Abstract

   For lupus nephritis (LN) management, it is very important to detect
   fibrosis at an early stage. Urinary exosomal miRNAs profiling can be
   used as a potential multi-marker phenotyping tool to identify early
   fibrosis. We isolated and characterised urinary exosomes and cellular
   pellets from patients with biopsy-proven LN (n = 45) and healthy
   controls (n = 20). LN chronicity index (CI) correlated with urinary
   exosomal miR-21, miR-150, and miR-29c (r = 0.565, 0.840, −0.559,
   respectively). This miRNA profile distinguished low CI from
   moderate-high CI in LN patients with a high sensitivity and specificity
   (94.4% and 99.8%). Furthermore, this multimarker panel predicted an
   increased risk of progression to end-stage renal disease (ESRD).
   Pathway analysis identified VEGFA and SP1 as common target genes for
   the three miRNAs. Immunohistochemistry in LN renal biopsies revealed a
   significant increase of COL1A1 and COL4A1 correlated with renal
   chronicity. SP1 decreased significantly in the high-CI group (p =
   0.002). VEGFA levels showed no differences. In vitro experiments
   suggest that these miRNA combinations promote renal fibrosis by
   increasing profibrotic molecules through SP1 and Smad3/TGFβ pathways.
   In conclusion, a urinary exosomal multimarker panel composed of miR-21,
   miR-150, and miR-29c provides a non-invasive method to detect early
   renal fibrosis and predict disease progression in LN.

   Keywords: lupus nephritis, renal fibrosis, urinary exosomes,
   multi-marker panel

1. Introduction

   Fibrosis, or tissue scarring, is the result of an excessive,
   persistent, and destructive accumulation of extracellular matrix
   components (ECM) in response to chronic tissue injury in the kidney.
   Renal fibrosis represents the final stage of most chronic kidney
   diseases and contributes to a progressive and irreversible decline in
   kidney function. The continuous accumulation of ECM causes a disruption
   of the epithelium and/or endothelium integrity that results in the
   activation of a complex cascade of molecular and cellular events
   [[32]1]. Initially, there is an inflammatory response with the release
   of profibrotic cytokines, chemokines, and growth factors, which in turn
   promote the proliferative phase of the scarring process characterised
   by the recruitment and activation of fibroblasts into ECM-secreting
   myofibroblasts and the formation of a permanent fibrotic scar
   [[33]2,[34]3].

   Lupus nephritis (LN) occurs in ~40–75% of systemic lupus erythematosus
   (SLE) patients [[35]4] and continues to be one of the major causes of
   morbi-mortality in those patients. Despite improvements in its
   management, up to 20% of patients will progress to end-stage renal
   disease (ESRD) [[36]5]. So far, renal biopsy continues to be the
   gold-standard technique to evaluate the degree of fibrosis, but its
   invasiveness makes it unsuitable for serial monitoring [[37]6].
   Detection of early stages of fibrosis could contribute to identify
   patients at risk of progressing to ESRD who can benefit from new
   therapeutic approaches. Therefore, there is the need to identify
   non-invasive biomarkers to detect early fibrosis and to monitor its
   progression.

   Urine samples are obtained relatively easily and cost-efficiently
   compared with other biological samples such as blood, cerebrospinal
   fluid, or tissue biopsy. The analysis of urinary miRNAs provides a
   measure of the health of the excretory system and is detected
   noninvasively from urinary cellular pellet or inside urinary exosomes
   [[38]7]. Most studies use urinary cellular pellet for study of miRNA
   expression [[39]8,[40]9,[41]10]. In recent years, urinary exosomes
   gathered strength in the field of biomarkers due to being small
   membranous vesicles secreted by a variety of living cells and
   implicated in cell-to-cell communication [[42]11,[43]12]. Inside them,
   there are small non-coding RNAs, the miRNAs, which regulate many
   molecular and cellular processes by repressing translation or causing
   breakdown in target gene mRNAs [[44]13]. Urinary exosomal miRNAs can
   accurately reflect renal dysfunction and structural damage, making them
   good targets for the exploration of biomarkers for urinary tract
   diseases [[45]14,[46]15].

   miRNA expression profiling in renal fibrosis has been evaluated in
   kidney tissue samples [[47]16], blood [[48]17], and urine [[49]18] from
   various chronic kidney diseases. Despite a large number of miRNAs being
   identified, a recent meta-analysis study only showed seven of them to
   be highly related to fibrosis [[50]19]. Of those, miR-29c, miR-21, and
   miR-200a, regulators of the TGF-β/Smad3 signalling pathway, have been
   described in urinary exosomes [[51]20,[52]21,[53]22,[54]23].
   Furthermore, three miRNAs have been associated with renal fibrosis in
   LN patients: miR-410, which contributes to renal fibrosis by inhibiting
   interleukin-6 [[55]24], miR-29c as predictor of early renal fibrosis
   [[56]25], and miR-150, which promotes renal fibrosis by downregulation
   of SOCS1 [[57]26].

