Part 2| Genetic Variation Implicates Plasma Angiopoietin-2 in The Development Of Acute Kidney Injury Sub-phenotypes
Mar 03, 2022
For more information please contact emily.li@wecistanche.com
Cistanche can improve kidney function
Discussion
The search for susceptibility genes in AKI has been hindered by heterogeneity within the clinical disease phenotype. Using AKI sub-phenotypes, we have discovered that a genetic variant near the ANGPT2 gene, rs2920656, is protective against the development of AKI-SP2. Causal inference analysis suggests that plasma ANG-2 concentrations mediated 41.5% of the genetic association between rs2920656 and AKI-SP2 risk in subjects of European ancestry. Furthermore, in-vitro experiments demonstrated that the minor allele of rs2920656 may lead to lower ANG-2 protein release by human kidney endothelial cells. In addition, plasma ANG-2 is minimally really cleared, suggesting that elevated plasma ANG-2 concentrations are unlikely to result from kidney injury. Overall, these findings provide.
evidence that plasma ANG-2 plays a mechanistic role in the host’s response to critical illness leading to AKI-SP2. Efforts to target the Ang-Tie2 axis may limit AKI severity and result in poor clinical outcomes [35]. The ANPT2 gene is 100% nested within the microcephalin (MCPH1) gene. Mutations in the MCPH1 gene have been associated with diseases of neurogenesis and renal cell carcinoma [36]. We have shown that rs2920656 is an intronic variant in MCPH1 that regulates plasma ANG-2 concentrations. Previous studies have identified genetic variants in MCPH1 [37] and ANGPT2 [38] that are associated with ANG-2 concentrations. It is also conceivable that rs2920656 influences MCPH1 expression. However, to our knowledge, no role for MCPH1 in acute or chronic kidney disease or vascular injury has been described. In contrast, multiple pre-clinical and clinical studies have demonstrated the important role of plasma ANG-2 in the development of AKI [16, 39, 40].
Multiple reports have implicated plasma biomarkers of endothelial function in the pathophysiology of AKI [39, 41, 42]. ANG-1 and -2 are vascular endothelial growth factors that both bind to the endothelial tyrosine kinase receptor (Tie-2) but have context-dependent activities [43]. ANG-1 is released by pericytes and platelets and is an agonist for the Tie-2 receptor. ANG-1 is protective by stabilizing the endothelium and preventing microcirculatory capillary leakage, a hallmark of AKI [44]. In contrast, ANG-2 typically acts as an antagonist to the Tie-2 receptor and promotes endothelial permeability [45] and inflammation [46, 47]. The ANGPT2 gene encodes for circulating ANG-2, which is released from endothelial cells during an inflammatory stimulus. Animal studies have shown that inhibition of ANG-2 binding, augmenting ANG-1 concentrations [41, 44], or activation of Tie-2 [46] decreases endothelial leak and protects against AKI. Taken together these studies implicate a mechanistic role of the ANG-Tie2 axis in AKI. Here we demonstrate genomic regulation of plasma ANG-2 as another piece supporting the causative role of ANG-2 in the development of a severe form of AKI, AKI-SP2.
The strong association between plasma ANG-2 and the development of AKI-SP2 raises the question of whether ANG-2 is similar to creatinine: filtered by the kidney and elevated levels are simply reflections of decreased renal filtration. To demonstrate that plasma ANG-2 concentrations are not simply a marker but causal in the development of severe AKI, we provide two lines of evidence. First, using genetic causal inference analysis, we have shown that genetic variation near the ANGPT2 gene is associated with ANG-2 plasma concentrations and the development of AKI-SP2. Second, unlike creatinine (113 Da), ANG-2 (57,000 Da) is a large molecule that is unlikely to be regularly filtered at the glomerulus. In a critically ill population, we have demonstrated minimal renal clearance of ANG-2. Thus, elevations in plasma ANG-2 concentrations are unlikely to be due to differences in the renal filtration and, instead, may be involved in the pathophysiology of AKI in critically ill patients.
It is important to note that individual genetic variants likely have small overall effects on disease development because AKI is likely a polygenic disease. The strength of this analysis is the identification of a genetic variant that supports ANG-2 as causal in the development of AKI. Even variants with modest effect sizes provide opportunities for the investigation of potential novel causal pathways using genetic medication analysis. For example, cardiovascular disease, similar to AKI, is a polygenic trait with many genetic variants each explaining a small proportion of the risk. Regardless, three SNPs that explained only 0.4 to 2% of the variance in c-reactive protein levels allowed the determination that c-reactive protein was not causal in the development of ischemic vascular disease [48], and these findings were confirmed in subsequent studies [49].
