Urinary Podocyte Markers in Kidney Diseases

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Lingfeng Zeng, et al

Abstract

Podocytes play an important role in the maintenance of kidney function, and they are the primary focus of many kidney diseases. Podocyte injury results in the shedding of podocyte-derived cellular fragments and podocyte-specific molecular targets into the urine, which may serve as biomarkers of kidney diseases. Intact podocytes, either viable or dead, and podocyte-derived microvesicles could be quantified in the urine by various centrifugation, visualization, and culture methods. Podocyte-specific protein targets from the nucleus, cytoplasm, slit-diaphragm, glomerular capillary basement membrane, and cytoskeleton, as well as their corresponding messenger RNA (mRNA), in the urine, could be quantified by western blotting, ELISA, or quantitative polymerase chain reaction. Although some of these techniques may be expensive or labor-intensive at present, they may become widely available in the future because of the improvement in technology and automation. The application of urinary podocyte markers for the diagnosis and monitoring of various kidney diseases has been explored but the published data in this area are not sufficiently systematic and lack external validation. Further research should focus on standardizing, comparing, and automizing laboratory methods, as well as defining their added value to routine clinical tests.

Keywords: Podocyte, Biomarker, mRNA, miRNA, Nephrin, Podocin, Podocalyxin, Synaptopodin, Chronic kidney disease

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1. Introduction

1.1. Epidemiology of chronic kidney disease

Chronic kidney disease (CKD) is an important and costly non-communicable disease [1]. With the availability of renal replacement therapy, the healthcare expenditure used for the treatment of CKD (Chronic kidney disease) rose sharply after the 1960s [2,3]. In 2015, there were over 2.5 million patients receiving renal replacement therapy worldwide, and this number is projected to double by 2030 [4]. Effective means for diagnosis, treatment, and monitoring of kidney disease are much needed.

1.2. Podocyte as the focus of kidney disease

The primary function of the kidney is the excretion of metabolic wastes and excessive body water [5], which is achieved by three processes: (1) filtration of the circulating blood through the glomerulus to form an ultrafiltrate in the Bowman’s space; (2) selective reabsorption of useful substances via the renal tubule; and (3) selective secretion other metabolic wastes from peritubular capillary to the tubular fluid [6]

Although the kidney consists of multiple cell types that are arranged in a delicate 3-dimensional architecture, podocyte plays a key role in the maintenance of normal kidney function and is the primary focus of many kidney diseases. Podocytes are highly specific terminally differentiated cells that, together with the endothelial cells and glomerular capillary basement membrane (GCBM), constitute the glomerular filtration barrier [7]. Podocytes have a voluminous cell body that is separated from the GCBM by a sub-podocyte space [8]. The cell body gives rise to long primary processes that extend towards the GCBM and are affixed to the latter by an extensive array of foot processes [8].

Podocytes have several physiological functions. They maintain the glomerular vascular loop morphology, regulate glomerular filtration, and participate in the local immunological and inflammatory response. In addition, podocytes are partly responsible for the synthesis and turnover of the GCBM [9], as well as the production of paracrine factors such as vascular endothelial growth factor (VEGF), which is important in the regulation of permeability and proliferation of endothelial cells [10]. Podocyte dysfunction plays a pivotal role in the pathogenesis and progression of many kidney diseases. Mutations of various podocyte-related genes result in kidney diseases, which typically present clinically as steroid-resistant nephrotic syndrome and histologically as focal glomerulosclerosis (FGS). On the other hand, acquired podocyte dysfunctions, including a reduction in podocyte number or density and fusion of podocyte foot processes, lead to secondary endothelial and GCBM injury, loss of GCBM negative charge, Hiatal membrane protein abnormality, and finally proteinuria through various mechanisms [11,12].

A characteristic consequence of podocyte injury is the weakened interaction with GCBM, which cumulated into the detachment of podocytes from GCBM. Since podocytes have a limited capacity for regeneration and functional compensation, the remaining podocytes are not able to maintain the mechanical and charge barrier functions of GCBM, which would be irreversibly damaged, and the end result is proteinuria [13]. Depending on the severity of the insult, damage to podocytes could lead to loss of foot processes, vacuole and pseudocyst formation, dedifferentiation, and apoptosis. Neighboring structures would be affected subsequently, including the proliferation of parietal epithelial cells, shrinkage of GCBM, loss of glomerular capillary tuff, and finally glomerulosclerosis [14].

