Genetics Of Osteopontin in Patients With Chronic Kidney Disease: The German Chronic Kidney Disease Study Ⅲ

rs4253311; 4:187174683 (KLKB1 intronic)

The 2nd replicated index SNP rs4253311 (MAF = 0.50) on chromosome 4 is an intronic variant of the KLKB1 gene. Again, whether this variant is the causal variant responsible for the observed association cannot be answered by this study. KLKB1 encodes prekallikrein (PK) a single-chain zymogen that, after activation to kallikrein (KAL), a serine protease, becomes involved in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation, and inflammation. Diseases associated with KLKB1 include PK deficiency and malignant essential hypertension [51,52]. PK and subsequently KAL are part of the kallikrein-kinin system (KKS). The main function of KAL includes the release of bradykinin (BK) [53]. Genome-wide association studies in the past identified associated SNPs in KLKB1 with vasoactive peptides or precursors of vasoactive peptides (BK [54,55], active renin [56], B-type natriuretic peptide [57], aldosterone/renin ratio [57], mid regional adrenomedullin and C-terminal-pro-endothelin-1 [58], L-arginine [59]), and apolipoprotein A IV [60], but not OPN.

Organic Supplements For Kidney Health

Pathways related to this gene include complement and coagulation cascades, as well as degradation of the extracellular matrix [61,62]. An important paralog of KLKB1 is the gene F11. Since our analysis revealed colocalizations between OPN association signal for rs4253311 and expression in multiple tissues for F11, it could be presumed that rs4253311 is linked to F11 rather than KLKB1. Interestingly, OPN contains several protease cleavage sites that regulate its activity [31]. Some OPN interaction sites require cleavage by thrombin, another serine protease, to become fully functional. In return, OPN is a substrate for other proteases, that regulate its activity [31]. One might speculate that inflammatory processes within the kidney of CKD patients bring together, on the one hand, an activated KKS and, on the other hand, higher OPN levels, thus it might be plausible that new bioactive OPN fragments could be generated by KAL.

Conditional colocalization analyses with SNPs located around KLKB1 resulted in positive results for rs1593, mapping ironically into the F11 gene, and DVT. DVT is a serious disease influenced by both genetic and environmental risk factors, but 60% of the variation in risk for DVT has been attributed to genetic risk factors in the past [63]. Genetic studies of DVT have reported several common SNPs in the 4q35.2 locus to be associated with DVT [63]. These common SNPs were localized within KLKB1 and F11 amongst others [63].

Other colocalization results showed association signals between the OPN locus at KLKB1 and 87 plasma proteins. These proteins showed enrichment for proteins of the neuronal cell body in plasma and cerebrospinal fluid of patients. BK, the principal effector of the plasma KKS, is generated systemically and locally (vessel wall) and acts in a paracrine or autocrine way influencing vascular tone and ultrastructure via two G protein-coupled receptors [64,65]. Components of the KKS and in particular BK have been shown to have important functions in the central nervous system by regulating cerebrovascular resistance, vessel capacity, and permeability of the blood-brain barrier. Maintenance of a vascular permeability equilibrium in the central nervous system is critical for maintaining brain integrity. Associations of the KKS in CNS pathology include several disease states among which are neuropsychiatric lupus, Alzheimer's disease, schizophrenia, and epileptic syndrome [66]. These facts in turn explain the enrichment analysis results of the molecular functions: hormone activity via signaling receptor binding as well as receptor regulatory activity

rs2731673; 5:176839898 (F12/GRK6 intergenic) 

Finally, the index SNP rs2731673 (MAF = 0.25) that could not be replicated in the population-based YFS cohort is located between the genes F12 and GRK6. While this non-confirmation may indicate a false positive result of our GWAS, replication may have failed for some unknown reason such as being a specific result relevant for CKD patients. In the absence of another cohort (whether population-based or CKD cohort) with necessary data on OPN and genetics, we were unable to validate this any further. The gene nearest to the locus is the F12 gene that encodes coagulation factor XII, which, together with plasma PK, belongs to the contact activation system [67,68]. KAL can activate factor XII (factor XIIa: active enzyme of factor XII) that, in turn, promotes inflammation via the KKS, including PK [69]. Since CKD patients markedly have more inflammation and fibrosis as the joined common final path of kidney disease progression these insights and connection may encourage further validation of this locus. Inflammatory processes also play a major role in CVD and CKD patients, who are well known to suffer from excessive CVD procausing higher morbidity and mortality. 

In the human cardiovascular system, OPN is primarily expressed in endothelial cells, macrophages, and smooth muscle-derived foam cells and can also be detected in human atherosclerotic plaques of the arterial system [70,71]. Higher serum OPN levels were found in patients with acute coronary syndrome vs chronic coronary syndrome. In coronary artery disease (CAD) patients high OPN levels were associated with rapid coronary plaque progression and in-stent restenosis [72]. OPN has been known to be associated with adverse outcomes in patients with CVD [73–75], but its function in CVD is diverse. Acute increases of OPN in CVD are associated with wound healing and neovascularization [76,77]. Chronically increased OPN, however, is associated with a poor prognosis of major adverse cardiovascular events [78]. GRK6 on the other hand is involved in blood pressure regulation and was found to have a decreased kidney expression in spontaneously hypertensive rats, providing a similar rationale [79].

Strengths and limitations 

The presented analyses have several strengths and limitations: Firstly, our analyses are based on a CKD patient population of European ancestry and mostly CKD stage 3 under regular nephrologist care. While biological mechanisms may be upregulated in impaired kidney function and thus detected more easily in CKD patients, results potentially compromise generalizability to the general population as well as to other ethnicities. Secondly, we could replicate two of three identified loci in a population-based cohort of the YFS, which also applied an ELISA technique to measure OPN, thus confirming the potential to transfer findings from a CKD cohort to the general population. Regarding the third, non-replicated finding, one should await further validation of this result as regarding it as false-positive would be a premature conclusion. Thirdly, serum OPN was measured in the GCKD study from baseline samples using a state-of-the-art ELISA assay. Although serum has been validated for use in the used OPN assay, it is not the recommended sample type, because of proteolytic cleavage by thrombin during the clotting process. In contrast, OPN measurements were obtained from plasma samples in the YFS cohort. While a comparable assay was used, differences between levels in CKD patients and YFS participants might thus be explainable by more than disease status.

Conclusions 

In this first GWAS of serum OPN levels in a large CKD cohort, two replicated associations on chromosome 4 were detected. One locus closest to the SPP1 gene, as well as a locus mapping into the KLKB1 gene, connecting OPN to its production and the KKS. Further studies are needed to fully explain OPN's role in kidney (patho)physiology and elucidate functions of OPN in connection with the KKS and possibly inflammatory processes during kidney fibrosis. 

Material and methods Ethics statement The German Chronic Kidney Disease (GCKD) study was approved by all Ethics Committees of participating institutions in Germany that also cover the present project. It was registered in the national registry for clinical studies (DRKS 00003971; S1 Information). Written informed consent was obtained for all participants. 

Study population The GCKD study cohort consists of 5,217 adult CKD patients of European ancestry with (i) an eGFR between 30–60 mL/min per 1.73m2 or (ii) an eGFR >60 mL/min/1.73 m2 and 'overt' albuminuria/proteinuria at baseline [20]. At the baseline visit (2010–2012), trained personnel obtained data using a standardized questionnaire and physical examinations. Biosamples were obtained, directly processed, and then stored at -80˚C in a central biobank [80]. Study procedures and main baseline findings have been reported before [20,81].