Urine albumin or albuminuria is one of the most important biomarkers of kidney damage in individuals with or without diabetes. In healthy individuals, the glomerular filter in the kidneys retains most of the albumin, although a small amount can usually pass through to the tubular system (1). Reabsorption of albumin is facilitated by the kidney’s proximal tubular cells (PTCs), ensuring that almost no albumin is excreted in urine under normal conditions (2, 3). Elevated excretion of albumin in the urine - initially coined as “microalbuminuria” - is one of the earliest signs of chronic kidney disease (CKD) and may be the kidney-related manifestation of general endothelial damage, where scarring of the glomerulus causes chronic leakiness through the filter of albumin and other proteins (4).

Over the past decades, the number of people with diabetes mellitus has more than doubled to a global prevalence of 537 million in 2021 (5), with serious consequences for the healthcare system and society. According to a recent European study (6), one in four hospitalized patients has diabetes. Up to 40% of individuals with diabetes develop diabetic kidney disease (DKD), which is associated with elevated cardiovascular morbidity and mortality and progresses to dependency on kidney replacement therapies such as dialysis and transplantation and is a leading cause of CKD (7).

In the recent years, studies have begun to unravel genetic aspects of albuminuria. Recently, we and others identified that genetic variants (single nucleotide variants (SNVs)) in the gene encoding for cubilin (CUBN) – the main albumin-transporter in PTCs (1, 8) – are associated with microalbuminuria and higher urine albumin levels in populations with and without diabetes (8–14). Four variants in the C-terminal end of cubilin have been of particular interest (rs141640975 (c.5069C>T; p.Ala1690Val), rs144360241 (c.6469A>G; p.Asn2157Asp), rs45551835 (c.8741C>T; p.Ala2914Val), and rs1801239 (c.8950A>G, p.Ile2984Val)); these are functional (missense) variants that have been proposed to alter the function of cubilin, leading to a form of albuminuria that may reflect a lack of tubular reabsorption of albumin (i.e., tubular albuminuria) (8). In silico structural and damage prediction analyses of the variants indicate their potential to change secondary or even tertiary structure(s) in the cubilin protein and to have different degrees of damaging effects on protein function, disease, or both (8). Our recent study further suggests that the effect of some of these variants on urine albumin levels is 2-3 times higher in diabetes compared to non-diabetes (11).

However, the role of these CUBN variants in relation to estimated glomerular filtration rate (eGFR), a clinically used marker of kidney function, is largely unexplored, and most genetic studies have focused on the general population (8, 9, 11). Recent efforts to uncover the role of these variants specifically in diabetes – and to clearly separate the effect seen here from the effect in the non-diabetes-proportion of the general population – have been performed as relatively small secondary analyses without including rs144360241 or diabetes subtypes (8). Thus far, only rs45551835 has been connected to higher levels of eGFR in type 2 diabetes and rs141640975 in non-diabetes (8). Therefore, we investigated the relationship between the four CUBN variants and eGFR in different contexts: First, we meta-analyzed studies of SNV-eGFR_creatinine regressions in Europeans with type 1 (T1D) or type 2 diabetes mellitus (T2D). We then examined single- and aggregate-variant associations separately in diabetes and non-diabetes populations of a large, nationally representative cohort facilitating application of identical phenotype definitions, including the dependency of albuminuria-stage in SNV-eGFR_creatinine associations, generation of a CUBN-specific genetic risk score (GRS), and identification of associations between individual SNVs and cystatin C-based measures of kidney function. Together, these analyses both seek to replicate previous associations in DM and NDM populations and to provide novel insights into the link between CUBN and eGFR.

Recently, we demonstrated that individuals carrying the minor allele of the CUBN missense variant rs141640975 had higher albuminuria-levels than non-carriers. The effect of this variant was stronger in individuals with diabetes (DM) compared to those without diabetes (NDM) (11). In continuation of these findings, Bedin et al. (8) performed secondary lookups for CUBN-variants in the CKDGen eGFR GWAS study population, reporting that missense variants in CUBN may also be associated with higher levels of eGFR in the general population. Our current large-scale study aimed to examine the effect of minor alleles of three rare CUBN missense variants (rs144360241 (c.6469A>G; p.Asn2157Asp), rs45551835 (c.8741C>T; p.Ala2914Val) and rs141640975 (c.5069C>T; p.Ala1690Val)) and one common variant (rs1801239 (c.8950A>G; p.Ile2984Val)) on eGFR_creatinine levels separately in people with and without diabetes (n_DM ~ 34,000 individuals, n_NDM ~ 370,000 individuals), including stratification for diabetes-type and supplemented by tests on circulating cystatin C levels, the recently updated eGFR-equation based on creatinine and cystatin C (30), and aggregate-variant tests. We were able to replicate the association between creatinine-based eGFR and rs141640975 in NDM and report new insightful connections with the alternative measures of kidney function for all four SNVs.

