Senescence marker activin A is increased in human diabetic kidney disease: Association with kidney function and potential implications for therapy

Xiaohui Bian; Tomás P. Griffin; Xiangyang Zhu; Md Nahidul Islam; Sabena M. Conley; Alfonso Eirin; Hui Tang; Paula M. O'Shea; Allyson K. Palmer; Rozalina G. McCoy; Sandra M. Herrmann; Ramila A. Mehta; John R. Woollard; Andrew D. Rule; James L. Kirkland; Tamar Tchkonia; Stephen C. Textor; Matthew D. Griffin; Lilach O. Lerman; La Tonya J. Hickson

doi:10.1136/bmjdrc-2019-000720

Senescence marker activin A is increased in human diabetic kidney disease: Association with kidney function and potential implications for therapy

Xiaohui Bian, Tomás P. Griffin, Xiangyang Zhu, Md Nahidul Islam, Sabena M. Conley, Alfonso Eirin, Hui Tang, Paula M. O'Shea, Allyson K. Palmer, Rozalina G. McCoy, Sandra M. Herrmann, Ramila A. Mehta, John R. Woollard, Andrew D. Rule, James L. Kirkland, Tamar Tchkonia, Stephen C. Textor, Matthew D. Griffin, Lilach O. Lerman, La Tonya J. Hickson

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

Abstract

Objective Activin A, an inflammatory mediator implicated in cellular senescence-induced adipose tissue dysfunction and profibrotic kidney injury, may become a new target for the treatment of diabetic kidney disease (DKD) and chronic kidney diseases. We tested the hypothesis that human DKD-related injury leads to upregulation of activin A in blood and urine and in a human kidney cell model. We further hypothesized that circulating activin A parallels kidney injury markers in DKD. Research design and methods In two adult diabetes cohorts and controls (Minnesota, USA; Galway, Ireland), the relationships between plasma (or urine) activin A, estimated glomerular filtration rate (eGFR) and DKD injury biomarkers were tested with logistic regression and correlation coefficients. Activin A, inflammatory, epithelial-mesenchymal-transition (EMT) and senescence markers were assayed in human kidney (HK-2) cells incubated in high glucose plus transforming growth factor-β1 or albumin. Results Plasma activin A levels were elevated in diabetes (n=206) compared with controls (n=76; 418.1 vs 259.3 pg/mL; p<0.001) and correlated inversely with eGFR (r s =-0.61; p<0.001; diabetes). After eGFR adjustment, only albuminuria (OR 1.56, 95% CI 1.16 to 2.09) and tumor necrosis factor receptor-1 (OR 6.40, 95% CI 1.08 to 38.00) associated with the highest activin tertile. Albuminuria also related to urinary activin (r s =0.65; p<0.001). Following in vitro HK-2 injury, activin, inflammatory, EMT genes and supernatant activin levels were increased. Conclusions Circulating activin A is increased in human DKD and correlates with reduced kidney function and kidney injury markers. DKD-injured human renal tubule cells develop a profibrotic and inflammatory phenotype with activin A upregulation. These findings underscore the role of inflammation and provide a basis for further exploration of activin A as a diagnostic marker and therapeutic target in DKD.

Original language	English (US)
Article number	e000720
Journal	BMJ Open Diabetes Research and Care
Volume	7
Issue number	1
DOIs	https://doi.org/10.1136/bmjdrc-2019-000720
State	Published - Dec 15 2019

Keywords

adipocytokine
clinical aspects of diabetes
clinical nephrology
renal fibrosis

ASJC Scopus subject areas

Endocrinology, Diabetes and Metabolism

Access to Document

10.1136/bmjdrc-2019-000720

Cite this

Bian, X., Griffin, T. P., Zhu, X., Islam, M. N., Conley, S. M., Eirin, A., Tang, H., O'Shea, P. M., Palmer, A. K., McCoy, R. G., Herrmann, S. M., Mehta, R. A., Woollard, J. R., Rule, A. D., Kirkland, J. L., Tchkonia, T., Textor, S. C., Griffin, M. D., Lerman, L. O., & Hickson, L. T. J. (2019). Senescence marker activin A is increased in human diabetic kidney disease: Association with kidney function and potential implications for therapy. BMJ Open Diabetes Research and Care, 7(1), Article e000720. https://doi.org/10.1136/bmjdrc-2019-000720

