Urinary Protein And Peptide Markers in Chronic Kidney Disease Part 2

3. Minimal Change Disease and Focal Segmental Glomerulosclerosis 

Minimal-change disease (MCD) and primary focal glomerulosclerosis (FSGS) are diseases with primary podocyte injury (primary podocytopathies), which are manifested as high proteinuria and nephrotic syndrome [82,83]. However, morphological studies of a kidney biopsy in the early stages of FSGS can miss segmental sclerosis in individual glomeruli and may misclassify the disease as MCD [84]. Primary FSGS pathogenesis is associated with circulating permeability factors (such as soluble urokinase-type plasminogen activator receptor (SuPAR), corticotrophin-like protein-1, and anti-CD40 antibody CD-80 expression), which leads to the development of nephrotic syndrome [85–91]. In general, compared to FSGS, MCD has a more favorable prognosis regarding the progression of renal dysfunction; FSGS is more likely to develop therapy resistance and result in rapid renal dysfunction and is also more likely to need an aggressive and persistent therapeutic strategy [83,92,93]. In addition, the presence of secondary FSGS complicates diagnosis and disease treatment. Due to its non-immune nature, this form of disease does not require immunosuppressive therapy [1].

Several studies have aimed at identifying proteomic differences between these two nephropathies. In particular, it was shown that the calretinin and UBA52 levels were higher in FSGS [48,49], while the 39S ribosomal protein L17 was higher in MCD [48] (Table 2). Significantly higher levels of cathepsin B, cathepsin C, and annexin A3 were shown in cases of the collapsing variant of FSGS (characterized by glomerular collapse and a rapid loss of renal function) than in MCD, MN, and other FSGS variants [94]. Several potential markers specific only for FSGS include increased levels of cadherin-like 26, RNase A family 1, DIS3-like exonuclease 1 [50], matrix-remodeling protein 8 [51], CD59, insulin-like growth factor-binding protein 7, and roundabout homolog 4 [52], as well as a decrease in the polymeric immunoglobulin receptor and Golgi-associated olfactory signaling regulator [54] or the complete absence of dipeptidase 1 (DPEP1) [52]. Increased CD14 levels were found to be specific only for MCD [50] and were not identified in any other nephropathy (Table 2). At the same time, increases in transferrin and histatin-3 may distinguish both FSGS and MCD [48] from other types of kidney disease.

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Among the revealed potential markers, the overrepresentation of ribonuclease 2 and underrepresentation of haptoglobin may suggest the worst FSGS prognosis, whereas apolipoprotein A1 and matrix-remodeling protein 8 (MXRA8) showed significant changes between steroid-sensitive and steroid-resistant forms of FSGS [51]. 

In general, the presence of most of the aforementioned proteins in the urine and increases in their levels may reflect massive cell death and the release of intracellular contents during the podocytes’ separation from the glomerular membrane. These results may also suggest special roles for immunity, inflflammation, and apoptosis in the development of FSGS. Cell proliferation, differentiation, and death may be involved in MCD development [95]. Dynamic studies performed using a focal segmental glomerulosclerosis rat model (adriamycin (ADR)-induced nephropathy) revealed a gradual increase in afamin and ceruloplasmin, as well as a gradual decrease in cadherin-2 and aggrecan core protein in FSGS, and suggested that decreased levels of fetuin-B, α-1-microglobulin, and α-2-HS-glycoprotein may be promising markers for the early detection of FSGS [96]. Other promising markers include CD44, MXRA8, cathepsins, and apolipoprotein A1. CD44 reflects the activation of parietal epithelial cells, which triggers glomerulosclerosis. MXRA8 cathepsins are involved in fibrosis accumulation and disease progression. Apolipoprotein A1 reflects oxidative stress, is associated with hyperlipidemia, and represents one of the pathogenetic factors in the development of FSGS. 

4. Membranous Nephropathy 

Membranous nephropathy (MN) is a leading cause of nephrotic syndrome (NS) in adults. This disease has an autoimmune nature, which was confirmed by the presence of autoantibodies to podocyte antigens, including antibodies to phospholipase A2 receptors (aPLA2R) and thrombospondin 1 domain-containing 7A (THSD7A) [97,98]. The secondary causes of MN include drug use, infections, autoimmune diseases, and cancer [99]. The primary mechanism of MN is podocyte autoimmune damage by phospholipase A2 receptor antibodies, leading to massive proteinuria. The diagnosis and treatment of this disease are currently based on the determination of the aPLA2R antibody titer. The search for additional markers seems promising in the aPLA2R-negative type of idiopathic MN.

