Application of Exfoliated Podocytes from Urine in CKD: Comparison
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Please note this is a comparison between Version 2 by Sirius Huang and Version 1 by Henry H.L. Wu.

Chronic kidney disease (CKD) is a global health issue, affecting more than 10% of the worldwide population. It is defined by structural and functional changes to the kidney. Urinary exfoliated podocytes and podocyte-specific markers have demonstrated value for the early diagnosis of CKD and prognosticating CKD progression.

  • exfoliated kidney cells
  • chronic kidney disease
  • non-invasive
  • early diagnosis

1. Introduction

Chronic kidney disease (CKD) is a progressive disease that is defined by structural and functional changes to the kidney [1]. CKD is considered to be a global issue, one with a substantial public health burden which is exponentially growing [2]. With more than 10% of the adult population currently affected by CKD, it is projected to become the fifth leading cause of mortality worldwide by 2040 [3]. There are multiple causes of CKD, some of which are more common and clearly defined (e.g., diabetes mellitus, hypertension, glomerulonephritis and polycystic kidney disease), whilst others are not fully understood (e.g., Mesoamerican nephropathy) [4,5,6,7][4][5][6][7]. CKD progresses differently in each individual, depending on the primary cause of CKD, as well as other co-morbidities [8]. CKD is typically identified by a reduction in kidney function, an estimated glomerular filtration rate (eGFR) of less than 60 mL/min/1.73 m2, and supported by markers of kidney tissue damage (albuminuria and hematuria), as well as other laboratory-based and imaging investigations that are present for at least 3 months [9]. The identification of early CKD, particularly in younger patients, remains challenging. Asymptomatic individuals living with CKD can lose up to 90% of their kidney function, at which point CKD is irreversible, given the advanced pathological damage [10]. Early diagnosis of CKD is clinically important, given that therapies are now available to stabilize kidney function from an early stage of the disease [11,12][11][12].
Histopathological examination of the kidney is the gold standard for the diagnosis and prognostication of CKD [13]. However, the risk of adverse events for patients after kidney biopsy has been well-documented. Post-biopsy risks include bleeding, excess pain and occasionally nephrectomy. For most individuals, bleeding usually resolves spontaneously following kidney biopsy, although for a small percentage of individuals, blood transfusion may be required [14,15][14][15]. Recently, the stress for the physician of routinely performing kidney biopsies has been addressed, with the increasing workload and time pressures of the modern-day clinical environment contributing to this issue [16]. There is a suggestion that the quality of training in kidney biopsy has reduced in recent years [17]. With the continuous development of non-invasive diagnostic and prognostication tools in CKD, it is questioned whether other diagnostic methods can complement or replace kidney biopsy in the near future.

