Progress in the Early Detection of Risk of End-Stage Kidney Disease in Diabetes at Last?

Diabetes mellitus is the most common cause of end-stage kidney disease (ESKD) in the US and Europe. Approximately one-third of type 1 diabetes patients and 40% to 50% of patients with type 2 diabetes develop ESKD. At this stage, dialysis or renal transplantation is required to manage these patients. Diabetic kidney disease (DKD) frequently results in a progressive decline in the renal glomerular filtration rate and loss of the ability of the kidney to filter and retain plasma proteins, which produce albuminuria and proteinuria. Kidney Disease: Improving Global Outcomes (KDIGO) and other international medical organizations that publish expert guidelines for management of chronic kidney disease (CKD) have recommended monitoring these patients by testing both estimated glomerular filtration rate (eGFR) and for persistent albuminuria. These 2 measures provide reasonable estimates of the renal perfusion and filtration functions and the ability of the kidney to retain circulating proteins as part of its normal function (1).

For more than a decade eGFR and albuminuria have been the key biochemical parameters for monitoring patients with CKD. More recently, methods for estimating GFR have been updated and improved by incorporating measurement of plasma or serum cystatin C as a replacement for, or in addition to, measurement of serum creatinine (2). Furthermore, the equations for estimating GFR have been updated to remove the race-based modifiers which introduced unintentional bias to these tests for populations most at risk (3). Application of these improved tools for assessment of kidney function allows physicians to predict risk of death and progression to ESKD, offering the best available information regarding prognosis and adequacy of renal care. Nevertheless, these tests are limited because they reflect the sequelae of kidney disease after the damage is done; what is urgently needed are biomarkers that reflect the pathophysiology of kidney disease, so that we may interrogate the mechanisms of disease progression and find novel additional targets for slowing the progression of kidney failure.

Among the multiple candidate biomarkers that may reflect risk of progression of CKD and may contribute directly to the pathophysiology are the transforming growth factor (TGF)-β/bone morphogenic protein/activin family of signaling proteins and their receptors. Numerous published studies have implicated TGF-β1 and its downstream signaling partners in the dysfunctional process of renal interstitial fibrosis (4). In a new study published in Science Translational Medicine, Kobayashi et al. (5) present intriguing new findings interrogating the associations between circulating levels of TGF-β1 family members and the risk of progression of DKD.

The authors selected 4 independent, well-characterized cohorts of subjects from the Joslin Kidney Study and Pima Indian Kidney Study: a discovery cohort, a replication cohort, and 2 validation cohorts. Patients were at different stages of DKD and had either type 1 or type 2 diabetes. Initially, 1129 plasma proteins in 113 (56 with ESKD) subjects with type 1 diabetes were measured on the SOMAscan proteomic assay platform. Concentrations of 25 circulating proteins that modulate TGF-β signaling were subsequently measured in 754 subjects. These experiments revealed significant associations between progression to ESKD and increased concentrations of neuroblastoma suppressor of tumorigenicity 1 protein (NBL1), a secreted protein known to inhibit some bone morphogenic protein-signaling proteins. The 10-year risk of progression to ESKD in patients with a 1-SD increase in NBL1 concentrations was nearly 3-fold higher than in controls. Importantly, subjects in the highest NBL1 quartile had a significantly greater risk of ESKD at 10 years than those in the lowest quartile. The association was strong and significant in all 4 cohorts, in both type 1 and 2 diabetes, and occurred during both early and late stages of DKD. Furthermore, although NBL1 concentrations were associated with baseline levels of albuminuria, mediation analysis found that only 27% of the association between NBL1 and future risk of DKD progression could be explained by albuminuria at baseline. These data indicated that NBL1 concentrations were independent predictors of progression. Even more intriguingly, from a mechanistic standpoint, examination of 105 kidney biopsies revealed that circulating NBL1 concentrations were significantly correlated with renal histopathologic markers of disease progression, including podocyte damage, mesangial expansion, glomerular filtration barrier disruption, and kidney fibrosis. Cell culture experiments showed that incubating cultured human podocytes, renal tubular cells, or mesangial cells with NBL1 caused significant cell death, albeit at µg/mL concentrations, which are significantly higher than the pg/mL concentrations in the blood.