Previously, a borderline association between rs45551835 and higher eGFR-levels has been reported in a smaller type 2 diabetes (T2D) population from Denmark (8, 11), a finding which we could not replicate in our meta-analysis of up to 34,432 individuals with diabetes and its subtypes. Like the initial study (8), we could not establish a link between eGFR and the three other variants within the diabetes group. As for rs45551835, it was surprising to be unable to replicate the earlier findings as the current study has a larger sample size compared to earlier efforts. Our post-hoc power assessment indicated that insufficient power might be at play, even with a larger sample size for the diabetes group (8). We also speculated whether the apparent lack of association between CUBN and eGFR in our diabetes meta-analysis could be due to use of Angiotensin receptor blockers (ARBs) or Angiotensin-converting enzyme inhibitor (ACEi) medication which is frequently used in diabetes treatment. As part of our sensitivity analyses, we included models adjusted for systolic blood pressure (a proxy for such medication) and did not find evidence that this could explain why no association was found in the diabetes group. Another reason could be the allele frequency of the variants may differ between Danish and UK populations. We need further validation in well-powered populations to confirm the relationship between the rs45551835 and eGFR in diabetes, especially in T2D. In case of a true lack of association, CUBN may be associated with higher levels of urine albumin (11) with no pleiotropic effect to eGFR in this population.

We proceeded to single- and aggregate-variant analyses in the UK Biobank (UKBB), shifting focus to non-diabetes populations. For all four CUBN variants, we report significantly higher eGFR_creatinine-levels in individuals without diabetes harboring more copies of the minor alleles compared to individuals with fewer or no copies of the minor alleles in the same group. For rs141640975, we observed the strongest association with eGFR_creatinine (P = 2.2 × 10^-9) with replication in the Icelandic study (deCODE, P = 7.7 × 10^-4), confirming what has previously been observed for this SNV in NDM (8) – but also a significant interaction between the SNV and albuminuria stages (P_INT< 0.05). Taken together with the already known associations of the minor alleles with higher albuminuria (11), this not only demonstrates genetic pleiotropy of CUBN for albuminuria and eGFR in non-diabetes but also implies that these two associations are intertwined for this SNV, where the effect on eGFR is even higher for more elevated albuminuria-levels. Here, CUBN demonstrates a classic genetic pleiotropy phenomenon where a DNA variant influences multiple traits, usually in the same domain with concordant or sometimes discordant effects as observed earlier in complex disorders (32). Further validation of independent biological or related causal effects might be required in additional follow up studies.

This finding is unusual as there is no obvious clinical or pathophysiological explanation for such an albuminuria-eGFR pattern in the context of non-diabetes. It has been suggested that the tubular albuminuria observed in presence of C-terminal variants in CUBN has a benign or even slightly protective effect on kidney function in chronic kidney disease if glomerular albuminuria is also present (8, 33, 34). Another recent study on chronic isolated proteinuria suggests that different C-terminal CUBN variants uncouple proteinuria from glomerular filtration barrier through declined cubilin expression accompanied by aberrant amnionless (AMN) localization in renal tubules. AMN is part of the receptor complex (along with cubilin and megalin) necessary for tubular reabsorption of albumin. This is suggested to create a benign condition, not requiring any further proteinuria lowering treatment (35). In non-diabetes, where the population can be assumed to consist mostly of healthy individuals, a concept of such protectiveness is less relevant. However, it is possible that an undetected subpopulation with relevant comorbidities exists in the non-diabetes group.

Our CUBN aggregate-variant method – which was defined as a genetic risk score (GRS) combining the four variants – showed that a higher number of C-terminal CUBN risk alleles is associated with higher urine albumin and eGFR_creatinine levels and confirms both the single-variant association with higher urine albumin levels reported previously in diabetes and non-diabetes (11, 14), and the consistency of the overall effects on urine albumin levels being greater in diabetes compared to non-diabetes (10, 11). Through GRS _CUBN , we also saw that a higher number of minor alleles across the four variants was associated with higher eGFR_creatinine-levels in the UKBB population without diabetes, which is in line with our single-variant findings and the previous findings for rs45551835 (8). Using aggregate-variant methods is an optimal way to examine combined genetic effects and has been used extensively for polygenic traits (13, 36). Using GRS is highly relevant here as three of the four variants are rare and mostly present as heterozygous variants in our populations. This might substantiate with some additional power to detect effects and adds further certainty to the presence of a CUBN-eGFR relationship in non-diabetes. Nevertheless, we still do not find an association with eGFR in T2D, even when the variants are combined in a GRS.

