1 Renal Service, and 2 Haematology Service, Hospital Universitario, Universidad del Zulia, Medical School and 3 Instituto de Investigaciones Biomédicas (INBIOMED), Maracaibo, Venezuela
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Abstract |
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Methods. Studies were done in 16 patients with homozygous SCA who had normal or supranormal glomerular filtration rate (GFR) and in 20 normal controls of similar median age (23 years). The tubular stress test (TST) consisted of 30-min clearance periods ([125I]iothalamate and creatinine) done before (baseline) and after (three successive post-stimulation periods) the intravenous infusion of 88.4 µmol (10 mg) of creatinine per kg of body weight.
Results. Baseline studies showed that the SCA patients had higher GFR and lower serum creatinine concentration. After stimulation there were no changes in GFR. In contrast, creatinine clearance increased 2.3 times in normal but not in SCA patients (P<0.001) and the TScr in the first post-stimulation period was 161±83 nmol/kg/min in SCA patients vs 286±93.2 in normal controls (P<0.001). The mean TScr post-stimulation was also reduced in patients with SCA (123±52 nmol/kg/min vs 179±50 in normal controls, P<0.001). Since SCA patients had lower Pcr values, separate analysis was made of post-load clearance periods in which Pcr was comparable in patients and in normal controls (range 177265 µmol/l or 23 mg/dl) and the reduction in TScr was also present in SCA patients in these study periods.
Conclusion. Patients with SCA have impaired response to the TST before there are reductions in glomerular filtration. Therefore, a reduction in the tubular secretory reserve capacity represents an early event in the nephropathy of this condition.
Keywords: creatinine secretion; sickle cell anaemia; tubular stress test
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Introduction |
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A group of patients in whom it could be explored if the reserve capacity for creatinine secretion is impaired in hyperfiltering kidneys is the group of patients with sickle cell anaemia (SCA). The patients with homozygous sickle haemoglobin (SS) disease present a variety of renal haemodynamic, functional and structural abnormalities [recently reviewed in 35], but, pertinent to this investigation, these patients have increased glomerular filtration rate (GFR) and renal plasma flow, and reduced serum creatinine levels in childhood and adolescence [67], followed by a subsequent deterioration of renal function that is usually abnormal after the age of 40 [8,9]. When significant proteinuria or azotaemia are present, approximately half of the patients with SCA will go on to end-stage renal disease in less than 2 years [10]; renal failure still accounts for 18% of the deaths in adult patients with SCA [11]. Therefore, the detection of abnormalities in renal function that precede proteinuria and changes in GFR may not only give some insight on the mechanisms of progression of renal damage but also may have clinical importance.
The present study was done to answer a specific question: is the capacity to increase creatinine secretion in response to a creatinine load impaired in patients with SCA who still have a normal or supranormal levels of GFR? To address this issue we evaluated the response to the TST in young patients with SS haemoglobin disease.
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Patients and methods |
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The normal controls were 20 volunteers, median age 23.5 years (range 1843 years) with normal haematological and biochemical blood profiles and normal urine analysis. Fourteen of these normal subjects had been reported as a control group in a previous study of the TST [2]. All individuals studied denied voiding difficulties and complete bladder emptying by voluntary voiding was documented by ultrasound sonography.
For at least 2 weeks prior to the studies, the patients and the controls were not allowed to take medications that modify renal haemodynamics or block the renal response to a protein or creatinine load, such as non-steroidal anti-inflammatory drugs, nor drugs that interfere with the tubular secretion of creatinine, such as trimethoprin sulfamethoxasol or cimetidine, nor drugs that interfere with the Jaffe's reaction, such as cephalosporins or ascorbic acid. The urine of all subjects in the study was strip test negative for ketones.
The protocol of the study was approved by the Ethics Committee of the Department of Medicine.
Tubular stress test (TST)
The patients arrived at 8 am to the Renal Laboratory. The last meal was eaten 12 h before the studies (89 pm the previous night) and after this meal only free water intake was permitted. Diuresis was stimulated by the ingestion of 20 ml/kg body weight of water and maintained by drinking every 30 min the amount eliminated in the previous 30-min period. Upon arrival, 30 µCi of [125I]iothalamate (Gliofil-125, Cypros Pharmaceutical Corp., Carlsbad, CA), in 0.05 ml saline solution with 0.02 ml 1:1000 epinephrine solution was injected subcutaneously in the forearm. As reported previously [2], this resulted in stable serum cpm counts throughout the study. Forearm veins were cannulated and 1 h after the subcutaneous injection of [125I]iothalamate, three 30-min clearance periods were done as baseline (unstimulated, PRE) studies. All urinary collections were done by supervised voluntary voiding. Then, 88.4 µmol of creatinine (Sigma Chemical Co. St. Louis, MO) per kg body weight were dissolved in 100 ml of 5% dextrose and water sterile solution for intravenous use, passed through a single-use millipore filter (sterile acrodisk 0.2 µm, Gelman Sciences) and administered intravenously in 10 min. The creatinine infusion was well tolerated and there were no pyrogenic reactions.