   Unfortunately, it is now generally accepted that single markers do not
   achieve sufficient sensitivity and specificity for diagnosis and
   routine clinical practices. A novel trend based on the combination of
   several signatures has improved clinical performance
   [[58]27,[59]28,[60]29]. In this study, we compared miR-29c, miR-200a,
   miR-21, miR-410, and miR-150 expression levels between urinary pellet
   and exosomes. Then, we estimated a combinatory panel of urinary
   exosome-derived miRNAs from active LN patients to identify early
   fibrosis and predict progression to ESRD.

2. Materials and Methods

2.1. Patients and Samples

   Patients with biopsy-proven active LN were recruited from the Lupus
   Unit at Vall d’Hebron Hospital (N = 45). The study was ethically
   approved by Vall d’Hebron Hospital Ethics Committee and all patients
   provided written informed consent prior inclusion. All patients
   fulfilled at least 4 of the American College of rheumatology (ACR)
   revised classification criteria for SLE [[61]30]. Healthy donors were
   used as controls (N = 20). Repeated renal biopsy was performed if
   required according to clinical protocol (N = 7). Urine samples were
   collected from each patient 1 day before renal biopsy and processed
   immediately to be stored at −80 °C (more details in [62]SI). SLE
   disease activity was assessed by the SLE Disease Activity Index 2000
   update (SLEDAI-2Ks; range 0–105) [[63]31]. Patients were classified
   according to the chronicity index (CI) at renal biopsy: Low CI (<2, N =
   18), moderate CI (2–4, N = 21), and high CI (≥4, N = 6). ESRD
   progression was defined by an estimated glomerular filtration rate
   (eGFR) <15 mL/min/1.73 m^2, the initiation of renal replacement therapy
   or receiving kidney transplantation, or 40% reduction of baseline eGFR
   [[64]32]. Estimated glomerular filtration ratio (eGFR) was calculated
   using the chronic kidney disease epidemiology collaboration (CKD-EPI)
   formula [[65]33].

2.2. Renal Histology and Immunohistochemistry

   Renal biopsies were examined by light and immunofluorescence microscopy
   following standard methods and categorised according to the
   International Society of Nephrology/Renal Pathology Society
   classification (2003) [[66]34] and rated for activity (AI) and
   chronicity (CI) [[67]35]. Renal chronicity was measured by Gömöri
   trichrome staining from paraffin-embedded renal samples. For
   immunohistochemistry, rabbit anti-VEGF-A antibody (Abcam, ab46154,
   dilution of 1:50) and a diaminobenzidine (DAB) chromogen kit (DAKO)
   were used. Histology and immunohistochemistry samples were blinded and
   scored by the Vall d’Hebron pathologist unit (more details in [68]SI).

2.3. Exosome and Pellet Purification and Characterisation

   Fresh urine samples (50 mL) to obtain the urinary cellular pellet and
   supernatant were centrifuged at 3900× g for 30 min and kept for urinary
   exosome isolation. Following the manufacturer’s instructions, urinary
   exosomes were isolated using the miRCURY™ Exosome Isolation Kit—Cells,
   urine and CSF (Exiqon, Woburn, Massachusetts, USA). In order to
   validate the exosome purification protocol, urinary exosome morphology,
   shape, and size were analysed by Cryo-transmission electron microscopy.
   NanoSight and Western blot analysis were used to characterise exosome
   isolation (more details in [69]SI). Quantification of Urinary exososmes
   was performed byFluorCet Exosome Quantitation Kit (SBI, more details in
   [70]SI) according to the manufacturer’s instructions.

2.4. RNA Isolation from Urinary Cell Pellet and Exosomes

   RNA from urinary pellets or exosomes were extracted using the miRCURY™
   RNA Isolation Kit-Cell & Plant (Exiqon, Woburn, MA, USA, more details
   in [71]SI), following the manufacturer’s instructions. Quantification
   and evaluation of the RNA quality was assessed by Bioanalyzer PicoChip
   analysis.

2.5. Individual Assay qPCR-RT

   Initially a first-strand cDNA synthesis reaction was made to provide a
   template for all microRNA real-time PCR assay using the miRCURY LNA™
   Universal RT microRNA PCR (Exiqon, Woburn, MA, USA, more details in
   [72]SI). MiRNAs were quantified with specific miRCURY LNA primer set
   and ExiLENT SYBR Green master mix (Exiqon, [73]Table S1) using the ABI
   PRISM 7000 following the manufacturer’s instructions. Data were
   normalised based on the expression of RNU6 and relative quantification
   was calculated (RQ = 2^−ΔΔCt) using the Livak method [[74]36].

