To our knowledge, this is the first study of uromodulin concentrations undertaken in Japanese patients with chronic kidney disease. The results of this study are consistent with previous reports of a strong correlation between the serum uromodulin concentration and kidney function [3-5] and suggest that the serum uromodulin could be a unique biomarker in clinical nephrology. It had been thought that uromodulin is expressed only in renal tubules and that almost all of it is excreted in urine after undergoing proteolytic cleavage. However, in 1981, Dawnay et al. investigated blood uromodulin concentrations and identified trends between the serum uromodulin concentration and kidney disease, such as a level in the range of 70–540 ng/mL in patients with normal kidney function and undetectable serum uromodulin in patients who are anephric [16]. The first report of a good correlation between blood uromodulin concentration and kidney function was published by Thornley et al. in 1985 [17]. However, since then, there have been no detailed studies on blood uromodulin levels. In 2010, Prajczer et al. investigated the relationship between the serum uromodulin concentration and kidney function in healthy volunteers and CKD patients and did not find a significant correlation [18]. Although the authors analyzed serum samples using a custom-made ELISA, those concentrations were extremely low compared with others [3-5, 17]. It is possible that they were not fully evaluated. However, Steubl et al. [3] and Fedak et al. [4] in 2016 and Scherberich et al. [5] in 2018 reported a good correlation between the plasma/serum uromodulin concentration and kidney function using commercially available ELISA kits manufactured by BioVendor (Brno, Czech Republic) and Euroimmun AG (Lübeck, Germany).
We started by obtaining monoclonal antibodies de novo and then established a custom-made ELISA kit. This kit was prepared as a sandwich-type ELISA using two types of monoclonal antibodies recognizing different antigenic epitopes. As a preanalytical investigation, we confirmed that the accuracy of the kit was acceptable. Subsequently, we confirmed that the uromodulin protein was stable with no change in the measured value despite some variation in storage conditions; however, blood samples were handled suitably in this study. Scherberich et al. also reported the stability of uromodulin protein [5]. Stability is a very important issue when considering a protein for use as a biomarker. If the entity to be measured decomposes easily, its clinical application is not realistic. In this regard, uromodulin meets one of the requirements for use as a biomarker. Next, the uromodulin concentration was found to vary between serum and plasma samples. Previous studies have evaluated serum [4,5] and plasma samples [3] and both have shown a good correlation between blood uromodulin and GFR. In this study, we only analyzed serum samples.
Kidney function is indicated by the GFR, accurate measurement of which is best achieved by the inulin clearance test. However, this test is rarely performed because of its complexity. Kidney function has recently been evaluated by eGFR values calculated from creatinine and/or cystatin C. Although equations from the Modification of Diet in Renal Disease Study [19] and the Chronic Kidney Disease Epidemiology Collaboration [20] have been used internationally, they tend to overestimate kidney function in the Japanese population. Therefore, we Japanese should use the eGFR equations established for Japanese population [14,15]. eGFR is convenient for clinical use but not without problems. Given that eGFR is calculated from creatinine and/or cystatin C, its value is affected by numerical fluctuations in these parameters, which may not necessarily be directly related to kidney function. It has been shown that the creatinine concentration is affected by muscle mass and fluid volume and that the cystatin C concentration is affected by thyroid function [21] and several drugs [22]. Kazama et al. reported a 72% positive rate below 50 mL/min of GFR by creatinine measurement and an 85% positive rate below 70 mL/min of GFR by cystatin C measurement when assessing the detection power of decreased kidney function [23]. Therefore, cystatin C is more useful than creatinine for detecting an early decline in kidney function. In contrast, it is known that cystatin C, unlike creatinine, has a ceiling effect at about 5–6 mg/L, and if it exceeds 2 mg/L, the clinical significance of estimating kidney function by cystatin C is low.
Interestingly, the serum uromodulin concentration is positively correlated with GFR whereas conventional kidney function markers, such as creatinine, cystatin C and blood urea nitrogen, are negatively correlated with GFR. Moreover, the serum uromodulin concentration has a linear regression relationship with eGFR values. Intuitively, it would seem more advantageous to use a number with a positive correlation and linear regression relationship than a number with an inverse correlation and nonlinear regression relationship.
Given the difficulties involved in evaluating residual kidney function in patients on maintenance dialysis, their data were excluded from the scatter diagram in this study. However, there is no doubt that the GFR is <5 mL/min in these patients. In the present study, the mean eGFR-cre value in our patients on maintenance dialysis was 4.2 mL/min/1.73m2. Taking into account the error in the eGFR value itself and the patterns of uromodulin concentration, that is, high in subjects with good kidney function and low in those with decreased kidney function, it might not necessarily be a mistake to incorporate data for patients on maintenance dialysis into a scatter plot. When these data were included, the correlation coefficient for serum uromodulin concentration and the eGFR value naturally increased (data not shown).
