The pathophysiological significance between autosomal dominant polycystic kidney disease and neutrophil gelatinase-associated lipocalin

Pathophysiology of ADPKD and NGAL

Article information

Kidney Res Clin Pract. 2025;44(2):238-248

Publication date (electronic) : 2025 March 6

doi : https://doi.org/10.23876/j.krcp.23.339

Pallavi Devapatla ¹

, Wen-Yih Jeng ²

, Wen-Tai Chiu ³

, Hsiu Mei Hsieh-Li^,¹

¹Department of Life Science, National Taiwan Normal University, Taipei, Taiwan

²University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan

³Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan

Correspondence: Hsiu Mei Hsieh-Li Department of Life Science, National Taiwan Normal University, 162 Section 1, Heping E. Rd., Taipei City 106, Taiwan. E-mail: hmhsieh@ntnu.edu.tw

Received 2023 December 6; Revised 2024 March 3; Accepted 2024 March 22.

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of polycystic kidney disease (PKD) and is a typical adult-onset multisystem disorder. It is a progressive disease characterized by the disruption of renal tubular integrity, involving the modulation of cellular proliferation and apoptosis. Most ADPKD results from a mutation in either the PKD1 or PKD2 gene encoding polycystin-1 and polycystin-2, respectively. With the inconsistent disease course of ADPKD, biomarkers that can predict the treatment efficacy and rapid progression of the disease are needed. Studies have identified neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for predicting the progression of ADPKD patients. The NGAL protein is expressed at a low level in the kidneys, which helps to regulate iron transport and participates in epithelial differentiation, inflammation, and cell proliferation. NGAL level also increases in serum and urine during renal detrimental conditions such as ischemia and acute and chronic kidney diseases. On the other hand, some studies have also demonstrated that NGAL may act as a tubulogenic factor controlling cell growth and that the upregulation of the Ngal gene hinders tubular cell proliferation, resulting in significantly reduced cyst growth in cellular and murine models of ADPKD. This review attempts to correlate ADPKD and NGAL based on available research findings to evaluate the therapeutic potential of NGAL in ADPKD.

Keywords: Autosomal dominant polycystic kidney; Biomarkers; Neutrophil gelatinase-associated lipocalin; Polycystic kidney diseases; Autosomal dominant polycystic kidney, Biomarkers, Neutrophil gelatinase-associated lipocalin, Polycystic kidney diseases

Introduction

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by the development and progressive enlargement of fluid-filled cysts with subsequent renal and extra-renal manifestations. The disease characteristics vary by age of onset and rate of progression. The common disease progression includes cyst infections and hemorrhage, hematuria, intracranial aneurysms, hepatic cysts, hypertension, urinary tract infections, etc. [1,2]. Although the precise mechanism behind the cystogenesis of ADPKD is not fully understood, a notable number of studies have shown its association with different molecular signaling pathways controlled by the protein products of PKD1 and PKD2, namely polycystin-1 (PC1) and polycystin-2 (PC2), respectively, that are localized in primary cilia of renal tubules [3].

Individuals affected with mutations in the PKD2 gene have milder kidney disease with fewer kidney cysts, and experience delayed onset of hypertension, kidney failure, and long survival compared to those with mutations in the PKD1 gene [4]. Nevertheless, similar renal and extra-renal manifestations are seen in patients with mutations in both genes [5]. These mutations lead to disruption of downstream signaling pathways and cystogenesis as a result of altered G-protein and cyclic adenosine monophosphate (cAMP) signaling [6].

In ADPKD, kidney function loss is often unnoticed until the fourth decade when cyst development is advanced [4]. Several clinical studies and applications have proved that tolvaptan, a selective vasopressin-2-receptor antagonist can effectively inhibit renal cyst growth and slow down the worsening of renal function in ADPKD patients [7,8]. Identifying patients at higher risk of progression is crucial, especially as a drug like tolvaptan may slow deterioration [9]. Predictors include PKD1 mutations, hypertension, proteinuria, and kidney volume [10]. Height-adjusted total kidney volume is considered the best predictor, but its capacity is limited [11]. Additionally, certain molecules in urine, linked to tubular injuries or low blood supply, could serve as indicators of ADPKD-related renal function decline; these include kidney injury molecule-1, neutrophil gelatinase-associated lipocalin (NGAL), liver fatty acid-binding protein, β2-microglobulin, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) [12,13].