1.3. Method of literature review

We conducted a literature review with search terms including “podocyte”, “podocyte-associated molecules”, or “biomarker”, together with “chronic kidney disease”, “acute kidney injury”, “glomerulonephritis”, “diabetic nephropathy”, or “diabetic kidney disease”, were used to search relevant literature from Medline, Embase and Cochrane Library. References of relevant publications were examined for additional potential papers. Eligible studies included human and animal studies published before July 2020. Publications with irrelevant topics, such as podocyte cell biology, membrane physiology, and molecular manipulation of podocytes, were excluded.

1.4. Podocyte-specific targets as biomarkers

Podocyte injury leads to the shedding of various podocyte-derived molecules into the urinary space and may serve as a biomarker of kidney diseases (Fig. 1). The podocyte cell bodies could be divided into three anatomically and functionally distinct compartments: the base part, the top part, and the slid-diaphragm (SD) part [15]. Specific cell membrane proteins are present in each part, and their interaction with the cytoplasmic cytoskeleton system is responsible for the stability of podocyte structure and function [16]. The major podocyte-derived molecules that are potential targets for biomarker development are summarized in Table 1.

Fig. 1. Podocyte injury and potential podocyte-derived markers in urine. Injury to podocytes results in 3 inter-related pathological processes: morphological change, apoptosis, and detachment. Podocytes may remain viable but manifest as morphological changes that have functional downstream consequences, such as proteinuria and tubulointerstitial fibrosis. Podocyte apoptosis may develop as a result of cumulated morphological changes or directly from specific insults. Loss of podocytes in the glomeruli (i.e. podocytopenia) leads to architectural changes in the glomerular capillary loop and results in glomerulosclerosis. Podocytes, either apoptotic or viable ones due to loss of α3β1 integrin, may detach to the urinary space, be identified in the urine, and serve as markers of kidney disease. Vesicles, microparticles, or podocyte-specific molecules derived from injured or apoptotic podocytes may also be detected in the urine and serve as biomarkers. (GCBM, glomerular capillary basement membrane; AgII, angiotensin II; TGF-β, transforming growth factor-beta; ROS, reactive oxygen species).

1.5. Nuclear and cytoplasmic targets

Wilms’ tumor suppressor gene-1 (WT1) is a zinc finger-like transcription factor in the podocyte nucleus and is probably the most commonly used podocyte-specific marker for histological study [17]. The WT1 gene consists of 10 exons, of which exons 7 to 10 encode the 4 zinc fingers of the DNA-binding domain. WT1 is specifically expressed in podocytes and it probably regulates the expression of nephrin [18]. Heterozygous mutations in exons 8 and 9 of the WT1 gene lead to Denys-Drash syndrome, which presents with nephrotic syndrome, male pseudohermaphroditism, and Wilms tumor [19].

Other nuclear and cytoplasmic proteins of podocytes are less well studied as biomarkers. The aarF domain-containing kinase-4 (ADCK4) is specifically present in the mitochondria within the foot processes of rat podocytes [20]. ADCK4 is responsible for stabilizing the CoQ complex and it is required for normal podocyte homeostasis function [20]. Podocyte-specific ADCK4 ablation in mice results in foot process effacement, disorganization of the filtration slit, enlarged and dysfunctional mitochondria, and reduced CoQ10 [21], and treatment restored CoQ10, mitochondrial function, improved podocytes morphology, and prevented foot process effacement [21,22].

