*2.2. Label Free Analysis of the Urine Proteome for Patients with FSGS and MCD*

Comparison of proteomic profiles of patients with FSGS and and MCD showed no significant differences in the protein levels (Figure 2).

**Figure 2.** Hierarchical clustering of proteins identified in urine samples of patients with MCD and FSGS. The box denotes log2-transformed values of peak intensity.

However, the FSGS group in total showed a high variability between the patients inside the group. Thus for a comprehensive assessment of this cohort, a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second—depending on the severity of proteinuria, the third—steroid resistance of the nephrotic syndrome. Steroid-resistance was defined as the absence of a decrease in proteinuria levels after 16 weeks of prednisolone therapy or a decrease by less than 50% of the baseline level.

The renal function was considered "saved", if the estimated glomerular filtration rate, determined by the CKD-EPI formula (eGFR CKD-EPI), was above 60 mL/min/1.73 m2; and "impaired"—if it was less.

Using this index the patients with FSGS were subdivided into two groups: with a sum of scores of less than 3—mild FSGS (1), and with 3 or higher—severe progressive FSGS (2) (Table 1).

These two subgroups did not differ significantly in the severity of the nephrotic syndrome and renal dysfunction at the onset of the disease. However, in the follow-ups, the patients of the second group were characterized by a more severe FSGS course, meaning impaired renal function and steroid resistance. A wide range of urine proteins was detected at elevated levels in group 2 (Figure 3). For example, an increase in urinary excretion of complement components C3, C4B, factor B, as well as components of the membrane attack complex C8a and C9 were found. The detection of retinol-binding protein 4 and vitamin Dbinding proteins in the urine is a consequence of tubulo-interstitial inflammation and injury of the tubular epithelium secondary to glomerular proteinuria [18–21]. Simultaneously with the interstitial inflammation, the accumulation of extracellular matrix (ECM) components and tubulo-interstitial fibrosis are also activated. Alpha-2-HS-glycoprotein can be attributed to the group of proteins responsible for active processes of ECM accumulation, expression of receptors on cells, and ECM protein metabolism (Figure 3).

**Figure 3.** Hierarchical clustering of proteins identified in urine samples of patients with FSGS in group 1 and group 2. The box denotes log2-transformed values of peak intensity.

Considering patients with FSGS separately, we found some minor differences in the protein profiles of patients with saved and impaired renal function. In particular, patients with impaired renal function showed higher levels of thyroid hormone-binding protein, β2 microglobulin, vitamin D-binding protein, alpha-2-HS-glycoprotein (fetuin A) (Figure 4).

Proteins that differ between FSGS group 2 and MCD are by 83% identical to those differentiating the two FSGS groups (Figure 5). It can also be seen that like FSGS group 1 patients samples from MCD patients also have elevated levels of osteopontin and the inhibitor of phosphoinositide-3 kinase, while complement proteins, apolipoproteins, hemopexin, vitronectin, and other proteins in urine remain low (Figure 6).

**Figure 4.** Hierarchical clustering of proteins identified in urine samples of FSGS patients with saved and impaired renal function. The color gradient denotes the log2-transformed ratio of the mean peak intensity values measured in the two groups.

**Figure 5.** Proteins that differ between FSGS sub groups (1 and 2) and MCD.

Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups (Supp. Table S1). Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons (Table 3). Also all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Moreover, it is worth to note that the absolute values of the measured proteins fold changes between groups for the two quantitation methods (label-free vs. MRM SIS) in their orders of magnitude are in good agreement (Supp. Table S1).

**Figure 6.** Hierarchical clustering of proteins identified in urine samples of patients with FSGS in group 2 and MCD. The color gradient denotes the log2-transformed ratio of the mean peak intensity values measured in the two groups.

The most important function and source of proteins are presented in Table 4. The levels of the most significant proteins in arbitrary units in FSGS group 1, FSGS group 2 and MCD are shown in Figures 7–9.

*Int. J. Mol. Sci.* **2022**, *23*, 12607




**Table 4.** Description of urinary proteins selected in this study for differentiating patients with MCD and FSGS (group 1—FSGS 1 and group 2—FSGS

 2).




**Figure**  patients (group group 2) (**a**) Vitronectin, (**b**) Apolipiprotein(**c**) Hemopexin, (**d**) Inter-alfa-trypsin inhibitor heavy chain H2; (**e**) Vitamin-D-binding protein; (**f**) Retinol-binding protein.

*Int. J. Mol. Sci.* **2022**, *23*, 12607

*Int. J. Mol. Sci.* **2022**, *23*, 12607

C4-b, (**c**)

alfa-2-HS-glycoprotein,

 (**d**)

Complement

component

 C9; (**e**)

Complement

 factor B; (**f**)

 group

Complement

 factor I.

inhibitor,

 (**c**)

galectin-3-binding

 protein.
