A MULTIDRUG, ANTIPROTEINURIC APPROACH TO ALPORT SYNDROME:
A TEN-YEAR COHORT STUDY
SUPPLEMENTAL MATERIAL
Treatment Period
MONTH-1: ACE-INHIBITOR THERAPY – Eligible patients were given 10 mg/day of benazepril. If
treatment was well tolerated (see safety evaluations), one week later the dose was up-titrated to 20
mg/day and then maintained up to completion of the treatment period (Figure 1).
MONTH 2: ARB ADD-ON THERAPY – Patients were given 80 mg of valsartan. If treatment was well
tolerated, one week later the dose was up-titrated to 160 mg/day and then maintained up to completion
of the treatment period.
MONTH 3:
I. NDCCB ADD-ON THERAPY – If systolic/diastolic BP was >120/80 mmHg, the heart rate was
>50/min and PQ<20 msec, patients were given 60 mg/day of diltiazem. If treatment was well
tolerated, the dose was up-titrated to 120 mg/day and eventually to 180 mg/day and then
maintained up to completion of the treatment period.
II. HMGCOA REDUCTASE INHIBITOR ADD-ON THERAPY – Patients not receiving diltiazem therapy were
given 40 mg/day of fluvastatin. If ten days later liver transaminases and creatinphosphokynase
serum levels were in normal range, the dose was up-titrated to 80 mg/day. In case of liver,
muscular or renal toxicity, fluvastatin was back-titrated to 40 mg/day or withdrawn as deemed
appropriate.
MONTH 4: HMGCOA REDUCTASE INHIBITOR ADD-ON THERAPY – Fluvastatin was introduced also to
patients receiving diltiazem.
Recovery period
At completion of the 4-month treatment period, all study treatments (benazepril, valsartan, diltiazem
and fluvastatin) were withdrawn and patients were actively followed up to completion of the onemonth Recovery period (Figure 1).
Extension
After the recovery period, - patients were actively followed up until July, 2014 or until progression to
an endpoint (ESRD or last available visit for those prematurely withdrawing from the study) by 3-6
monthly evaluations of. blood pressure, routine laboratory tests including safety parameters, serum
lipids, urinary albumin excretion and spot urine A/C ratio (Table 1). During this long-term observation
period all of them were maintained on the same treatment regimen that during the Treatment Period
was associated with the largest antiproteinuric effect (Month 4, Figure 1).
Measurements
Blood pressure was recorded as the mean of 3 values taken 2 min apart, after 5-min rest in the
sitting position, on the same (dominant) arm by a standard sphygmomanometer. Albuminuria
was measured in three consecutive overnight urine specimens collected during the three days
prior the day visit. The average value of the three collections was considered for the analyses.
Serum creatinine was measured using a modified Jaffe’s method.
CLEARANCE
STUDIES
- Before treatment initiation (month 0), at the end of the Treatment
(month 4) and Recovery (month 5) periods, patients were admitted at the Department of
Renal Medicine of the Clinical Research Center to have their GFR and RPF measured by the
mean of three consecutive inulin and para-aminohippuric acid (PAH) renal clearances [1],
respectively. Inulin and PAH concentrations in plasma and urine samples were determined
using previously described colorimetric assays [2,3]. Clearance data were adjusted per 1.73
2
m2 of body surface area. Filtration fraction (FF) and renal vascular resistances (RVR) were
calculated by standard formulas:
FF = GFR/RPFx100; RVR = MAP/RPF
To account for possible hematocrit changes associated with RAS inhibitor therapy, we also
calculated the effective renal blood flow (eRBF) and the RVR as a function of eRBF (eRVR)
by the following formulas:
eRBF = RPF/ (1-hematocrit); eRVR = MAP/eRBF.
SERUM AND URINARY PROTEIN MEASUREMENTS - Albumin and IgG concentrations in plasma and
urine samples were determined using a sensitive enzyme-linked immunosorbent assay
(ELISA). Briefly, diluted samples were placed in polystyrene wells coated with antibodies to
human albumin (Sigma Chimica, Milan, Italy), IgG or IgM (Boehringer Mannheim, Milan,
Italy). Goat anti-human albumin (American Qualex, La Miranda, CA) or IgG (Sigma
Chimica) conjugated with alkaline phosphatase were added, followed by alkaline phosphatase
substrate (Sigma Chimica), and optical density of wells read using a micro-ELISA reader.
Fractional clearances of albumin-IgG were computed as:
JAlb or IgG = (U/P) Alb or IgG/(U/P)IN
Where (U/P) Alb or IgG and (U/P)IN are the urine-to-plasma concentration ratios of Albumin
or IgG and inulin respectively. Other laboratory measurements were performed using routine
laboratory techniques.
Safety Evaluations and Concomitant Medications
Blood pressure, serum potassium and creatinine and venous pH were measured within 7-10 days after
first benazepril or valsartan administration and after each drug up-titration. Additional measurements
were performed whenever deemed clinically appropriate. In case of symptomatic hypotension,
hyperkalemia (serum potassium ≥5.5 mEq/L despite adequate correction of metabolic acidosis) or
serum creatinine increase by more than 30% after any benazepril or valsartan up-titration, the dose of
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the drug was back-titrated to the previous step or withdrawn. Throughout the whole treatment period,
treatment was aimed at achieving and maintaining systolic and diastolic BP <120 and <80 mmHg,
respectively. Concomitant BP lowering treatment with medications that do not directly affect
glomerular sieving function (i.e., carvedilol, clonidine, doxazosine or diuretics) and therefore were not
expected to interfere with the primary efficacy variable of the study was allowed. Treatment could be
back-titrated or withdrawn to avoid symptomatic hypotension during the Remission Clinic treatment
period and could be up-titrated to avoid BP values exceeding the safety threshold of 130/90 mmHg
during the Run-in and Recovery periods, respectively.
References
1. Campbell R, Sangalli F, Perticucci E, Aros C, Viscarra C, Perna A, Remuzzi A, Bertocchi
F, Fagiani L, Remuzzi G, Ruggenenti P: Effects of combined ACE inhibitor and
angiotensin II antagonist treatment in human chronic nephropathies. Kidney Int
2003;63: 1094-1103.
2. Perico N, Remuzzi A, Sangalli F, Azzollini N, Mister M, Ruggenenti P, Remuzzi G: The
antiproteinuric effect of angiotensin antagonism in human IgA nephropathy is
potentiated by indomethacin. J Am Soc Nephrol 1998;9: 2308-2317.
3. Remuzzi A, Perticucci E, Ruggenenti P, Mosconi L, Limonta M, Remuzzi G: Angiotensin
converting enzyme
inhibition
improves
glomerular
size-selectivity in
IgA
nephropathy. Kidney Int 1991;39: 1267-1273.
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