1.6. Slit-diaphragm protein complex

Proteins components of the podocyte slit-diaphragm have been extensively studied as biomarkers of kidney diseases. The major proteins in this group include nephrin, podocin, CD2-associated protein (CD2AP), and podoplanin [23]. Nephrin is a transmembrane protein with extracellular domains that form the core of the slit diaphragm. It acts both as a physical sieve barrier and a signaling scaffold [24]. Mutations of the nephrin gene were the first reported cause of congenital nephrotic syndrome in humans [25]. Podocin and CD2AP are responsible for connecting nephrin to the actin cytoskeleton under the cell membrane. Podocin is a hairpin-like protein of the stomatin family and is exclusively expressed in podocytes [26]. It is encoded by the NPHS2 gene that consists of eight exons and is located on the chromosome 1q25-q31 region [27]. Mutations of the NPHS2 gene cause an autosomal recessive steroid-resistant nephrotic syndrome in both pediatric and adult patients [27,28]. CD2AP is a scaffolding molecule that directly interacts with filamentous actin and other cell membrane proteins and is implicated in dynamic actin remodeling and membrane trafficking [29]. Podoplanin is a small transmembrane glycoprotein with a large number of O-glycoside chains. Selective loss of podoplanin in podocytes leads to proteinuria, followed by alterations in podocyte morphology in a rat model of spontaneous proteinuria [30].

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1.7. Targets linked to the GCBM

The major protein components of the podocyte basal region are the α3β1 integrin and α-dystroglycan (α-DG). These proteins anchor podocytes to the GCBM and play an important role in maintaining the correct position of the podocyte as well as the integrity of the filtration membrane [31,32]. The α3β1 integrin is a transmembrane protein with the extracellular domain linked to the laminin G-like domain G (LG) of laminin 521 in the GCBM, while its cytoplasmic region is connected to the podocyte cytoskeleton by the α-actinin-4. The glycosylated protein α-DG is connected to the cytoskeleton via utrophin, and to the podocyte and GCBM through its interaction with laminin 521.

1.8. Targets at the top membrane

Podocalyxin is the major transmembrane protein on the apical side of the podocyte. It is a negatively charged sialic acid protein that contributes to the charge barrier of glomerular filtration [33]. Podocalyxin has three functions: (1) to prevent negatively charged protein from leaking into the urine; (2) to maintain the separation of adjacent podocytes; and (3) to prevent the adhesion between epithelial cells and capillary loops [34]. Podocalyxin is connected to the actin cytoskeleton by ezrin and sodium-proton exchanger regulatory factor 2 (NHERF2).

1.9. Actin-related targets

A number of podocyte-specific proteins are linked to the actin cytoskeleton and maintain the 3-dimensional structure of the podocyte. The best-studied targets of this group are synaptopodin and α-actinin-4. Both of them bind to the actin skeleton by interacting with the membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1) [35]. Synaptopodin is a proline-rich linear protein encoded by the SYNPO gene [36] and is expressed in differentiating podocytes when they develop the foot processes. As a result, synaptopodin is considered to be a marker of mature podocytes [37].

2. Methods of study

2.1. Intra-glomerular podocyte markers

Although the focus of this review is on urinary podocyte markers, it is logical to discuss briefly the detection of these markers in the kidney tissue. Podocyte depletion can be absolute (loss of podocytes) or relative (reduced number of podocytes per volume of glomerulus) [38]. The gold standard method of quantifying podocytes in kidneys is stereological methodologies (light or electron microscopes, confocal laser scanning microscopes, ultrasound, computed tomography, or magnetic resonance imaging) [39]. However, all of them are time-consuming and labor-intensive. Recently, Venkatareddy et al. evaluated the method for the estimation of podocyte density by single paraffin-embedded formalin-fixed sections, in which podocyte nuclei were visualized by indirect immunofluorescence with antibodies against WT1 or transducin-like enhancer of split [40]. A subsequent study showed that this method provided accurate podocyte enumeration in a transgenic mouse model of selective podocyte depletion, confirming that this approach provides an efficient quantitative tool for the analysis of podocyte depletion in the whole glomeruli [41]. Nonetheless, this method still requires kidney biopsy and is therefore not suitable for serial monitoring.

