EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Proteinuria is associated with decreased graft and patient survival after kidney transplantation. Increasing evidence shows that vitamin D has antiproteinuric and renoprotective effects. The aim of our study was to assess the influence of 25-hydroxyvitamin D levels on proteinuria after kidney transplantation.

Material and Methods: Between May 1, 2012, and November 30, 2012, we tested 395 kidney transplant recipients for 25-hydroxyvitamin D levels during their regular visits to our transplant center together with routine blood sampling and proteinuria testing. Patients within 12 months of transplant, who had undergone parathyroidectomy, had unstable graft function, had concomitant intake of calcineurin inhibitors and mammalian target of rapamycin inhibitors were not included in the study. Subjects with advanced liver disease, or receiving vitamin D supplementation were also excluded. All laboratory, clinical, and therapeutic factors for proteinuria were taken into consideration. Statistical analyses included descriptive statistics and univariate and multivariate log-log regression with backward selection (SPSS version 22.0; SPSS Inc., Chicago, IL, USA), with significance at P < .05. Determination of total 25-hydroxyvitamin D levels was performed by a validated liquid chromatography-tandem mass spectrometry method.

Results: Our study group included 230 patients (148 men, 82 women). Positive association was established between proteinuria and history of diabetes mellitus, rejection episode 12 months within testing for 25-hydroxyvitamin D levels, and use of mammalian target of rapamycin inhibitors (P < .05). Significant negative relations were detected for patient age, graft function, and 25-hydroxyvitamin D concentrations (P < .05).

Conclusions: Our study established that better vitamin D status is associated with lower proteinuria. However, further research is needed to clarify the possible renoprotective properties of vitamin D.

Key words : Kidney transplantation, Urinary protein excretion, Vitamin D

Proteinuria after kidney transplantation is associated with poor graft and patient survival.^1,2 Increasing evidence suggests that vitamin D has effects beyond calcium-phosphorus metabolism, including renoprotection, renin-angiotensin system suppression, immunomodulatory effects, diabetes mellitus control, and neoplasia prevention.^3,4 Vitamin D insufficiency is widespread worldwide.⁵ A poor vitamin D status is more frequently encountered in kidney transplant recipients due to several reasons, including sun exposure avoidance, persistent uremia, immunosuppressive treatment, and reduced oral intake.⁶ Reports have linked better vitamin D status with better outcomes after renal transplantation (improved graft survival and reduced incidence of diabetes mellitus and proteinuria).⁷ The aim of our study was to assess the possible influence of serum levels of 25-hydroxyvitamin D [25(OH)D], the generally accepted marker of vitamin D status, on proteinuria in Bulgarian kidney transplant recipients.⁸

Materials and Methods

Tests for 25(OH)D levels, proteinuria, and other laboratory measurements were performed during regular visits of patients to our transplant center between May 1 and November 30, 2012. In addition to laboratory measurements, blood pressure, body weight, and body mass index were assessed during the follow-up visits. Kidney transplant recipients with unstable kidney function, having kidney transplant for less than 12 months, with active/advanced liver disease (Child-Pugh score B and over), and with performed parathyroidectomy, vitamin D supplementation were excluded from the study. Patients with concomitant intake of calcineurin inhibitors and mammalian target of rapamycin (mTOR) inhibitors were also excluded, as were outliers for 25(OH)D level, proteinuria, age, body mass index, and blood pressure (absolute value for z score > 3.29). All possible laboratory, clinical, and therapeutic factors influencing proteinuria were taken into consideration.

The study was approved by the Institutional Ethics Committee and was in accordance with the Helsinki Declaration of 1975 (as revised in 2000). All participants gave their informed consent.

Routine blood and urine clinical chemistry measurements
Serum creatinine, calcium, phosphate, alkaline phosphatase, proteinuria, and other routine laboratory tests were performed on a standard clinical chemistry analyzer. For assessment of proteinuria, we used 24-hour urine specimens. The Chronic Kidney Disease Epidemiology Collaboration equation was used to estimate glomerular filtration rate.

Analyses of immunosuppressive drugs (cyclosporine, tacrolimus, sirolimus, and everolimus) in human whole blood
Cyclosporine, tacrolimus, sirolimus, and everolimus levels were determined by a validated liquid chromatography-tandem mass spectrometry method with documented selectivity and matrix effect, accuracy, and precision within 8.9%. The testing method had extraction recoveries that averaged 65% to 76% with linearity range of R² > 0.99 and had standards ensured by the UK Analytical Services International Proficiency Testing Scheme.

Vitamin D testing
Determination of 25(OH)D was performed by a validated liquid chromatography-tandem mass spectrometry method developed in house, utilizing extraction with hexane. The method was calibrated with the use of the US National Institute of Standards and Technology Standard Reference Material 972 and was validated according to US Food and Drug Administration guidance requirements, with documented selectivity and matrix effects, accuracy and precision within 7.5%, extraction recoveries averaging 57% to 73%, and linearity range of 3.0 to 300.0 nmol/L (R² > 0.99). The method was certified by the UK Vitamin D External Quality Assessment Scheme, with certification obtained in 2012.

Statistical analyses
Descriptive statistics and univariate and multivariate log-log regression with backward elimination were used to investigate the association between proteinuria and the measured variables. Spearman correlation coefficients were used to express associations between continuous parameters. Significance was set at P < .05. Statistical analyses were performed with SPSS version 22.0 software (SPSS Inc., Chicago, IL, USA).