Uromodulin is a protein derived from renal tubular cells. Although most of the uromodulin is excreted into the urine, a very small amount of uromodulin that is expressed on the basolateral side of the tubular cells is not cleared and enters the circulation via blood vessels in the interstitial area. Thornley et al. reported that urinary excretion of uromodulin decreases in patients with decreased kidney function and tubular damage, and suggested that the serum uromodulin concentration decreases with worsening CKD stage because of a decreased expression level in the renal tubules [17].
Serum uromodulin could be a useful biomarker for evaluation of kidney functional reserve in all stages of CKD. However, little is known about the factors that regulate its protein expression and affect uromodulin concentrations. Furthermore, it is not known if renal and serum uromodulin protein expression levels show individual or racial differences. Given that no standard reference protein for uromodulin measurement has been established, we cannot directly compare our data with those in the past reports. Nonetheless, similar results have been reported using other ELISA kits or method [3-5, 17], and suggest that there is a certain value in the usefulness of uromodulin measurement.
Analysis of mean serum uromodulin concentrations using the eGFR-cre value-based staging system, a significant decrease in serum uromodulin concentration was observed below an eGFR-cre value of 60 mL/min/1.73m2 (Figure 2A). On analysis of mean serum uromodulin concentrations using the eGFR-cys value-based system, a significant decrease in serum uromodulin concentration was observed in 60–89 mL/min/1.73m2 of eGFR-cys (Figure 2B). Furthermore, when the classification according to the eGFR-cys value was subdivided and analyzed, a significant decrease in the serum uromodulin concentration was observed below eGFR-cys 90 mL/min/1.73m2 (Figure 2C). These results suggest that early decline of kidney function could be detected by serum uromodulin measurement as in previous reports [3-5].
When we evaluated the scatter plot as a whole, there was a strong positive correlation between the serum uromodulin concentration and the eGFR value but there is also considerable variation in the serum uromodulin concentration in the population with relatively good kidney function, that is, an eGFR >60 mL/min/1.73 m2. This trend is similar to the past reports [3-5]. Thornley et al. found a good correlation between serum uromodulin concentration and kidney function in patients with CKD but not in normal subjects (17). Risch et al. investigated the serum uromodulin concentration in patients with good kidney function, whose average eGFR-cys value was 85 mL/min/1.73m2, and found only a weak correlation of r = 0.38 [24].
It is widely known that the neonatal glomerulus is immature and that the GFR at birth is one-fourth to one-fifth that of an adult. Furthermore, the nephron number in an adult has been estimated to be one million per kidney; however, recent studies have shown that the nephron number in a Japanese adult is about 0.7 million per kidney [25] and that the glomerular density in a low-birth-weight infant is significantly low [26]. These reports suggest that there may be racial and individual differences in both the nephron number and their development. However, because the glomerulus is basically equipped with a hyperfiltration mechanism, a normal glomerulus can perform more than twice as much work as usual. Therefore, a slight variation in nephron number is unlikely to affect the GFR value. Furthermore, uromodulin is a tubule-derived protein and is not filtered by the glomeruli because of its high molecular weight. Variation of serum uromodulin concentration in groups with relatively good kidney function may reflect the number of functioning nephrons.
The eGFR value is an estimate of kidney function and also indicates the current “residual” kidney function. Looking at uromodulin concentration from the perspective of evaluating residual kidney function, we believe that uromodulin can be used not only as a biomarker for predicting kidney function but also as a novel tool in the field of preventive medicine. It should be possible to measure uromodulin levels at a young age, when kidney function is normal and before the onset of conditions such hypertension, diabetes, and chronic glomerulonephritis, all of which increase the risk of renal impairment. It is still unclear whether individuals with a high uromodulin concentration have high renal reserve and those with a low uromodulin concentration have poor renal reserve. However, there are two case reports on patients who were found to have a decreased uromodulin concentration related to allograft failure and graft loss after kidney transplantation [27,28]. These reports suggest that the higher the uromodulin concentration, the greater the renal reserve.
This study has some limitations, the main one being that we compared only the serum uromodulin concentration with the eGFR value and did not include an inulin clearance test. The eGFR is only an estimated value and does not necessarily guarantee equivalence with the inulin clearance value; however, evaluation of kidney function based on the eGFR value is well accepted in daily clinical practice. We evaluated the relationship between the uromodulin concentration and the eGFR-cre and eGFR-cys values and found that both were highly correlated.