Fiabilidad

increased in the glomeruli in all diseased kidneys. For the DS domain defi ned by GD3A12 there was no diff erence in expression in the interstitium and the glomerulus between the normal and the kidneys with transplant rejection, and between the normal kidneys and renal biopsies with FSGS, MGN and SLE. These data suggest a role for the increased expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 in renal diseases with early fi brosis, but not of the IdoA-Gal-NAc4S DS domain recognized by GD3A12. A variation of the phage display technique was described in Chapter 6, i.e. the in vivo phage display technique. Here, we injected a phage display library enriched for GAG binding scFv-expressing phages into living rats. Selection of kidney reactive anti-GAG antibodies was performed by harvesting kidney-bound phages and using them in subsequent rounds of panning and amplifi cation. Using the in vivo panning approach, two distinct anti-GAG antibodies were selected that showed a good staining in the normal kidney. Further characterization of the two selected anti-GAG antibodies showed a very high similarity of one of them (JLiv18) at the base pair level with the known antibody HS4C3 (only one amino acid diff erence). Using an ELISA approach with well characterized GAGs, there was a clear diff erence between the known antibody HS4C3 and the two in vivo selected anti-GAG antibodies, but also between the two in vivo selected anti-GAG antibodies. This new in vivo phage display approach might be used for targeting blood facing GAGs and provide a quick access to them.

Future perspectives

In this thesis the phage display technique was used to generate antibodies directed against specifi c GAG domains and the role of these GAG domains in normal renal (patho)physiology was investigated. There are indications that the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 plays an important role in early fi brotic renal diseases. To study the structure-function relationship in renal diseases of the DS domain defi ned by LKN1, and of other GAG domains as well, GAGs should be extracted from renal tissue of a range of fi brotic renal diseases as well as from diff erent transplant rejection kidneys and control kidneys. Separate isolation of GAGs from the glomerular compartment and the tubular compartment is recommended, since for example the DS domain defi ned by LKN1 is only expressed in the diseased glomeruli and not in the normal glomeruli. The extracted GAGs should be analyzed for their chemical make-up using disaccharide analysis by mass spectrometry. For this

GAGs are fl uorescently labeled, followed by fractionating using reverse- phase ultra-performance liquid chromatography and then analyzed by electrospray ionization mass spectrometry. To get more homogenous GAG preparations, partially digested GAGs derived from the kidney may be affi nity purifi ed using for example the anti-DS antibody LKN1, followed by structural analysis. Furthermore, the presence of specifi c DS domains, but also HS domains, can be measured in the blood and urine over time in animal models of renal fi brotic disease. In addition, a patient study can be designed using the urine and blood of kidney transplant patients over time and correlating the presence of specifi c DS/HS domains with renal biopsy data and other clinical parameters such as urinary albumin and glomerular fi ltration rate. If clinical parameters could be correlated to the presence of specifi c DS/HS domains in the urine and/or blood, this might be used as non-invasive diagnostic and prognostic marker.

To gain more insight into the fundamental role of DS and GAGs in the development of inherited renal disease, animals should be sampled starting in utero with follow up at diff erent time points until there is prominent renal disease. Comparing the expression of specifi c DS and/ or GAG domains during normal renal development versus inherited renal diseases, may provide a basis for the identifi cation of critical processes in the development of the kidney in general and of inherited renal diseases in particular that are related to specifi c DS and/or HS domains. Ultimately, this kind of studies could lead to the rational development of defi ned DS or HS-based therapeutics. The potential role of such DS or HS- based glycomimetics in treating renal disease can be evaluated in in vivo experimental models of renal disease.

Recently, a novel hypothesis has been put forward suggesting a key role for DS in autoimmunity1_{. This adds to the already highly diverse roles of DS in}

health and disease. The tools generated in this thesis may be instrumental in further dissecting the pathophysiological consequences of (altered) DS domain structures.

1. Rho JH, Zhang W, Murali M, Roehrl MH, Wang JY: Human proteins with affi nity for dermatan sulfate have the propensity to become autoantigens. Am J Pathol. 178:2177-2190, 2011

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