2.6. Pathway Analysis

   To gain insight into the functions of miRNA target genes, we performed
   gene ontology (GO) classification and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway enrichment analysis using the online tools of
   DNA ntelligent Analysis (DIANA)-miRPath v3.0 software [[75]37]. To
   present the regulation between miRNA and gene, experimentally validated
   targets were extracted for identified miRNAs from miRTarBase [[76]38]
   and critical miRNA–target interactions were constructed using miRNet
   ([77]www.mirnet.ca) [[78]39].

2.7. Immunofluorescence in Renal Biopsy

   Immunofluorescence was performed on paraffin-embedded (FFPE) renal
   biopsies during renal flare (N = 3 for each subgroup) following the
   protocol described by Mason et al. [[79]40]. Staining was performed
   with 1:50 rabbit anti-SP1 (Abcam, ab124804), 1:100 mouse anti-COL1A1
   (Abcam, ab6308), or 1:100 mouse anti-COL4A1 (Abcam, ab6311) overnight
   at 4 °C (more details in [80]SI).

2.8. Human Kidney Cells Culture

   Primary human renal mesangial cells (RMCs) and renal tubular epithelial
   cells (RTCs) were purchased from InnoProt (Derio, Spain) and cultured
   in the recommended media provided by the manufacturer. Cells were grown
   at 37 °C in a humidified 5% CO[2] atmosphere. Cell passes were
   performed using TrypLE™ Express (Gibco^® ThermoFisher Scientific,
   Waltham, MA, USA).

2.9. Overexpression of miR-21/miR-150 and Inhibition of mir-29c in Human
Kidney Cells

   Cells plated on 24-well plates were transfected with mimic miR-21 and
   mimic miR-150 (ThermoFisher Scientific, Waltham, MA, USA) or with
   miR-29c inhibitor (ThermoFisher Scientific, Waltham, MA, USA) using
   Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) following the
   manufacturer’s instructions. After 48 h, cells were stimulated with
   TGFβ1 citokine (10 ng/mL, ThermoFisher Scientific, Waltham, MA, USA).
   After 24 h, the total RNA was extracted using miRCURY RNA Isolation Kit
   (Exiqon, Woburn, MA, USA) or the immunofluorescence was performed (more
   details in [81]SI, Table S2).

2.10. Luciferase Activity Analysis

   Human kidney cells were plated in 96-well plates for 24 h and then were
   cotransfected using DharmaFECT Duo transfect reagent (ThermoFisher
   Scientific, Waltham, MA, USA) with 40 ng of pEZX-MT06-SP1 3′UTR plus 10
   nM of miR-21, miR-150, or miR-29c analog or miR negative control (5
   duplicates per group). The pEZX-MT06-SP1 3′UTR plasmid contains firefly
   luciferase and miR binding site from SP1 3′UTR, which is inserted
   between firefly luciferase cDNA and PolyA (more binding site details in
   [82]Figure S1). This plasmid also includes Renilla luciferase as an
   internal control (GeneCopoeia, Rockville, MD, USA). Transfected cells
   were lysed by reporter lysis buffer (Promega, Madison, WI, USA). Dual
   luciferase activity was measured with the Dual-Luciferase Reporter
   Assay System (Promega, Madison, WI, USA) by FLUOstar Omega (BMG
   Labtech, Oternberg, Germany).

2.11. Statistical Analysis

   Statistical analyses were performed by GraphPad Prism version 6
   (GraphPad Software, La Jolla, CA, USA) and SAS version 9.3 (SAS
   Institute Inc., Cary, NC, USA). The mean expression of miRNA levels was
   compared using Mann–Whitney U/Kruskal–Wallis H tests, as appropriate.
   The relationship between miRNA expression and histological/clinical
   parameters of LN patients were analysed using the Spearman correlation
   coefficient. Risk of progression to ESRD and renal survival rate across
   urinary exosomal miRNAs were analysed and compared using the
   Kaplan–Meier analysis and the log-rank test. The diagnostic performance
   of biomarkers was evaluated by calculating their sensitivity and
   specificity using receiver operating characteristic (ROC) curves. A
   combinatorial analysis of multiple biomarker signatures was carried out
   using the CombiROC method [[83]41] (more details in [84]SI).

3. Results

3.1. Patients

   Patient demographic characteristics and laboratory measurements are
   summarised in [85]Table 1. Most patients included had a type IV GMN and
   had a moderate chronicity index degree (N = 21) at inclusion
   ([86]Figure S2). No significant clinical differences were observed
   between subgroups such as age, gender, serum creatinine, proteinuria,
   blood urea nitrogen (BUN), and eGFR ([87]Table 1). Patients were
   followed up for a median of 8 years (range: 3.5 to 12.5) after renal
   biopsy.

