We describe the case of a 60-year-old Caucasian woman with a positive familial history for dyslipidemia and nephropathy.
The patient was first hospitalized in February 2001 with: hypertension (160/100 mmHg), high lipidic values (cholesterol 372 mg/dl, LDL cholesterol 267 mg/dl, triglycerides 239 mg/dl), creatininemia within the standard limits (0.9 mg/dl), a corresponding GFR of 63 ml/min calculated according to Cockroft and Gault, albuminemia of 3.15 gr/dl, total proteins of 5.7 g/dl, and a micro-hematuria, proteinuria (200 mg/dl) and proteinuria and/or albuminuria ratio of 2 g/g. The main immunological studies, ASO, rheumatoid factor, C3-C4, VDRL, serum immuno-fixation and serum immunoglobulin were normal. The serum markers for HBV, HCV and HIV were negative. The leucocyte formula was normal.
A physical examination ruled out the presence of xanthelasma and corneal arc in the absence of edemas. An ultrasound revealed kidneys which were regular in dimension and structure.
A renal biopsy showed the presence of lipid deposits in the glomerular lumens under the optical microscope after staining with Red-O oil. The interstitium showed a diffuse fibrous edema and tubular atrophy in the areas of fibrosis with a moderate infiltration of inflammatory cells. An arteriole showed hyaline deposits. An immunofluorescence revealed the presence of deposits of IgM and C3 in the sub endothelial seat. An electronic ultrastructural analysis confirmed the presence of thrombi in the lumen of the glomerular capillary loops. The histopathological diagnosis suspected LPG, but ApoE typing identified the polymorphism (E2/2) with a diagnosis of familial type III dyslipoproteinemia.
The patient started treatment with atorvastatin 40 mg/day. We did not use fibrates in association with statin to avoid possible side effects. After one year of therapy we observed a good control of lipid values (cholesterol 250 mg/dl, LDL cholesterol 114 mg/dl) and a reduction of the proteinuria (120 mg/dl). Hypertension was controlled (145/90 mmHg) by means of ACE-inhibitors (irbesartan 300 mg/day, ramipril 5 mg/day), beta-blockers (carvedilole 25 mg/day) and Ca antagonists (nifedipine 60 mg/day).
Four years later in April 2005, the patient was again hospitalized for clinically overt nephrotic syndrome with proteinuria (5 g/24 h) and renal failure (creatinine 2.2 mg/dl). Arterial hypertension (180/100 mmHg) was hardly responsive to treatment. The patient had a low response (cholesterolemia 445 mg/dl, LDL 318 mg/dl, triglycerides 292 mg/dl) to lipid-lowering drugs (atorvastatin 40 mg/day + cholestyramine 4 g/day + Omega3 fatty acids 2 g/day) with the presence of periocular thelasmas.
A renal biopsy revealed the presence of lipids with a fibrous growth of the interstitium and a percentage increase of sclerotic glomeruli with a diagnosis of LPG evolved into nephrotic syndrome (histology will be published in a manuscript in preparation and when published the reference will be provided).
In view of the patient's poor response to pharmacological treatment alone, in July 2005 we decided to associate for the first time in a patient with LPG an LDL-Apheresis protocol (three aphereses in the first two weeks, then one weekly session for two months followed by one apheresis every 15 days) with dextran sulfate columns on the basis of the evidence present in literature for the treatment of the steroid-resistant nephrotic syndrome. [4–6]
During the first LDL-apheresis (July 2005) with dextran sulphate columns (Liposorber System - Kaneka™), an anaphylactoid reaction occurred. This complication, as described by Olbricht in 1992 in patients undergoing treatment with ACE inhibitors and an LDL-apheresis regimen with dextran sulphate columns, is due to the inhibition of kininases that metabolize bradikynin [7].
To avoid stopping the anti-hypertension treatment with ACE-inhibitors we decided to use another LDL-apheresis procedure: the extracorporeal precipitation of LDL-cholesterol induced by heparin in acid pH (HELP System, B. Braun Avitum™ Melsungen Germany). This procedure does not involve complications linked to the use of ACE-inhibitors, following the same protocol previously described.
The effects of a single HELP-apheresis on vascular homeostasis is a simultaneous, drastic reduction within two hours of cholesterol (-52%), LDL-cholesterol (-56%), VLDL-cholesterol (-52%), oxLDL (-47%), Lp(a) (-55%), triglycerides (-50%), fibrinogen (-56%), thrombin (-55%), von Willebrand factor (-56%), factor V (-57%), factor VII (-35%), PCR (-56%), plasma viscosity (-14%), erythrocyte aggregability (-60%), and thrombocyte aggregability (-60%). Also reported in the long-term treatment was an increase of HDL-cholesterol (+14%), peripheral muscle oxygenation (+33-50%), coronary flow reserve (+14%), and cerebral CO2 reactivity (+14%) [8].