MN patient studies provide comparative cross-sectional analyses of the proteome in MN compared to that in other nephrotic types of nephritis and healthy controls. The panel of specific urinary protein markers distinguishing MN from other nephropathies includes decreased levels of zinc finger protein ZFPM2, E1A-binding protein, and microtubule-associated protein tauAP-3 complex subunit delta-1 [54], as well as increased levels of thyroxine-binding globulin (SERPINA7) [50], lysosome membrane protein-2 (LIMP-2) [56], plasminogen [54], LDB3, PDLI5 [100], and afamin [55,57]. A comparison of samples from patients with APLA2R-positive MN and APLA2R-negative MN, as well as healthy individuals, revealed significantly higher levels of A1AT and famine in the positive-MN group [101]. A combination of urinary retinol-binding protein 4 and SH3 domain-binding glutamic acid-rich-like protein 3 can differentiate MCD from DN. Similarly, a combination of urinary afamin and complement C3 urine/plasma ratio can differentiate MN from DN [55]. 

In general, markers found in MN play a role in the classical pathway of complement activation and immune responses, cell adhesion, receptor-mediated endocytosis, platelet degranulation, and the coagulation cascade [57]. LIMP-2 plays a pivotal role in inflammatory immune-response regulation in kidney tissue [56] and reflects tissue infiltration by immune cells. LIMP-2 may also help to determine disease activity. The LDB3 and PDLI5 proteins play a role in the modification of the podocyte cytoskeleton, which can lead to proteinuria. Afamin, whose elevation is associated with idiopathic MN, is the most promising specific MN marker, as its significance was confirmed in several studies (Table 2). 

5. IgA Nephropathy 

IgA nephropathy (IgAN) is the most common form of chronic glomerular disease in adults. In Europe, the frequency of IgAN ranges from 19 to 51% of the renal biopsies performed for glomerular diseases [102–104]. Patients with IgAN often have increased levels of IgA1 with galactose-deficient O-glycans in the hinge region. The blood levels of an aberrantly glycosylated IgA1 are higher in IgAN than in healthy controls or patients with other kidney diseases. The production of galactose-deficient IgA1 antibodies, immune-complex formation, and the accumulation of these complexes in the mesangium were shown to initiate renal injury [105]. Moreover, the activation of alternative complement pathways potentiated tissue injury [106]. Transferrin receptor (CD71) on human mesangial cells can bind immune complexes containing galactose-deficient IgA [107].

About 40 urinary protein markers differentiating IgAN have been described, >20 of which are specific only for IgAN (Table 2). The levels of complement C9, Ig kappa chain C region, and three cytoskeleton keratins (type I(10) and type II (1 and 5)) changed synchronously in the glomeruli (biopsy sample) of IgAN patients compared to the intact renal-tissue areas of patients with tumors [59]. Altered levels of 30 urine proteins and four potential markers (intercellular adhesion molecule 1 (ICAM1), metalloproteinase inhibitor 1, antithrombin III, and adiponectin) were revealed in IgAN with low proteinuria (<1 g/L) and stable renal function (glomerular filtration rate: 57.3 (23–106) mL/min). A larger multicenter study suggested that a decreased number of collagen fragments in the urine (specifically type I collagen) might be most informative in progressive IgAN, due to decreased collagen degradation and collagenase inhibition in kidney fibrosis [62].

Other potential IgAN-specific markers include increased levels of adiponectin [60], α2-macroglobulin, complement C4a, prothrombin [63], antithrombin III [60,63], α-1Bglycoprotein [64], glycoprotein 2, epidermal growth factor, CMRF35-like molecule, protocadherin, uteroglobin, dipeptidyl peptidase IV, NHL repeat-containing protein 3, and CD84 [36] and decreased levels of rifabutin-5, YIP1 family member 3, proposing [108], aminopeptidase N [65], and the LG3 fragment of endorepellin [64]. The last was the only decreased protein in heavier IgAN with a lower glomerular filtration rate [64]. At the same time, high LG3 levels could inhibit angiogenesis and be responsible for renal function loss in some other IgAN patients [64]. Although data on changes in the level of vasorin are inconsistent [36,65], it can also be considered a specific IgAN marker. Antithrombin III is especially noteworthy as the only specific IsAN marker confirmed in two independent studies [60,63].

6. Diabetic Nephropathy 

Diabetic nephropathy (DN) affects about 30–40% of diabetes mellitus (DM) patients and is the leading cause of CKD and end-stage renal disease (ESRD) all over the world, especially in high- and middle-income countries. DN leads to glomerular mesangial expansion; the thickening of the basement membrane; and, characteristically, the progression of nodular glomerulosclerosis due to glomerular hyperfiltration [109]. 

The array of potential specific DN markers in the urine includes >10 proteins (Table 2), with increased levels of vitamin D–binding protein, calgranulin B, hemopexin [71], zinc- α2-glycoprotein [71,74], 408 N-linked glycoproteins [73], cystatin C, ubiquitin, α-1-acid glycoprotein 1, pigment epithelium-derived factor [74], Clara cell protein CC16 [76], and fibronectin [110], as well as decreased levels of transthyretin [71,74] and differently changing levels of the α-1 microglobulin/bitumen precursor (AMBP) [71,74,75].