Table 1.

   Clinical and laboratory characteristics of the study subjects.
   Characteristics Lupus Nephritis (n = 45) Healthy Control (n = 20)
   Low CI (N = 18) Moderate CI (N = 21) High CI (N = 6)
   Demographic
   Age, year 33 ± 7 29 ± 5 30 ± 4 28 ± 5
   Sex, male/female 7/11 8/13 3/3 8/12
   Race/ethnicity, n (%)
   Caucasian 18 20 6 19
   Hispanic 0 1 0 1
   Laboratory Parameters
   Serum creatinine, mg/Dl 0.9 ± 0.3 1.1 ± 0.8 1.1 ± 0.6 0.7 ± 0.2
   eGFR (mL/min) 92.3 ± 26.7 78.6 ± 29.3 78.8 ± 42.6 97.2 ± 31.7
   BUN (mmol/l) 4.7 ± 1.2 4.6 ± 2.9 4.3 ± 1.7 5.0 ± 1.5
   Anti-dsDNA Abs, IU/mL 333 ± 104 343 ± 107 357 ± 143 n.d.
   Serum C3, mg/dL 69.5 ± 16.1 87.5 ± 12.1 74.5 ± 16.1 n.d.
   Serum C4, mg/dL 8.3 ± 2.1 10.4 ± 3.5 11.2 ± 2.7 n.d.
   Proteinuria, g/24 h 4.2 ± 2.4 3.6 ± 2.5 3.9 ± 4.7 n.d.
   Disease Index (SLEDAI-2K)
   Total SLEDAI score 18 ± 2 14 ± 2 16 ± 3 n.d.
   Renal Biopsy, n (%)
   Class, n (%)
   III 4 6 0 n.d.
   IV 14 12 4 n.d.
   V 0 3 1 n.d.
   Activity Index 6.3 ± 3.7 5.3 ± 4.1 6.3 ± 3.2 n.d.
   Chronicity Index 0.6 ± 0.4 3.5 ± 0.5 5.8 ± 1.2 n.d.
   [88]Open in a new tab

   Values are means ± SE. BUN, blood urea nitrogen; eGFR, estimated
   glomerular filtration rate; anti-dsDNA, anti-double-stranded DNA; n.d.,
   not determinate.

3.2. Isolation and Characterization of Urinary Exosomes and Cellular Pellet

   Urinary exosomes and pellets were isolated as described in the
   methodology. Exosomes were examined using cryo-transmission electron
   microscopy, nanoparticle tracking, and Western blot analysis. Results
   revealed vesicles of 85.5 ± 33.4 nm diameter with the characteristic
   cup-shaped exosome morphology and the presence of the urinary exosome
   TSG101 protein ([89]Figure 1A). The number of urinary exosomes was
   similar among the different LN groups (mean 2.3 × 10^7, [90]Figure S3).
   The urinary cellular pellet was confirmed by the negativity of TSG101
   protein, confirming the absence of contamination from other cellular
   compartments.

Figure 1.

   [91]Figure 1
   [92]Open in a new tab

   Characterisation of urinary exosomes and expression levels of urinary
   exosomal miR-21, miR-150, miR-410, and miR-29c in lupus nephritis (LN)
   patients’ urinary pellets. (A) Cryo-transmission electron micrograph,
   nanoparticle tracking analysis and Western blotting of TSG101 protein
   confirmed that the isolated small vesicles are urinary exosomes (size
   of 52–119 nm in diameter). P1–P3: Pellet urinary samples. E1–E3:
   Exosome urinary samples. C-: Negative control. (B) Values of miRNAs
   expression from healthy controls and LN patients at flare time were
   represented as individual dots. (C) Values of miRNAs expression when LN
   patients were classified according to the chronicity index at renal
   biopsy (low CI (<2, N = 18), moderate CI (2–4, N = 21), high CI (≥4, N
   = 6)). Values were normalised using RNU6 and fold change was calculated
   over healthy control group. * p < 0.05, ** p < 0.005, and *** p <
   0.0005.

