INTRODUCTION

Chronic kidney disease (CKD) is one of the disease entities causing the greatest economic and social burden in Mexico¹. The main cause of CKD in the world is diabetes mellitus (DM). In underdeveloped countries, the frequency of CKD of "unknown origin" is increasing in young adults. In 2020, the incidence of end-stage renal disease (ESRD) in the United States was estimated in 374.8 cases per million/population (pmp), with a prevalence of 2,242 cases pmp². Although there are no registries in Mexico that can be used to conduct an epidemiological analysis of CKD, in 2018 the state of Aguascalientes created a CKD registry that reported a prevalence of 1,883 pmp and those patients on replacement therapy, placing this entity in the ninth position in the world². Unlike reports on global observations, the most frequent cause of ESRD is of "unknown origin," followed by DM. Its prevalence is highest in the 30-40-year-old age group, and 70% of cases were classified as renal disease of "unknown origin"³,⁴. Based on these observations, in 2020, we conducted CKD screening in the state of Aguascalientes; 513 adolescents were included, and the prevalence of persistent albuminuria (pA) was 3.7% (95% CI 2.1-5.3), a much higher value than that reported internationally (1.9%)⁵-⁸.

The histological and clinical characteristics of the adolescents with pA in Aguascalientes, reported by Díaz et al. in 2022, suggest that oligonephrony may be associated with environmental factors and genetic predisposition⁵,⁹. Fibrosis, either in the glomerular space or in the tubulo-interstitium, is the histological hallmark of CKD. The development of fibrosis is triggered by the incursion of inflammatory cells and cytokines that locally increase fibroblasts and the release of growth factors, that is, transforming growth factor-β and connective tissue growth factor, that lead to the loss of nephrons and promote the epithelial-mesenchymal transition¹⁰.

TFG-β and proteinuria are markers of CKD progression but they reflect late functional changes more than early structural kidney abnormalities and may also vary depending on the patient's sex, age, race, muscle mass, and metabolism, among other factors¹¹. The scarcity of minimally invasive sensitive and specific markers for the timely detection and prediction of CKD course has led to current intense research on possible markers, using urine as the ideal source¹².

We have recently identified in our laboratory a promising CKD biomarker, SerpinA3 (alpha-1-antichymotrypsin). SerpinA3 is a member of the serine protease inhibitor superfamily. Cathepsin G is the main protease target of SerpinA3; it is expressed in neutrophil azurophilic granules and increases in inflammatory processes, although its role in the kidney is not clear¹³. We have previously shown that uSerpinA3 levels increase in animals and patients with CKD of various etiologies, and that in experimental CKD, increases in uSerpinA3 precede fibrosis and renal dysfunction, suggesting that it could potentially detect CKD in a timely manner. In the same context, we have shown that heat-shock protein-72 (HSP-72) is a reliable biomarker in the detection of acute kidney injury, since it significantly increases in urine (uHSP72) up to 48 h before serum creatinine increases or urinary output decreases¹⁴.

Urinary podocalyxin (PCX) is a transmembrane protein located at the apical surface of glomerular podocytes, and it is the main component of the glomerular basement membrane. PCX plays an important role in normal glomerular development and in podocyte architecture; furthermore, several authors have reported that PCX is released from injured podocytes into urine and can be detected in patients with direct podocyte injury and thus, it facilitates the detection of several glomerular diseases¹⁵,¹⁶. Finally, nephrin, a protein encoded by the NPHS1 gene, is a key structural molecule of the slit diaphragm of podocytes, whose function hinges on its interaction with the actin cytoskeleton. It has been identified as a potential biomarker of podocyte injury, and it appears before the development of clinically significant microalbuminuria. Some studies have suggested that podocyte dysfunction may correlate with the number of podocytes in urine and an abnormal expression of nephrin in renal tissue, and therefore, may be associated with renal dysfunction¹⁵,¹⁷. Both markers correlate with renal parameters such as microalbuminuria, serum creatinine, and the estimated glomerular filtration rate, and may therefore become biomarkers in the early detection of CKD¹⁸.

The high prevalence of CKD of unknown origin in young adults in Aguascalientes, Mexico, and the high incidence of early CKD (G1A2) in adolescents encouraged the search for non-invasive strategies that can detect CKD in early stages and delay its progression into full-blown ESRD. Our work has the hypothesis that the presence of albuminuria and its persistence will condition a higher expression of SerpinaA3 and Hsp72 and a lower presence of nephrin and PCX. Hence, the aim of this study was to identify the presence of potential biomarkers of early renal injury in patients with pACR, and that coincide with the characteristic nephropathy profile that prevails in this entity.