Our work has several strengths. First, we used AKI sub-phenotypes to leverage precision in the phenotype definition and to maximize sample size to discover genetic variants. Second, causal inference analysis suggests that 41.5% of the rs2920656-associated risk for developing AKI-SP2 is explained by plasma ANG-2 levels. This provides clinical evidence, to build on work from animal studies, that modulation of plasma ANG-2 concentrations may improve outcomes in critical illness-associated AKI. Third, to account for potential residual confounding and to link Ang-2 production specifically to kidney endothelial cells, we completed in-vitro experiments using HKMECs. Fourth, using a unique ICU cohort with timed urine collection samples and before and after plasma samples, we were able to demonstrate that minimal amounts of plasma ANG-2 are filtered by the kidney. Thus, the strong association of ANG-2 with kidney-specific outcomes is likely not confounded by issues of reverse causation.
Our study has limitations. Our sample size was relatively small. However, our well-defined quantitative trait (AKI-SP2) allowed us to identify a genetic association with the limited number of critically ill patients with AKI. Second, analyses were limited to patients of European ancestry in order to reduce genetic admixture, maximize power, and because of differences in allelic frequencies among ethnic backgrounds. Future work is warranted to study alternative ethnic populations to determine if similar genomic variation influences plasma ANG-2 concentrations. Third, while there was a trend in lower ANG-2 measurements in PTECs with the minor allele, the results were not statistically significant. However, the human samples are difficult to obtain, and even with a small sample size we saw a consistent direction. Fourth, due to the uniqueness of this dataset, we were unable to find a similar patient population to replicate our findings. Our dataset included well-phenotyped patients with AKI, with genomic, plasma, and clinical outcome data. However, within the four individual populations included in iSPAAR there was a consistent direction in effect between rs2920656 and the development of AKI-SP2. Future work is warranted to understand the influence of rs2920656 on sub-phenotype development and AKI-specific clinical outcomes.
Conclusion
In summary, we identified a genetic variant near the ANGPT2 gene that is associated with plasma ANG-2 concentrations and the development of AKI-SP2 among a critically ill population. We also tested this association through studies completed in HKMECs. Our findings suggest that plasma ANG-2 plays a causal role in the development of AKI-SP2 and believe efforts to target the Ang-Tie2 axis may prevent the development of poor clinical outcomes.
References
1. Clermont G, Acker CG, Angus DC, Sirio CA, Pinsky MR, Johnson JP. Renal failure in the ICU: comparison of the impact of acute renal failure and end-stage renal disease on ICU outcomes. Kidney Int. 2002;62:986–96.
2. Ishani A, Xue JL, Himmelfarb J, Eggers PW, Kimmel PL, Molitoris BA, et al. Acute kidney injury increases the risk of ESRD among elderly. J Am Soc Nephrol JASN. 2009;20:223–8.
3. Coca SG, Yusuf B, Shlipak MG, Garg AX, Parikh CR. Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis. Am J Kidney Dis Off J Natl Kidney Found. 2009;53:961–73.
4. Chawla LS, Eggers PW, Star RA, Kimmel PL. Acute kidney injury and chronic kidney disease as interconnected syndromes. N Engl J Med. 2014;371:58–66.
5. Stafford-Smith M, Li Y-J, Mathew JP, Li Y-W, Ji Y, Phillips-Bute BG, et al. Genome-wide association study of acute kidney injury after coronary bypass graft surgery identifies susceptibility loci. Kidney Int. 2015;88:823–32.
6. Zhao B, Lu Q, Cheng Y, Belcher JM, Siew ED, Leaf DE, et al. A genome-wide association study to identify single-nucleotide polymorphisms for acute kidney injury. Am J Respir Crit Care Med. 2016;195:482–90.
7. Barasch J, Zager R, Bonventre JV. Acute kidney injury: a problem of definition. Lancet Lond Engl. 2017;389:779–81.
8. Copeland KT, Checkoway H, McMichael AJ, Holbrook RH. Bias due to misclassification in the estimation of relative risk. Am J Epidemiol. 1977;105: 488–95.
9. Sioux V, González JR, Bouzigon E, Curjuric I, Boudier A, Imboden M, et al. Genetic heterogeneity of asthma phenotypes identified by a clustering approach. Eur Respir J. 2014;43:439–52.
10. Bhatraju PK, Zelnick LR, Herting J, Katz R, Mikacenic C, Kosamo S, et al. Identification of acute kidney injury sub-phenotypes with differing molecular signatures and response to vasopressin therapy. Am J Respir Crit Care Med. 2018;199:863–72.
11. Russell JA, Walley KR, Singer J, Gordon AC, Hébert PC, Cooper DJ, et al. Vasopressin versus norepinephrine infusion in patients with septic shock. N Engl J Med. 2008;358:877–87.