Bian, X, Griffin, TP, Zhu, X, Islam, MN, Conley, SM, Eirin, A, Tang, H, O'Shea, PM, Palmer, AK, McCoy, RG , Herrmann, SM, Mehta, RA, Woollard, JR, Rule, AD , Kirkland, JL , Tchkonia, T , Textor, SC, Griffin, MD, Lerman, LO & Hickson, LTJ 2019, 'Senescence marker activin A is increased in human diabetic kidney disease: Association with kidney function and potential implications for therapy', BMJ Open Diabetes Research and Care, vol. 7, no. 1, e000720. https://doi.org/10.1136/bmjdrc-2019-000720

@article{7c57d0723822442b9a046a3d7fe8e4e1,

title = "Senescence marker activin A is increased in human diabetic kidney disease: Association with kidney function and potential implications for therapy",

abstract = "Objective Activin A, an inflammatory mediator implicated in cellular senescence-induced adipose tissue dysfunction and profibrotic kidney injury, may become a new target for the treatment of diabetic kidney disease (DKD) and chronic kidney diseases. We tested the hypothesis that human DKD-related injury leads to upregulation of activin A in blood and urine and in a human kidney cell model. We further hypothesized that circulating activin A parallels kidney injury markers in DKD. Research design and methods In two adult diabetes cohorts and controls (Minnesota, USA; Galway, Ireland), the relationships between plasma (or urine) activin A, estimated glomerular filtration rate (eGFR) and DKD injury biomarkers were tested with logistic regression and correlation coefficients. Activin A, inflammatory, epithelial-mesenchymal-transition (EMT) and senescence markers were assayed in human kidney (HK-2) cells incubated in high glucose plus transforming growth factor-β1 or albumin. Results Plasma activin A levels were elevated in diabetes (n=206) compared with controls (n=76; 418.1 vs 259.3 pg/mL; p<0.001) and correlated inversely with eGFR (r s =-0.61; p<0.001; diabetes). After eGFR adjustment, only albuminuria (OR 1.56, 95% CI 1.16 to 2.09) and tumor necrosis factor receptor-1 (OR 6.40, 95% CI 1.08 to 38.00) associated with the highest activin tertile. Albuminuria also related to urinary activin (r s =0.65; p<0.001). Following in vitro HK-2 injury, activin, inflammatory, EMT genes and supernatant activin levels were increased. Conclusions Circulating activin A is increased in human DKD and correlates with reduced kidney function and kidney injury markers. DKD-injured human renal tubule cells develop a profibrotic and inflammatory phenotype with activin A upregulation. These findings underscore the role of inflammation and provide a basis for further exploration of activin A as a diagnostic marker and therapeutic target in DKD.",

keywords = "adipocytokine, clinical aspects of diabetes, clinical nephrology, renal fibrosis",

author = "Xiaohui Bian and Griffin, {Tom{\'a}s P.} and Xiangyang Zhu and Islam, {Md Nahidul} and Conley, {Sabena M.} and Alfonso Eirin and Hui Tang and O'Shea, {Paula M.} and Palmer, {Allyson K.} and McCoy, {Rozalina G.} and Herrmann, {Sandra M.} and Mehta, {Ramila A.} and Woollard, {John R.} and Rule, {Andrew D.} and Kirkland, {James L.} and Tamar Tchkonia and Textor, {Stephen C.} and Griffin, {Matthew D.} and Lerman, {Lilach O.} and Hickson, {La Tonya J.}",

