Relationship between Urinary Podocytes and Kidney Diseases

Abstract

Podocyte loss is an important component of disease progression in glomerular diseases. To some extent, the loss of podocytes can predict the degree of damage and the advancement of renal disease. Detecting the loss of podocytes in the urine could be a valuable, noninvasive method for obtaining information about the activity of the disease or the disease type, allowing the early diagnosis of glomerular diseases. One of the most robust markers that has been successfully used for urinary podocyte diagnostics is podocalyxin (PDX). PDX is a sialoprotein that is expressed on podocytes and on a variety of nonrenal cells as well as on glomerular endothelial and parietal epithelial cells. Therefore, podocyte loss can be detected by the amount of PDX in the urine. The relationship between urinary podocytes and renal diseases is supported by the detection of podocytes in patients with immunoglobulin A (IgA) nephropathy, Henoch–Schönlein purpura nephritis, lupus nephritis, diabetic nephropathy, and focal segmental glomerulosclerosis. The use of technology for detecting podocytes in the urine would have broad implications for the evaluation of disease activity, the degree of dedifferentiation, and the possibility of regeneration.

• podocyte
• urine
• kidney diseases
• podocalyxin
• diagnostic

INTRODUCTION

Currently, a large amount of experimental and clinical evidence supports the role of podocyte damage in the development of glomerulosclerosis (GS) and the progression of renal diseases.^1–3 The podocyte is the primary target of injury in several immunologic and nonimmunologic forms of glomerulonephritis.^4,5 Podocyte loss and the determination of urinary messenger RNA (mRNA) levels have been shown to be an important component of the progression of glomerular diseases.⁶ One of the best markers that has been successfully used for urinary podocyte diagnostics is podocalyxin (PDX), which is a sialoprotein that is found on podocytes and on a variety of nonrenal cells as well as on glomerular endothelial and parietal epithelial cells. The loss of podocytes reflects, to some extent, disease activity and the degree of dedifferentiation or regeneration. The detection of podocytes in the urine could be a valuable, noninvasive method for obtaining information on the activity of the disease or the disease type and could be used as a follow-up after a biopsy in an outpatient setting. Many studies have summarized the available information on marker proteins that have been successfully used in the diagnosis of podocytes in the urine. In addition, podocytes are also seen as a potential target for the evaluation of the effect of treatment for various renal diseases. This article summarizes the presumed relationship between urinary podocytes and renal diseases.

FUNCTION OF PODOCYTES IN KIDNEY DISEASES

Podocytes are highly specialized epithelial cells that line the urinary surface of the glomerular capillary tuft. They are part of the filtration barrier together with capillary endothelial cells and the glomerular basement membrane (GBM), which ensures the selective permeability of the glomerular capillary wall.⁷ The turnover rate of podocytes is very low under normal and various pathologic conditions compared with that of other glomerular cells. In addition, podocytes contribute by acting as a filter for macromolecules. To a certain extent, podocytes establish the fundamental principle of selective permeability in the GBM. Furthermore, podocytes synthesize components of the GBM and provide structural stability to the glomerular tuft.⁸ They have a specialized configuration, the slit diaphragm, which is an electron-dense membrane that spans the 30 nm space between their interdigitating foot processes. The transmembrane protein nephrin is the dominant component of the slit diaphragm and forms a complex that is relevant for intracellular survival signaling and mechanosensation.

The loss of podocytes appears to play an important role in the progression of glomerular disease and the development of GS. Recently, studies have found podocytes not only in the urine of patients with glomerular disease but also in the urine of healthy subjects. It has also been proven that the number of podocytes in the urine of healthy controls and patients with inactive disease was significantly lower than in patients with active glomerular disease. The rate of podocyte excretion appears to reflect both disease activity and disease type.

It has been well established that a decrease in podocyte number is associated with the development of GS in most forms of nephrotic syndrome. Currently, there are several explanations that can account for the decreased number of podocytes in immune-mediated disease. The potential mechanisms of podocyte injury include foot process effacement, hypertrophy, detachment, apoptosis, and perhaps epithelial-to-mesenchymal transition.^9–11 One explanation is that the podocytes are unable to proliferate and replace those that are lost because they are terminally differentiated.¹² Another explanation is podocyte apoptosis, which has been shown in experimental and human glomerular diseases.^13,14 Some findings suggest that apoptosis and nonapoptotic cell death also accompany the shedding of podocytes from the glomerulus into the urine. A third plausible mechanism underlying podocyte loss is the detachment of cells from the GBM. To summarize, further research is needed to illuminate the mechanism of podocyte loss.