12. Wu X, Guo R, Chen P, Wang Q, Cunningham PN. TNF induces caspase-dependent inflammation in renal endothelial cells through a rho- and myosin light chain kinase-dependent mechanism. Am J Physiol Renal Physiol. 2009;297:F316–26.
13. Xu C, Chang A, Hack BK, Eadon MT, Alper SL, Cunningham PN. TNF- mediated damage to the glomerular endothelium is an important determinant of acute kidney injury in sepsis. Kidney Int. 2014;85:72–81.
14. Cunningham PN, Dyanov HM, Park P, Wang J, Newell KA, Quigg RJ. Acute renal failure in Endotoxemia is caused by TNF acting directly on TNF Receptor-1 in the kidney. J Immunol. 2002;168:5817–23.
15. Han S, Lee S-J, Kim KE, Lee HS, Oh N, Park I, et al. Amelioration of sepsis by TIE2 activation-induced vascular protection. Sci Transl Med. 2016;8:335ra55.
16. Parikh SM. The Angiopoietin-Tie2 signaling Axis in systemic inflammation. J Am Soc Nephrol JASN. 2017;28:1973–82.
17. Wei Y, Tejera P, Wang Z, Zhang R, Chen F, Su L, et al. A missense genetic variant in LRRC16A/CARMIL1 improves acute respiratory distress syndrome survival by attenuating platelet count decline. Am J Respir Crit Care Med. 2017;195:1353–61.
18. Trinder M, Genga KR, Kong HJ, Blauw LL, Lo C, Li X, et al. Cholesteryl Ester transfer protein influences high-density lipoprotein levels and survival in Sepsis. Am J Respir Crit Care Med. 2019;199:854–62.
19. Reilly JP, Wang F, Jones TK, Palakshappa JA, Anderson BJ, Shashaty MGS,
et al. Plasma angiopoietin-2 as a potential causal marker in sepsis-associated ARDS development: evidence from Mendelian randomization and mediation analysis. Intensive Care Med. 2018;44:1849–58.
20. Bhatraju PK, Mukherjee P, Robinson-Cohen C, O’Keefe GE, Frank AJ,
Christie JD, et al. Acute kidney injury subphenotypes based on
creatinine trajectory identify patients at increased risk of death. Crit Care Lond Engl. 2016;20:372.
21. National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome (ARDS) Clinical Trials Network, Matthay MA, Brower RG, Carson S, Douglas IS, Eisner M, et al. Randomized, placebo-controlled clinical trial of an aerosolized β2-agonist for treatment of acute lung injury. Am J Respir Crit Care Med. 2011;184:561–8.
22. National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome (Ards) Clinical Trials Network, Wheeler AP, Bernard GR, Thompson BT, Schoenfeld D, Wiedemann HP, et al. pulmonary-artery versus central venous catheter to guide treatment of acute lung injury. N Engl J Med 2006;354:2213–2224.
23. Rice TW, Wheeler AP, Thompson BT, de Boisblanc BP, Steingrub J, Rock P, et al. Enteral omega-3 fatty acid, gamma-linolenic acid, and antioxidant supplementation in acute lung injury. JAMA. 2011;306:1574–81.
24. Gong MN, Zhou W, Williams PL, Thompson BT, Pothier L, Christiani DC. Polymorphisms in the mannose-binding lectin-2 gene and acute respiratory distress syndrome. Crit Care Med. 2007;35:48–56.
25. Liu KD, Glidden DV, Eisner MD, Parsons PE, Ware LB, Wheeler A, et al. Predictive and pathogenetic value of plasma biomarkers for acute kidney injury in patients with acute lung injury. Crit Care Med. 2007;35:2755–61.
26. Liu K-L, Lee K-T, Chang C-H, Chen Y-C, Lin S-M, Chu P-H. Elevated plasma thrombomodulin and angiopoietin-2 predict the development of acute kidney injury in patients with acute myocardial infarction. Crit Care Lond Engl. 2014;18:R100.
27. Siew ED, Matheny ME, Ikizler TA, Lewis JB, Miller RA, Waitman LR, et al. Commonly used surrogates for baseline renal function affect the classification and prognosis of acute kidney injury. Kidney Int. 2010;77:536–42.
28. Howie BN, Donnelly P, Marchini J. A flexible and accurate genotype imputation method for the next generation of genome-wide association studies. PLoS Genet. 2009;5:e1000529.
29. Marchini J, Howie B. Genotype imputation for genome-wide association studies. Nat Rev Genet. 2010;11:499–511.
30. Lee PH, Bergen SE, Perlis RH, Sullivan PF, Sklar P, Smoller JW, et al. Modifiers and subtype-specific analyses in whole-genome association studies: a likelihood framework. Hum Hered. 2011;72:10–20.