2.2. Urinary podocyte quantification and culture

Since podocytes are fixed to their physiological position only by the foot processes, they are vulnerable to being lost as viable cells in the urine [42]. In fact, intact podocytes are detectable in the urine of healthy people and patients with kidney diseases [43], and urinary loss is probably a major mechanism of podocytes depletion during the progression of CKD (Chronic kidney disease) [42]. The traditional method for the identification of podocytes in urine involves cytospin techniques and immunofluorescence study with specific anti-podocalyxin antibodies [44], for which automation is partly feasible. However, the technique has limited sensitivity and specificity because of contaminating cellular debris in the urinary sediment.

Alternatively, podocytes could be identified by the detection of podocyte-specific tryptic peptides with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Although the equipment cost of this technique is high, it has the advantages of being operator-independent and highly reproducible [45], and will probably have growing applicability in clinical laboratory practice.

Previous studies showed that with the cytospin immunofluorescence technique, healthy people excrete less than 0.5 podocytes/mg creatinine, whereas patients with active glomerular disease excrete up to 400 podocytes/mg creatinine [43]. The majority of urinary podocytes are viable when tested with propidium iodide exclusion and TUNEL staining [43]. In theory, the culture of viable podocytes ex vivo would therefore improve the specificity of podocyte identification by removing dead and nonspecific cells. However, podocytes do not normally proliferate in vivo, and special experimental conditions are required for their culture ex vivo [46]. Previous reports showed that podocytes could first be grown under growth-permissive conditions, where immortalized podocytes replicate [46]. The cell culture media for this purpose generally consists of Roswell Park Memorial Institute (RPMI) 1640 or Dulbecco’s modified Eagle’s medium, with 10% fetal bovine serum supplemented with 20–100 U/ml of mouse interferon-gamma (INF-γ), and the culture dishes are usually coated with type I collagen to promote podocyte proliferation [46]. This method has great value in translational study and therapeutic target identification but is too complicated for routine clinical use.

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2.3. Urinary podocyte-derived fragments

Extracellular vesicles (EVs) are spherical membranous bodies released by various cell types [47]. In the urinary system, EVs containing apical membrane and intracellular fluid are shed from all nephron segments, including podocytes, renal tubular epithelial cells, and uroepithelial cells into the urine. Urinary EVs contain protein markers of dysfunction and structural injury of the kidney. Recent studies further suggest that EVs may be relevant for intercellular communication [48]. In addition, previous studies also showed that apical cell membranes fragments are shed from injured podocytes into the urine [49], and could be identified as podocalyxin-positive granular structures (PPGS) by electron microscopy [49]. In diabetic mice models, urinary exosomes from podocytes could be isolated by serial centrifugation, and microparticles could be quantitated by flow cytometry using annexin V antibody (which detects all microparticles), followed by antibody to podocalyxin or podoplanin (which specifically identify podocytederived microparticles) [50,51]. The additional light-scattering analysis could be used to further determine the size of the microparticles [52]. With these sophisticated techniques, it has been shown that podocyte-derived microparticles are increased in mice upon exposure to high glucose [51], as well as in diabetic mice before the onset of albuminuria [52]. In the former case, the abnormality was suppressed by a Rho-kinase inhibitor, indicating that cytoskeletal reorganization is the major trigger of microparticle release [51]. However, the application of these techniques to human kidney disease has not been explored.

Rather than measuring the number and size of podocyte-derived EVs and microparticles in the urine, there is recently a growing interest in measuring podocyte-specific molecules in urinary exosomes. For example, podocyte-derived signal transduction factors (PDSTFs) in urinary EVs have been proposed as a potential candidate for the assessment of podocyte injuries [52]. However, their clinical applications remain under development.

2.4. Urinary podocyte-specific protein targets

The urinary levels of several podocyte-specific protein targets are readily measured and their role as biomarkers of kidney disease has been studied. Urinary podocalyxin level can be measured by indirect immunofluorescence, ELISA, or flow cytometry [53], and has been proposed as a marker for urinary podocyte count. However, a previous study showed that urinary podocalyxin mostly originated from the apical membrane of injured podocytes rather than intact ones shredded into the urine, and its level is increased in early kidney injury [49]. Podocalyxin and nephrin, another important podocyte-specific protein target in urine, are not detectable in the urine of healthy people by traditional Western blotting [54] but are detectable by conventional ELISA in patients with pre-eclampsia [55]. Moreover, urinary nephrin level correlates with the severity of proteinuria [56] and kidney function [57]. Plasma nephrin level could also be measured [57], but its biological or clinical relevance has not been determined. The major problem of using the urinary levels of podocyte-specific protein targets as a clinical biomarker is the low concentration (usually in the range of ng/ml) and the confounding effect of urine concentration-dilution.

