Mass Spectrometric Identification of Urinary Biomarkers of Chronic Kidney Disease: A Proteomic-related Preliminary Report

Introduction

Chronic kidney disease (CKD)¹ has a global prevalence of 11–13%.² Progression of CKD depends on the primary disease and also other influencing factors such as smoking, diet, or coexisting metabolic diseases.³ Inflammatory response associated with oxidative stress promotes renal damage by inducing necrosis, apoptosis, and fibrosis, which may play a role in the progression of CKD.⁴ Several markers of various risk factors of CKD can be measured in the tissue or serum to monitor the disease and its progression.

Investigation of alternative biofluids such as urine as a potential non-invasive biomarker⁵ would provide an easier means of disease monitoring. Urinary peptides identified through liquid chromatography–mass spectrometry (LCMS) could help in monitoring the disease progression and treatment response.

The study aims to identify a urinary proteome-based panel of novel protein biomarkers of CKD.

Materials and Methods

This preliminary study consisted of 32 patients with CKD stage V recruited at the Division of Nephrology, Department of General Medicine, Aarupadai Veedu Medical College and Hospital, Puducherry, India. The study was approved by the Institutional Human Ethics Committee of AVMC & H (IHEC No: AV/IEC/2021/010), and informed consent was obtained from each participant. The participants were asked to answer questionnaires regarding anthropometric measurements and recorded their laboratory and clinical examinations. The sample collection and preparation workflow are represented in Figure 1.

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Figure 1:

Urine sample collection and preparation work flow.

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Results

The data on the baseline characteristics of the study subjects are presented in Table 1. We identified 135 proteins and segregated them based on gene ontology enrichment analysis, shown in Supplementary Data 1. Out of 135 proteins, 35 showed higher expression in urine [Table 2] and are involved in pathophysiological processes of kidneys such as protein sorting, ion and/or iron transport, metal binding, hemostasis, protease and antiprotease activity, transcriptional regulation, stress response, and apoptosis. We chose 10 candidates, namely, CD59, ANPEP, APOH, HPX, LMAN2, ANXA4, ANXA5, TPP1, ANXA11, and ATP1A1, having significance in the progression of CKD to end-stage renal disease.

A protein interaction network was constructed to evaluate any functional and/or physical interactions between the urinary protein candidates for 135 proteins using STRING and functional ontology (GO) enrichment analysis [Figure 2a-2b, Figure S1 and S2]. This analysis revealed that the majority of urinary protein candidates were membrane proteins associated with biological processes of heme homeostasis, protein trafficking and transport, and many of them were involved in the process of gene expression and cell signaling. Gene ontology analysis provides a comprehensive resource related to genes and gene products. The figure represents the list of proteins; proteins and peptides are interconnected in PPI networks, which can be altered in diseased conditions [Figure 2].

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Figure 2:

Protein interaction network analysis. (a) Protein interaction analysis of 135 proteins identified in urine. (b) Protein interaction analysis of 35 proteins identified in urine.

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Glycoproteins (CD59, HPX, LMAN2, and APOH) showed increased expression in urine. SERPINA1 identified in urine samples of patients with CKD acts on SERPINE1, which is a potent fibrosis-promoting glycoprotein. Endonucleases are unique and may be assembled with a variety of enzymes typically associated with DNA damage and/or repair (e.g. XRCC3, FANCG, ATP23, FEN1, APTX, and SFN) [Supplementary Data 1]. Results showed the presence of the most active apoptotic endonucleases of the kidneys such as DNAse 1 and EndoG in urine. We identified F- and P-type ATPases in urine, which include ATP1A1, ATP2A3, ATP2B2, ATP7B, ATP2A1, and ATP5IF1; these are involved in sodium/potassium, calcium, and copper transport. ATP5IF1 acts as an inhibitor of endogenous F1F(o)-ATPase. The zinc finger proteins are involved in transcriptional regulation. These zinc finger proteins (e.g. SNAI3, ZNF621, ZNF511, ZNF205, ZNF331, ZNF787, and DNLZ) have a promising role in the development of podocyte damage or glomerular diseases.