NGAL, a 22-kDa protein released from tubular cells in pathological conditions, has been extensively explored as a biomarker because of a rapid rise in its levels in distinct settings such as acute kidney injury, cardiac surgery, and kidney transplantation [14,15]. It is involved in the innate immune response to bacterial infection and kidney disease development [16,17]. NGAL belongs to a family of small secreted proteins that bind various ligands, including bacterial siderophores, soluble extracellular macromolecules, growth factors, and cell surface receptors, such as megalin [18,19]. For example, acts as an iron transporter and is involved in the embryonic and adult development of the renal epithelium through the induction of iron-dependent cellular signals, thus hindering the apoptotic process [20]. NGAL inhibits bacterial growth by sequestering iron and delivering intracellularly where it is required for the regulation of iron-dependent enzymes, as it can bind to the siderophore [18]. In addition, increased NGAL production and its release from tubular cells following several kinds of unfavorable stimuli may indicate a self-defensive mechanism dependent on specific iron-dependent pathways [21].

NGAL has often been shown to play a protective role against renal injuries, amelioration of ischemia [22], and suppression of cyst progression [23]. Multiple studies have established a correlation between the levels of serum or urinary NGAL and renal residual function, indicating that the concentration of this protein might be influenced by the extent of underlying renal dysfunction [4,24]. These findings suggest that NGAL could potentially serve as a marker for renal failure, and offer valuable insights into the severity of kidney impairment. Advancements in diagnosis, prognosis, and therapeutic interventions for ADPKD patients are hindered by the incomplete understanding of the pathophysiological significance between ADPKD and NGAL. This review comprehensively explores the literature to elucidate the relationship between ADPKD and NGAL and provides insights into their pathophysiological significance and potential clinical implications.

Genetic basis and molecular mechanisms of autosomal dominant polycystic kidney disease

Gene mutations and autosomal dominant polycystic kidney disease

The primary cause of ADPKD is mutations in PKD1 and PKD2 genes. The PKD1 gene is located on chromosome 16p13.3, and accounts for 85% of ADPKD cases, while the PKD2 gene, on chromosome 4q22, accounts for 15% of ADPKD cases [25,26]. Nevertheless, PKD2 mutations may be more prevalent because of the extended time leading to end-stage renal disease in community-based studies [2]. In 2016, in genetically unresolved cases of ADPKD, mutations in the gene GANAB, encoding glucosidase II subunit α (Gllα), were identified [26]. Other reported mutations include those in the DNAJB11 gene, causing a type of cystic disease in which kidney failure may develop but without any marked kidney enlargement [25]. ADPKD cystogenesis can be induced by miRNAs, such as miR-15a [27] and miR-21 [28], which affect several target genes involved in cell proliferation, and directly regulate PKD gene expression [27,29]. The forthcoming miRNA analysis in human polycystic kidney tissue is expected to reveal new insights into ADPKD development.

PC1 and PC2, respectively, are the protein products of PKD1 and PKD2 genes. They are found in renal tubular epithelia, hepatic bile ducts, and pancreatic ducts [30]. These proteins form the polycystin complex within the primary cilia and other organelles and contribute to intracellular calcium regulation [30]. PC1 is a large integral membrane protein comprising 4,303 amino acids [31] located in the distal nephrons and collecting ducts of the kidney, bone, brain, and muscles [5]. The structure of PC1 includes an N-terminal 11 transmembrane G-protein coupled receptor (GPCR) domain and a small cytoplasmic C-terminus domain that interacts with PC2 [31]. The interaction between PC1 with PC2 is implicated in the regulation of ion transport and mainly affects Ca²⁺ signaling [32]. Researchers suggest that in ADPKD, the G-protein α subunit binds to the G-protein binding domain of the GPCR of PC1, thereby capturing and preventing free β/γ subunits from binding to the α subunit [33,34]. The interaction between the subunits may be responsible for the heightened disease severity observed in patients with PKD1 mutations compared to those with PKD2 mutations. This may also explain why therapies targeting GPCRs have shown potential in treating ADPKD [6].

