Renal Mast Cell-Specific Proteases in the Pathogenesis of Tubulointerstitial Fibrosis

Abstract

Chronic kidney disease is detected in 8–15% of the world’s population. Along with fibrotic changes, it can lead to a complete loss of organ function. Therefore, a better understanding of the onset of the pathological process is required. To address this issue, we examined the interaction between mast cells (MCs) and cells in fibrous and intact regions, focusing on the role of MC proteases such as tryptase, chymase, and carboxypeptidase A3 (CPA3). MCs appear to be involved in the development of inflammatory and fibrotic changes through the targeted secretion of tryptase, chymase, and CPA3 to the vascular endothelium, nephron epithelium, interstitial cells, and components of intercellular substances. Protease-based phenotyping of renal MCs showed that tryptase-positive MCs were the most common phenotype at all anatomic sites. The infiltration of MC in different anatomic sites of the kidney with an associated release of protease content was accompanied by a loss of contact between the epithelium and the basement membrane, indicating the active participation of MCs in the formation and development of fibrogenic niches in the kidney. These findings may contribute to the development of novel strategies for the treatment of tubulointerstitial fibrosis.

Introduction

Chronic kidney disease is detected in 8–15% of the world’s population and leads to a gradual deterioration in organ function up to complete loss. ¹ There are already 800 million people with specific symptoms of chronic kidney disease, and this number is predicted to increase in the future. ² Renal fibrosis is a common final pathway in chronic and progressive nephropathy and is characterized by the production of renal myofibroblasts and the accumulation of extracellular matrix (ECM) components, such as collagen, fibronectin, laminin, and glycoproteins, in the tubulointerstitium. Accumulation of these components, and thus the deposition of ECM, results in the loss of tubules and peritubular capillaries and later to detectable and diagnosed renal function and failure.^3,4

Several cytokines and molecules play a role in maintaining a balance in the turnover of accumulation or removal of ECM components. In line with this, transforming growth factor-beta (TGF-β), platelet-derived growth factor, and fibroblast growth factor 2 act in favor of accumulation of ECM components, whereas hepatocyte growth factor and bone morphogenetic protein 7 antagonize TGF-β, thus inhibiting the overexpression of ECM components.^5–8

A profibrotic phenotype developing at certain loci in a specific tissue microenvironment results in the formation of intraorgan tissue niches with features of the functional architecture of the immune and stromal landscapes, selectively accumulating a wide range of pro-inflammatory factors, such as extracellular vesicles, metabolites, cytokines, chemokines, and growth factors, including those in the kidneys.^9–11 The fibrogenic-specific tissue microenvironment has pronounced inductance and long-term autonomy to stimulate fibroblast proliferation, nephron epithelial damage, microvascular endothelial alteration, and macrophage polarization.^11–13

Mast cells (MCs) play a key regulatory role in the orchestration of innate and specific immune responses and in ECM remodeling in a particular tissue milieu.^14–16 Possessing a wide-ranging repertoire of surface-expressed receptors and releasing mediators, MCs are involved in the pathogenesis of allergic and non-allergic pathologies and contribute to physiological processes, including fertility or wound healing.^8,17,18 Concerning fibrosis, MCs become activated and release mediators, mainly TGF-β ¹⁹ or matrix metalloproteinases (mainly gelatinases), which are imperative in the turnover of the ECM components^20,21 and support the overexpression of ECM components.

Human MCs are classified according to their protease content into MC_T (expressing tryptase) and MC_TC (expressing tryptase and chymase) subpopulations. ²² The latter subset selectively produces carboxypeptidase A3 (CPA3). ¹⁵

MCs have complicated crosstalk with other cells in fibrotic regions, including myofibroblasts, which are known as the main source of ECM components and are, thus, the key cells in fibrosis. A significant colocalization of MCs and α-smooth muscle actin (α-SMA, a marker of activated myofibroblasts) positivity provides evidence for such an interaction.^23–25 In addition, MCs are the main source of the pro-inflammatory profibrotic interleukin (IL)-17A at the fibrosis site. ²⁵

