucidate the mechanism for increased plasma Lp(a) concentrations in HD patients [44]. PRs of apo(a) and apoB, the two apolipoproteins contained in Lp(a), were normal, when compared to control subjects with similar plasma Lp(a) concentrations (Figure 4). The FCR of these apolipoproteins was, however, significantly reduced compared to controls resulting in a much longer plasma RT for apo(a) of almost 9 days, compared to only 4.4 days in controls. Since the PR of Lp(a) did not differ between HD patients and controls, its decreased

clearance in HD patients leads to increased Lp(a) plasma concentrations and is likely the result of loss in kidney function [44]. A role of the kidney in the catabolism has been previously supported by the observation of renovascular arteriovenous differences in Lp(a) concentrations [112] as well as apo(a) fragments in urine [113, 114].

Comparing kinetic data in HD patients [44] with those in patients with nephrotic syndrome [69] points to fundamental differences in the metabolism of Lp(a) and other proteins between these two patient groups. Patients with nephrotic syndrome do not differ with respect to the FCR of Lp(a) compared to controls but have increased Lp(a) PRs [69]. It is well known that nephrotic patients show a generally increased lipoprotein synthesis of lipoproteins [115]. Since kidney function is relatively well preserved in nephrotic syndrome, a decreased clearance of Lp(a) in these patients is not likely to be expected. Metabolic differences between nephrotic and dialysis patients are not only evident for Lp(a) but also for albumin. Whereas the FCR of albumin in HD patients is similar or even reduced compared to controls, the FCR in patients with nephrotic syndrome is increased [116, 117].

#### *3.2.4. Consequences of the impaired metabolism of atherogenic lipoproteins in HD patients*

The observation of markedly decreased FCRs of apoB of LDL and IDL as well as apo(a) and apoB in Lp(a) causes a prolonged RT of these highly atherogenic lipoproteins. Due to the long retention period, "aged" lipoprotein complexes are thus more susceptible for alterations such as oxidation damage, which was shown to be associated with accelerated atherogenesis in HD patients [118]. Previous kinetic studies investigated the metabolism of the two LDL subclasses, "buoyant" LDL1 and the smaller cholesterol-poor "dense" LDL2, in subjects with familial defective apoB-100 (FDB). The authors found a more than four-fold longer RT for small dense LDL2 in those patients as compared to normolipidemic controls [99]. It was therefore suggested that oxidative damage of an "aged" LDL2, which is present in large concentrations in both blood and the subendothelial space, may be an important mechanism for the development of premature atherosclerosis in patients with familial defective apoB-100. Since the LDL-like particle of Lp(a) is compositionally similar to LDL2 [41], it is tempting to speculate that the increased RT of circulating Lp(a) might pose an additional risk factor for the increased incidence of cardiovascular disease in HD patients.

#### *3.2.5. Influence of statin treatment on kinetic parameters in hemodialysed patients*

In the general population, therapy with HMG-CoA-reductase inhibitors (statins) which inhibit endogenous cholesterol biosynthesis has shown to improve outcome in several atherosclerotic diseases [119, 120]. The inhibition of cholesterol biosynthesis subsequently leads to up-regulation of LDL receptors and therefore increased clearance and thus reduced RT of circulating LDL [103]. Statins also have a beneficial role as antiinflammatory agents, which is independent of their lipid-lowering effect. Inflammation is highly prevalent in patients with CKD and is consistently associated with cardiovascular morbidity and mortality. In line with this metabolic background, the first studies in HD patients demonstrated a substantial normalisation of the dyslipidemic plasma profile and reduced progression of renal disease [121, 122] and in one study also reduced mortality [123] in these patients.

34 Lipoproteins – Role in Health and Diseases

clearance in HD patients leads to increased Lp(a) plasma concentrations and is likely the result of loss in kidney function [44]. A role of the kidney in the catabolism has been previously supported by the observation of renovascular arteriovenous differences in Lp(a)

Comparing kinetic data in HD patients [44] with those in patients with nephrotic syndrome [69] points to fundamental differences in the metabolism of Lp(a) and other proteins between these two patient groups. Patients with nephrotic syndrome do not differ with respect to the FCR of Lp(a) compared to controls but have increased Lp(a) PRs [69]. It is well known that nephrotic patients show a generally increased lipoprotein synthesis of lipoproteins [115]. Since kidney function is relatively well preserved in nephrotic syndrome, a decreased clearance of Lp(a) in these patients is not likely to be expected. Metabolic differences between nephrotic and dialysis patients are not only evident for Lp(a) but also for albumin. Whereas the FCR of albumin in HD patients is similar or even reduced compared to controls, the FCR in patients with nephrotic syndrome is increased [116, 117].