3.3. miR-29c, miR-200a, miR-21, miR-410, and miR-150 Expression Levels
Measured by Quantitative Reverse Transcription-Polymerase Chain Reaction
(qPCR-RT) in Urinary Exosomal Preparations and Cellular Pellet

   A deep sequencing analysis was performed to compare the miRNA
   composition in the urinary exosomes and cellular pellets obtained from
   three patients with LN ([93]Table S3). The highest percentage of miRNA
   was extracted from exosomes compared with the cell pellet (total reads
   41,994,100 vs. 15,545,900, [94]Figure S4A), suggesting that there is an
   enrichment of miRNA in exosomes. Next, we compared the expression
   levels of the five miRNAs (miR-29c, miR-200a, miR-21, miR-410, and
   miR-150) in urinary exosomes and cell pellet using qPCR-RT. Expression
   levels of the study miRNAs were only detected in urinary exosomes
   ([95]Figure S4B). For these reasons, we decided to focus on urinary
   exosomes as the source to identify miRNA biomarkers for LN renal
   fibrosis.

   We found miR-21 and miR-150 to be significantly overexpressed in the LN
   group compared with healthy controls (6.6- and 2.3-fold change,
   respectively) and miR-410 and miR-29c to be significantly reduced
   (−1.8- and −2.2-fold change, respectively, [96]Figure 1B). No
   significant differences were observed in miR-200a-3p expression levels
   ([97]Figure S5). When results were evaluated according to the CI
   subgroups, miR-410 expression levels did not differ between subgroups
   ([98]Figure S6). However, miR-21-5p, miR-150, and miR-29c were strongly
   correlated with renal chronicity (r = 0.565, r = 0.840, and r = −0.559,
   respectively, [99]Figure S7). Both miR-21 and miR-150 expression levels
   increased progressively according to the degree of CI, being most
   highly expressed in the high CI group (13.3- and 4.8-fold change,
   respectively). However, miR-29c showed a progressive downregulation,
   being the lowest expression levels found in the high CI group
   (−18.7-fold change, [100]Figure 1C). No correlations were found between
   miR-21, miR-150, and miR-29c with traditional clinical parameters of
   damage progression, such as serum creatinine levels, proteinuria or
   eGFR, and the activity index score. However, significant correlation
   was found with tubular atrophy, interstitial fibrosis, glomerular
   sclerosis, and fibrous crescent (p < 0.05, [101]Figures S8–S11).

3.4. CombiROC Performance Analyses for Optimal Marker Combinations

   We next tested miR-21, miR-150, and miR-29c prognostic value for the
   early detection of renal fibrosis in LN. The diagnostic/prognostic
   accuracy of individual and of multiple marker combinations was
   calculated by uploading the original data of analyte concentrations to
   the CombiROC tool. A test cut off value of 1.80 (control mean + 3SD)
   was used. All possible marker combinations to distinguish low CI from
   moderate-high CI group were obtained separately by setting the minimum
   sensibility and specificity values in the next step and finally the
   best performing individual markers and combinations were obtained via
   ROC analysis ([102]Figure 2A,B).

Figure 2.

   [103]Figure 2
   [104]Open in a new tab

   Correlation of urinary miRNAs with chronicity index and risk of
   end-stage progression disease (ESRD). (A) Receiver operating
   characteristic (ROC) curve analysis of individual urinary exosomal
   miR-29c, miR-21, and miR-150 expression to distinguish low CI group
   from moderate-high CI group. Opt cutoff: Optimal cutoff according
   Youden’s index. (B) Multiple ROC curves showed that
   miR-29c/miR-21/miR-150 multimarker panel has the best ROC curve
   profile. They were obtained using CombiROC online program. eGFR:
   Estimated glomerula filtration rate. (C) Kaplan–Meier curve of renal
   survival rate stratified by urinary exosomal miRNAs multimarker panel.
   There were significant differences of the renal survival rate between
   positive and negative patients to multimarker panel by the long-rank
   test. (D) Relative expression levels of miR-150, miR-21, and miR-29c
   were evaluated in seven patients with two kidney renal biopsies. Values
   were normalised using RNU6. * p < 0.05.

   As an individual biomarker, miR-150 had the best profile to distinguish
   the two CI groups (AUC = 0.970, cut-off > 0.903 with 96% sensibility
   and 83% specificity) whereas miR-21 ROC curve was the least predictive
   (AUC = 0.742, cut-off > 4.15 with 81% sensibility and 72% specificity).
   ROC analysis of other clinical parameters such as creatinine, eGFR, or
   proteinuria levels as predictors of early renal fibrosis did not show
   better predictive value ([105]Figure 2B).

   Interestingly, the multimarker panel of miR-29c/mir-150/miR-21
   expression levels exhibited the highest sensitivity and specificity
   values (94.4% and 99.8%, AUC = 0.996, respectively, [106]Figure 2B).
   The pie chart of three miRNAs multimarker panel shows a low percentage
   of false positive cases for this model ([107]Figure S12).