Molecular mechanisms

Functional loss of polycystin expression causes alterations in several cellular pathways, such as those involved in apoptosis, proliferation, differentiation, fluid secretion, and cell adhesion [35,36]. The interaction between PC1 and PC2, a non-selective calcium channel, regulates the cAMP pathway and Ca²⁺ homeostasis (Fig. 1A) [37,38]. The renal expression of Janus kinase-signal transducer and activator (JAK-STAT) of transcription is abnormally high in ADPKD. There is clear evidence that ADPKD can result from reduced expression of PC1, which disrupts the JAK-STAT signaling and leads to kidney epithelial cell proliferation (Fig. 1B) [39]. In ADPKD, the activation of the rat sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf) signaling pathway is attributed to a reduced expression of PC1, which results in cellular proliferation (Fig. 1C) [1].

Figure 1.

PC1/PC2 signaling pathways in autosomal dominant polycystic kidney disease.

A: Cyclic adenosine monophosphate (cAMP) signaling [1]. Loss of polycystin function causes low intracellular calcium levels, which leads to elevation of cAMP. It stimulates the Ras-dependent activation of B-Raf and ERK, which leads to increased cell proliferation. B: JAK-STAT signaling [39]. Mutations in polycystins cause the dysregulation of JAK-STAT signaling which fails to arrest the cell cycle and leads to unabated cell proliferation. C: Ras/Raf MEK/ERK signaling [1]. Mutation in PC2 causes reduced intracellular calcium levels, leading to decreased Akt formation. This failure to inhibit the B-Raf proliferation factor leads to uncontrolled cell proliferation.

Akt, serine/threonine-protein kinase; cdk2, cyclin-dependent kinase 2; ERK, extracellular signal regulated kinase; JAK, Janus kinase; MEK, mitogen-activated protein kinase; PC1, polycystin-1; PC2, polycystin-2; PI3K, phosphoinositide 3-kinase; Raf, rapidly accelerated fibroblastoma; Ras, rat sarcoma; STAT, signal transducer and activator of transcription.

Renal cystogenesis in ADPKD is a complex process, distinguished by cell proliferation, extracellular matrix formation, fluid secretion, and cell polarity. This makes identifying biomarkers that can forecast disease progression a paramount important issue [40]. According to several studies, NGAL is involved in tumor development [41] and cyst development [24], while also playing a defensive role against renal injuries and in the suppression of cyst progression [42]. Therefore, the association of NGAL and ADPKD needs further characterization.

Gene structure and protein conformation of neutrophil gelatinase-associated lipocalin

The NGAL gene was first found by Northern blotting in human bone marrow as an 850 bp transcript [43]. The NGAL gene structure includes a 63-bp 5'-untranslated region, a 591-bp open reading frame, and a 142-bp 3'-untranslated region [43,44]. The overall structure was found to have seven exons and six introns spanning a 3696-bp genomic region [44]. This NGAL gene is similar to other lipocalin genes in its genomic structure [45]. While NGAL on chromosome 9 is near other lipocalin genes [46], mouse lipocalins are located on chromosome 2. Previous research has shown that NGAL is present in three distinct isoforms: monomeric (25 kDa), homodimeric (45 kDa), and heterodimeric (145 kDa, a homodimer that is covalently attached to gelatinase, matrix metalloproteinase 9) [47,48]. Renal tubular epithelial cells primarily synthesize the monomeric isoform, with a minor synthesis from the heterodimeric isoform [49]. Neutrophils are composed entirely of isomers, with the dimeric configuration being unique to these white blood cells [47,49]. The NGAL from urine is composed of a molecular weight range of 24.9–25.9 kDa, including various isoforms with isoelectric points between 5.9 and 9.1. On the other hand, the NGAL from CHO (Chinese Hamster Ovary) cells showed a molecular weight range of 25.9–27.9 kDa and comprises multiple isoforms with isoelectric points spanning from 5.6 to 9.1 [50].