It is well known that an increased number of intraorganic MCs in loci with fibrotic changes, including the kidneys, has not yet been provided with specific details of their integration into the pathogenetic links of the disease.^26–29 Further studies are needed to explore the mechanisms underlying protease-dependent MC participation in ECM remodeling and the development of excessive fibrous structures in renal fibrosis. In this study, we focused on the role of specific kidney MC proteases (tryptase, chymase, and CPA3) in the pathogenesis of tubulointerstitial fibrosis in six patients diagnosed with nephrosclerosis with a non-functioning upper pole of the duplex right kidney at the junction of the refluxing megaureter of the upper pole of the duplex right kidney.

Materials and Methods

Case Report

The study involved kidney biomaterials obtained from six patients aged 3–5 years as a result of heminephroureterectomy of pathologically altered areas of the kidney at The Institute of Pediatrics and Pediatric Surgery, named after Academician Yu.E. Veltishchev, Ministry of Health of the Russian Federation (hereinafter—Veltishchev Institute). Each child was comprehensively examined, and each medical case was individually validated to determine the required volume of surgical intervention, including incomplete duplication, hydronephrosis, atrophic signs, nephrosclerosis, and recurrent pyelonephritis. According to the static nephroscintigraphy, the integral capture index in the pathologically altered areas of the kidney was less than 10%. This parameter was a pivotal clinical decision point regarding the volume of surgical intervention. The study was conducted in accordance with the World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects and was approved by the local ethics committee of the Veltischev Institute. Informed consent was obtained from the children’s legal representatives. The samples were de-identified.

Tissue Probe Staining

The tissue probes left after the routine diagnostic procedure were fixed in buffered 4% formaldehyde and embedded in paraffin. Paraffin tissue sections (5 and 2 µm thick for histochemical and immunohistochemical staining, respectively) were deparaffinized with xylene and rehydrated with graded ethanol according to a standard procedure.^30,31 Tissue probes of approximately 1 mm³ were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide solutions and analyzed using electron microscopy. ³⁰

Immunohistochemistry and Histochemistry

For the immunohistochemical assay, we subjected deparaffinized sections to antigen retrieval by heating the sections in a steamer with R-UNIVERSAL Epitope Recovery Buffer (Aptum Biologics Ltd., Southampton, UK) at 95C × 30 min. ³² After antigen retrieval and, when required, endogenous peroxidase quenching, the sections were incubated with primary antibodies. The list of primary antibodies used in this study is presented in Table 1, and the list of secondary antibodies and other reagents is presented in Table 2. The staining design for kidney sections from all patients, including the multiplex immunohistochemical staining protocols, is listed in Table 3. Immunohistochemical visualization of bound primary antibodies was performed either with a Ventana Slide Stainer or manually according to the standard protocol. ³² For manually performed immunostaining, primary antibodies were applied in concentrations from 1 to 5 µg/mL and incubated overnight at 4C.

Table 1.

Primary Antibodies Used in This Study.

Antibodies	Host	Catalog Number	Dilution	Manufacturer
Tryptase	Mouse monoclonal Ab	#ab2378	1:3000	Abcam, Cambridge, UK
Tryptase	Rabbit monoclonal [EPR9522]	#ab151757	1:1000	Abcam, Cambridge, UK
Carboxypeptidase A3	Rabbit polyclonal Ab	#ab251696	1:2000	Abcam, Cambridge, UK
Chymase	Mouse monoclonal Ab[CC1]	ab2377	1:2000	Abcam, Cambridge, UK
α-Smooth muscle actin	Mouse monoclonal [1A4]	ab7817	1:2000	Abcam, Cambridge, UK
Vimentin	Rabbit monoclonal [EPR3776]	ab92547	1:1000	Abcam, Cambridge, UK
Transforming growth factor-β	Rabbit monoclonal	#ab215715	1:500	Abcam, Cambridge, UK
CD31	Rabbit monoclonal	#ab182981	1:500	Abcam, Cambridge, UK

Table 2.

Secondary Antibodies and Other Reagents.