*3.2.4. Consequences of the impaired metabolism of atherogenic lipoproteins in HD patients* 

The observation of markedly decreased FCRs of apoB of LDL and IDL as well as apo(a) and apoB in Lp(a) causes a prolonged RT of these highly atherogenic lipoproteins. Due to the long retention period, "aged" lipoprotein complexes are thus more susceptible for alterations such as oxidation damage, which was shown to be associated with accelerated atherogenesis in HD patients [118]. Previous kinetic studies investigated the metabolism of the two LDL subclasses, "buoyant" LDL1 and the smaller cholesterol-poor "dense" LDL2, in subjects with familial defective apoB-100 (FDB). The authors found a more than four-fold longer RT for small dense LDL2 in those patients as compared to normolipidemic controls [99]. It was therefore suggested that oxidative damage of an "aged" LDL2, which is present in large concentrations in both blood and the subendothelial space, may be an important mechanism for the development of premature atherosclerosis in patients with familial defective apoB-100. Since the LDL-like particle of Lp(a) is compositionally similar to LDL2 [41], it is tempting to speculate that the increased RT of circulating Lp(a) might pose an additional risk factor for the increased incidence of cardiovascular disease in HD patients.

*3.2.5. Influence of statin treatment on kinetic parameters in hemodialysed patients* 

In the general population, therapy with HMG-CoA-reductase inhibitors (statins) which inhibit endogenous cholesterol biosynthesis has shown to improve outcome in several atherosclerotic diseases [119, 120]. The inhibition of cholesterol biosynthesis subsequently leads to up-regulation of LDL receptors and therefore increased clearance and thus reduced RT of circulating LDL [103]. Statins also have a beneficial role as antiinflammatory agents, which is independent of their lipid-lowering effect. Inflammation is highly prevalent in patients with CKD and is consistently associated with cardiovascular morbidity and mortality. In line with this metabolic background, the first studies in HD patients demonstrated a substantial normalisation of the dyslipidemic plasma profile and

concentrations [112] as well as apo(a) fragments in urine [113, 114].

In contrast and quite surprisingly, three previously conducted large, randomized, placebocontrolled trials on statin treatment in CKD patients had not led to significant benefits regarding their primary cardiovascular outcome. Two of those studies, the German Diabetes Dialysis (4D) Study and the Study to Evaluate the Use of Rusovastatin in Subjects on Regular Hemodialysis (AURORA) were performed on HD patients, one study, the Assessment of Lescol in Renal Transplantation (ALERT) Study on patients who had undergone kidney transplantation. Their primary endpoints (death of cardiovascular cause, nonfatal myocardial infarction or nonfatal stroke) were virtually unchanged [124-126]. However, there has been a promising risk reduction in the secondary endpoint 'all cardiac events combined' in one study [126]. A simulated study of exactly the same trial using a large historical database with more than 10.000 patients also demonstrated that statin use was associated with some benefit [127]. A comprehensive review of outcome data from the 4D and AURORA trials found no benefit of statin therapy in either the whole study group of HD patients or after stratification for inflammatory marker levels [128]. More recently, another, much larger trial including 9270 patients with chronic kidney disease, the Study of Heart and Renal Protection (SHARP) could show a significant risk reduction in cardiovascular events in a mixed population of patients with kidney disease including 2/3 predialysis and 1/3 HD patients treated with a combination of a statin and ezitimibe. This effect did not differ between HD and predialysis patients [129].

Based on our previous studies on lipoprotein kinetics in HD patients and the abovedescribed conflicting results regarding their cardiovascular risk profile after statin treatment, we examined by stable-isotope technology the in vivo kinetics of apoB-containing particles in HD patients before and after treatment with atorvastatin (Schwaiger et al., unpublished).

In this study we described for the first time effects of HMG-CoA reductase inhibition on apoB metabolism in CKD patients treated with HD. Low-dose atorvastatin, given for three months to six male patients, lowered, as expected, concentrations of VLDL- and LDL-apoB, both accompanied by a significant increase of their FCR, while hepatic production of both apolipoproteins was not altered. This led, as expected, to a lower RT of these atherogenic apoB-containing particles comparable to RT values of healthy subjects with normal kidney function. The observed findings therefore argue for a beneficial effect of statin therapy regarding cardiovascular events in HD patients similar to described for the general population.

To understand why statins have surprisingly failed to reduce cardiovascular events in HD patients, the basic mechanisms underlying the pathophysiology of CVD in CKD must be critically considered. In contrast to the general population, CKD patients suffer, in addition to dyslipidemia, from several further complex comorbid conditions including diabetes mellitus, hypertension, oxidative stress, inflammation, insulin resistance, anemia and disturbances in mineral metabolism. Lipid lowering therapy by statins have the potential to

ameliorate only some but no all of those conditions (see review [130]). Taken together, statin therapy in CKD maybe recommended based on our kinetic studies on apoB-containing lipoproteins, optimally combined with medication to treat atherogenic non-lipid factors in HD patients.