To address the residuals heteroscedasticity, we used the natural logarithms of the quantitative values for proteinuria, 25(OH)D levels, age, body mass index, and systolic and diastolic blood pressure levels. To avoid distortion of parameter and statistical estimates, we screened for outliers of 25(OH)D, proteinuria, age, body mass index, and blood pressure measurement using the z score method, with cut-off values lower than -3.29 and higher than +3.29.

We enrolled 230 patients who met our inclusion criteria. All kidney transplant recipients were Caucasians, with 148 male and 82 female patients. Table 1 shows basic characteristics of patients, including their 25(OH)D and proteinuria results (provided as means ± standard deviation). Most patients were on a triple immunosuppressive regimen (calcineurin inhibitors, mTOR inhibitors, plus azathioprine or mycophenolate plus steroids).

Kidney transplant recipients were assessed for proteinuria according to their vitamin D status (sufficient levels, mild insufficiency, severe insufficiency, or deficiency). A trend for higher proteinuria was detected in those with lower vitamin D levels (Figure 1).

Pearson correlation analyses revealed significant positive correlations between proteinuria and systolic and diastolic blood pressure measurements. Negative significant coefficients were detected between proteinuria and vitamin D and graft function (Table 2).

A large number of variables influenced proteinuria, and we used multivariate regression with backward selection to detect the significant factors. Proteinuria was inversely associated with glomerular filtration rate, serum levels of 25(OH)D, and patient age. History of diabetes mellitus, intake of mTOR inhibitors, and rejection 12 months before or after testing for 25(OH)D were associated with higher proteinuria (Table 3).

No other significant associations were detected, including treatment with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (P = .542) and dihydropyridine (P = .975) and nondihydropyridine calcium channel blockers (P = .239).

Proteinuria is associated with poorer graft and patient survival, especially in the long run. Long-term survival has not improved over the past decades in graft recipients, despite the significant success in short-term results.⁹ Therefore, influencing proteinuria could lead to improved posttransplant outcomes.

Our research confirmed the antiproteinuric effect of vitamin D detected in several studies. Levels of 25(OH)D are inversely related to increased prevalence of albuminuria in nontransplant patients.¹⁰ Improved renoprotection and decreased proteinuria were established after treatment with vitamin D and its analogues, independent of the use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers.^11,12 The relation between vitamin D and proteinuria after kidney transplant has been less studied. However, Lee and associates reported a significant association between vitamin D insufficiency and proteinuria in kidney transplant recipients.¹³ The few studies that investigated treatment with vitamin D and its influence on proteinuria and renal function have shown conflicting results. O’Herrin and associates demonstrated that calcitriol treatment had beneficial effects on renal graft function.¹⁴ Another study failed to demonstrate renoprotection after vitamin D supplementation in kidney transplant recipients, but there were certain limitations of that study, including a small number of patients involved and failure to maintain 25(OH)D levels above 25 ng/mL.¹⁵ In addition, both studies did not have proteinuria as the primary endpoint.

Several mechanisms have been proposed for the antiproteinuric properties of vitamin D. Animal studies have revealed a negative association between 25(OH)D and the renin-angiotensin-aldosterone system.^3,16 An inverse relation between serum 25(OH)D and angiotensin levels was also detected in healthy normotensive volunteers, without significant association with renin levels.¹⁷ Suppression of the renin-angiotensin-aldosterone system however is not the only possible mechanism for proteinuria reduction. Vitamin D was found to inactivate nuclear factor-κΒ by inhibiting its binding to DNA.¹⁸ Nuclear factor-κΒ is associated with inflammation, proliferation, and fibrosis. Furthermore, vitamin D analogues were found to suppress nuclear factor-κΒ in obstructive nephropathy, cyclosporine, and gentamycin toxicity.^19,20 Suppression of the Wnt/β-catenin pathway has also been suggested as a mechanism for the antiproteinuric effect of vitamin D in experimental models.^21,22 The results were confirmed in experimental models where vitamin D analogues were applied.²³ Finally, vitamin D could influence proteinuria by upregulating the expression of the slit-diaphragm proteins, especially nephrin.^24,25 Therefore, vitamin D may reduce proteinuria by keeping the slit-diaphragm intact. Our study revealed no significant influence of angiotensin-converting enzyme inhibitor or angiotensin receptor blocker treatment on proteinuria, indicating that the antiproteinuric properties of vitamin D may span beyond renin-angiotensin-aldosterone system suppression. It must be noted that the above-mentioned studies were performed in experimental models or in nontransplant individuals.

Our study is a retrospective observational one, which is its major disadvantage. However, most reports on vitamin D after kidney transplant share this limitation.²⁶

In conclusion, there are many factors influencing proteinuria in native kidneys, and there are even more after renal transplantation - use of mTOR inhibitors, allograft nephropathy, and history of rejection. . More data are needed about the pleiotropic effects of vitamin D, including target serum levels, target supplementation dose, and treatment duration. In kidney transplant recipients, there are few studies addressing this issue, with predominantly retrospective, single center reports, and only small interventional studies, dealing predominantly with calcium-phosphorus metabolism, are available. Therefore, further prospective and larger trials are needed to clarify the association between vitamin D status and proteinuria after kidney transplant.

Volume: 14 Issue: 6 December 2016

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