   We further investigated whether the combinatory multimarker panel could
   predict disease progression in LN. Kaplan–Meier survival analysis
   showed that multimarker positive LN patients (miR-29c/miR-150/miR-21)
   had 5- and 10-year renal survival rates of 73.6% and 63.2%,
   respectively, in contrast with 100% and 89.4% for negative patients.
   The difference in renal survival rate was statistically significant
   according to log-rank test ([108]Figure 2C, p = 0.027).

   Seven patients had a repeated biopsy in a median time of 23.8 months
   (range from 12 to 96 months) ([109]Table S4). We observed a significant
   increase in miR-21 and miR-150 expression levels (2.7- and 3.1-fold
   change, respectively) and miR-29c reduction (−6.2-fold change, p <
   0.05, [110]Figure 2D) between baseline and repeated samples that
   correlated with a CI increase.

3.5. Pathway Enrichment Analyses

   We next hypothesised the biological pathways those selected miRNAs may
   regulate by targeting multiple pathway-specific mRNAs. For each miRNA,
   we retrieved validated targets from miRTarBase, ([111]Table S5). An
   interaction network was constructed using miRNet ([112]Figure 3A). Only
   two mRNAs were common to the three miRNAs, SP1 and VEGFA. Since LN
   patients with renal fibrosis overexpressed miR-150 and miR-21 but
   downregulated miR-29c, we also performed GO and KEGG pathway enrichment
   analysis using miRPath (p value < 0.01, [113]Figure 3B). Significant
   enrichment of target genes was detected for seven functional GO
   pathways associated with extracellular matrix and collagen formation
   ([114]Figure 3B). In KEGG pathway analysis, three pathways were
   detected where ECM-receptor interaction was the most significant
   ([115]Figure 3B). The predicted interactions of this pathway showed
   that COL1A1 and COL4A1 were one of the most significant modified mRNA
   genes common for the three miRNAs ([116]Table S6).

Figure 3.

   [117]Figure 3
   [118]Open in a new tab

   Role of urinary exosomal miRNAs in LN renal fibrosis formation. (A)
   Venn diagram representing overlap of validated targets of miR-29c-3p,
   miR-150-5p, and miR-21-5p. Network of miRNA–target interaction was
   obtained using miRNet tool. (B) Enriched gene ontology (GO) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathways using DIANA miRPatch
   v3.0 software. We focus in pathways with downregulation of miR-29c and
   upregulation of miR-150 and miR-21. GO: Gene Ontology. KEGG: Kyoto
   Encyclopedia of Genes and Genomes. CI: chronicity index. (C)
   Immunofluorescence of SP1 (green), COL1A1 and COL4A1 (red) in renal
   biopsy from LN chronicity subgroups (low CI, moderate CI, and high CI).
   DAPI staining was used to label cell nuclei. Scale bar = 100 µm. DAPI:
   4’,6-diamidino-2-phenylindole. (D) Cotransfection of miR-21, miR-150,
   or Mir-29c and SP1 3’ UTR luciferase reporter significantly decreases
   the luciferase activity compared with miR-control (five replicates per
   group). MRC: Renal mesangial cells; TRC: Renal tubular cells. * p <
   0.05, ** p < 0.005, and *** p < 0.0005.

3.6. Confirmation of SP1 as Common Urinary miRNAs Target and Associated
Profibrotic Molecules in Kidney Biopsies

   Next, we sought to confirm the pathway analysis in the kidney biopsies
   from LN patients. Therefore, we examined the protein levels of SP1 and
   VEGFA in the different CI subgroups. Immunohistochemical staining of
   VEGFA in the glomeruli was similar in all LN groups ([119]Figure S13).
   However, high tubular staining of SP1 protein was observed in low and
   moderate CI patients but SP1 decreased significantly in high CI
   patients (p = 0.004 and 0.002, respectively, [120]Figure 3C). COL1A1
   and COL4A1 proteins were predominantly localised in the tubular
   epithelial cells and extracellular matrix, increasing the staining
   progressively with the degree of chronicity ([121]Figure 3C).

   To confirm SP1 as a target for the three studied miRNAs, we performed
   luciferase assay studies using primary human renal mesangial cells
   (RMCs) and renal tubular epithelial cells (RTCs). We co-transfected
   RMCs and RTCs with a plasmid containing a luciferase gene under the
   control of SP1 3′ untranslated region (UTR) and with either miR-29c,
   miR-150, or miR-21 analogue. MiR analogue negative was used as control.
   Luciferase activity decreased in RMCs by 41.2%, 63.9%, and 54.9% 48 h
   after the transfection in the presence of miR-29c, mir-150, and miR-21
   analogue compared with the negative control, respectively ([122]Figure
   3D). In RTCs, the luciferase activity reduction was more pronounced in
   all the three (68.8%, 66.8%, and 67.9%, respectively, [123]Figure 3D).