The N-terminal of the NGAL consists of a signaling peptide composed of 20 amino acids [51]. This is cleaved and the N-terminal domain plays a key role in binding to specific ligands [52]. NGAL proteins also contain disulfide bonds, which are important for maintaining their structure [53]. There are high similarities among NGALs from different species. For example, the human and mouse NGALs are 61.5% identical and 79.5% similar, while the human and rat NGAL proteins are 63.6% identical and 78.3% similar. The mouse and rat NGALs are even more similar, with 80.5% identity and 90.0% similarity (Fig. 2A). These similarities suggest that they may have similar functions. However, there is a unique property only shown in human NGAL, which has three cysteines (Cys), while only two Cys in mice and rats. The two Cys of mouse and rat are located in the same positions as humans and could form an intramolecular disulfide bond. At the same time, a third Cys was identified on human NGAL at amino acid 107 (Fig. 2B). Whether this extra Cys would form intramolecular disulfide bonds with Cys at position of 96 or 195, or even form intermolecular disulfide bond is not well studied. However, a report suggests that it is for homodimerization and heterodimerization with gelatinase (matrix metalloproteinase 9) [49,54]

Figure 2.

Structure and sequence of NGALs in three species.

(A) Amino acid sequence profiles of human, mouse, and rat NGALs. (B) Protein structures of human, mouse, and rat NGALs.

NGAL, neutrophil gelatinase-associated lipocalin.

NGAL is a protein of the lipocalin superfamily, comprising eight β-strands that form a β-barrel defining a calyx. Lipocalins have been reported to be involved in the modulation of cell growth and metabolism, retinol transport, pheromone transport, prostaglandin synthesis, immune response regulation, tissue development, and animal behavior regulation [53]. The biological activity of lipocalins is thought to be defined by the calyx, which binds and transports low molecular weight molecules, including iron, retinoids, arachidonic acid, prostaglandins, fatty acids, and steroids [55].

NGAL was primarily identified in neutrophils. Cystic tubular epithelia are the predominant source of NGAL produced. Furthermore, several studies have identified HIF-1α as a fundamental mediator between estimated glomerular filtration rate and NGAL upregulation [56]. Additionally, NGAL expression is also elevated in the kidney and other tissues in many conditions, including inflammation, ischemia, infection, intoxication, neoplastic transformation, and acute kidney injury (Table 1) [19,35,56–70 ]. According to previous studies, NGAL serves as a crucial component of innate immunity, especially during bacterial infection [16,17]. It has been implicated that in addition to its antimicrobial effect, NGAL has other complex activities. This is given its rapid appearance in the urine and serum in response to renal tubular injury, which can be useful as an early biomarker of renal failure [71].

Table 1.

Sources and expression of NGAL in different diseases

NGAL also plays a critical role in regenerating the renal epithelium during embryonic and adult stages. This is achieved through the induction of iron-dependent cellular signals, which subsequently retards the apoptotic processes [20]. Certain cancer cell lines with high NGAL levels have documented resistance to radiotherapy and chemotherapy [72]. Further, increased NGAL expression in prostate cancer cell lines boosts their growth and colony-forming ability [73]. Only low levels of NGAL are detectable in urine at baseline given that NGAL is immediately reabsorbed in the proximal tubule via a megalin-dependent pathway during the filtration in the glomerulus and luminal surface [19,74]. Several studies have documented that urinary or serum NGAL levels influence renal function and are recommended to be used as a marker for renal failure [21].

Role of neutrophil gelatinase-associated lipocalin in the prognosis of autosomal dominant polycystic kidney disease

The malfunctioning of renal tubule calcium channels in ADPKD can lead to disturbances in calcium homeostasis, resulting in various effects on renal function and the growth of cysts [75]. A few vital cellular defects involved in cystogenesis comprise increased proliferation and apoptosis, abnormal polarization of membrane proteins, fluid and ion secretion, and changes in cell-matrix interactions [76].

