Urine alpha-1-microglobulin protein is a sensitive biomarker for detecting damage in the proximal renal tubules. Free kappa light chains may also be elevated in tubular damage of any origin. The aim of this study was to evaluate whether the detection of free kappa light chains in urine could show earlier tubular damage than alpha-1-microglobulin.
Patients and methodsA total of 330 analyses performed between 2020 and 2024 were selected, corresponding to patients who had an estimated glomerular filtration rate between 8−131 mL/min/1.73 m2. Those samples that were normal in first morning urine were selected and the alpha-1-microglobulin adjusted for creatinine was calculated. Taking into account the total population, 2 groups were formed: group with normal alpha-1-microglobulin adjusted for creatinine in first morning urine –NA1MG– and group with elevated alpha-1-microglobulin adjusted for creatinine in first morning urine –EA1MG–.
ResultsThe third quartile of kappa free light chain/creatinine in NA1MG was 15.32 (mg/L)/creatinine (g/L), and 39 samples had a higher value. All of them were within the interquartile range of the kappa free light chain/creatinine value of the EA1MG group (17–98.04 [mg/L]/creatinine [g/L]). In turn, 36 samples from the EA1MG group had a kappa free light chain/creatinine value < 15.32 (mg/L)/creatinine (g/L), and the remaining 126 urine samples from this group had elevated levels of both parameters, alpha-1-microglobulin and kappa free light chains.
ConclusionsFree kappa light chain/creatinine ratio (mg/L)/(g/L) > 15.32 in first morning urine shows tubular damage even with normal alpha-1-microglobulin/creatinine values (<7.08 alpha-1-microglobulin [mg/L]/creatinine [g/L]). Tubular damage would begin with isolated elevation of alpha-1-microglobulin or kappa light chains, with earlier elevation of the latter observed in younger patients. Both are more elevated when tubular damage is more severe.
La proteína alfa-1-microglobulina en orina es un biomarcador sensible para detectar daño en los túbulos renales proximales. Las cadenas ligeras libres kappa también pueden estar elevadas en casos de daño tubular de cualquier origen. El objetivo de este estudio fue evaluar si la detección de cadenas ligeras libres kappa en orina podría evidenciar un daño tubular más temprano que la alfa-1-microglobulina.
Pacientes y métodosSe seleccionaron 330 análisis realizados entre 2020 y 2024, correspondientes a pacientes con una tasa de filtrado glomerular estimada entre 8 y 131 mL/min/1,73 m2. Se seleccionaron las muestras normales en la primera orina de la mañana y se calculó la alfa-1-microglobulina corregida por creatinina. Considerando la población total, se formaron 2 grupos: grupo de orina de primera hora de la mañana con alfa-1-microglobulina normal –GA1MN– y grupo de orina de primera hora de la mañana con alfa-1-microglobulina elevada –GA1ME–.
ResultadosEl tercer cuartil de cadenas ligeras libres kappa/creatinina en la primera orina de la mañana dentro del GA1MN fue de 15,32 (mg/L)/creatinina (g/L), y 39 muestras presentaron un valor superior, encontrándose dentro del rango intercuartílico del valor de cadenas ligeras libres kappa/creatinina del grupo GA1ME (17-98,04 [mg/L]/creatinina [g/L]), estando diagnosticadas en la mayoría de las enfermedades que producen daño tubular. A su vez, 36 muestras del grupo GA1ME presentaron un valor de cadenas ligeras libres kappa/creatinina < 15,32 (mg/L)/creatinina (g/L), y las 126 orinas restantes de este grupo tenían elevados ambos parámetros, alfa-1-microglobulina y cadenas ligeras libres kappa.
ConclusionesUn cociente de cadenas ligeras libres kappa (mg/L)/creatinina(g/L) > 15,32 aislado en la primera orina de la mañana evidencia daño tubular incluso con valores normales de alfa-1-microglobulina/creatinina (<7,08 alfa 1-microglobulina [mg/L]/creatinina [g/L]). El daño tubular comenzaría por la elevación aislada de alfa-1-microglobulina o cadenas ligeras libres kappa de forma aislada, observándose una elevación más temprana de estas últimas en pacientes más jóvenes. Ambas están más elevadas cuando el daño tubular es más intenso.