Studies have shown that podocytes exist not only in the urine of patients with glomerular disease but also in the urine of healthy subjects. Podocytes were identified by expressing several specific markers. The most inclusive marker on cytospin was PDX. At present, PDX is one of the most robust markers that has been successfully applied for urinary podocyte diagnostics. PDX is a sialoprotein that is expressed not only on podocytes but also on a variety of nonrenal cells as well as on glomerular endothelial and parietal epithelial cells. Furthermore, more podocyte-specific marker proteins, such as slit diaphragm-associated molecules, are often inferior to PDX in their diagnostic value. In addition, PDX has also been observed in subsets of breast, prostate, liver, pancreatic and kidney cancer, as well as leukemia.¹⁵

PDX is likely the most prevalently applied marker protein for podocyte diagnostics in the urine. It is a 140 kDa polyanionic sialoprotein and is localized to plasma membrane of podocytes.¹⁶ As a result of its localization throughout the cell body, PDX remains well preserved in many glomerulopathies independent of the integrity of the slit diaphragm. PDX has played a key role in detecting podocyte excretion in many different renal diseases [i.e., membranous nephropathy (MN), membranoproliferative glomerulonephritis, focal segmental glomerulosclerosis (FSGS), immunoglobulin A (IgA) nephropathy (IgAN), Henoch–Schönlein purpura nephritis (HSPN),¹⁷ lupus nephritis (LN), diabetic nephropathy (DN),¹⁸ and preeclampsia¹⁹]. Several studies have also indicated that the number of PDX-positive cells in the urine falls after various therapeutic interventions in patients with FSGS, LN, HSP, IgAN, PSGN, and DN.^20–26 Compared with other markers, PDX proved to be the most reproducible marker for podocyte assessment in the urine of these patients. According to Vogelmann et al.,²⁷ the cells that stained positive for PDX also stained positive for 30–40% of the other podocyte-specific markers. In contrast, the cells that were positive for different podocyte-specific markers always stained positive for PDX. Further research is needed into the specific mechanisms of the loss of viable podocytes from the glomerular tuft.

The rate of podocyte excretion appears to reflect both disease activity and disease type. Quantitative podocyturia tended to be greater in patients with active LN than it was in those patients with FSGS. Podocyturia was significantly greater in active disease than it was in inactive disease. Specific staining with markers, such as PDX, confirmed the identity of the cultured urinary cells as podocytes. The amount of PDX-positive cells correlates well with disease activity in various glomerular diseases, especially FSGS, MN, and IgAN.²⁸ Therefore, determining PDX-positive cells in the urine using cytospins might still be a valid tool for estimating glomerular disease activity in these diseases. The method of culturing the urine sediments has led to the selection of viable cells that attach to collagen.

The excretion of glomerular epithelial cells might indicate destructive glomerular disease; therefore, detection of such cells could be helpful when deciding on whether to biopsy or whether it is justifiable to wait for a response to the treatment of glomerular disease. Proteinuria is not a completely reliable readout of the ongoing disease. The detection of PDX-positive cells in urine could be a valuable tool in assessing the disease activity of certain glomerular diseases.

APPLICATION OF URINARY PODOCYTE DETECTION IN DIAGNOSING AND TREATING ALL TYPES OF KIDNEY DISEASES

IgA Nephropathy

Lemley et al.²⁹ showed that podocyte loss occurs concomitantly with increasing disease severity in IgAN. In their study, the patients with the most severe glomerular dysfunction had a reduced number of podocytes per glomerulus. The degree of podocytopenia was related to the extent of GS and the degree of permselectivity impairment, and glomerular filtration rate (GFR) disease progression in IgAN can be associated with both prolonged excretion of urinary podocytes and repeated episodes of acute glomerular injury. Research has reported that the urinary excretion of viable podocytes has a positive correlation with the level of proteinuria. The shedding of podocytes into urine might, therefore, be an important contributor to albuminuria.³⁰ Meanwhile, the total number of PDX-positive cells in the urine could be used as a marker of disease activity. Several studies have also shown that the number of PDX-positive cells in the urine decreases after various therapeutic interventions in patients with IgAN, and it is beneficial for patients with IgAN to reduce the loss of PDX-positive cells in the urine.

Henoch–Schönlein Purpura Nephritis

Prolonged excretion of urinary podocytes in HSPN suggests either that the initial acute glomerular injury is severe enough to persist after acute injury or that the acute glomerular injury can persist for prolonged periods. A significantly higher number of urinary podocytes were found at the acute state of glomerular diseases, including HSPN.²⁰ We found that hematuria, proteinuria, and urinary podocytes all had significant correlations with acute renal lesions. In contrast, there was no significant correlation between urinary studies and any of the renal chronicity scores. A prospective study by Hara et al.¹⁷ examined patients with HSPN and showed a strong correlation between the cumulative burden of the excretion of PDX-positive cells (the amount excreted in 6 consecutive months) and the development of GS in follow-up biopsies. The number of podocytes in the urine was found to be clinically useful as a diagnostic tool for glomerular versus nonglomerular diseases, inflammatory versus noninflammatory diseases, and as a marker for the severity of active glomerular injury.