note = "Funding Information: This study is the first comprehensive study we are aware of focused on the relationship between circulating activin A levels and kidney function in adults with diabetes and metabolically healthy controls. Importantly, an inverse relationship was identified between activin A concentrations and kidney function in two independent diabetes cohorts over a wide eGFR range. Low serum albumin levels, but not metabolic status (eg, hemoglobin A1c) or the inflammatory marker CRP, appeared to be linked to activin A concentration. The independent relationship between albuminuria and activin A potentially indicates that proteinuric kidney disease may be characterized by a distinctive injury with increased senescence phenotype compared with DKD without albuminuria. Finally, a co-association with biomarkers (eg, TNFR-1) that are more directly linked to inflammatory response and renal tubular injury was demonstrated both in adults with diabetes and in a cell culture model of diabetic tubulopathy. Collectively, these findings provide novel evidence that maladaptive inflammation and cellular senescence track together in DKD and that therapies designed to target both pathways (eg, anti-activin, senolytic and regenerative cell-based therapies) could be pursued alongside informative biomarker assays. Activin A is produced by a variety of tissues, including inflamed adipose tissue and injured kidney cells. It is postulated that in the normal, non-injured kidney, glomerular filtered activin A is reabsorbed by renal tubules via endocytosis, which in the setting of tubular dysfunction, contributes to increased urine concentrations. 19 This could explain why higher urine activin A levels (not normalized for creatinine) in individuals with AKI did not strongly correlate with plasma activin A levels. 19 However, animal studies also suggest that activin A originates from injured kidney cells. In experimental studies, elevated activin A was derived directly from renal tubule epithelial cells in streptozotocin-treated 38 and UUO 25 rat models, from myofibroblasts in Alport CKD mice, 22 25 renal interstitial cells in Ldlr –/– high fat-fed mice with ablative CKD, 22 infiltrating macrophages in lupus nephritis mice 23 and glomerular culture supernatants in an anti-Thy1 glomerulonephritis rat model. 39 Furthermore, while UUO induced activin A gene expression in the kidney and its release into the circulation, these findings were not observed in the plasma, contralateral kidney or remnant kidney of unilaterally nephrectomized rats having similar fall in total GFR, suggesting the obstructed kidney as the source. 24 In our human diabetes studies, albuminuria, reflective of kidney injury, directly correlated with activin A in plasma and urine. Plasma activin A also independently and directly correlated with TNFR-1, a robust biomarker for progressive DKD. 28 30 40 41 Notably, an upsurge in urinary activin A levels was only detected in those with significant proteinuria, whereas the relationship between its plasma levels and UACR was more continuous ( figure 4 ), supporting a link between albumin-induced tubular injury and urinary activin A. Moreover, given that in our study urine activin A concentrations rose as plasma levels increased in the setting of reduced GFR, high plasma activin A levels in DKD may be the result of multiple mechanisms, but are unlikely to reflect reduced clearance. Cumulatively, these studies provide insight into the influence of kidney injury, rather than impaired renal handling alone, on circulating activin A levels. This study is strengthened by use of the largest cohort of individuals with DKD (or CKD) to date to undergo measurement of plasma and urine activin A. Harada et al 42 previously found higher circulating activin A concentrations in older individuals and those with cirrhosis, advanced solid cancer, pregnancy and CKD (n=41) compared with non-diseased controls. Nonetheless, despite the pathophysiological relevance and potential for clinical application, subsequent studies assessing the relationship between kidney function and activin A levels have been sparse. Recently, in over 400 older community dwelling adults in Taiwan (19% diabetes; mean eGFR 82.5 mL/min/1.73 m 2 ), Peng et al 43 showed that eGFR was lowest in the highest tertile of plasma activin A. After adjustment for confounding variables, however, only older age, metabolic syndrome and uric acid remained associated with higher tertile status. In our study of over 200 individuals with diabetes (55% with eGFR <60 mL/min/1.73 m 2 ; mean eGFR 41.4 mL/min/1.73 m 2 ), we identified an inverse association between circulating activin A and eGFR. This finding may reflect increased tissue inflammation, cellular senescence burden and/or activin A production by injured kidney cells. While kidney injury related closely to circulating activin A levels in our study, other systemic processes have been associated with activin A. Despite the known relationship with inflammation, in our study, the inflammatory marker CRP did not correlate with activin A concentrations. Nonetheless, serum albumin, a marker of nutritional status and inflammation in CKD, 44–46 correlated inversely with activin A. Surprisingly, metabolic status, indicated through current glycemic control (hemoglobin A1c), was not a significant influencer of activin A levels beyond kidney function. Our in vitro studies explored the effects of glucose, albumin and TGF-β1 on activin A gene expression and protein release by human kidney cells. Notably, high glucose exposure alone was insufficient to activate release of activin A by HK-2 cells. Yet, the addition of albumin to high glucose-containing medium evoked this response. Moreover, our diabetic tubulopathy model of high glucose plus TGF-β1 led to marked upregulation of inflammatory and profibrotic EMT markers followed by release of activin A robustly into cell culture supernatant. Together, the associations between serum albumin, urine protein excretion rate (albuminuria) and activin A suggest proteinuric kidney disease relates to more robust inflammatory signatures and increased senescent cell abundance compared with CKD without albuminuria. Based on these relationships, we tested gene expression of senescence markers in HK-2 cells and found a modest rise in p16 levels after DKD injury. Possibly, a 48-hour exposure was insufficient to upregulate these genes in HK-2 cells and activin A represents an early mediator of the pro-inflammatory, profibrotic state associated with DKD senescence. Nonetheless, our studies support the notion that activin A relates to kidney injury associated with changes in kidney function, increased cellular senescence and possibly adipose tissue dysfunction in individuals with DKD. Beyond being a potential biomarker of DKD injury or cellular senescence, activin A plays an active role in kidney damage and might thus constitute a therapeutic target. In a prior study, we demonstrated that clearance of senescent cells in DIO mice improved podocyte function. 