2.5. Urinary podocyte-specific mRNAs

Measurement of urinary podocyte-specific mRNA level has been proposed as an alternative method for the determination of urinary podocyte count. The amount of podocyte-specific mRNA in urine is usually not sufficient for the traditional northern blot study but is readily measured by real-time quantitative polymerase chain reaction (RT-QPCR). Two previous studies showed that urinary nephrin mRNA levels had a close correlation with urinary podocyte count [56,58]. In a mouse model of anti-glomerular basement membrane disease with a serial kidney biopsy, urinary podocyte-specific mRNA levels correlated with the rate of glomerular podocyte loss [52].

Recent studies, however, have shifted the focus to derived urinary mRNA indices as surrogate markers for podocyte stress or relative podocyte injury. In a rat model, the progressive glomerular disease was associated with decreased nephrin as compared to podocin mRNA levels, and urinary podocin-to-nephrin mRNA ratio correlated with the severity of histological progression [59]. In a subsequent study, the mean arterial pressure of healthy people was shown to correlate with the urinary podocin mRNA to creatinine ratio (a marker of podocyte detachment), podocin-to-nephrin mRNA ratio (a marker of podocyte stress), and urinary podocin-to-aquaporin-2 mRNA ratio (a marker of relative podocyte injury versus tubular injury) [60]. Glomerular injury is specifically associated with increased urinary podocin-to-aquaporin-2 and nephrin-to-aquaporin-2 mRNA ratios [61].

2.6. Urinary miRNA markers specific for podocytes

Micro-RNAs (miRNAs) are short non-coding RNAs that regulate many biological pathways by targeting specific mRNAs [62]. Similar to mRNA, urinary miRNA is readily quantified by RT-qPCR [63]. From the perspective of biomarker development, miRNA has the advantage of being resistant to degradation, which facilitates its clinical use as well as the study of archive samples [63]. A number of specific miRNA changes have been observed in kidney diseases [9]. For example, podocyte-specific loss of functional miRNAs, including miR-23b, miR-24, and miR-26a, was found to result in rapid glomerular and tubular injury [64]. Specifically, miR-27b regulates podocyte survival through targeting adenosine receptor 2B [65]. Similarly, urinary miR-21, miR-124, and miR-192 levels were reported to correlate with the severity of kidney dysfunction (albuminuria and estimated GFR) and urinary podocyte markers (nephrin, synaptopodin, and podocalyxin) in diabetic patients [66]. Recent studies further identified specific downstream pathways for several miRNAs that may be relevant for the pathogenesis or progression of kidney diseases. Notably, miR-193a suppresses the expression of WT- 1, which affects podocyte differentiation [67] and may play a role in the pathogenesis of the glomerular disease [68]. On the other hand, miR-21 inhibits the expression of tissue inhibitor of metalloproteinase 3 (TIMP3) [69,70], miR-26a inhibits transforming growth factor beta-1 (TGF-β1) expression [71,72], miR-23b targets Ras GTPase-activating protein SH3 domain-binding protein 2 (G3BP2) [73], miR-29c targets Sprouty homolog 1 (Spry1) [74]; all of which are involved in the progression of diabetic nephropathy.