After the creatinine bolus, urinary collections were done, first from time 0 (end of the creatinine infusion) to 15 min and then, in three successive collections periods of 30 min each. Clearance calculations were done in these three collection periods using blood samples that bracketed each urinary collection.
As described previously [2], the initial 15 min after the end of the creatinine infusion are unreliable for clearance studies because during this time there is a steep exponential decrease in serum creatinine (Pcr), but after the initial 15 min the Pcr decreases slowly in almost linear fashion and, as reported previously [2], clearance calculations using the averaged Pcr(a-Pcr)=[Pcri+Pcrf]/2, where i and f are initial and final, respectively, creatinine concentrations of the corresponding clearance period) are practically similar to the calculations using the exponential expression (e-Pcr=exp[(lnPcri+lnPcrf)/2]. Therefore, calculations of GFR, creatinine clearance (Ccr), urinary creatinine excretion rate (UcrV) and tubular secretion of creatinine (TScr) were done in 30-min successive clearance periods 1545 min post-load (Period A), 4575 min post-load (Period B) and 75105 min post-load (Period C). Data was evaluated separately for Period A and for the mean of the three stimulated periods ([A+B+C]/3).
[125I]iothalamate clearance and Ccr were calculated by the standard formula (UxV/Px). [125I]iothalamate clearance was considered equivalent to the GFR. The TScr was estimated by the difference between the UcrV and the filtered creatinine (GFRxPcr): TScr=UcrV-(GFRxPcr).
The clearance of creatinine by secretion is CTScr=TScr/Pcr.
The clearances were corrected by 1.73 m2 surface area. UcrV and TScr were corrected for body weight.
The per cent excreted of the exogenous creatinine load was estimated subtracting the baseline (pre-load) urinary creatinine excretion from the post-load urinary creatinine excretion in the corresponding period: (Stimulated UcrV-Baseline UcrV)x100/creatinine load.
Chemical determinations
I125 radioactivity in plasma and urine was counted with a gamma counter (LKB 1282 Compugamma). Creatinine determinations were done by autoanalyser methodology (Express Ultra Plus, Ciba-Corning) since studies in our laboratories with oral creatinine loads had previously shown that the ratios of true (chromogen free) Ccr/Autoanalyser Ccr were not significantly different from 1 [1].
Statistical calculations
Comparisons between the patient and control groups were done by unpaired analysis of variance (ANOVA) followed by multiple comparisons Tukey-Kramer post-tests. Changes from baseline determinations of Ccr and Cin in SCA patients and in controls were evaluated with repeated measures ANOVA followed with Dunnett post-tests. A commercial statistical package (GraphPad Instat, San Diego, CA) was used for statistical analysis. Data are expressed as mean values±SD. Two-tailed P values <0.05 were considered statistically significant.
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Results |
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The GFR did not change during the TST in SCA patients nor in controls (Figure 1). In contrast, the Ccr increased significantly in SCA patients and in controls with respect to pre-test baseline levels, but the increment in Ccr found in the SCA patients (Period A/Pre=1.17±0.19) is significantly less (P<0.05) than the increment observed in controls (Period A/Pre=1.30±0.14) (Figure 2
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Discussion |
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In agreement with previous studies, our baseline data showed that SCA patients had normal or supranormal levels of GFR and lower than normal Pcr levels (Table 1).
The baseline TScr was lower in the SCA patients than in controls. This finding is contrary to the increased values that are quoted in literature reviews [5,18] and deserve some considerations. To our knowledge, the only study that evaluated the tubular secretion of creatinine (unstimulated) is the study of de Jong et al. [7]. This elegant investigation explored the haemodynamic effects of prostaglandin synthesis inhibition in SCA and found that the baseline tubular secretion of creatinine was increased in these patients. However, the discrepancy in the conclusions regarding the baseline TScr in their work [7] and in this investigation may be due, at least in part, to the method of estimation of creatinine secretion. De Jong et al. [7] evaluated the fractional creatinine excretion [(Ccr/Cin)x100] and their data showed that it was 134% in SCA patients and 108 in controls. We estimated TScr as the difference between urinary creatinine excretion minus filtered creatinine (see methods) and found it decreased in SCA patients. Ccr/Cin ratios in our baseline studies indicate that fractional creatinine excretion in our patients was 118±24% and 124±19% in our control subjects, and this difference is not statistically different. Furthermore, if we compare our baseline Ccr/Cin ratios with those of de Jong et al. [7], we find that the values in SCA patients are not statistically different (de Jong et al. =134±31, present work =118±24, P=0.13), while in contrast, the control group in de Jong et al.'s work [7] had fractional creatinine clearances lower than in the present studies (de Jong et al. =108±14, present work =124±19, P=0.02). Therefore, it appears that when our data is calculated in a similar manner as in de Jong et al.'s, the difference is in the control subjects, rather than in the SCA patients.