15 More in-depth studies of activin A-induced kidney dysfunction have been performed by others. Notably, activin A-induced kidney dysfunction in a Ldlr −/ − CKD mouse model (fibrosis and proteinuria) and in a polycystic kidney disease mouse model (fibrosis and cyst formation) was decreased by blocking activin A effect via the activin receptor type IIA ligand trap, RAP-011, and a soluble activin receptor IIB fusion protein. 22 47 Anti-activin therapy tested in early phase clinical trials of muscular diseases, osteoporosis, postmenopausal anemia and cancer showed variable side effects and efficacy. 48 49 Additional efforts are also underway to modify the CKD-associated SASP in clinical trials of DKD (Clinicaltrials.gov: NCT02848131 ; NCT03325322 ), 12 50 with recent evidence illustrating that senolytics reduce senescent cell abundance in humans. 51 Our study had limitations. First, the cross-sectional study design did not permit assessment of causal relationships among diabetes, kidney function and circulating activin A concentrations. Reverse causality still remains a possibility as reduced GFR may increase activin A levels via reducing their renal clearance thus prospective studies are needed. Second, the study population was mostly white, and the sex distribution was unequal between controls and diabetes, limiting generalizability. However, we did not detect an association between sex and activin A levels in these cohorts. Furthermore, the Galway cohort was specifically enrolled from general diabetes, nephrology and diabetic nephropathy clinics to parallel the environment found in community-dwelling individuals, therein improving generalizability. Third, while this is the first and largest study in a predominantly DKD population, it had a modest sample size, limiting statistical power. Nonetheless, findings were predominantly consistent between the two distinct cohorts. In conclusion, we observed in two independent cohorts elevated circulating activin A levels in individuals with diabetes, which correlated inversely with kidney function and directly with albuminuria and other kidney injury markers. In vitro studies demonstrated that activin A is produced directly in copious amounts by renal tubular cells exposed to high glucose plus TGF-β1 or albumin, likely contributing to overproduction of circulating activin A. Ongoing prospective follow-up of these findings will allow us to assess the power of plasma activin A as a marker of DKD progression and therapeutic success in the future. The authors would like to thank all study participants and study coordination team members of Mayo Clinic: Donna K Lawson CCRP, LPN, Jennifer M Manggaard CCRP, Tammie L Volkman RN, Erin Wissler Gerdes, Marcia K Mahlman CCRP, Beverly K Tietje CCRP and Tamara K Evans. The authors would like to thank Shannon K Meier for secretarial support. The authors would also like to thank the scientific, nursing and medical staff at the Centre for Endocrinology, Diabetes and Metabolism and the Department of Clinical Biochemistry, Saolta University Health Care Group (SUHCG), Galway University Hospitals. Contributors All authors contributed in the following: substantial contributions to the conception or design of the work, or the acquisition, analysis or interpretation of data. Drafting the work or revising it critically for important intellectual content. Final approval of the version published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Funding This project was supported by funding from the Extramural Grant Program of Satellite Healthcare (LJH), Regenerative Medicine Minnesota (RMM 091718; LJH); National Institute of Health (NIH) grant K23 DK109134 (LJH), UL1 TR002377 (LJH; Mayo Clinic), UL1TR000135 (LJH; Mayo Clinic) and Mayo Center for the Science of Health Care Delivery (LJH, Kern Scholar Program). Additional support was provided by NIH grants R01 DK102325 (LOL), R01 DK120292 (LOL), R01 DK100081 (SCT), T32 DK07013 (SMC), DK106427 (AE), DK118120 (SMH), K23 DK114497 (RGMcC) and AG13925 (JLK), the Ted Nash Long Life and Noaber Foundations (JLK), the Connor Group (JLK) and Robert J. and Theresa W. Ryan (JLK). XB is supported by Natural Science Foundation of Liaoning Province (No. 2015020490). TPG is supported by a Hardiman Scholarship from the College of Medicine, Nursing and Health Science, National University of Ireland Galway and a bursary from the Irish Endocrine Society/Royal College of Physicians of Ireland. The Galway authors are supported by grants from the European Commission (Horizon 2020 Collaborative Health Project NEPHSTROM) (grant number 634086; TPG, MNI, MDG), Science Foundation Ireland (REMEDI Strategic Research Cluster (grant number 09/SRC-B1794; MDG), C{\'U}RAM Research Centre (grant number 13/RC/2073; MDG)), and the European Regional Development Fund. Competing interests None declared. Patient consent for publication Not required. Ethics approval Mayo Clinic Institutional Review Board, Clinical Research Ethics Committees, Galway University Hospitals (Ref: C.A. 1404) and the National University of Ireland Galway, Research Ethics Committee (Ref: 16-July-05) approved these studies. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information. Publisher Copyright: {\textcopyright} Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.",