3.7. Over-Expression of miR-21-5p/miR-150 and Inhibition of miR-29c Increase
Profibrotic Proteins

   In order to better investigate the roles of miR-21/miR-150/miR-29c in
   LN renal fibrosis formation, we overexpressed both miR-21/miR-150 and
   inhibited miR-29c in TGFβ-stimulated RMCs and RTCs. Overexpression of
   miR-21/miR-150 under TGFβ-stimulated RMCs induced a significant
   reduction of SP1 expression (−3.8-fold change) whereas no changes in
   expression levels were observed with miR-29c inhibition in this cell
   type ([124]Figure 4A). The overexpression of miR-21/Mir-150 or the
   inhibition of miR-29c induced COL4A1 formation via Smad3/TGFβ pathway
   (3.8- and 4.6-fold change, respectively, [125]Figure 4A).

Figure 4.

   [126]Figure 4
   [127]Open in a new tab

   Overexpression of miR-21/miR-150 and inhibition of miR-29c increase
   profibrotic proteins. (A) Quantitative real-time RT-PCR analysis shows
   the relative mRNA levels of SP1, COL1A1, COL4A1, TGFβ, Smad3 in
   TGFβ-stimulated renal mesangial cells (RMCs) and renal tubular
   epithelial cells (RTCs). Values were normalised using GADPH and fold
   change was calculated over control condition (mimic miR-control or anti
   miR-control). (B) Immunofluorescent staining exhibits COL1A1 expression
   in MRCs and TRCs with overexpression of miR-21/miR-150 and inhibition
   of miR-29c. SP1 staining was inhibited completely with the
   overexpression of miR-21/miR-150 in both type of cells. Scale bar = 50
   µm. * p < 0.05, ** p < 0.005, and *** p < 0.0005. (C) Proposed
   mechanism for urinary exosomal miRNAs in early and later stages of LN
   renal fibrosis.

   However, overexpression of SP1 was only observed in miR-29c-inhibited
   RTCs along with an increased expression of the study profibrotic
   molecules. Overexpression of miR-21/miR-150 in RTCs induced a
   significant reduction in SP1 expression levels, whereas COL1A1, COL4A1,
   TGFβ, and Smad3 were significantly increased suggesting a Smad3/TGFβ
   fibrosis-dependant pathway, independent of SP1.

   Immunofluorescence staining of SP1 was significantly increased in
   miR-29c inhibited RTCs (p = 0.003, [128]Figure 4B). In contrast, SP1
   was inhibited in miR-21/mir-150 transfected renal cells ([129]Figure
   4B). COL1A1 protein levels increased in all conditions and did not
   correlate with SP1 levels ([130]Figure 4B).

4. Discussion

   We have shown that urinary exosomes are the best source of urinary
   miRNA biomarkers in LN patients. The comparison between urinary pellet
   and exosomes showed a better yield of miRNAs from exosomes than from
   the cellular pellet. One possible explanation is the fact that inside
   urinary exosomes, miRNAs can be preserved and protected from the RNase
   activity in the kidney urinary tract [[131]42].

   We simultaneously measured five urinary exosomal MiRNAs, previously
   associated with fibrosis: miR-21 and miR-200a, which directly regulate
   multiple collagen isoforms and extracellular matrix components via TGFβ
   pathway [[132]23]; and miR-410, miR-29c, and miR-150, which have been
   reported in renal fibrosis in LN [[133]24,[134]25,[135]26]. Of those,
   we only found miR-21, miR-150, and miR-29 to be differentially
   expressed in urinary exosomes from patients with LN and to correlate
   with chronicity scores in the renal biopsy. High degree of fibrosis was
   characterised by a significant upregulation of miR-21, miR-150, and
   miR-29c downregulation.

   FibromiRs are essential downstream components of both fibrogenic and
   fibrosis-suppressive signalling pathways, and changes in their
   expression directly affect the biological response following activation
   of these pathways [[136]3]. As a consequence, several reports studied
   them separately, as individual miRNAs assay. However, fibrogenesis
   results from alterations or imbalances in multiple interconnected
   molecular pathways [[137]43]. We report here, for the first time, a
   multimarker urinary exosomal miRNA panel to diagnose the degree of
   chronicity in lupus nephritis patients at flare using the CombiROC
   tool. This methodology is available as an easy-to-use web application
   to accurately determine optimal combinations of markers and takes
   advantage of combinatorial analysis and ROC curves [[138]41]. It has
   been used to screen multimarker signatures for autoimmune hepatitis
   [[139]44], biomarker panels for cancer [[140]45], and Parkinson disease
   [[141]46]. In our study, the individual miRNAs discriminate low from
   moderate-high chronicity in lupus nephritis; however, their combination
   as multiple biomarker signatures showed the best specificity and
   sensitivity profile to detect early fibrosis. In addition, our results
   also showed the presence of the miR-29c/miR-150/miR-21 combinatory
   panel as a predictor of progression to ESRD and its predictive value
   was superior to routine conventional biomarkers such as creatinine and
   eGFR. If these data are confirmed in larger studies, this biomarker
   panel could be used as a diagnostic marker of renal fibrosis and a
   prognostic biomarker of progression to ESRD.