METHODS

Participant selection and group assignment

This is a cross-sectional, analytical, and comparative study. Study participants were classified into four groups (negative control, isolated albuminuria, pA, and positive control). The selection of the first three groups was based on the results of CKD screening among adolescents between the ages of 10 and 17, and without a previous history of CKD, in the municipality of Calvillo (Aguascalientes, Mexico)⁵. We obtained the first morning urine and peripheral blood serum to determine the albumin-creatinine ratio (ACR), and estimated the GFR with Schwartz equation. Patients with an ACR < 30 mg/g were classified as negative controls, those with an ACR≥30 mg/g were programmed to repeat the laboratory tests. In the second sample, after a median of 5 months interquartile range (IQR 0.36-5.3), patients with an ACR < 30 mg/g were classified as isolated ACR (iACR), while those with an ACR≥30 mg/g were assigned to the pACR group. The positive control group included patients with established glomerular disease confirmed by biopsy, and they were recruited from the Centenario Hospital Miguel Hidalgo glomerulopathy consult. This study is based on the guidelines of the STROBE statement (Fig. 1; Supplementary data, material 1 and material 2).

Figure 1 Flow chart for sample collection and group assignment. 

RESULTS

Between February 2020 and April 2021, we recruited 91 patients. Sixty-six patients detected in the CKD screening process were classified as follows: negative control (n = 25), iACR (n = 22), and pACR (n = 19). Twenty-five cases with biopsy-confirmed glomerular disease were assigned to the positive control group.

SerpinA3

The densitometric analysis corrected for urinary creatinine showed a statistically significant difference in urinary protein excretion between groups (p < 0.001). Protein expression was significantly higher in the positive control group than in negative controls (0.70 ng/mL vs. 0.08 ng/mL, p < 0.001), and in the iACR group (0.19 ng/mL, p = 0.005). Likewise, we found greater SerpinA3 urinary excretion in patients with pACR, a very different value than that found in the negative control group (0.08 vs. 0.25 ng/mL, p = 0.037) (Fig. 2A).

Figure 2 Biomarkers of tubulointerstitial injury. (A) Urinary excretion of SerpinA3 quantified by WB in each classification group, and corrected for urinary creatinine. Significant difference between the positive control and negative control groups (p < 0.001), positive control and isolated albuminuria (p = 0.005), and negative control and persistent albuminuria (p = 0.037). (B) Urinary excretion of HSP-72 quantified by WB in each classification group, and corrected for urinary creatinine. Significant difference between the negative control and positive controls (p = 0.027). Statistical analysis with the Kruskal-Wallis and the post hoc Dunn test. A p < 0.05 value was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 

PCX

Data analysis revealed variability in urinary PCX levels according to the group classification, as well as a statistically significant difference (p = 0.028) due to lower concentrations in the positive control group. Patients in the positive control groups excreted less urinary PCX than those in the iACR group (105.1 vs. 186.9 ng/mL, p = 0.017), but the difference between the negative controls and the pACR patients was not significant (152.5 vs. 162.0 ng/mL, p = 0.999) (Fig. 3A).

Figure 3 Biomarkers of podocyte injury. (A) Podocalyxin urinary levels quantified by sandwich ELISA in each classification group. Significant difference between the positive control group and the isolated albuminuria group (p = 0.017). (B) Urinary Nephrin levels quantified by sandwich ELISA in each classification group. No significant differences were found between groups (p = 0.432). Statistical analysis with ANOVA or Kruskal-Wallis test according to their distribution, and with post hoc Dunn or Tukey test. A value of p < 0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 

DISCUSSION

This study showed that SerpinA3 was the only possible biomarker associated with pA, while HSP-72, PCX, and nephrin were not significantly associated. The usefulness of a biomarker of kidney injury is directly related with the type of injury and the objectively injured area²⁰. Therefore, it is important to take into account that the group of adolescent patients with pA was recruited in a region with a high prevalence of CKD of unknown origin; that is, the triggering factor remains to be elucidated. The histological characteristics suggest that oligonephrony may be the primary cause, a hypothesis that is supported by the observed glomerulomegaly, the decrease in kidney volume by ultrasound, the partial fusion of the podocyte processes, and the absence of fibrosis reported by Díaz et al.⁵ Oligonephrony may also be secondary to nephrogenesis abnormalities or to the loss of nephrons as a result of any post-natal pathological process. In this group of patients, the absence of fibrosis and inflammation suggests that its origin may be pre-natal.