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However, podocyte contributes to only a small proportion of urinary miRNA. Any specific miRNA alterations observed in the podocytes may not be readily discernable in the urine, while any alteration in the urinary miRNA levels may reflect the pathological change in other renal cell types [75,76]. For example, although urinary levels of miR-155, miR-663, and miR-1915 are significantly different between patients with FGS or minimal change nephropathy (MCN) and healthy controls [77], it has not been shown that miR-155, miR-663, or miR-1915 specifically originates from podocytes. Significant correlations between many miRNA levels and tubular injury markers, including urinary levels of N-acetyl-β-D-glucosaminidase (NAG) and kidney injury molecule-1 (KIM-1) [66], have also been reported. In other words, it is difficult to confirm their specificity as podocyte injury markers. The notable exception to this rule is miR-26a. A recent study showed that urinary exosomal miR-26a levels were significantly higher in lupus nephritis than in healthy control, miR-26a was the most abundantly expressed miRNA in the glomerulus of normal C57BL/6 mouse, and miR-26a was responsible for the regulation of podocyte differentiation and cytoskeletal integrity [78]. Nonetheless, the role of urinary miR-26a as a podocyte-specific marker needs to be validated.

2.7. Circulating podocyte markers

Many of the above-mentioned podocyte markers, notably podocyte-specific protein targets and mRNAs, could be measured in systemic circulation [56,79,80], and their plasma levels as a biomarker of kidney diseases have been explored [55,81–84]. However, a full discussion on this topic is beyond the scope of this review. The key advantages and disadvantages of the above-mentioned methodologies are summarized in Table 2.

3. Urinary podocyte markers in specific kidney diseases

3.1. Diabetic kidney disease

Diabetic kidney disease (DKD) is the most common cause of CKD (Chronic kidney disease) around the world, and there is a wealth of literature on the use of various urinary podocyte markers for the diagnosis and monitoring of DKD. For example, Jim et al [14] reported that urinary nephrin levels were detectable in all DKD patients with albuminuria, but only 54% in those without albuminuria. Urinary nephrin levels correlated with the level of albuminuria, and inversely with kidney function [14]. Ma et al reported that the urinary angiopoietin-like-4 (Angptl4) level was increased in DKD [85]. Urine podocalyxin level was increased in diabetic patients before the onset of microalbuminuria and was probably more sensitive than microalbuminuria for the early detection of DKD [86]. In established DKD, urine podocalyxin level positively correlated with HbA1c level and the albumin-to-creatinine ratio [87,88]. Taken together, available results indicate that urinary levels of several podocyte-specific targets are early indicators of DKD, and urinary podocalyxin level is the most promising marker in this regard [54].

The levels of the podocyte-specific target in individual urine components have also been explored. Urinary podocyte microparticle levels, as determined by flow cytometry, were higher in type 1 diabetes than in healthy controls, and the level further increased with hyperglycemic clamp [89]. Urinary exosomal WT-1 levels correlated with the severity of proteinuria, kidney function, glomerular damage, and the rate of renal function decline in diabetic patients [58,90]. In another study, glycogen synthase kinase (GSK)3β levels in urinary exfoliated cells were more accurate than albuminuria in discriminating diabetic patients with and without progressive renal impairment [91]. However, the methodology of this assay is cumbersome and may not be applicable to routine clinical practice.

As to podocyte-specific mRNA markers in urine, a previous study showed that urinary mRNA levels of nephrin, podocin, synaptopodin, WT-1, and α-actin-4 were higher in patients with DKD than in normal controls [92]. There was a significant difference in urinary mRNA levels of nephrin, podocin, α -actinin-4, and CD2-associated protein in diabetic patients with various severity of albuminuria [93]. Urinary mRNA levels of nephrin, podocin, synaptopodin, WT-1, and α-actin-4 also correlated with urinary podocyte count, urinary nephrin level, albuminuria, and the severity of renal impairment [93]. More recently, urinary podocyte-specific mRNA levels were found to precede the onset of microalbuminuria in patients with type 2 diabetes [94], and urinary podocin mRNA-to-creatinine ratio, a marker of podocyte detachment, significantly correlated with the subsequent rate of kidney function decline [94]. Furthermore, urinary synaptopodin mRNA level was lower after 12 weeks of angiotensin-converting enzyme (ACE) inhibitor and angiotensin receptor blocker (ARB) combination therapy as compared to ACE inhibitor monotherapy [95]. Taken together, available evidence suggests that the measurement of podocyte-specific mRNA levels in urine may be valuable for risk stratification and monitoring of therapeutic response in DKD.