year = "2019",

month = dec,

day = "15",

doi = "10.1136/bmjdrc-2019-000720",

language = "English (US)",

volume = "7",

journal = "BMJ Open Diabetes Research and Care",

issn = "2052-4897",

publisher = "BMJ Publishing Group",

number = "1",

}

TY - JOUR

T1 - Senescence marker activin A is increased in human diabetic kidney disease

T2 - Association with kidney function and potential implications for therapy

AU - Bian, Xiaohui

AU - Griffin, Tomás P.

AU - Zhu, Xiangyang

AU - Islam, Md Nahidul

AU - Conley, Sabena M.

AU - Eirin, Alfonso

AU - Tang, Hui

AU - O'Shea, Paula M.

AU - Palmer, Allyson K.

AU - McCoy, Rozalina G.

AU - Herrmann, Sandra M.

AU - Mehta, Ramila A.

AU - Woollard, John R.

AU - Rule, Andrew D.

AU - Kirkland, James L.

AU - Tchkonia, Tamar

AU - Textor, Stephen C.

AU - Griffin, Matthew D.

AU - Lerman, Lilach O.

AU - Hickson, La Tonya J.

N1 - Funding Information: This study is the first comprehensive study we are aware of focused on the relationship between circulating activin A levels and kidney function in adults with diabetes and metabolically healthy controls. Importantly, an inverse relationship was identified between activin A concentrations and kidney function in two independent diabetes cohorts over a wide eGFR range. Low serum albumin levels, but not metabolic status (eg, hemoglobin A1c) or the inflammatory marker CRP, appeared to be linked to activin A concentration. The independent relationship between albuminuria and activin A potentially indicates that proteinuric kidney disease may be characterized by a distinctive injury with increased senescence phenotype compared with DKD without albuminuria. Finally, a co-association with biomarkers (eg, TNFR-1) that are more directly linked to inflammatory response and renal tubular injury was demonstrated both in adults with diabetes and in a cell culture model of diabetic tubulopathy. Collectively, these findings provide novel evidence that maladaptive inflammation and cellular senescence track together in DKD and that therapies designed to target both pathways (eg, anti-activin, senolytic and regenerative cell-based therapies) could be pursued alongside informative biomarker assays. Activin A is produced by a variety of tissues, including inflamed adipose tissue and injured kidney cells. It is postulated that in the normal, non-injured kidney, glomerular filtered activin A is reabsorbed by renal tubules via endocytosis, which in the setting of tubular dysfunction, contributes to increased urine concentrations. 19 This could explain why higher urine activin A levels (not normalized for creatinine) in individuals with AKI did not strongly correlate with plasma activin A levels. 19 However, animal studies also suggest that activin A originates from injured kidney cells. In experimental studies, elevated activin A was derived directly from renal tubule epithelial cells in streptozotocin-treated 38 and UUO 25 rat models, from myofibroblasts in Alport CKD mice, 22 25 renal interstitial cells in Ldlr –/– high fat-fed mice with ablative CKD, 22 infiltrating macrophages in lupus nephritis mice 23 and glomerular culture supernatants in an anti-Thy1 glomerulonephritis rat model. 39 Furthermore, while UUO induced activin A gene expression in the kidney and its release into the circulation, these findings were not observed in the plasma, contralateral kidney or remnant kidney of unilaterally nephrectomized rats having similar fall in total GFR, suggesting the obstructed kidney as the source. 24 In our human diabetes studies, albuminuria, reflective of kidney injury, directly correlated with activin A in plasma and urine. Plasma activin A also independently and directly correlated with TNFR-1, a robust biomarker for progressive DKD. 28 30 40 41 Notably, an upsurge in urinary activin A levels was only detected in those with significant proteinuria, whereas the relationship between its plasma levels and UACR was more continuous ( figure 4 ), supporting a link between albumin-induced tubular injury and urinary activin A. Moreover, given that in our study urine activin A concentrations rose as plasma levels increased in the setting of reduced GFR, high plasma activin A levels in DKD may be the result of multiple mechanisms, but are unlikely to reflect reduced clearance. Cumulatively, these studies provide insight into the influence of kidney injury, rather than impaired