Maximal stimulation of the creatinine secretory capacity has been found to be impaired in conditions associated with subclinical reduction in nephron mass and a tubular stress test, consisting in the sequential evaluation of the TScr after a single intravenous bolus of creatinine, has been proposed to test the tubular functional reserve [2]. The clinical usefulness of this test depends on the premise that renal patients have abnormal responses before their Pcr is elevated and, ideally, before their glomerular filtration rate is reduced. Since young SCA patients frequently have supranormal GFR before many of them develop progressive segmental glomerulosclerosis [19], we decided to test if TST would be impaired in these patients.
The administration of a standardized dose of 88.4 µmol of creatinine per kg (TST) given intravenously resulted in similar levels of Pcr in controls and SCA patients at the end of the infusion (Time 0 in Table 2). Nevertheless, the SCA patients had significantly lower Pcr levels than controls in the clearance periods of the TST due to their increased GFR.
Several observations were made with the TST. The first observation was that GFR remained unchanged during the TST at values that were uniformly higher in the SCA patients than in controls (Figure 1). Reasonably stable GFR levels during the TST were expected from the initial description of the test [2]. In contrast to the GFR findings, a clear difference existed in the response of Ccr to the TST (Figure 2
): while a supranormal Ccr increased slightly in the SCA patients, the normal controls increased sharply the Ccr during period A, at which time their Ccr reached levels that were similar to those of the SCA patients (Table 2
and Figure 2
) and, subsequently, decreased towards pre-stimulation values.
The principal parameter explored with the TST is the TScr. As shown in the serial values of TScr in Figure 3 and in the summarized data in Table 2
, the patients with SCA had about 40% reduction in their creatinine secretory rate in Period A with respect to the normal response. A reduction of the TScr in the TST could be due to decreased tubular reserve capacity or to insufficient stimulation of TScr during the TST. While it is conceivable that maximal tubular secretion of creatinine in SCA nephropathy would require higher levels of Pcr or longer periods of creatinine administration, the data obtained in normal subjects and in subjects with reduced renal mass indicates that maximal Ucr/Pcr ratios are associated with a bolus of intravenous creatinine that produces, at the end of the infusion, Pcr values of 500700 µmol/l [2]. In fact, Pcr levels at the end of the creatinine infusion (time 0, Table 2
) were in the desired range. Nevertheless, the subsequent reduction of Pcr was more pronounced in SCA patients (period A and mean post-stimulation values in Table 2
), likely resulting from their higher filtration rate. Therefore, the possibility of insufficient stimulation of creatinine secretion has to be considered in SCA patients. It should be realized that while the Pcr are lower in SCA patients in Period A (Table 2
), the increment with respect to baseline values (Period A/Baseline) is similar in patients and controls (SCA patients =3.15±0.75, controls 3.07±0.57). Nevertheless, it is possible that the absolute level of Pcr, rather than its increment, is the determining stimulus for the secretion of creatinine. Therefore, to evaluate the TScr at comparable levels of Pcr, we analysed the individual clearance periods in which both the SCA patients and the normal controls had Pcr in a similar range: 176.8256.2 µmol/l (23 mg/dl). These data are shown in Figures 5A
and 5B
, and demonstrate that in this analysis the TScr and the CTScr were also significantly reduced in SCA patients. In addition, tubular secretory response was not related to the GFR levels in early SCA nephropathy since patients with GFR >120 ml/min did not have significantly different stimulated TScr than patients with lower GFR values.
The cause of impaired TST in SCA cannot be defined from the present studies. The nephropathy of SCA is a complex condition in which certain tubular functions, such as negative water clearance, potassium secretion and urinary acidification are impaired and other tubular functions, such as renal plasma flow and uric acid secretion are augmented. It must be emphasized that our findings concern specifically the capacity of the tubule to respond to creatinine stimulation, rather than to the unstimulated status of tubular creatinine secretion. In this sense, a TST is more alike to a water deprivation test that uncovers impairment in the capacity to maximally concentrate the urine. Such a defect is also an early characteristic of SCA nephropathy and was in fact demonstrated in our patients who had a mean urinary osmolarity of 427 mOsm/l after 12-h dehydration. Recent investigations indicate that repeated periods of ischaemia are associated with local inflammatory reaction in tubulointerstitial areas in the transgenic sickle cell mice [20]. If similar episodes occur in the human they could conceivably affect earlier the capacity to respond to stimulation than the baseline function.
In conclusion, the TST demonstrates that young SCA patients with normal or supranormal GFR have a reduced capacity to increase their TScr. A diminished tubular secretory functional reserve represents an early event in the nephropathy of patients with haemoglobin SS disease. While it is temping to consider that abnormal TST could predict subsequent deterioration of glomerular filtration in the subset of SCA patients that develop end-stage renal disease, there is no evidence at the present time to support this possibility.
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Acknowledgments |
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Notes |
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References |
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