Renal tubules are the main component of the renal interstitium.1 Low molecular weight proteins that normally pass through the glomerulus are reabsorbed by the megalin and cubilin receptors of tubular cells.2 In the event of tubular damage, these receptors decrease their reabsorptive capacity, leading to increased urinary excretion of these proteins. Among these proteins, alpha-1-microglobulin (A1M) offers an interesting alternative for evaluating tubular function,3 because it is almost completely reabsorbed (99%) in the proximal convoluted tubules by the megalin receptor, resulting in very low urinary concentrations.4 It is a sensitive biomarker for detecting tubular dysfunction even in the early phase of injury, when no histological damage is detected. It may be elevated in diabetic nephropathy, even in normoalbuminuric patients, demonstrating that tubular injury is an early event in this disease.5–9 Furthermore, the decline in renal function correlates better with tubulointerstitial damage than with glomerular injury.10–12
Other low molecular weight proteins filtered by the glomerulus, which are routinely quantified to rule out Bence Jones proteinuria (BJP), are free light chains. Although polyclonal free light chains are not routinely used as a marker of tubular damage, they are often elevated in chronic kidney disease,13 and also in polyclonal hypergammaglobulinemia primarily due to autoimmune and inflammatory diseases,14 and in patients with diabetes mellitus (DM) before the onset of overt renal failure, suggesting a possible role in predicting early diabetic nephropathy.15–17 These polyclonal free light chains are ligands for both receptors (megalin and cubilin).2,18
Although elevated urinary A1M is considered one of the most reliable parameters for detecting tubular damage, the aim of our study was to evaluate whether the increase in polyclonal kappa free light chains (pKFLC) in first morning urine (FMU) constitutes an earlier and more specific marker of incipient tubular damage than A1M.
Material and methodsPopulationThis was an observational, retrospective, analytical study, based on data extracted from our laboratory information system during the years 2020–2024, selecting 330 analyses for which the glomerular filtration rate (GFR), albumin, A1M, and pKFLC had been requested in FMU and 24-h urine (24hU). Creatinine and specific gravity (SG) were measured only in FMU. The GFR of these analyses ranged between 8–131 mL/min/1.73 m2. Analyses belonging to patients diagnosed with any monoclonal component in serum or urine (multiple myeloma, monoclonal gammopathy of undetermined significance, BJP, amyloidosis, etc.) were excluded. After performing the correlation between A1M and pKFLC adjusted for creatinine (A1Mcr, pKFLCcr) in FMU and 24hU, 95 normal samples in FMU were selected; these belonged to patients with a GFR > 90 mL/min/1.73 m2, without microalbuminuria, oliguria, or polyuria, with SG > 1015, and without renal or systemic diseases demonstrated to cause tubular damage, with an A1M concentration < 4.3 mg/L (lower limit of detection).
Considering the total population and the normal A1Mcr value, we formed 2 groups: NA1MG (normal A1Mcr) and EA1MG (elevated A1Mcr), comprising 168 and 162 samples, respectively. The clinical data of the groups are shown in Table 1, which includes age, sex, GFR, and the albumin/creatinine ratio, divided into patient groups according to normal levels of A1Mcr and pKFLCcr.
Clinical data of the patients.