3.2. Minimal change in nephropathy and focal glomerulosclerosis

MCN and FGS are often regarded as podocyte diseases, and the role of urinary podocyte targets has been tested. Zhou et al. [96] reported that urinary exosomal WT-1 level, as measured by immunoblotting technique, was significantly higher in FGS patients than in those with steroid-sensitive nephrotic syndrome (SSNS) or healthy volunteers. Urinary exosomal WT-1 levels decreased significantly following corticosteroid treatment and remission of FGS or SSNS [96]. In another study, urinary nephrin level, as measured by a conventional ELISA assay, was significantly higher in adults with FGS than in other causes of nephrotic syndrome [97].

Urinary levels of podocyte-specific mRNA have also been studied. An early study showed that urinary nephrin and podocin mRNA levels were not significantly different between patients with MCN and healthy volunteers, and their levels were significantly lower than DKD patients [98]. A subsequent study showed that urinary nephrin and podocin mRNA levels were lower in patients with MCN than in FGS or healthy controls, and the magnitude of reduction correlated with the degree of proteinuria [99]. In this study, urinary synaptopodin mRNA levels were found to correlate with the subsequent rate of renal function decline in FGS patients [99], suggesting that a qualitative alteration in FGS podocytes could be identified in the urine.

3.3. Membranous nephropathy

Although the principal pathological change of membranous nephropathy (MGN) is the deposition of an immune complex in the subepithelial space, which results in the alteration of the glomerular filtration barrier, studies on urinary podocyte markers in MGN are scarce. Urinary sediment mRNA levels of nephrin, podocin, and synaptopodin were significantly elevated in MGN, but the levels did differentiate MGN reliably from other causes of nephrotic syndrome [98]. More recently, Lu et al [100] reported that the number of urinary podocyte-derived microparticles decreased simultaneously with the improvement in clinical parameters after immunosuppressive therapy, suggesting a role in the monitoring of MGN.

3.4. IgA nephropathy

Although immunoglobulin A nephropathy (IgAN) is primarily a mesangial disease, the role of urinary podocyte count as a biomarker has been explored. Notably, Shen et al [101] found that IgAN patients had an increase in urinary podocyte loss, fewer podocytes in the glomeruli, and more severe foot process fusion in the kidney biopsy specimens. In this study, urinary podocyte was identified by indirect immunofluorescence study of podocalyxin, and the urinary podocyte count correlated with serum creatinine and proteinuria [101]. A subsequent study showed that the number of urinary podocytes in IgAN patients with segmental sclerosis was higher than in those without segmental sclerosis [102]. In children with IgAN or Henoch-Sch¨ online purpura nephritis, Hara et al [103] showed that the cumulative loss of podocytes in urine correlated with the severity of histology damage and the degree of glomerulosclerosis in kidney biopsy, and patients with persistent urinary podocyte excretion had rapid histological progression.

The studies on podocyte-specific molecules in urine as biomarkers of IgAN are fragmented and incomplete. Urinary podocalyxin level significantly correlated with the severity of acute extra capillary lesions in adult IgAN [102]. Urinary podocin mRNA level has been reported to reflect the severity of active glomerular injury in kidney biopsy and provides prognostic information complementary to the level of proteinuria [104]. Since IgAN is the most common primary glomerulonephritis around the world, more studies are certainly needed in this area.

In addition to IgAN, the role of urinary podocyte markers has been explored in other mesangial glomerulopathies. For example, urinary podocyte loss is markedly increased in both hereditary and acquired forms of diffuse mesangial sclerosis [102]. Nonetheless, this area is less well studied, probably because of the heterogeneity in its pathological classification.

3.5. Lupus nephritis

Urinary podocyte shedding in lupus nephritis has been studied. A previous report showed that the major of urinary podocytes in patients with active lupus nephritis were viable but dedifferentiated, and the proportion of apoptotic podocytes in urine was significantly lower than that in healthy controls [105]. In this study, urinary podocalyxin, synaptopodin, podocin, nephrin, and WT-1 levels (as measured by western blotting) were significantly increased in systemic lupus, especially in active lupus nephritis, and their levels significantly correlated with the severity proteinuria and histological activity [105]. A subsequent study further reported that urinary mRNA levels of nephrin, podocin, and synaptopodin were significantly higher in patients with active lupus nephritis than in quiescent lupus [106]. Specifically, urinary nephrin mRNA level correlated with proteinuria and systemic disease activity, but not the histological class of lupus nephritis, while urinary podocin mRNA level was an independent predictor of subsequent renal function decline [107].