For example, researchers studied the protective effects of NGAL in rats with kidney injury caused by ischemia/reperfusion. Rats treated with NGAL showed reduced kidney damage and lower levels of tubular cell death compared to those without NGAL treatment. This study suggests that NGAL may protect the kidneys by preventing cell death processes through the inhibition of caspase-3 activation and reduction of Bax expression [22].

Findings of preceding studies conducted on ADPKD mouse models have emphasized that cyst development is further associated with tubulointerstitial abnormalities. This is given that cystic tubular epithelia exhibit increased levels of tubular stress proteins, like osteopontin, MCP-1, and NGAL, which is suggestive of their roles in cystogenesis [21]. Furthermore, studies on cell lines lacking PC1 revealed enhanced proliferation and cyst formation, which was counteracted by the addition of NGAL, suggesting its potential as a therapeutic target for slowing cyst growth. Additionally, Ngal overexpression in Pkd1 mutant mice suppressed cyst enlargement without improving renal function [23].

It has been proposed that NGAL can also be involved to a certain extent in cystogenesis given that serum and urinary NGAL levels are remarkably higher than in control ones in ADPKD patients [21]. It was also found that the excretion of urinary NGAL and interleukin (IL)-18 was continually increased in ADPKD and changed over time, though this did not correspond with an increase in total kidney volume or worsening in kidney function [77]. A finding that NGAL was correlated with the glomerular filtration rate over creatinine and cystatin C suggests that serum NGAL might have the potency to be used as a new marker of renal function [78].

According to a study in a murine model, Ngal gene inactivation inhibited tubular cell proliferation, leading to a significant decrease in cyst growth [55]. Our animal study demonstrated that the overexpression of exogenous kidney-specific mouse Ngal elicited a protective effect against polycystic kidney disease (PKD) progression through a series of molecular pathways functioning in proliferation, apoptosis, and fibrosis [79]. The NGAL receptor (NGAL-R, Slc22a17) is expressed in the apical membranes of the distal tubules and collecting ducts and is known to be involved in endocytic iron delivery [79]. The association of NGAL and NGAL-R leads to the depletion of intracellular iron, proliferation reduction, and apoptosis stimulation [18,78]. Furthermore, the balance between proliferation and apoptosis in the renal tubular epithelia in PKD progression emerges as crucial (Fig. 3) [26,37,65,80–82 ].

Figure 3.

Effects of NGAL on apoptosis and cell growth pathways.

A: Apo-NGAL binds to its potential receptor, 24p3R, entering the cell and interacting with an intracellular siderophore to release chelated iron. This reduces intracellular iron levels, leading to the induction of apoptosis through increased expression of the pro-apoptotic protein Bcl2-interacting mediator of cell death (BIM) [26]. B, C: NGAL has also shown anticancer effects by inhibiting cancer-promoting factors, hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor [81], and also inhibits focal adhesion kinase (FAK) phosphorylation [81] in colon [82], ovarian [65], and pancreatic [82] cancers. Though it has not directly been mentioned, these anticancer properties of NGAL could have implications in autosomal dominant polycystic kidney disease given its role in cell growth and apoptosis.

NGAL, neutrophil gelatinase-associated lipocalin.

We also found that adding recombinant mouse Ngal in ADPKD cells could inhibit cell proliferation where the molecular analysis documented that Ngal reduced proliferation, and induced apoptosis and autophagy pathways [83]. Furthermore, overexpression of secreted Ngal enhanced the inhibition of cyst enlargement in an ADPKD 3D cell culture [83].

Challenges of using neutrophil gelatinase-associated lipocalin as an autosomal dominant polycystic kidney disease biomarker

The differences in NGAL levels between the cyst epithelium and urine may be attributed to the lack of communication between the urinary collecting system and the cystic space [83]. It is also important to consider that the response of NGAL when interpreting clinical results given that its response to kidney injury and systemic inflammation is somewhat separate. Further, NGAL is not recommended for predicting the prognosis at an early stage when renal function is maintained given that NGAL levels can rise only in advanced disease [12]. Although several findings have highlighted the close relationship between NGAL and ADPKD, the basic pathophysiological significance of this relationship has not yet been elucidated [24].