2. Application of Exfoliated Podocytes in CKD

Urinary exfoliated podocytes and podocyte-specific markers have demonstrated value for the early diagnosis of CKD and prognosticating CKD progression (Table 1). Diabetic kidney disease (DKD) is the most common cause of CKD worldwide. In a post-hoc exploratory analysis comparing archived urine samples from normoalbuminuric patients with uncomplicated type 1 diabetes and healthy controls, urinary podocyte microparticle levels were found to be higher in the cohort with type 1 diabetes [29][18]. Interestingly, the elevation of urinary podocyte microparticle levels was well in advance of changes to other more well-established biomarkers of CKD such as albuminuria and nephrin, suggesting its potential utility as an early biomarker of glomerular injury in uncomplicated type 1 diabetes [29][18]. There is evidence demonstrating significant differences in urinary podocyte mRNA levels of nephrin, podocin, synaptopodin, Wilms Tumor-1 (WT-1) and α-actinin-4 between DKD and non-DKD patients [30][19]. These markers were found to precede the clinical appearance of microalbuminuria in patients with type 2 diabetes [31][20]. Urinary synaptopodocin mRNA levels were used to measure therapeutic response to angiotensin-converting enzyme inhibitor and angiotensin-receptor blocker treatment in DKD [32][21]. Urinary podocyte-derived indices such as podocin mRNA-to-creatinine ratio are a strong marker of podocyte detachment from GBM and are shown to project the rate of kidney functional decline in DKD [30][19]. For minimal change disease (MCN) and focal segmental glomerulosclerosis (FSGS), urinary nephrin and podocin mRNA levels were lower in patients with MCN and FSGS compared to healthy controls, and urinary nephrin and podocin mRNA levels correlated with the degree of proteinuria within this context [33][22]. Urinary synaptopodin mRNA levels were found to correlate with kidney function decline in FSGS [33][22]. In membranous nephropathy (MN), the number of urinary podocyte-derived microparticles displayed an inverse relationship with clinical parameters of MN, decreasing with improving clinical parameters following immunosuppression treatment [34][23]. The urinary podocyte mRNA levels of nephrin, podocin, and synaptopodin were all elevated in MN, with these levels clearly differentiating MN from other causes of nephrotic syndrome [35][24].
Table 1. Clinical utility of urinary exfoliated podocytes and podocyte-specific markers for early diagnosis and prognostication of CKD.
Etiology of CKD Clinical Utility of Urinary Exfoliated Podocytes and Podocyte-Specific Markers
DKD
  • Exfoliated podocyte microparticles in early DKD may prognosticate kidney function decline and disease progression [29][18]
  • ELISA of nephrin, podocalyxin and PCR-based testing of nephrin, podocin, synaptopodin mRNA, WT-1 and α-actinin 4 in DKD can identify early disease, assess degree of podocyte loss and monitor treatment response [30,31,32][19][20][21]
  • Podocin mRNA-to-creatinine ratio is a strong podocyte-derived index of podocyte detachment from GBM, and is shown to project the rate of kidney functional decline in DKD [30][19]
MCN and FSGS
  • PCR-based testing of nephrin and podocin mRNA in MCN and FSGS to differentiate between MCN and FSGS, nephrin and podocin mRNA levels correlated with the degree of proteinuria in MCN and FSGS [33][22]
  • PCR-based testing of urinary synaptopodin mRNA levels found correlations with kidney function decline in FSGS [33][22]
MN
  • Exfoliated podocyte microparticles can monitor treatment response in MN [34][23]
  • Urinary podocyte-specific nephrin, podocin, and synaptopodin all usually markedly elevated in MN following PCR-based testing. These markedly elevated levels allow for differentiation of MN from other causes of nephrotic syndrome [35][24]
IgA nephropathy
  • Indirect IF of exfoliated podocyte count in IgA nephropathy can be utilized to prognosticate histological severity [36,37][25][26]
Lupus nephritis
  • A greater proportion of apoptotic urinary exfoliated podocytes in patients with lupus nephritis compared to those without is typically found following flow cytometry [38][27]
  • Western blotting of nephrin, podocin, WT-1, podocalyxin, synaptopodocin and PCR-based testing of nephrin, podocin, synaptopodocin mRNA in lupus nephritis to prognosticate disease severity and kidney function decline [39][28]
ANCA-associated vasculitis
  • PCR-based testing of podocin mRNA in ANCA-associated vasculitis to assess histological severity and predict disease progression [40][29]
  • Urinary podocin-to-nephrin mRNA ratio, a surrogate marker of intra-glomerular podocyte stress, correlated with the extent of crescent formation in ANCA-associated vasculitis [40][29]
General CKD
  • Western blotting of urinary podocyte-specific synaptopodin protein expression demonstrated significant correlations with kidney function in all forms of CKD, regardless of the degree of albuminuria [41][30]
  • Application of scRNA-seq techniques for exfoliated podocytes for early identification and prognostication of CKD require further development [26][31]
ANCA: anti-neutrophil cytoplasmic antibody; CKD: chronic kidney disease; DKD: diabetic kidney disease; ELISA: enzyme-linked immunosorbent assay; FSGS: focal segmental glomerulosclerosis; GBM: glomerular basement membrane; IF: immunofluorescence; IgA: immunoglobulin A; MCN: minimal change disease; mRNA: messenger ribonucleic acid; MN: membranous nephropathy; PCR: polymerase chain reaction; scRNA-seq: single cell ribonucleic acid sequencing; WT-1: Wilms Tumor-1.