3.6. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis

Although podocytes are not the primary target of kidney injury in ANCA-associated glomerulonephritis, intra-glomerular podocyte density is characteristically reduced [108]. Zou et al [108] found that the rate of podocyte detachment to urine predicts subsequent renal function loss in ANCA-associated glomerulonephritis, and Minakawa et al [109] reported that urinary podocin to nephrin mRNA ratio, a surrogate marker of intra-glomerular podocyte stress, correlated with the percent of crescent formation [109]. In the latter study, patients with high levels of urinary podocyte-derived mRNA had a favorable renal outcome, presumably because of better glomerular podocyte reserve and the potential for reversibility [109].

3.7. Other CKD

The utility of urinary podocyte markers in specific kidney diseases is summarized in Table 3. A number of podocyte-specific markers in urine have also been explored as generic markers of CKD (Chronic kidney disease). For example, urinary podocyte-derived extracellular microvesicles, as quantified by flow cytometry, were significantly increased in hypertensive patients with impaired kidney function than in those without renal impairment [110]. Urine synaptopodin level, as determined by Western blotting, correlated with kidney function in both diabetic and non-diabetic CKD (Chronic kidney disease), regardless of the degree of albuminuria, suggesting that urine synaptopodin is a generic marker of podocyte damage [110]. Among a panel of podocyte-specific urinary mRNA targets, urinary brain-derived neurotrophic factor (BDNF) mRNA level had the best correlation with urinary kidney injury molecule-1 (KIM-1), which is a generic marker of kidney damage [111]. For miRNA targets, urinary exosomal miR-21 levels were increased in animal models of podocyte injury as well as CKD (Chronic kidney disease) patients [112], but the cellular origin of miR-21 was not determined in this study.

Nonetheless, it is important to note that not all kidney diseases have podocyte injury. An increase in urinary podocyte loss has been reported in various glomerular diseases but not in autosomal dominant polycystic kidney disease (ADPKD) [113]. More importantly, the association between podocyturia and proteinuria varied markedly between different glomerular diseases, suggesting that urinary podocyte markers should better be regarded as disease-specific [113].

4. Conclusion

Podocyte injury plays an important role in the pathogenesis and progression of many kidney diseases. Podocyte-derived cellular fragments and podocyte-specific molecular targets in the urine have a great potential to be developed as biomarkers for the diagnosis and monitoring of kidney diseases. The process of biomarker development includes the identification of specific markers, determination of the methodology of measurement, and decision on the clinical context for application (Fig. 2). With the advance in our understanding of podocyte biology and the availability of new technologies, urinary podocyte markers are expected to have an expanding scope of application, both because of the identification of new targets and the development of novel methods for their quantification. In return, the validation of urinary podocyte markers may shed light on our understanding of the pathophysiology of kidney diseases. There is a wealth of methods for the quantification of podocyte and various podocyte-specific markers in urine. In the coming decade, research efforts should be focused on standardizing, comparing, and automizing laboratory methods, as well as defining their added value to routine clinical tests.

Fig. 2. Aspects to be considered for the development of urinary podocyte-related biomarkers for kidney diseases. (GCBM, glomerular capillary basement membrane; CKD, chronic kidney disease; DKD, diabetic kidney disease; FGS, focal glomerulosclerosis).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This study was supported by the Richard Yu Chinese University of Hong Kong (CUHK) PD Research Fund, and CUHK research accounts 6905134 and 8601286. The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. The authors declare no other conflict of interest. The results presented in this paper have not been published previously in whole or part.

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From: 'Urinary podocyte markers in kidney diseases' by Lingfeng Zeng, et al

---Clinica Chimica Acta 523 (2021) 315–324