renal handling alone, on circulating activin A levels. This study is strengthened by use of the largest cohort of individuals with DKD (or CKD) to date to undergo measurement of plasma and urine activin A. Harada et al 42 previously found higher circulating activin A concentrations in older individuals and those with cirrhosis, advanced solid cancer, pregnancy and CKD (n=41) compared with non-diseased controls. Nonetheless, despite the pathophysiological relevance and potential for clinical application, subsequent studies assessing the relationship between kidney function and activin A levels have been sparse. Recently, in over 400 older community dwelling adults in Taiwan (19% diabetes; mean eGFR 82.5 mL/min/1.73 m 2 ), Peng et al 43 showed that eGFR was lowest in the highest tertile of plasma activin A. After adjustment for confounding variables, however, only older age, metabolic syndrome and uric acid remained associated with higher tertile status. In our study of over 200 individuals with diabetes (55% with eGFR <60 mL/min/1.73 m 2 ; mean eGFR 41.4 mL/min/1.73 m 2 ), we identified an inverse association between circulating activin A and eGFR. This finding may reflect increased tissue inflammation, cellular senescence burden and/or activin A production by injured kidney cells. While kidney injury related closely to circulating activin A levels in our study, other systemic processes have been associated with activin A. Despite the known relationship with inflammation, in our study, the inflammatory marker CRP did not correlate with activin A concentrations. Nonetheless, serum albumin, a marker of nutritional status and inflammation in CKD, 44–46 correlated inversely with activin A. Surprisingly, metabolic status, indicated through current glycemic control (hemoglobin A1c), was not a significant influencer of activin A levels beyond kidney function. Our in vitro studies explored the effects of glucose, albumin and TGF-β1 on activin A gene expression and protein release by human kidney cells. Notably, high glucose exposure alone was insufficient to activate release of activin A by HK-2 cells. Yet, the addition of albumin to high glucose-containing medium evoked this response. Moreover, our diabetic tubulopathy model of high glucose plus TGF-β1 led to marked upregulation of inflammatory and profibrotic EMT markers followed by release of activin A robustly into cell culture supernatant. Together, the associations between serum albumin, urine protein excretion rate (albuminuria) and activin A suggest proteinuric kidney disease relates to more robust inflammatory signatures and increased senescent cell abundance compared with CKD without albuminuria. Based on these relationships, we tested gene expression of senescence markers in HK-2 cells and found a modest rise in p16 levels after DKD injury. Possibly, a 48-hour exposure was insufficient to upregulate these genes in HK-2 cells and activin A represents an early mediator of the pro-inflammatory, profibrotic state associated with DKD senescence. Nonetheless, our studies support the notion that activin A relates to kidney injury associated with changes in kidney function, increased cellular senescence and possibly adipose tissue dysfunction in individuals with DKD. Beyond being a potential biomarker of DKD injury or cellular senescence, activin A plays an active role in kidney damage and might thus constitute a therapeutic target. In a prior study, we demonstrated that clearance of senescent cells in DIO mice improved podocyte function. 15 More in-depth studies of activin A-induced kidney dysfunction have been performed by others. Notably, activin A-induced kidney dysfunction in a Ldlr −/ − CKD mouse model (fibrosis and proteinuria) and in a polycystic kidney disease mouse model (fibrosis and cyst formation) was decreased by blocking activin A effect via the activin receptor type IIA ligand trap, RAP-011, and a soluble activin receptor IIB fusion protein. 22 47 Anti-activin therapy tested in early phase clinical trials of muscular diseases, osteoporosis, postmenopausal anemia and cancer showed variable side effects and efficacy. 48 49 Additional efforts are also underway to modify the CKD-associated SASP in clinical trials of DKD (Clinicaltrials.gov: NCT02848131 ; NCT03325322 ), 12 50 with recent evidence illustrating that senolytics reduce senescent cell abundance in humans. 