The role of exfoliated podocytes from urine as markers to prognosticate the progression of mesangial diseases such as IgA nephropathy has been explored. Previous studies noted that urinary podocyte counts correlated with serum creatinine and proteinuria in IgA nephropathy [36][25]. Those with segmental sclerosis, which was confirmed histologically, had greater numbers of urinary podocytes compared to those without segmental sclerosis [37][26]. Evidence is incomplete regarding the use of podocyte-specific mRNA and miRNA levels in IgA nephropathy as biomarkers for early diagnosis and prognostication, and this requires further study. For other mesangial glomerulopathies outside of IgA nephropathy, urinary podocyte counts are increased in hereditary and acquired diffuse mesangial sclerosis [37][26]. The application of non-invasive risk assessment techniques is not as well studied for these conditions, likely explained by a lack of clarity in pathological classification. Utilizing urinary podocytes as markers of disease activity in lupus nephritis has been evaluated. Studies have noted that the majority of urinary podocytes in patients with lupus nephritis are viable but dedifferentiated, with a greater proportion of apoptotic urinary podocytes being much lower in patients without kidney disease [38][27]. Urinary podocyte-specific mRNA markers such as podocalyxin, synaptopodin, podocin, nephrin, as well as WT-1 levels, are significantly elevated in patients with active lupus nephritis compared to those without systemic lupus or active lupus nephritis [39][28]. Within this context, urinary nephrin mRNA levels are shown to correlate with the degree of proteinuria and systemic lupus disease activity, but not with the histological progression of lupus nephritis [42][32]. Meanwhile, urinary podocin mRNA levels are demonstrated to be an independent predictor of kidney function decline in lupus nephritis [38,39,42][27][28][32]. There is minimal data regarding the use of exfoliated podocytes from urine for anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. It was previously found that the rate of podocyte detachment to urine predicted kidney function loss in ANCA-associated vasculitis [43][33]. Reports have also suggested that urinary podocin-to-nephrin mRNA ratio, a surrogate marker of intra-glomerular podocyte stress, correlated with the extent of crescent formation [40][29]. Paradoxically, patients with higher urinary podocyte-specific mRNA levels achieved better outcomes, as this indicated a stronger glomerular podocyte reserve for the reversibility of vasculitic disease [40][29]. Ultimately, there are urinary podocyte-specific biomarkers which have shown universal prognostic value for all forms of CKD. Urine synaptopodin levels are described as a generic marker of podocyte damage. Synaptopodin protein expression, determined by Western blot, has demonstrated significant correlations with kidney function in CKD, regardless of the degree of albuminuria [44][34]. Urinary podocyte-specific mRNA targets such as urinary brain-derived neurotrophic factor mRNA level had the best correlation with urinary kidney injury molecule-1 (KIM-1), which is recognized as a generic marker to prognosticate CKD progression [41][30]. The recent development of single-cell RNA sequencing (scRNA-seq) is a revolutionary technique in providing an unbiased genome-wide characterization of individual exfoliated cells from urine at scale [45][35]. Both animal and human studies have demonstrated that scRNA-seq is able to generate an initial map of gene expression for most kidney cells, thereby allowing us to advance from a morphology-based cell characterization through cell shape, color and location to the more objective method of cellular definition through transcriptomics [46,47][36][37]. In CKD, scRNA-seq may be able to define cell type-specific changes, cell fractions and cell-to-cell interactions [26,48,49][31][38][39]. This information can be useful for the diagnosis and risk stratification of CKD. In a combined analysis between urinary exfoliated podocytes, bladder single cells and human kidney tissue nucleus (extracted from DKD and control patients) scRNA-seq datasets, a strong correlative relationship was found between exfoliated podocyte and kidney tissue nucleus scRNA-seq datasets, where together they formed a strong cluster [26][31]. Urinary scRNA-seq has particularly shown a strong expression of monogenic nephrotic syndrome genes in podocytes [26][31]. Nevertheless, these results were obtained in a pilot study with few patients and controls. Abedini and colleagues ensured urine was collected at different time points and through different methods to confirm the reproducibility and feasibility of this approach [26][31]. Other confounding factors include the differences of scRNA-seq in capturing efficiency between the male and female urine samples, and perhaps a subgroup analyses between them is required to reduce sex-associated bias in the interpretation of results. Furthermore, there was significantly higher ambient RNA contamination in 24-h urine collections [26][31]. Meticulous arrangement to optimize the environment of urinary cell storage may mitigate the risks of cellular degradation during extended storage. As the authors noted, large prospective cohort studies are needed to validate the diagnostic and prognostic utility of urinary scRNA-seq in CKD.

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