In the LDL/fibrinogen apheresis procedure (HELP-apheresis), the plasma is obtained by filtration of the whole blood through a 0.55 μm pore-size filter and then mixed continuously in a 1:1 ratio with a solution of a 0.2 M sodium acetate buffer (pH 4.85) containing 100 IU/ml (300,000 UI) of heparin. The pH of the plasma-buffer solution reaches 5.12 and, at this value, the heparin binds the LDL-cholesterol, Lp(a), fibrinogen and triglycerides forming aggregates. These aggregates precipitate and are retained by the precipitate filter. Precipitate filter is a 0.4 μm pore-size polycarbonate filter from which plasma free from LDL-cholesterol, Lp(a) and fibrinogen is obtained and then passed through an anion exchange filter (heparin absorber) to adsorb excess heparin. The last stage of the system (dialysis filter) restores the physiological pH of the plasma and the balance of the liquids, removing excess fluid by ultrafiltration. The liquid used for dialysis is a sterile solution with a bicarbonate concentration of 35 mmol/l. After the dialysis process, the plasma, purified of lipids and fibrinogen, is returned to the patient mixed with the haematic cell components.
We processed 3000 ml of plasma. That means about 1.4 patient plasma volumes in two hours, with HELP machine (Plasmat Futura®, B. Braun Avitum, Melsungen Germany) and a disposable kit. There were no side effects with the HELP. method, except that the patient had problematic vascular accesses leading to flow problems, So, it became necessary to modify some software parameters (most notably the "PA minimum" threshold; software version 2.06.01) to be able to use a 17G fistula needle for the blood withdrawal and a 20G cannula needle for the blood reinfusion during the procedure. With this configuration, the flows were relatively moderate for an LDL-apheresis (blood flow of 50 ml/min and plasma flow of 20 ml/min), to complete the treatment target of 3000 ml. After some procedures we proceeded with the use of an 18G fistula needle in re-entry, maintaining a 17G fistula needle for drawing, obtaining flows that were definitely higher (blood flow of 80-100 ml/min and plasma flows of 25-30 ml/min). By increasing the flows, there was a marked reduction in the duration of the individual procedure, super-imposable to that of a patient with good vascular accesses.
After two months of treatment with LDL-apheresis (10 procedures), the laboratory data showed a progressive increase of albuminemia (from 3.39 to 3.70 g/dl) with partial remission of proteinuria (from 3.3 to 2 expressed as urine protein to creatinine ratio. A ratio of 0.1 is normal - protein and creatinine are expressed in mg/dl); and a progressive decline in creatininemia (from 1.9 to 1.6 mg/dl). The patient's anemia was improving and her arterial pressure was well under control with a reduction of the pharmacological dose, compared to two months earlier. There was also a significant drop in the mean values of TG (from 285 to 231 mg/dl) and LDL-Cholesterol (from 178 to 158 mg/dl) with reduction of xanthelasma. Pre-apheresis fibrinogen and Lp(a) mean values did not show a significant reduction. Considering the patient's clinical condition improvement, we decided to reduce the statins dosage (from 40 to 20 mg/die) continuing LDL-apheresis protocol of one session every two weeks.
Four months after aphaeretic treatment (15 procedures), the patient's albuminemia values were maintained (3.70 g/dl), her creatinine (1.3 mg/dl) and proteinuria (ratio 1.3) were reduced and her arterial pressure was stabilized (120-80 mmHg). There was a further reduction of LDL-cholesterol (105 mg/dl) and TG (134 mg/dl) mean values, with a suspension of cholestyramine and Omega-3 fatty acids treatment and a further reduction of the xanthelasmas. The LDL-Apheresis session was reduced to one session every three weeks.
After ten months (25 procedures) the laboratory data were as follows: creatinine 1.6 mg/dl, albuminemia 4.30 g/dl, proteinuria (ratio 0.13), LDL-cholesterol 106 mg/dl and TG 154 mg/dl. The values of Lp(a) and fibrinogen are unchanged (Figure 1, 2).
After two years of follow up, with a maintenance treatment of one LDL-apheresis session every three weeks in the last period, the mean biochemical values were: cholesterol 199 mg/dl, LDL-cholesterol 110 mg/dl, HDL-cholesterol 76 mg/dl, TG 112 mg/dl, fibrinogen 381 mg/dl, creatininemia 1.5 mg/dl, Proteinuria ratio 0.9, arterial pressure 120-80 mmHg. The pharmacological therapy is unchanged.
The sequence of the ApoE gene made in 2007 showed that the patient was homozygous for epsilon-2 allele (polymorphism E2/2 with Cys112 and Cys158 in the mature protein) and heterozygous for a novel mutation in exon 4: c.502 C>T [Arg 150>Cys of mature protein]. The mutant Apo E (ApoEMODENA) expresses a new residue of Cys in place of an Arg. This new cysteine residue could form a disulphide bridge with the other Cys residue of the E2 isoform resulting in ApoE polymerization (dominant negative effect). This phenomenon is probably the cause of both dyslipidemia and lipid thrombi in the glomerular capillaries [9].
