Hypocitraturia as a risk factor for nephrocalcinosis after kidney transplantation
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- Stapenhorst, L., Sassen, R., Beck, B. et al. Pediatr Nephrol (2005) 20: 652. doi:10.1007/s00467-005-1831-y
Calcium-oxalate crystal deposition in kidney transplant biopsy specimen led us to investigate the impact of calcineurin inhibitor treatment on urinary excretion of lithogenic and stone inhibitory substances in 53 children after successful kidney transplantation (KTx) receiving cyclosporine A (CsA) or tacrolimus. We compared the values obtained with those of 12 patients with recurrent nephrotic syndrome under CsA and of 6 patients with Rasmussen encephalitis (RE) under tacrolimus therapy. Renal ultrasound examinations were repeatedly performed. Hypocitraturia was found in 69% of patients, with KTx patients having a significantly lower urinary citrate excretion than those receiving calcineurin inhibitors for other reasons. Secondly, we found hyperoxaluria in 35% of patients, again especially in those after KTx. No significant difference in urinary substances was seen comparing CsA with tacrolimus treatment. Urolithiasis was found in one and calcium-oxalate crystal deposition in biopsy specimen of three KTx patients. Calcineurin inhibitor treatment can lead to significant hypocitraturia, especially in patients after KTx receiving the highest dose of medication. Hyperoxaluria is primarily the result of a removal of significant body oxalate stores, deposited during dialysis, but may not be suspected as a specific side effect of calcineurin inhibitor therapy. Both findings can increase the risk for urolithiasis or nephrocalcinosis.
KeywordsCalcineurin inhibitorsHypocitraturiaHyperoxaluriaNephrolithiasisNephrocalcinosisKidney transplantation
Nephrocalcinosis and kidney stones are rare complications following renal transplantation, with an incidence of 2–5% in adults [1, 2, 3, 4]. The etiology of both is multifactorial, and major risk factors for stone disease and nephrocalcinosis after KTx are renal tubular acidosis (RTA), low urinary pH, recurrent urinary tract infections, ureteral obstructions or reflux, and foreign bodies such as suture material . Whether an imbalance of the urinary excretion and concentrations of lithogenic (e.g., oxalate, calcium) or stone inhibitory substances (e.g., citrate) plays an important role has not yet been adequately examined after kidney transplantation.
Cyclosporine A (CsA) is known to cause hypercalciuria either due to a high bone turnover or to RTA caused by a decrease in urinary ammonium excretion [5, 6, 7]. In RTA intracellular acidosis leads to a higher proximal tubular reabsorption of citrate, resulting in significant hypocitraturia [7, 8]. As citrate secretion is very low, urinary citrate excretion is regulated by the rate of proximal tubular reabsorption [8, 9]. Citrate acts in the tubular lumen by binding to calcium to form a nondissociable but soluble complex, which prevents development of insoluble calcium-oxalate crystals.
We were surprised to see obvious crystal deposition (nephrocalcinosis) in the kidney biopsy specimen of three patients. Microscopic hematuria was present in one patient, while ultrasound was not typical for nephrocalcinosis in any of the patients. This prompted us to investigate the effect of calcineurin inhibitor treatment as potential risk factors for nephrolithiasis and nephrocalcinosis in pediatric patients after successful kidney transplantation. We collected and analyzed 24-h urine specimens for lithogenic and stone inhibitory substances with a specific interest in urinary citrate excretion. Patients with calcineurin inhibitor medication for other conditions such as nephrotic syndrome (NS) and RE served as a control group. The values obtained were also compared to normal levels of urinary constituents, previously determined in an age matched group of 473 healthy boys and girls .
We studied 71 children (46 boys, 25 girls; age range 2–28 years, mean 11.7): 53 after KTx, 6 with Rasmussen encephalitis (RE), and 12 with relapsing NS. Immunosuppressive therapy in the KTx group consisted of corticosteroids (n=53), mycophenolate mofetil (n=43), and CsA (n=36) or tacrolimus (n=17). Four patients were treated with an interleukin-2 inhibitor (basiliximab n=2, daclizumab n=2), four patients had antithymocyte globulin induction treatment, and two received OKT3 at rejection episodes. Patients in the non-KTx group received either tacrolimus (RE) or CsA (NS). CsA through-levels ranged between 100 and 200 µg/l (EIA) in the KTx group and between 50 and 100 µg/l in the NS group. Tacrolimus levels were within 6–10 µg/l (EIA) in all patients. Angiotensin-converting enzyme (ACE) inhibitors were given in 15 of the 53 KTx patients but in none of the non-KTx patients.
At least one 24-h urine was collected in all patients, at a median of 16 months after KTx (1–132 months). The urine was directly voided into sampling bottles, containing 10 ml thymol 5% (in isopropanol) per liter of urine for preservation. An aliquot (50 ml) was later stored at −20°C and analyzed within 1 month. The 24 h urine collections of all patients were analyzed for pH, specific gravity, sodium, potassium, chloride, calcium, oxalate, uric acid, phosphate, sulfate, magnesium and citrate excretion.