Finally, we examined the association between the study SNVs and two alternative markers of kidney function. In non-diabetes, the minor alleles of rs141640975 and rs144360241 were associated with higher levels of eGFR_{creatinine-cystatin C}. This measure was estimated using a recent update to the equation, CKD-EPI₂₀₂₁, which does not include ethnicity and is a more precise indicator of kidney function in comparison to the CKD-EPI_{creatinine(2012)} equation which is based only on creatinine. Our results using the conventional eGFR_creatinine equation are concordant with our results from the updated equation in terms of directionality of effect and with our finding that rs141640975 is associated with lower cystatin C levels, which is another indicator of kidney function. It should be noted, though, that considering Table 1 and Supplementary Tables 4 – 6 , the 0.1% – 2.02% higher mean eGFR we report for each minor allele is modest and may reflect that individual harboring these genetic variants have normal kidney function rather than a better kidney function.

A strength of our study is the restriction to specifically diabetes- and non-diabetes-only subgroups so that effects from mixed diabetes-status are minimized. Heterogeneity is likely to be present in meta-analyses of a diverse set of cohorts originally used for different research purposes. Indeed, some of the cohorts included in our meta-analyses differ regarding available covariates and/or kidney disease status. However, we did not observe heterogeneity in our meta-analyses. In addition to this, we could minimize heterogeneity in the remainder of our analyses by using data from the UKBB, which is a nationally representative cohort facilitating application of identical phenotype definitions across subgroups. Another strength is the broad spectrum of additional analyses that we explored in the UKBB population to nuance our findings on the relationship between eGFR and CUBN. The judicious use of UKBB leveraging individual-level genotype information to investigate interaction-analyses based on albuminuria groupings is a great strength of the current study, especially for rare variants.

A major limitation is that we did not have sufficient statistical power for our meta-analyses in the diabetes group due to the limited availability of suitable datasets. Consequently, interpretations of T2D findings should not be overstated and we thus could not demonstrate, nor disprove, the presence of a CUBN-eGFR relationship in this population. Although we demonstrate that C-terminal missense variants in CUBN are associated with different measures of normal (or even higher) kidney function in non-diabetes, we emphasize that the current study is insufficient to establish causality. Finally, using multiple-testing-corrected significance thresholds might be too conservative when testing a very small number of variants from the same locus as it may remove true associations. In genome-wide studies, a conservative threshold of 5 × 10⁻⁸ is generally agreed upon for novel associations. There is less consensus on when and how to appropriately apply multiple testing correction in smaller-scale genetic studies dealing with a mixture of new and known associations. Nevertheless, we deemed that it would be fair to apply FDR-correction of the significance threshold to our primary analyses in DM and NDM.

In conclusion, the current study identifies the existence of pleiotropic genetic effects of CUBN on two facets of kidney function – albuminuria and eGFR – by reporting SNV-eGFR associations in a large study population without diabetes. The interaction between rs141640975 and albuminuria-status on eGFR_creatinine in this population and its associations with lower cystatin C and higher levels of eGFR_{creatinine-cystatin C} expands our knowledge of these variants in relation to measures of kidney function. The demonstration of a CUBN-focused GRS in relation to albuminuria and eGFR_creatinine further suggests an important role of CUBN-variants in the future personalization of chronic kidney disease management.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Ethical approval has previously been obtained locally for each individual study. The patients/participants provided their written informed consent to participate in this study.

NU, MS, PR, and TA contributed to conception and design of the study. NU wrote the first draft of the manuscript.NU, MC-G, CH, AN, AO, SS, D-AT, EA, MH, AM, EJS, MS, PR, and TA contributed to manuscript revision. D-AT, VS, KS, EA, MH, AM, PR and TA acquired data. NU, FA, MC-G, CH, AN, SS, LS, D-AT, and TA performed statistical analysis. NU, MC-G, LS, D-AT, AM, MS, PR, and TA contributed to interpretation of data. TA and MS acquired funding and TA administered this project. PR and TA supervised the project. LC and MG had other roles. All authors contributed to the article and approved the submitted version.