| Total | Age (years), median (IQR) | Sex, M/F | GFR (mL/min/1.73 m2), median (IQR) | A/cr (mg/g), median (IQR) | |
|---|---|---|---|---|---|
| NUG | 95 | 45 (32–60) | 51/44 | 100 (95–110) | 2.9 (1.7–5.8) |
| NA1MG | 168 | 46 (35.5–59.6) | 81/87 | 99 (90–108) | 3.1 (1.76–6.2) |
| pKFLCN | 129 | 46 (34–59.75) | 62/67 | 99 (90–108) | 2.9 (1.55–5.24) |
| pKFLCE | 39 | 49 (41–59) | 20/19 | 94.57 (84–107) | 3.8 (2.32–14.3) |
| EA1MG | 162 | 62 (53–72) | 73/89 | 75 (46–95) | 8.3 (3.3–26.7) |
| pKFLCN | 36 | 57.5 (47.25–63) | 7/29 | 93.5 (85.5–102) | 3.2 (2–6.1) |
| pKFLCE | 126 | 65 (55–73) | 66/60 | 63 (40.3–90.3) | 11.7 (4.1–41.6) |
A/cr: albumin/creatinine ratio; pKFLCE: elevated polyclonal kappa free light chains; pKFLCN: normal polyclonal kappa free light chains; NA1MG: group with normal alpha-1-microglobulin; EA1MG: group with elevated alpha-1-microglobulin; NUG: normal urine group; F: female; IQR: interquartile range; GFR: glomerular filtration rate; M: male.
Blood samples were collected between 08:00 and 09:00 h in the morning after 12 h of fasting. Serum was obtained by centrifugation at 3000 rpm for 5 min. The FMU sample was collected that same morning, and the 24hU was also delivered; both were centrifuged at 2000 g for 10 min.
Creatinine was determined by the Jaffé method on a cobas® c702 autoanalyzer, and urinary albumin by an immunoturbidimetric method (Roche Diagnostics GmbH, Mannheim, Germany). Normoalbuminuria was defined as an albumin (mg/L)/creatinine (g/L) ratio < 30 in FMU19 or <30 mg of albumin in 24hU.20 A1M and pKFLC were determined by immunoturbidimetric assay on an Optilite® analyzer (Binding Site, Birmingham, United Kingdom). For A1M determination, urine samples were aliquoted and stored at 2–8 °C for a maximum of 7 days, as stability is maintained for up to 21 days at 4 °C.21 A value of 4.3 mg/L was assigned to those results with an A1M concentration < 4.3 mg/L (detection limit provided by the manufacturer, Binding Site), in order to calculate the A1Mcr value in FMU or in mg/24 h.22 SG was measured in FMU using cobas® u pack strips on the cobas® u 601 urine analyzer (Roche Diagnostics GmbH, Mannheim, Germany). It reflects the kidney's ability to concentrate or dilute urine, with normal values of 1015–102020. Oliguria is defined as a 24hU volume < 400 mL/day,23 and polyuria as a urine volume > 3000 mL/day.24 For GFR estimation, the CKD-EPI 2021 equation was used.25
Statistical analysisStatistical analysis was performed using IBM® SPSS® Statistics software (version 20.0). The correlation between A1Mcr and pKFLCcr in FMU and 24hU was evaluated using Pearson's correlation coefficient. The normal value of A1Mcr in FMU was determined by calculating the P97.5. The Kolmogorov–Smirnov test was used to verify whether the variables were normally distributed (P < .05). After normality was rejected, the Mann–Whitney U test was used to determine whether there were statistically significant differences between the NA1MG and EA1MG groups, and between normal and elevated pKFLC (pKFLCN and pKFLCE, respectively) in both groups.
Informed consentDue to the retrospective nature of the study, informed consent was not required. The study was conducted in accordance with the ethical principles set forth in the Declaration of Helsinki and the procedures established by the Ethics Committee of our hospital. Some medical records were reviewed to confirm the correct diagnosis; these data were processed anonymously, ensuring confidentiality at all times.
ResultsThere was no significant correlation between pKFLCcr and A1Mcr values with age or sex in the normal FMU group (P > .05).
The correlation of A1Mcr and pKFLCcr in FMU with A1M and pKFLC in 24hU was r = 0.884 and r = 0.92, respectively. The median and interquartile range (IQR) of pKFLCcr within the NA1MG group were 6.53 (3.25–15.32), and in the EA1MG group, 45.92 (17–98.04), with statistically significant differences between the 2 groups (P < .01) (Fig. 1).
Interquartile range of pKFLCcr between the NA1MG and EA1MG groups. pKFLCcr: polyclonal kappa free light chains adjusted for creatinine (mg/L)/(g/L); EA1MG: group with elevated alpha-1-microglobulin adjusted for creatinine in first morning urine > 7.08 (mg/L)/creatinine (g/L); NA1MG: group with normal alpha-1-microglobulin adjusted for creatinine in first morning urine < 7.08 (mg/L)/creatinine (g/L).