Discussion

Proteomics provide good tools with clinically relevant diagnosis and prognosis models as translational research. Our study showed 35 urinary proteins with increased expression in urine in patients with CKD stage V. These proteins are involved in various physiological and pathophysiological activities of the kidneys including cell signaling, inflammation, oxidative stress, hypoxia, DNA damage/repair mechanism, cell cycle arrest, mitochondrial dysfunction, heme homeostasis, and chaperons. CD59, a glycosylphosphatidylinositol (GPI) is composed of a phosphatidylinositol group linked through carbohydrate groups acts as an anchored glycoprotein and is ubiquitously expressed on epithelial, endothelial and mesangial cells of glomeruli. A study used anti-CD59 to neutralize its activity observed reduced renal function with severe endothelial damage with increased platelet and also fibrin deposition in glomeruli.⁷ Scavenging of free heme with hemopexin (HPX) is expected to mitigate heme-induced cell injury. Increased free heme concentration and HPX deficiency indicate that increased expression of CD59 could defend against complement-dependent renal injury.⁸ Aminopeptidase N (APN) is identical to CD13, belongs to type I metalloproteases, and has a role in metastasis, tumor invasion, and angiogenesis. The potential use of ^99mTc-probestin SPECT as a new technique is imaging kidney APN expression.⁹ Increased urinary APN levels associated with microalbuminuria indicate tubular damage in diabetic nephropathy (DN).¹⁰

Annexins (Anx) can mediate a varied range of cellular processes including endo-, exocytosis, and membrane-cytoskeletal organization.¹¹ AnxA1 in renal tissue is negatively correlated with the degree of tubular damage.¹² AnxA1 levels correlated with normal kidney outcomes and showed higher expression during DN.¹³ AnxA4 regulates adenylyl cyclase type 5 and affects β-adrenoceptor/cAMP-dependent signaling.¹⁴ AnxA6 has a role in integrin recycling, aberrant Src, and FAK signaling, ECM cell surface delivery¹⁵ and IL-2 homeostasis-related T-cell proliferation.¹⁶ Progranulin (PGRN) is a cysteine-rich secretory protein used to predict the early signs of podocyte injury in DN¹⁷ and also indicates an endotoxin-induced septic acute kidney injury.¹⁸

Sodium/potassium-transporting ATPase (Na/K-ATPase) is an important ion pump of P-type ATPase. Reduced Na/K-ATPase α1 causes deficiency in signaling protein Src and NFkB activation. A study identified sodium/potassium-transporting ATPase subunit gamma in urine using capillary electrophoresis-coupled mass spectrometry in IgA nephropathy patients.¹⁹ Na/K-ATPase involved in CD40 receptor activation in the renal tubular epithelium during pathogenesis.²⁰ Tripeptidyl peptidase-I (TPP-I) is a lysosomal serine proteinase expressed in the kidneys. Urine TPPI was significantly increased in patients with CKD.²¹ Peroxiredoxins (PRDXs) is an antioxidant enzyme key regulator of apoptosis and cell survival, it can intermediate during H₂O₂-induced activation of c-Abl/MST1/FOXO signaling.²² Mic060 is an important factor involved in the regulation of cell apoptosis, mitochondrial biogenesis, and oxidative stress.²³ A study reported significantly decreased Mic60 levels in hemodialysis patients.²⁴

Lon protease homolog, mitochondrial (LONP1) is a serine peptidase that protects the kidneys by inhibiting mitochondrial dysfunction and podocyte apoptosis.²⁵ Radixin is one of the membrane-cytoskeletal crosslinkers, and increased levels of radixin were observed in dialysis patients.²⁶ Limitations of this preliminary study are smaller sample size and that the proteins identified herein require further external validation.

Conclusion

Protein identification using LC-MS/MS allowed us to identify 135 proteins expressed in the urine of CKD patients. Possibly further studies on EndoG, HPX, APN, AnxA1, Mic60, LONP1, and HYOU1 potential markers will enable a better understanding of the mechanism of CKD and may provide novel opportunities for therapy. Sufficiently powered cohort studies are required to confirm the usefulness of these candidates to help improve understanding of CKD progression.