Oligonephrony is a common finding in all nephropathies, and glomerular hyperfiltration is the pathogenic mechanism leading to kidney function impairment²¹. Structurally, this hyperfiltration is the result of disrupted vascular tone in afferent and efferent arterioles. Several factors, be they endocrine (renin-angiotensin system, aldosterone, and glucagon), vasoactive peptides (prostaglandins and vasopressin), and mediators of tubulo-glomerular feedback, are necessary to adequately maintain renal plasma flow and glomerular filtration²²,²³. Pharmacologic actions applied to this axis have proven to preserve and protect kidney function, mainly as a result, in a great measure, of a decrease in glomerular pressure²⁴-²⁷. Prematurity is the most studied process that can trigger congenital oligonephrony (not significant in Aguascalientes)⁵; it can lead to hyperfiltration, glomerulomegaly, albuminuria, and secondary focal and segmental glomerulosclerosis resulting in histologically evident CKD²⁸. The follow-up of an adult patient cohort (mean age, 23.1 years) was recently reported: one group had a history of prematurity, and the authors studied their copeptin levels, plasma renin activity, and aldosterone concentration as possible glomerular hyperfiltration markers. Cases with a history of prematurity had decreased kidney volumes and increased albuminuria. Copeptin was the only marker that was significantly associated with this group of patients. Copeptin is a vasopressin surrogate and could also be a potential biomarker of hyperfiltration in these patients²⁹.

There are very few reports on kidney injury markers in the early phases of kidney injury in which only microalbuminuria is detected. SerpinA3 has been associated with multiple physiopathological processes (e.g., inflammation and fibrosis)³⁰. The role of SerpinA3u in this group of patients has not been studied. Although pA is a clear manifestation of chronicity, albumin excretion per se and the neuro-humoral mechanisms associated with glomerular hyperfiltration, promote inflammatory injury to podocytes²¹,³¹,³². In a murine focal and segmental glomerulosclerosis model, SerpinA1 and SerpinA3 were found to be overexpressed in the extracellular matrix, suggesting their possible physiopathogenic role in podocyte injury in this primary entity³³. We also recently demonstrated that SerpinA3 relocates from the cytoplasm to the apical tubular membrane (ATM) in CKD, so it appears to be secreted into the luminal space in a physiopathological sense. We also showed that the expression and secretion of SerpinA3 increase in HEK-293 cells after transfection with a SerpinA3 clone tagged with FLAG and exposed to H₂O₂, or in cases of starvation³⁴. These overall results suggest that the presence of SerpinA3 in urine reflects a physiopathological state that could, in turn, mirror incipient kidney injury.

Although our group reported no fibrosis in the kidney biopsies, the presence of uSerpinA3 may be a manifestation of this protein's role in glomerular hypertension. SerpinA3 has also been reported to play an important role in the renin-angiotensin-aldosterone system. One of the main functions of SerpinA3 is the regulation of inflammation by inhibiting cathepsin G in neutrophils, and mastocyte chymase in the kidney; it assumes a putative inhibitory function of the angiotensin-converting enzyme, thus precluding the conversion of angiotensin I to active angiotensin II. Surprisingly, increased levels of the angiotensin-converting enzyme have been detected in the serum of patients lacking this protein³³,³⁵. Regardless, the specific role of SerpinA3 in renal physiopathology remains unknown. We encourage investigators to continue in-depth research on this variable, for a more thorough understanding of this molecule and its potential applications³⁰.

The main limitation of this study is its exploratory nature and the lack of a standardized quantification method to measure the biomarkers. Therefore, subsequent studies with complementary methods and more controlled patient's scenarios are necessary to clarify the potential role of the SerpinA3 protein expression quantification values that could represent an alternative diagnostic tool for early stages that may prevent irreversible kidney damage. At this moment, it is difficult to know whether the difference in SerpinA3 values between groups is sufficient for it to be used as a biomarker. This decreases its clinical applicability for now. It should be mentioned that we tested two different commercial ELISA kits to detect Serpin-A3; their sensitivity was low and there was great variability between tests, even when using the same sample.

The study's main strength lies on the fact that this is the first study searching for CKD biomarkers in this population. Although the nephropathy described in Aguascalientes has not been previously reported throughout the country, Mexico has a large disease burden due to CKD that may be explained by this newly described entity. It is therefore fundamental to create a national CKD registry and characterize its main etiologies, to establish whether the phenomenon that we have detected in Aguascalientes is local or has a nationwide presence.

We found that uSerpinA3 is a possible marker associated with pA in adolescents from Aguascalientes. The role of SerpinA3 as a mechanism of injury in this entity should be clarified in future studies, as well as its usefulness as a marker of incipient renal injury.