We considered as the normal A1Mcr value a level < 7.08 (P97.5 of the normal FMU group), and for pKFLCcr a level < 15.32, corresponding to the third quartile of FMU in the NA1MG group (Fig. 1).
When we selected pKFLCcr values > 15.3 and A1Mcr values > 7.08 from the total population and ranked them by GFR (Table 2), we observed that as GFR decreased and urinary albumin increased, urinary pKFLC also increased, as did A1M.
Relationship between A1Mcr and K/cr relative to A/cr as a function of GFR in patients with altered A1Mcr and pKFLCcr.
| GFR mL/min/1.73 m2 | A/cr Median | A/cr IQR | A1Mcr Median | A1Mcr IQR | A1Mcr r (A/cr) | A1Mcr r (GFR) | K/cr Median | K/cr IQR | K/cr r (A/cr) | K/cr r (GFR) |
|---|---|---|---|---|---|---|---|---|---|---|
| >90 | 6.73 | 3–11.4 | 8.5 | 5.6–14.6 | 0.14 | −0.07 | 19.2 | 10.5–39.4 | 0.21 | −0.08 |
| 60–89 | 4.1 | 2.2–12 | 13.1 | 8.3–18.8 | 0.00 | −0.24 | 24.2 | 15.9–61.4 | 0.06 | −0.10 |
| 30–59 | 22.7 | 10.3–60 | 30.26 | 16.1–54.4 | 0.27 | −0.48 | 67.8 | 39.3–116.6 | 0.30 | −0.36 |
| <30 | 44.8 | 10–739 | 62.7 | 32.2–105.6 | 0.42 | −0.77 | 115.2 | 83.4–241 | 0.41 | −0.53 |
A/cr: albumin/creatinine ratio; A1Mcr: alpha-1-microglobulin/creatinine ratio > 7.08; K/cr: polyclonal kappa free light chain/creatinine ratio > 15.3; IQR: interquartile range; r (A/cr): correlation of A1Mcr or K/cr with A/cr; r (GFR): correlation of A1Mcr or K/cr with GFR; GFR: glomerular filtration rate.
Of the patients with normal A1Mcr (NA1MG: 168), 23.2% (39) had elevated pKFLCcr, and most of these corresponded to patients who maintained a normal GFR, which would suggest an earlier increase of pKFLC in tubular damage (Fig. 2). There were statistically significant differences between elevated pKFLCcr values (>15.3) and normal values within the NA1MG group (P < .01).
pKFLCcr levels in the NA1MG group. A1Mcr: alpha-1-microglobulin adjusted for creatinine in the NA1MG group; pKFLCcr: polyclonal kappa free light chains adjusted for creatinine in the NA1MG group; Q3 pKFLC line: line indicating the third quartile of pKFLC in the NA1MG group: 15.3 (mg/L)/creatinine (g/L); GFR 90 line: line indicating the GFR of 90 mL/min/1.73 m2; GFR: glomerular filtration rate in the NA1MG group.
In turn, within the EA1MG group, the majority (77.8%) had elevated pKFLCcr values, while 36 (22.2%) had normal pKFLCcr (<15.3) (Fig. 3), with statistically significant differences between the pKFLC values of both subgroups (P < .01).
pKFLCcr levels in the EA1MG group. A1Mcr: alpha-1-microglobulin adjusted for creatinine in the EA1MG group; pKFLCcr: polyclonal kappa free light chains adjusted for creatinine in the EA1MG group; A1Mcr 7.08 line: line indicating the normal value of A1Mcr < 7.08 (mg/L)/creatinine (g/L); Q3 pKFLCcr line: line indicating the third quartile of pKFLCcr in the NA1MG group: 15.3 (mg/L)/creatinine (g/L).
No statistically significant differences were observed between normal pKFLC values in the NA1MG and EA1MG groups, but differences were significant between elevated pKFLC values in the NA1MG group (type I) and normal pKFLC values in the NA1MG and EA1MG groups (P < .01).