Separation and quantification of urinary oxalate was performed using a Dionex Series Dx500 gradient ion chromatography system (Dionex, Sunnyvale, Calif., USA) adopting a method for plasma oxalate determination . Urinary concentrations of calcium and magnesium were determined by atomic absorption spectroscopy, and phosphorus and sulfate by ion chromatography. Citric and uric acid were analyzed enzymatically using the citrate lyase and uricase method, respectively. All measurements of lithogenic and stone inhibitory substances, and urinary creatinine, sodium, chloride, potassium, the 24-h urine volume, pH, and specific gravity were entered in the PC based program EQUIL2 to calculate the model values of the relative urinary supersaturation in respect to the salts calcium-oxalate (βCaOx) brushite and uric acid . The values obtained were compared with normal values previously determined in 473 healthy boys and girls from our region . Normal 24-h urinary excretion values were defined as follows: oxalate, less than 0.5 mmol/1.73 m2 body surface area; citrate, more than 1.6 mmol/1.73 m2 in girls and more than 1.9 in boys; uric acid, 0.12 mmol/kg body weight or less; and calcium 4 mg/kg body weight or less. The normal relative urinary saturation levels in children aged 5–15 years were defined as: calcium-oxalate less than 5.5 (girls) and 6.3 (boys), and brushite less than 1 and uric acid less than 2.
All patients underwent routine serial ultrasound investigations of the kidney by two experienced pediatric radiologists in our department. Renal ultrasonography was performed using a real time sector scanner with a 5- or 7.5-MHz transducer (Accuson Computed Sonography, 128XP/10, Mountain View, Calif., USA). Statistical analysis were performed using SPSS software (Chicago, Ill., USA). Analysis of variance and the Mann-Whitney test were used to evaluate differences between the groups and P values less than 0.05 were considered significant. Data are presented as mean ±standard deviation or median with range, as appropriate. Informed consent from all patients or parents was obtained prior to the urine collections.
Urinary excretion of lithogenic and stone inhibitory parameters, urinary pH, and urinary saturation levels in the different patient populations. (KTx kidney transplantation, CsA cyclosporine A, Tacro tacrolimus, RE Rasmussen encephalitis, NS nephrotic syndrome, RS relative urinary saturation in respect to indicated salt)
KTx CsA (n=36)
KTx Tacro (n=17)
KTx metabolic acidosis (n=11)
Normal values 
Calcium (mg/kg per 24 h)
Oxalate (mmol/1.73 m2 per 24 h)
Citrate (mmol/1.73m2 per 24 h)
Uric acid (mmol/kg per 24 h)
RSCaOx (rel. units)
RSBrushite (rel. units)
RSUric acid (rel. units)
Urinary calcium excretion was elevated in 7 (9.8%) of the 71 patients examined. Hypercalciuria was found in 9.4% of KTx patients (11% of those with CsA and 6% of those with tacrolimus), but in only 1 of the 12 patients with NS and in none of the 6 RE patients. Hyperuricosuria was found in 2 patients, 1 after KTx and 1 with NS; both were on CsA medication. Urinary calcium and uric acid excretions were significantly higher in patients after KTx than in patients with RE or NS (P<0.05). Urinary CaOx saturation was elevated in three patients (one in each group). The brushite saturation was elevated in one patient after KTx and CsA medication, and the uric acid saturation was increased in one patient with RE.
Urinary pH values did not differ between either groups, with a mean of 5.5±0.25 in the KTx patients (range 5–8) and 5.71±0.84 in the NS patients (range 5–7; Table 1). Urinary pH values were not determined in the RE patients. Blood gas analysis, performed in all KTx and NS patients, showed metabolic acidosis in 11 of those with KTx. Mean citrate excretion was slightly but not significantly lower than in the other KTx patients. Patients with acidosis were later treated with sodium bicarbonate supplementation. There were no significant differences in the urinary analyses of patients receiving CsA or tacrolimus therapy. Ultrasound investigations showed nephrolithiasis in one patient after kidney transplantation but did not reveal nephrocalcinosis in any of the patients. Biopsy of the kidney graft was performed in 26 of 53 patients, with nephrocalcinosis (CaOx crystal deposition≥birefrigent crystals) found in three patients.
The main finding in our investigation was the high prevalence of hypocitraturia after KTx, in nearly 80% of patients, while only 40% of those under immunosuppressive therapy due to other reasons had hypocitraturia. In addition, hyperoxaluria was observed as another major risk factor for nephrocalcinosis or nephrolithiasis in the patients after KTx, especially in the first months after transplantation. However, the urines of our patients were not generally supersaturated for calcium-oxalate.