In addition to the association between increased plasma concentrations of NBL1 and progression of DKD, the authors observed significantly increased NBL1 levels in the urine of patients with progression, indicating that something was increasing the systemic synthesis and secretion of NBL1. The authors were not able to identify what organ or cell type was secreting NBL1 (healthy kidneys have minimal NBL1), but they were able to demonstrate that immunohistochemical accumulation of NBL1 proteins in glomerular and renal interstitial macrophages of diabetic patients was greater than that in non-diabetic controls, consistent with increased receptor binding and activity at these locations. They also showed that the 10-year risk of ESKD associated with NBL1 remained significant after adjusting for other known risk factors (eGFR, albumin/creatinine ratio, Hb A_1c) and improved the accuracy of predictive models that included these other risk factors (as indicated by C-statistic and NRI [net reclassification improvement] analyses). Taken together, these findings emphasize the significance of the association between NBL1 and risk, independent of other well-documented risk factors.

The study has several limitations that are particularly important if NBL1 is to be translated into a biomarker with clinical relevance. The SOMAscan aptamer assay platform used to measure NBL1 values reported in the main manuscript was analytically validated by comparison to a more traditional antibody-based sandwich immunoassay on the Meso Scale Discovery (MSD) analyzer. Although the validation demonstrated that the relative fluorescence units on the SOMAscan were significantly correlated with concentrations of NBL1 by immunoassay (Spearman correlation between log₁₀-transformed SOMAscan Relative Fluorescence Intensities and MSD protein concentrations in pg/mL r_s = 0.85, P = 2.8 × 10⁻²⁸), the correlation was somewhat diffuse and the relationship was non-linear (Supplemental Fig. S9 in Ref. (5)). These findings indicate that NBL1 relative fluorescence units measured by SOMAscan increased disproportionately in comparison to concentrations measured by MSD after the log transformation is removed. Furthermore, the SOMAscan measurements were reported in relative fluorescence units without calibrating the assay using purified standards, rendering it difficult to evaluate the analytical specificity of the assay or how high and low absolute NBL1 protein concentrations were in the cases compared to controls. Furthermore, while the SOMAscan assay may be a useful platform for multiplex proteomic screening experiments, it is not really suited for high-throughput economical quantitative measurements that would be required for clinical use. Although the MSD immunoassay platform (described in the Supplementary Materials in Ref. (5)) that the authors used for validation experiments is an alternative that in theory could be used for clinical testing, this method is somewhat labor-intensive, requires multiple separate steps, and includes overnight incubation at 4°C. Moreover, the description of the assay provided in the manuscript lacks sufficient characterization to consider it for clinical use. No information is provided about the antibodies used in the assay or standard assay characteristics, e.g., analytical specificity, linearity, imprecision, and interferences are not provided. Confirmation of the findings by other investigators using an accurate and specific assay is needed. Finally, the clinical component of their study was exclusively observational and needs to be validated in prospective studies.

In summary, Kobayashi and colleagues (5) adopted a targeted, multipronged strategy that combined “bench” and “bedside” techniques to identify an intriguing candidate biomarker for predicting the progression of DKD to ESKD. The findings appear to be applicable to type 1 and type 2 diabetes and to patients of different backgrounds. Importantly, the study opens up several avenues of exploration to further interrogate NBL1 as a potential early prognostic marker of ESKD in patients with diabetes. In order to conduct additional clinical studies, a well-characterized, cost-effective, high-throughput assay needs to be developed. In addition, the tantalizing early data support further studies to explore the role of this TGF-β family member in renal pathophysiology to determine whether it contributes to the development of DKD and if it is a potential therapeutic target.

Nonstandard Abbreviations

ESKD, end-stage kidney disease; DKD, diabetic kidney disease; eGFR, estimated glomerular filtration rate; TGF, transforming growth factor; NBL1, neuroblastoma suppressor of tumorigenicity 1 protein; MSD, Meso Scale Discovery.

Author Contributions

The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list.

Authors’ Disclosures or Potential Conflicts of Interest

Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership

D.B. Sacks, Clinical Chemistry, AACC.

Consultant or Advisory Role

None declared.

Stock Ownership

None declared.

Honoraria

None declared.

Research Funding

D.B. Sacks, Intramural Research Program of the National Institutes of Health.

Expert Testimony

None declared.

Patents

None declared.