Hypertension and/or DM constituted the largest proportion of diseases in which pKFLCcr was elevated in isolation in the NA1MG group and A1Mcr in the EA1MG group (Fig. 4).
Diseases in patients with tubular damage. A1Mcr: alpha-1-microglobulin adjusted for creatinine in first morning urine; pKFLCE: elevated polyclonal kappa free light chains adjusted for creatinine in first morning urine; pKFLCN: normal polyclonal kappa free light chains adjusted for creatinine in first morning urine; DM: diabetes mellitus; EA1MG: group with elevated A1Mcr in first morning urine; NA1MG: group with normal A1Mcr in first morning urine; HTN: hypertension; Tx: transplant.
Regarding SG, of the 39 FMU samples from the NA1MG group with pKFLCcr > 15.3, 87.2% had an SG > 1015, while 88.2% of the 36 urine samples from the EA1MG group with pKFLCcr < 15.3 had an SG < 1015 (Fig. 5).
Specific gravity of first morning urine. pKFLCcr: polyclonal kappa free light chains adjusted for creatinine in first morning urine; EA1MG: group with elevated alpha-1-microglobulin adjusted for creatinine in first morning urine; NA1MG: group with normal alpha-1-microglobulin adjusted for creatinine in first morning urine.
A1M detects tubular dysfunction even in the early phase before glomerular damage occurs with increased urinary albumin and histological damage is observed5–9. Although kidney biopsy is the gold standard for diagnosing renal parenchymal diseases, an elevated urinary A1M level correlates with interstitial fibrosis and tubular atrophy on kidney biopsy.26 However, there are also other proteins indicative of tubular dysfunction that are elevated in the absence of serum monoclonal components or BJP, such as pKFLC. In our study, we observed a significant correlation between pKFLC and A1Mcr (r = 0.75), with both being elevated in tubular damage; that is, the greater the increase in A1M, the greater the elevation of pKFLC. The increase in both urinary proteins is due to tubular damage affecting the receptors (megalin and cubilin).
Megalin is the main receptor according to Andersson et al.4 and other authors2,27,28 for A1M reabsorption, but it requires the contribution of the sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3), which is responsible for the majority of filtered Na+ (60%–75%).29 This exchanger is located in the proximal tubule and in the thick ascending limb of the loop of Henle.30,31 It contributes to protein reabsorption by directly interacting with the endocytic receptor megalin located on the apical membrane of epithelial cells of the proximal convoluted tubule and plays an important role in the regulation of urinary pH and volume.32 Furthermore, it has been demonstrated that in NHE3 knockout mice in the thick ascending limb of the loop of Henle, urinary SG is significantly decreased.33 Thus, an increase in A1Mcr in FMU indicates an alteration of the megalin and NHE3 receptors and is associated with a decrease in urinary SG due to impairment of tubular concentration mechanisms, including the NHE3 receptor (Fig. 5).
Regarding cubilin, it is the main receptor for pKFLC, since when there is a pKFLC overload, cubilin assumes the primary role due to the inability of megalin to handle the overload.2,18,34 A similar situation occurs with albumin: in the presence of cubilin deficiency, the excretion of physiologically filtered albumin in urine increases,35 with the megalin receptor being unable to compensate for the increased excretion. Therefore, when cubilin receptor damage occurs, urinary pKFLC will increase, but urinary SG will generally remain normal, as the NHE3 receptor remains intact.
However, in our study, discordant findings were observed, with patients presenting elevated A1M with normal pKFLC levels and vice versa. If we associate the values of these 2 markers with urinary SG and extrapolate to the functioning of the tubular transporters cubilin and megalin, the existence of 3 patient profiles can be inferred based on the type of tubular damage:
- •
Type I: this would include patients with diseases that can cause tubular damage who present elevated pKFLC levels in the absence of a serum monoclonal component (excluding multiple myeloma, monoclonal gammopathy of undetermined significance, or amyloidosis) or BJP, together with normal A1Mcr values (Fig. 3). In this group, the predominantly affected receptor would be cubilin, while the integrity of the megalin and NHE3 receptors would maintain normal A1M and urinary SG values (Fig. 5). This profile would correspond to 39 patients (23.2%) from the NA1MG group with elevated pKFLC (Fig. 2).