51 Our study had limitations. First, the cross-sectional study design did not permit assessment of causal relationships among diabetes, kidney function and circulating activin A concentrations. Reverse causality still remains a possibility as reduced GFR may increase activin A levels via reducing their renal clearance thus prospective studies are needed. Second, the study population was mostly white, and the sex distribution was unequal between controls and diabetes, limiting generalizability. However, we did not detect an association between sex and activin A levels in these cohorts. Furthermore, the Galway cohort was specifically enrolled from general diabetes, nephrology and diabetic nephropathy clinics to parallel the environment found in community-dwelling individuals, therein improving generalizability. Third, while this is the first and largest study in a predominantly DKD population, it had a modest sample size, limiting statistical power. Nonetheless, findings were predominantly consistent between the two distinct cohorts. In conclusion, we observed in two independent cohorts elevated circulating activin A levels in individuals with diabetes, which correlated inversely with kidney function and directly with albuminuria and other kidney injury markers. In vitro studies demonstrated that activin A is produced directly in copious amounts by renal tubular cells exposed to high glucose plus TGF-β1 or albumin, likely contributing to overproduction of circulating activin A. Ongoing prospective follow-up of these findings will allow us to assess the power of plasma activin A as a marker of DKD progression and therapeutic success in the future. The authors would like to thank all study participants and study coordination team members of Mayo Clinic: Donna K Lawson CCRP, LPN, Jennifer M Manggaard CCRP, Tammie L Volkman RN, Erin Wissler Gerdes, Marcia K Mahlman CCRP, Beverly K Tietje CCRP and Tamara K Evans. The authors would like to thank Shannon K Meier for secretarial support. The authors would also like to thank the scientific, nursing and medical staff at the Centre for Endocrinology, Diabetes and Metabolism and the Department of Clinical Biochemistry, Saolta University Health Care Group (SUHCG), Galway University Hospitals. Contributors All authors contributed in the following: substantial contributions to the conception or design of the work, or the acquisition, analysis or interpretation of data. Drafting the work or revising it critically for important intellectual content. Final approval of the version published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Funding This project was supported by funding from the Extramural Grant Program of Satellite Healthcare (LJH), Regenerative Medicine Minnesota (RMM 091718; LJH); National Institute of Health (NIH) grant K23 DK109134 (LJH), UL1 TR002377 (LJH; Mayo Clinic), UL1TR000135 (LJH; Mayo Clinic) and Mayo Center for the Science of Health Care Delivery (LJH, Kern Scholar Program). Additional support was provided by NIH grants R01 DK102325 (LOL), R01 DK120292 (LOL), R01 DK100081 (SCT), T32 DK07013 (SMC), DK106427 (AE), DK118120 (SMH), K23 DK114497 (RGMcC) and AG13925 (JLK), the Ted Nash Long Life and Noaber Foundations (JLK), the Connor Group (JLK) and Robert J. and Theresa W. Ryan (JLK). XB is supported by Natural Science Foundation of Liaoning Province (No. 2015020490). TPG is supported by a Hardiman Scholarship from the College of Medicine, Nursing and Health Science, National University of Ireland Galway and a bursary from the Irish Endocrine Society/Royal College of Physicians of Ireland. The Galway authors are supported by grants from the European Commission (Horizon 2020 Collaborative Health Project NEPHSTROM) (grant number 634086; TPG, MNI, MDG), Science Foundation Ireland (REMEDI Strategic Research Cluster (grant number 09/SRC-B1794; MDG), CÚRAM Research Centre (grant number 13/RC/2073; MDG)), and the European Regional Development Fund. Competing interests None declared. Patient consent for publication Not required. Ethics approval Mayo Clinic Institutional Review Board, Clinical Research Ethics Committees, Galway University Hospitals (Ref: C.A. 1404) and the National University of Ireland Galway, Research Ethics Committee (Ref: 16-July-05) approved these studies. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information. Publisher Copyright: © Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