31. Bhatraju P, Hsu C, Mukherjee P, Glavan BJ, Burt A, Mikacenic C, et al. Associations between single nucleotide polymorphisms in the FAS pathway and acute kidney injury. Crit Care Lond Engl. 2015;19:368.
32. Yeboah J, Delaney JA, Nance R, McClelland RL, Polak JF, Sibley CT, et al. Mediation of cardiovascular risk factor effects through subclinical vascular disease: the multi-ethnic study of atherosclerosis. Arterioscler Thromb Vasc Biol. 2014;34:1778–83.
33. Kosuke Imai, Luke Keele, Teppei Yamamoto. Identification, Inference, and Sensitivity Analysis for Causal Mediation Effects. JSTOR. 2010;25:51–71.
34. Skol AD, Scott LJ, Abecasis GR, Boehnke M. Joint analysis is more efficient than replication-based analysis for two-stage genome-wide association studies. Nat Genet. 2006;38:209–13.
35. Higgins SJ, Purcell LA, Silver KL, Tran V, Crowley V, Hawkes M, et al. Dysregulation of angiopoietin-1 plays a mechanistic role in the pathogenesis of cerebral malaria. Sci Transl Med. 2016;8:358ra128.
36. Pulvers JN, Journiac N, Arai Y, Nardelli J. MCPH1: a window into brain development and evolution. Front Cell Neurosci. 2015 [cited 2019 Mar 19];9.
37. Lieb W, Chen M-H, Larson MG, Safa R, Teumer A, Baumeister SE, et al. Genome-wide association study for endothelial growth factors. Circ Cardiovasc Genet. 2015;8:389–97.
38. Meyer NJ, Li M, Feng R, Bradfield J, Gallop R, Bellamy S, et al. ANGPT2 genetic variant is associated with trauma-associated acute lung injury and altered plasma angiopoietin-2 isoform ratio. Am J Respir Crit Care Med. 2011;183:1344–53.
39. Jongman RM, van Klarenbosch J, Molema G, Zijlstra JG, de Vries AJ, van Meurs M. Angiopoietin/Tie2 Dysbalance is associated with acute kidney injury after cardiac surgery assisted by cardiopulmonary bypass. PLoS One. 2015;10:e0136205.
40. Robinson-Cohen C, Katz R, Price BL, Harju-Baker S, Mikacenic C, Himmelfarb J, et al. Association of markers of endothelial dysregulation Ang1 and Ang2 with acute kidney injury in critically ill patients. Crit Care Lond Engl. 2016;20:207.
41. David S, Park J-K, van Meurs M, Zijlstra JG, Koenecke C, Schrimpf C, et al. Acute administration of recombinant Angiopoietin-1 ameliorates multiple- organ dysfunction syndromes and improves survival in murine sepsis. Cytokine. 2011;55:251–9.
42. Kümpers P, Hafer C, David S, Hecker H, Lukasz A, Fliser D, et al. Angiopoietin-2 in patients requiring renal replacement therapy in the ICU: relation to acute kidney injury, multiple organ dysfunction syndrome, and outcome. Intensive Care Med. 2010;36:462–70.
43. Yuan HT, Khankin EV, Karumanchi SA, Parikh SM. Angiopoietin 2 is a partial agonist/antagonist of Tie2 signaling in the endothelium. Mol Cell Biol. 2009; 29:2011–22.
44. Kim DH, Jung YJ, Lee AS, Lee S, Kang KP, Lee TH, et al. COMP-angiopoietin- 1 decreases lipopolysaccharide-induced acute kidney injury. Kidney Int. 2009;76:1180–91.
45. Felcht M, Luck R, Schering A, Seidel P, Srivastava K, Hu J, et al. Angiopoietin- 2 differentially regulates angiogenesis through TIE2 and integrin signaling. J Clin Invest. 2012;122:1991–2005.
46. Fiedler U, Reiss Y, Scharpfenecker M, Grunow V, Koidl S, Thurston G, et al. Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nat Med. 2006;12:235–9.
47. Higgins SJ, De Ceunynck K, Kellum JA, Chen X, Gu X, Chaudhry SA, et al. Tie2 protects the vasculature against thrombus formation in systemic inflammation. J Clin Invest. 2018;128:1471–84.
48. Zacho J, Tybjaerg-Hansen A, Jensen JS, Grande P, Sillesen H, Nordestgaard BG. Genetically elevated C-reactive protein and ischemic vascular disease. N Engl J Med. 2008;359:1897–908.
Emerging Risk Factors Collaboration; Kaptoge S, Di Angelantonio E, Lowe G, Pepys MB, Thompson SG, Collins R, Danesh J. C-reactive protein concentration and risk of coronary heart disease, stroke, and mortality: an individual participant meta-analysis. Lancet. 2010;375(9709):132-40