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Type II: this would comprise patients with diseases associated with tubular damage who present elevated A1M but with normal pKFLC levels. In these cases, the damage would primarily affect the megalin and NHE3 receptors, resulting in a decrease in urinary SG (Fig. 5). This group would include 36 patients (22.2%) from the EA1MG group with normal pKFLC.
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Type III: this would encompass patients with simultaneous elevation of A1M and pKFLC. Both receptors are damaged due to sustained and persistent injury over time, representing an evolution of the two previous types, with consequent glomerular involvement manifested by decreased GFR and the onset of albuminuria (Table 1). This group includes the 126 patients from the EA1MG group with elevated pKFLC.
In this last type of patients, we observed a significant correlation (r = 0.75) between the 2 proteins when both are elevated, whereas in types I and II this correlation is low, as only one of the 2 is increased (r = 0.25).
Considering that there are no statistically significant differences between normal pKFLCcr values in the NA1MG and EA1MG groups, but there are significant differences between elevated pKFLCcr values in the NA1MG group and normal pKFLCcr values in the NA1MG and EA1MG groups, we can infer that the elevated pKFLCcr values (>15.3) in the NA1MG group are similar to the values within the IQR of elevated pKFLCcr in the EA1MG group (where a significant correlation exists between elevated pKFLCcr and A1Mcr) (Fig. 1), and furthermore, they are observed in diseases that cause tubular damage, predominantly hypertension and DM, as is the case with isolated A1Mcr elevation (Fig. 4). Nevertheless, the isolated elevation of pKFLCcr in diseases that produce tubular damage in the NA1MG group (type I patients) occurs at a younger age than the isolated elevation of A1Mcr in the EA1MG group (type II patients).
The initial elevation of these 2 proteins in isolation will depend on how the disease affects the tubule, depending on its characteristics. When only one of the 2 proteins is elevated, as occurs in types I (NA1MG with elevated pKFLC) and II (EA1MG with normal pKFLC), tubular damage is not associated with a significant decrease in GFR or the presence of microalbuminuria (Table 1). However, when tubular injury persists over time, type I and II profiles could evolve toward a type III pattern, characterized by concomitant elevation of both proteins, with secondary glomerular involvement, decreased GFR, and the onset of albuminuria (Table 1).
Consequently, isolated elevated pKFLCcr in FMU could serve clinically as a very early marker of interstitial injury in at-risk groups such as type I patients, with A1Mcr values < 7.08, pKFLCcr > 15.3, and normal urinary SG, which moreover become elevated at a younger age than A1M in type II patients.
The limitations of this study were its observational design, precluding causal inference, and the clinical information obtained retrospectively. In addition, histological correlation was not available to confirm tubular damage, as in most cases these were patients with diseases (DM, hypertension, lithiasis) with a chronic course that do not usually require kidney biopsy unless they progress acutely. Larger prospective studies are needed to confirm the prognostic value of urinary pKFLCcr and to evaluate them in comparison with kidney biopsy. Molecular studies of the genes encoding primarily the cubilin, megalin, and NHE3 receptors are also needed to attempt to explain why tubular damage initially manifests with elevation of pKFLC or A1M, and why isolated elevation of pKFLC tends to occur earlier in tubular damage.
ConclusionpKFLCcr in FMU shows a good correlation with A1Mcr in established tubular damage and could act as an early marker in some patients depending on the pathophysiological characteristics of the tubule, in whom there is no A1Mcr elevation and renal function is preserved, at a younger age than that at which A1Mcr elevation begins to be detected.
Author contributionsJLG conceived the study. APM and MTCR designed the experiments and analyzed the data. NRR contributed to the experimental methodology and supervised the pathophysiological aspects of the work. BSE and MJFC drafted the manuscript. SGV and CXA collected and selected the data. All authors reviewed and approved the final manuscript.
FundingThis research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.
The authors declare no conflicts of interest of any kind.