PY - 2019/12/15

Y1 - 2019/12/15

N2 - Objective Activin A, an inflammatory mediator implicated in cellular senescence-induced adipose tissue dysfunction and profibrotic kidney injury, may become a new target for the treatment of diabetic kidney disease (DKD) and chronic kidney diseases. We tested the hypothesis that human DKD-related injury leads to upregulation of activin A in blood and urine and in a human kidney cell model. We further hypothesized that circulating activin A parallels kidney injury markers in DKD. Research design and methods In two adult diabetes cohorts and controls (Minnesota, USA; Galway, Ireland), the relationships between plasma (or urine) activin A, estimated glomerular filtration rate (eGFR) and DKD injury biomarkers were tested with logistic regression and correlation coefficients. Activin A, inflammatory, epithelial-mesenchymal-transition (EMT) and senescence markers were assayed in human kidney (HK-2) cells incubated in high glucose plus transforming growth factor-β1 or albumin. Results Plasma activin A levels were elevated in diabetes (n=206) compared with controls (n=76; 418.1 vs 259.3 pg/mL; p<0.001) and correlated inversely with eGFR (r s =-0.61; p<0.001; diabetes). After eGFR adjustment, only albuminuria (OR 1.56, 95% CI 1.16 to 2.09) and tumor necrosis factor receptor-1 (OR 6.40, 95% CI 1.08 to 38.00) associated with the highest activin tertile. Albuminuria also related to urinary activin (r s =0.65; p<0.001). Following in vitro HK-2 injury, activin, inflammatory, EMT genes and supernatant activin levels were increased. Conclusions Circulating activin A is increased in human DKD and correlates with reduced kidney function and kidney injury markers. DKD-injured human renal tubule cells develop a profibrotic and inflammatory phenotype with activin A upregulation. These findings underscore the role of inflammation and provide a basis for further exploration of activin A as a diagnostic marker and therapeutic target in DKD.

AB - Objective Activin A, an inflammatory mediator implicated in cellular senescence-induced adipose tissue dysfunction and profibrotic kidney injury, may become a new target for the treatment of diabetic kidney disease (DKD) and chronic kidney diseases. We tested the hypothesis that human DKD-related injury leads to upregulation of activin A in blood and urine and in a human kidney cell model. We further hypothesized that circulating activin A parallels kidney injury markers in DKD. Research design and methods In two adult diabetes cohorts and controls (Minnesota, USA; Galway, Ireland), the relationships between plasma (or urine) activin A, estimated glomerular filtration rate (eGFR) and DKD injury biomarkers were tested with logistic regression and correlation coefficients. Activin A, inflammatory, epithelial-mesenchymal-transition (EMT) and senescence markers were assayed in human kidney (HK-2) cells incubated in high glucose plus transforming growth factor-β1 or albumin. Results Plasma activin A levels were elevated in diabetes (n=206) compared with controls (n=76; 418.1 vs 259.3 pg/mL; p<0.001) and correlated inversely with eGFR (r s =-0.61; p<0.001; diabetes). After eGFR adjustment, only albuminuria (OR 1.56, 95% CI 1.16 to 2.09) and tumor necrosis factor receptor-1 (OR 6.40, 95% CI 1.08 to 38.00) associated with the highest activin tertile. Albuminuria also related to urinary activin (r s =0.65; p<0.001). Following in vitro HK-2 injury, activin, inflammatory, EMT genes and supernatant activin levels were increased. Conclusions Circulating activin A is increased in human DKD and correlates with reduced kidney function and kidney injury markers. DKD-injured human renal tubule cells develop a profibrotic and inflammatory phenotype with activin A upregulation. These findings underscore the role of inflammation and provide a basis for further exploration of activin A as a diagnostic marker and therapeutic target in DKD.

KW - adipocytokine

KW - clinical aspects of diabetes

KW - clinical nephrology

KW - renal fibrosis

UR - http://www.scopus.com/inward/record.url?scp=85077128763&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85077128763&partnerID=8YFLogxK

U2 - 10.1136/bmjdrc-2019-000720

DO - 10.1136/bmjdrc-2019-000720

M3 - Article

C2 - 31908790

AN - SCOPUS:85077128763

SN - 2052-4897

VL - 7

JO - BMJ Open Diabetes Research and Care

JF - BMJ Open Diabetes Research and Care

IS - 1

M1 - e000720

ER -

Senescence marker activin A is increased in human diabetic kidney disease: Association with kidney function and potential implications for therapy

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