   The exact mechanism of fibrosis in LN is unknown, but the persistence
   of wound-healing processes with prolonged production of growth factors,
   fibrogenic cytokines, and proteolytic enzymes, leading to increased
   synthesis and degradation of the extracellular matrix [[142]47] seems
   to play a role. Previous studies in LN focused on the fibrotic role of
   miR-150 by targeting SOCS1, a negative regulator of profibrotic
   proteins [[143]26] and miR-29c downregulation [[144]25]. Upregulation
   of miR-21 also induces collagen and other extracellular matrix
   components via the TGFβ/Smad3 pathway [[145]48]. Our pathway analysis
   identified VEGFA and SP1 as common targets of miR-21, miR-150, and
   miR-29c. The expression of VEGFA, SP1, and profibrotic proteins was
   then measured in the LN kidney biopsies. As expected, COL1A1 and COL4A1
   staining increased progressively and correlated with the renal
   chronicity index. This is not surprising, since renal interstitial
   fibrosis is the result of an increase in important ECM components, such
   as collagens [[146]49]. Although it is well-known that down regulation
   of VEGF is related with worse prognosis in lupus nephritis [[147]50],
   we did not find differences in the VEGFA staining in the CI subgroups.
   However, SP1 was found to be increased in patients with low and mainly
   moderate CI, but significantly decreased in patients with high CI.

   We next evaluated in vitro the physiologic relevance of the study
   miRNAs in promoting fibrosis. Transfection of PTCs with miR-150 and
   miR-21 mimics, significantly suppressed SP1 production while inducing
   an increase of profibrotic molecules after TGFβ stimulation. The same
   condition in mesangial cells mirrored the results obtained in the
   tubular cells suggesting a profibrotic effect of these miRNAs through
   the Smad3/TGFβ pathway, independent of SP1. Transfection of PTCs with a
   miR-29c inhibitor significantly increased the production of SP1,
   profibrotic molecules, and Smad3. In mesangial cells, there was a
   moderate production of COL4A1, TGFβ, and Smad3, whereas SP1 and COL1A1
   were not significantly expressed. Data suggest a role for miR-29c in
   the production of fibrosis in tubular epithelial cells through the
   SP1/Smad3/TGFβ pathway.

   Growing evidence demonstrates that SP1 plays an important regulatory
   role for the expression of several genes relevant to fibrosis,
   including TGFβ, vascular endothelial growth factor, type I collagen,
   and downstream targets of TGFβ, such as matrix metalloproteinases,
   plasminogen activator inhibitor-1, and fibronectin [[148]51]. Several
   studies have shown that SP1 mediates TGFβ fibrogenic factor through
   cooperation with Smads proteins and is an essential mediator for the
   production of type I collagen induced by miR-29c downregulation
   [[149]52]. SP1 has been shown in the glomeruli and proximal tubules of
   a broad spectrum of human glomerulonephritides, being more prominent in
   the glomeruli of secondary proliferative GNs [[150]51]. It has been
   shown that SP1 mediates MCP-1 expression in murine podocytes and could
   also be implicated in the induction of renal injury through the
   attraction of macrophages [[151]53]. Therefore, Sp1 overexpression in
   glomeruli of proliferative GNs may be as a result of the inflammatory
   process.

   Our data suggest that miR-150, miR-21, and miR-29c play an important
   role in renal fibrogenesis by increasing the synthesis of profibrotic
   molecules through several mechanisms. We demonstrated an early role of
   miR-29c and SP1 in regulating collagen production in tubular epithelial
   cells, while miR-150 and miR-21 expression contribute to the
   maintenance and amplification of the fibrotic process leading to ESRD
   through a Smad3/TGFβ pathway independent of SP1 at later stages
   ([152]Figure 4C).

   In summary, we have demonstrated that a urinary exosomal multimarker
   panel formed by miR-21, miR-150, and miR-29c provide a non-invasive
   method to detect early renal fibrosis and predict disease progression
   in LN patients. Although the exact molecular basis of the three urinary
   exosomal miRNAs during renal injury remains unclear, we have identified
   relevant pathways in the LN renal fibrosis mechanism, and a better
   understanding of the pathogenesis could contribute to develop new
   therapeutic approaches.

Acknowledgments