Significant increases in the urinary levels of α1B-glycoprotein (7-fold), zinc-containing α2-glycoprotein (5.9-fold), α2-HS-glycoprotein (4.7-fold), vitamin D-binding protein (4.8-fold), calgranulin B (3.9-fold), A1AT (2.9-fold), and hemopexin (2.4-fold) reliably distinguished DN with macroalbuminuria from DM without albuminuria [71]. Conversely, a significant decrease in transthyretin (4.3-fold), apolipoprotein A1 (3.2-fold), AMBP (1.6-fold), and retinol-binding plasma protein (1.52-fold) was observed in DN with macroalbuminuria [71]. A model study with selected proteins suggested the significance of cathepsin A, mucin 1, the GM2 ganglioside activator, SPARC-like protein 1, and lysosomal acid phosphatase in the poor prognosis of the early development of DN, as well as in kidney fibrosis [111]. A combination of 408 N-linked glycoproteins, A1AT, and ceruloplasmin were shown to be able to distinguish microalbuminuria and normoalbuminuria in DN patients [73]. Urinary haptoglobin and AMBP can differentiate between diabetic patients with and without DN [75]. The increased excretion of 15.8 kDa Clara cell protein CC16 was found to be associated with proximal tubule dysfunction in DM patients with micro- or macroalbuminuria compared to DM patients without albuminuria and healthy controls [76]. The levels of osteopontin and fibronectin were also higher in DN compared to those in DM, and increases in urinary neprilysin and VCAM-1 were observed after losartan treatment in DN [110].

A longitudinal study of type 2 DM revealed an increase in urine transthyretin/albumin and the Ig kappa C chain region within 0–5 years of the onset of DM; the appearance of cystatin C and ubiquitin after 5–10 years; and the detection of α-1-acid glycoprotein 1, apolipoprotein A1, AMBP, pigment epithelium-derived factor, and zinc α-2-glycoprotein after 10–20 years [74]. The nonenzymatic glycation of these proteins and their peptides interferes with normal tubular reabsorption and may lead to damage to the proximal tubules and the direct excretion of the proteins into the urine.

Overall, the aforementioned DN markers may reflect the processes of tubular atrophy and tubulointerstitial fibrosis, many of which are important for DN prognosis. Zinc-α2- glycoprotein, transthyretin, and AMBP should be specially noted, as their prognostic significance was confirmed in at least two independent studies [71,74,75]. 

7. Lupus Nephritis 

Lupus nephritis (LN) is one of the most common and severe complications of systemic lupus erythematosus and usually appears at least 3-5 years after the onset of the disease. The mechanisms of renal glomerulus damage can be found in the deposition of immune complexes or autoantibodies with subsequent complement activation [112]. LN leads to severe kidney damage that advances to end-stage renal disease if not treated adequately. The most important goal for LN treatment is to dynamically assess the degree of renal damage activity since the available activity markers (daily proteinuria, erythrocyturia, compliment, and antinuclear antibodies) are not informative. LN patients currently need to undergo several kidney biopsies to monitor LN activity during immunosuppressive therapy to determine where LN treatment should be continued or canceled. In this case, there is a need for highly sensitive and specific LN markers able to predict disease exacerbation or indicate insufficient effectiveness of the therapy. 

Only a few potential urinary protein markers specific to LN can be noted (Table 2). A pair of peptides, “3340” and “3980” (m/z), have made it possible to differentiate an acute LN condition from LN remission with 92% sensitivity and 92% specificity prior to any changes in clinical parameters (the urinary protein/creatinine ratio, antibodies to DNA, hematuria, serum creatinine, etc.). Moreover, these peptides were able to predict early relapse and remission [66]. 

Particular fragments of hepcidin, together with fragments of A1AT and albumin, were found to be more significant than systemic lupus erythematosus renal flflare cycle LN in a dynamic study on the urinary proteome [67]. The altered expression of hepcidin 20 might be a marker of renal flflare, whereas an increase in hepcidin 25 upon treatment could be used to estimate the effectiveness of therapy [67].

The classifier based on 172 peptides reliably differentiated 92 LN cases from the general CKD group (1180 patients) and identified the protein S100-A9 as another specific LN marker, whose increased level was found to be essential for LN differentiation in combination with increased levels of collagen peptides and uromodulin, as well as decreased levels of clusterin, β-2-microglobulin, and α-2-HS-glycoprotein [54]. 

α-1-Antichymotrypsin (SERPINA3) is another potential specific LN marker in urine and the only LN marker whose significance was confirmed in two independent studies [68,69]. Together with haptoglobin and retinol-binding protein, SEPINA3 was significantly increased in active LN compared to inactive LN [68]. Moreover, SERPINA3 demonstrated a moderately positive correlation with LN histological activity, which was confirmed via immunohistochemistry [69].

In general, the described LN markers make it possible to assess the activity of the disease and the accumulation of fibrosis in the kidneys, which are very important in clinical practice when managing patients. The increased levels of some proteins may suggest tubular dysfunction during the acute form of the disease [68]. 

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