Research into biomarkers for early detection of Autosomal Dominant Polycystic Kidney Disease

Promovendus/a: Asmin Andries

Promotor(en): Prof. dr. Ann Van Schepdael, Prof. dr. Djalila Mekahli

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease. It is mainly caused by mutations in either the polycystic kidney disease 1 gene (PKD1) or the PKD2 gene, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. ADPKD is characterized by the continuous growth of innumerable cysts in both kidneys, which leads to an enlargement of the kidneys themselves and a loss in the normal architecture of the kidney. During the years, the number of cysts increases and eventually, the normal kidney tissue is displaced by cyst tissue. Although the continuous growth of those cysts starts early in childhood, most of the patients develop symptoms starting from the third decade of life. Eventually, the latter leads to
end-stage kidney disease, which can only be treated with either kidney dialysis or a kidney transplantation.

Since cyst formation already starts from childhood, there is a high need for a good early biomarker, which may act as a tool for early diagnosis of the disease and for monitoring the disease progression. In addition, this could be evaluated as a novel therapeutic target as well. During this project, we developed and used various chromatographic methods to search for possible early oxidative stress (OS) biomarkers for ADPKD, since several reports already showed increased OS in patients with ADPKD. Nowadays, an increasing trend points towards the use of either allantoin, a metabolite of the purine catabolic pathway and an OS degradation product of uric acid, or the allantoin/uric acid ratio as an OS biomarker in humans.

We developed a new HPLC-UV method for the simultaneous quantification of allantoin and adenosine in human urine samples. After validation of this newly developed HPLC-UV method, the method meets the predetermined requirements of both the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. As a result, we could use our method to test our hypothesis, being more OS and thus more urinary allantoin levels and an increased urinary allantoin/uric acid ratio, in urine samples of children with ADPKD compared to pediatric healthy volunteers and in urine samples of adults with ADPKD compared to adults with another Chronic Kidney Disease. Unfortunately, our hypothesis was not confirmed by these experiments.

On the other hand, a HILIC-MS/MS method was developed for the determination of allantoin in human plasma samples. Since allantoin is endogenously present in plasma samples, and since the standard addition method – used in the previously developed HPLC-UV method – is time consuming, we decided to make activated charcoal stripped plasma. The procedure to make charcoal stripped plasma was fully optimized before we could continue with the validation of the HILIC-MS/MS method. The method does not fully meet the requirements of the FDA and EMA as the inter-day precision and the accuracy of the lower limit of quantitation are not within the required limits. Nevertheless, this method could be used to start a pilot study and to analyze plasma samples from adult ADPKD patients. The results of this pilot study were within our hypothesis, being more OS in plasma samples of patients with ADPKD.

In summary, we can conclude that this PhD project has succeeded to develop and validate novel methods in order to measure and evaluate OS in patients’ samples. Our preliminary data are promising. It is advisable to include a larger pediatric cohort for both the urinary and plasma determinations on the one hand, while the inclusion of healthy adults would also be beneficial.