Kidney grafts experience tubular damage due to the tubulotoxic side effects of calcineurin inhibitors, through ischemic injury during transplantation or due to chronic rejection [5, 13, 14, 15, 16, 17, 18]. The major problem of tubular dysfunction is renal tubular acidosis [5, 13]. RTA can, in our experience, result in a significantly reduced urinary excretion of citrate because intracellular acidosis leads to an increase in citrate reabsorption in the proximal tubule and hence to a reduction in urinary citrate excretion . This may be expressed by the slightly lower urinary citrate excretion in the group of KTx patients with metabolic acidosis. Furthermore, the acidic urinary pH levels in both the KTx and the NS group led to less calcium citrate binding, thus increasing the risk of both calcium oxalate and calcium phosphate precipitation [7, 9].
It has additionally been shown in rats that CsA lowers urinary citrate excretion irrespective of acidosis or hypokalemia, and therefore that hypocitraturia may be a specific side effect of treatment with calcineurin inhibitors . In contrast to the patients after KTx only 25% of those treated with CsA for the NS developed hypocitraturia, probably because the CsA dose and the C0 levels were lower than in the KTx group. In addition, the tubules of a grafted kidney may be more prone to toxic effects, as other parameters, for example, medications (ACE inhibitors) or acute/chronic rejections are also leading to tubular damage.
Converting enzyme inhibitors such as enalapril are known to cause hypocitraturia in humans and in rats . This is due at least in part to an increase in cytosolic citrate metabolism through ATP citrate lyase similar to that seen with chronic metabolic acidosis and hypokalemia. The effects of enalapril on urinary citrate and renal cortical ATP citrate lyase occur independently of acidosis or hypokalemia but may be due to intracellular acidosis that is common to all three conditions . Citrate excretion is essential for the prevention of urinary supersaturation (e.g., for CaOx or brushite), and as a result it is a major risk factor for nephrocalcinosis and/or nephrolithiasis . However, we did not find a specific difference in urinary citrate excretion patterns among KTx patients receiving ACE inhibitor therapy (see Fig. 1). Hypercalciuria, possibly due to steroid and CsA therapy, is also found in patients after KTx thus further increasing the risk of kidney stones.
We were surprised to find hyperoxaluria in a significant number of patients after KTx. Oxalate accumulates systemically in renal failure, especially in patients transplanted after a long time on dialysis. This finding contrasts with previous experience showing that systemic oxalate deposition occurs significantly only in patients with primary hyperoxaluria in end-stage renal disease . However, whether other effects are responsible cannot be answered. Either CsA or tacrolimus may change the intestinal absorption pattern of oxalate, thus leading to secondary, absorptive hyperoxaluria or may influence tubular oxalate secretion. Hence further studies and long-term follow-up are necessary.
Patients with hypocitraturia, hyperoxaluria, and hypercalciuria after KTx are at risk of urinary supersaturation leading to the development of stones or nephrocalcinosis. Urinary saturation was, however, not elevated in our patient population, possibly as a result of their high urinary volume (>1.5 l per day). Nevertheless, we observed hematuria in nine patients after KTx, urolithiasis in one patient (in situ) and CaOx crystal depositions in the renal parenchyma on routine biopsy examinations in three patients.
There is growing evidence that, in addition to the stone inhibitory potential, citrate may have further functions, for example, in protecting the kidney. In an animal model of autosomal-dominant polycystic kidney disease the male heterozygous Han:SPRD rat, intake of a solution of potassium citrate plus citric acid prevented a decline in glomerular filtration rate. Rats with polycystic kidney disease show abnormal renal handling of citrate. Citrate salts that have an alkalinizing effect preserved glomerular filtration rate and extended survival. Therefore potassium citrate might possibly be of benefit in slowing the progression of autosomal-dominant polycystic kidney disease in men, or even be beneficial in other renal diseases [22, 23, 24].
The incidence of urolithiasis after kidney transplantation was found to be equivalent or even lower than the numbers in the general population [1, 2, 3, 4]. However, a recent study reported 6% of adult and 5% of pediatric transplant recipients experiencing kidney stone disease . Our data support this finding, showing that the risk of urolithiasis/nephrocalcinosis is increased after kidney transplantation. This seems due particularly to hypocitraturia caused by the tubulotoxic effect of the calcineurin inhibitor therapy. In addition, significant hyperoxaluria especially in the first months after transplantation further increases that risk.
We currently treat our patients with alkaline citrate (0.5 mEq sodium-potassium citrate/kg body weight daily) to increase the urinary citrate excretion and, secondly the urinary solubility index, aiming to prevent the development of nephrocalcinosis, or even better, to possibly reduce the tubulotoxic effects of calcineurin inhibitors after KTx.
Parts of this study were presented at the IPNA conference 2001 in Seattle. We thank Prof. E. Leumann for his editorial comments, Prof. Dr. Gabriele Benz-Bohm for performing the ultrasound examinations and Mrs. B. Baer for her laboratory assistance.