The consideration of complexity of ADPKD is very important to explain the inconsistency between the experimental findings and clinical observations about NGAL in ADPKD. Some studies suggest that NGAL may act as a tubulogenic factor controlling cell growth [23]; however, upregulation of NGAL hinders tubular cell proliferation, resulting in significantly reduced cyst growth in cellular level and murine models of ADPKD [84]. Whereas, clinical studies show that urinary biomarkers, such as β2-microglobulin, VEGF, and MCP-1 are the significant biomarkers than NGAL that can predict the disease progression [13,85]. The noticed dissimilarities between experimental findings and clinical studies concerning NGAL in ADPKD can be explained by the complex nature of the disease. ADPKD is highly intricate and differs among individuals because of genetic and environmental factors. Experiment models might not completely replicate this intricacy. Disease progression in ADPKD is dynamic, and NGAL levels may fluctuate at different stages, making a single measurement less conclusive for predicting the progression. A comprehensive approach is required to tackle these challenges including longitudinal studies that trace NGAL levels over time in various patient cohorts. Subgroup analyses based on clinical phenotypes or genetic mutations may unveil certain contexts where NGAL is a more appropriate biomarker. Proposing future research directions, like exploring NGAL’s interactions with other markers, involving diverse and larger patient groups, and longer-term studies will help bridge the gap between experimental and clinical intuitions, providing a subtle understanding of the role of NGAL in ADPKD.

Summary and conclusion

ADPKD is a frequent hereditary monogenic disorder with a prevalence of 1 in 400 to 1 in 1,000 among newborns [86] and causes 8% to 10% of cases of end-stage kidney disease globally. This makes ADPKD one of the most active research areas in nephrology. The reason for individual variability is partially understood based on various genetic and environmental factors [21]. The pathogenesis of ADPKD is widely debated given that numerous proteins are involved in the development and progression of the disease. A critical challenge in the management of patients with ADPKD is predicting the prognosis. NGAL can be an attractive candidate to serve as a molecular imaging tool and as a diagnostic and follow-up marker in various diseases, including ADPKD. It is feasible that a combination of urinary biomarkers, as opposed to a single biomarker, could predict disease progression more accurately in PKD. Even though our review highlights the NGAL’s potential as a biomarker and therapeutic agent, it is important to consider that NGAL alone may not be a complete tool to speculate disease progression and treatment response. Further identifying the limitations of NGAL as a standalone marker can significantly improve our understanding and pave the way for more successful diagnostic and therapeutic strategies in ADPKD.

Notes

Conflicts of interest

All authors have no conflicts of interest to declare.

Funding

This work was supported by the National Science and Technology Council (112-2321-B-003-001).

Data sharing statement

The data presented in this study are available from the corresponding author upon reasonable request.

Authors’ contributions

Conceptualization: All authors

Funding acquisition: HMHL

Investigation: PD, HMHL

Writing–original draft: PD, HMHL, WYJ, WTC

Writing–review & editing: HMHL, WYJ, WTC

All authors read and approved the final manuscript.

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Tissue/cell	Conditions where NGAL is highly expressed	Reference
Kidney	Acute and chronic kidney disease, polycystic kidney diseases	[56,59]
Heart	Myocarditis, ischemia-reperfusion process, coronary heart disease	[60]
Skin	Psoriasis	[61]
Gums (alveolar mucosa)	Juvenile periodontitis	[62]
Adipose tissue	Obesity	[63]
Neutrophils and macrophages	Acute inflammatory response	[19]
Liver	Infection/post–partial hepatectomy, hepatocellular carcinoma	[64]
Astrocytes	Brain injury	[65]
Brain	Traumatic brain injury	[35]
Ovarian follicles	Ovarian tumors	[66]
Lungs	Inflamed lungs	[67]
Bone marrow	Osteoblast differentiation	[67]
Endometrial glands	Endometrial carcinoma	[68]
Spleen	Autoimmune encephalomyelitis	[69]
Synovial fluid	Rheumatoid arthritis and osteoarthritis	[70]