Lupus Nephritis

Nakamura et al.³¹ showed that the number of urinary podocytes in patients with LN had significant correlations with the proteinuria and hematuria excretion scores. Quantitative podocyturia tended to be greater in patients with active LN than in those patients with FSGS. The podocytes disappeared after the patients received hormone treatment. These data suggest that urinary podocytes may be an important sign of serious disease advancement in patients with LN, which can be repaired effectively by hormone therapy.

Focal Segmental Glomerulosclerosis

The loss of podocyte markers from glomeruli is associated with GS in human biopsy samples from patients with FSGS.^32,33 We can conclude that podocyte depletion is one mechanism by which mesangial expansion, FSGS, and GS can be triggered. The loss of podocytes increases the appearance of podocytes and podocyte constituents in urine. This is associated with GS and rapid deterioration of renal function in FSGS. The FSGS lesions can be seen as remedial for glomeruli because they can repair the injury caused by podocyte loss above a particular value. Research indicates that the detection of PDX could be a very useful tool as a marker of disease activity, even in an outpatient setting.

Diabetic Nephropathy

Previous studies of human DN have provided convincing evidence for a correlation between the loss of podocytes and the progression of glomerular diseases.³⁴ More recently, Su et al.³⁵ used PDX as a marker to evaluate podocyte damage and showed decreased podocyte number and density in patients with early stage of DN and found that podocyte damage became more dramatic as proteinuria progressed. The detection of urinary podocytes in various glomerular diseases, including IgAN and DN, is consistent with podocyte loss from the glomeruli.³⁶ Recent studies have shown that podocyte injury, an important pathogenetic process in DN,³⁷ might be a more sensitive means to assess the degree of glomerular damage.³⁸ Podocyte lesions in DN, including a decrease in podocyte density and an abnormal podocyte-specific protein expression pattern, have been extensively investigated. Recent studies have linked podocyte damage to increased renal angiotensin II (ANG II) levels in DN.³⁹ Furthermore, more recently, we demonstrated that podocytes presented with phenotypical change in the early stages of DN and that could be attenuated by irbesartan, an ANG II receptor antagonist.⁴⁰ The urinary expression of podocyte-associated molecules is not significantly affected by angiotensin-converting-enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) therapy. Some researchers have indicated that the serial measurement of podocyte-associated molecules in the urine may have additional value for monitoring the therapeutic response to ACEI or ARB treatment in patients with DN.⁴¹

Preeclampsia and Kidney Diseases

More recently, the excretion of anti-angiogenic proteins has been cited as a diagnostic feature of preeclampsia,⁴² and in this issue, Garovic and colleagues introduce us to podocyturia. Podocyturia is the excretion of glomerular visceral epithelial cells or podocytes into the urine of preeclamptic women. Research has shown that four podocyte markers (podocin, PDX, synaptopodin, and nephrin) have been detected in the cells excreted by proteinuric preeclamptics but not in the cells excreted by non-proteinuric normotensive gravidas. Podocin proved to be the most sensitive and specific marker. A larger number of studies have indicated that podocyturia seen in a variety of renal disorders can distinguish progression of preeclampsia from other glomerular disorders.

PERSPECTIVE

Despite the advancement of podocyte detection in the urine, many questions regarding the clinical application of this method must be studied and discussed. There have been a large number of studies that have supported excreted podocytes as a disease marker. The detection of urinary podocytes has been noninvasive and useful for assessing disease activity in glomerular diseases. Therefore, large-scale prospective studies are also required to further confirm the validity of the detection of podocytes in urine. If we have more evidence to prove that the detection of podocytes has significant correlations with renal glomerular disease, then one of the essential targets for the treatment of this disease would be the reduction in urinary excretion of viable podocytes. Is the detection of podocytes in urine indicative of renal glomerular diseases? We will need a massive amount of research to confirm it. Therefore, future research must focus on the development of observer-independent techniques for the detection of podocytes in the urine. At the same time, there are many possibilities for the use of technology in detecting podocytes in the urine to evaluate disease activity, the degree of dedifferentiation, and the probability of regeneration. Is the loss of podocytes able to predict the occurrence of renal glomerular disease early? Can the reduction in the number of podocytes provide evidence for the early prevention of renal glomerular disease? Overall, decreasing the loss of podocytes in the urine is of great importance for preventing further deterioration in renal diseases.

ACKNOWLEDGMENT

Funding. This work was supported by the Fund of the Educational Science Foundation of Jiangsu Province, China (no. 07KJB320121), the Project of the Science Foundation of Affiliated Hospital of Xuzhou Medical College, Qinglan Lan project (53051106), Special talents Fund of Dean of Xuzhou Medical College (2010KJZ23), the Project of Technology Development Foundation of Xuzhou City (XF10C76), the Project of 7th “Liu Da Ren Cai Gao Feng” of Jiangsu Province, China (2010-WS043), the Project of Technology Development Foundation of Kuitun City (201134), and the Project of “shi-er-wu ke-jiao-xing-wei” key medical personnel of Jiangsu Province (RC2011116).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.