Antibodies and Other Reagents	Source	Dilution	Label
Goat anti-mouse IgG Ab (#ab97035)	Abcam, Cambridge, UK	1/500	Cy3
Goat anti-rabbit IgG Ab (#ab150077)	Abcam, Cambridge, UK	1/500	Alexa Fluor 488
AmpliStain anti-Mouse 1-Step HRP (#AS-M1-HRP)	SDT GmbH, Baesweiler, Germany	Ready to use	HRP
AmpliStain anti-Rabbit 1-Step HRP (#AS-R1-HRP)	SDT GmbH, Baesweiler, Germany	Ready to use	HRP
Opal Polymer HRP Ms+Rb (#ARH1001EA)	Akoya Biosciences, Marlborough, MA, USA	Ready to use	Opal 480 Reagent Pack (#FP1500001KT)
Opal Polymer HRP Ms+Rb (#ARH1001EA)	Akoya Biosciences, Marlborough, MA, USA	Ready to use	Opal 540 Reagent Pack (#FP1494001KT)
Opal Polymer HRP Ms+Rb (#ARH1001EA)	Akoya Biosciences, Marlborough, MA, USA	Ready to use	Opal 570 Reagent Pack (#FP1488001KT)
Opal Polymer HRP Ms+Rb (#ARH1001EA)	Akoya Biosciences, Marlborough, MA, USA	Ready to use	Opal 620 Reagent Pack (#FP1495001KT)
Opal Polymer HRP Ms+Rb (#ARH1001EA)	Akoya Biosciences, Marlborough, MA, USA	Ready to use	Opal 690 Reagent Pack (#FP1497001KT)
10Х Spectral DAPI	Akoya Biosciences, Marlborough, MA, USA	Ready to use	#FP1490
4′,6-Diamidino-2-phenylindole (DAPI, #D9542-5MG)	Sigma, Hamburg, Germany	5 µg/mL	w/o
VECTASHIELD Mounting Medium (#H-1000)	Vector Laboratories, Burlingame, California	Ready to use	w/o
DAB Peroxidase Substrate Kit (#SK-4100)	Vector Laboratories, Burlingame, California	Ready to use	DAB
Toluidine blue (#07-002)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o
Giemsa solution (#20-043/L)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o
Silver impregnation (#21-026)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o
Mayer’s hematoxylin (#HK-G0-DL01)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o
Picro Mallory trichrome (#21-036)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o
Heidenhain’s Azan trichrome (#21-041)	Biovitrum, ErgoProduction LLC, St. Petersburg, S Russia	Ready to use	w/o
Giemsa (#20-023)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o
May–Grünwald Giemsa (#21-068)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o
Alcian blue pH 2.5 and PAS (#21-069/L)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o
Eosin Y 1% aqueous (#HK-EV-A250)	Biovitrum, ErgoProduction LLC, St. Petersburg, Russia	Ready to use	w/o

Table 3.

Design of Immunohistochemical and Histochemical Staining of Histological Sections of the Kidney.

Technology	Detection Targets	Label	Counterstaining of Nuclei
Monoplex immunohistochemical staining	Tryptase	HRP	Mayer’s hematoxylin
	Chymase	HRP
	CPA3	HRP
Multiplex immunohistochemical staining	Tryptase + chymase	Cy3, Alexa Fluor 488	DAPI (Sigma, Germany)
	Tryptase + CPA3	Cy3, Alexa Fluor 488
	Tryptase + TGF-β	OPAL690, OPAL 480	DAPI (Akoya Biosciences)
	Chymase + CD31 + CPA3 + Tryptase	OPAL 480, 540, 620, and 690
	Chymase + CD38 + CD163 + CD68 + α-SMA	OPAL 480, OPAL 540, OPAL 570, OPAL 620, and OPAL 650
Histochemical staining	Toluidine blue, Giemsa solution, Silver impregnation, Picro Mallory trichrome, Heidenhain’s Azan trichrome, May–Grünwald Giemsa, Alcian blue pH 2.5—PAS, Mayer’s hematoxylin—Eosin Y 1% aqueous

Abbreviations: CPA3, carboxypeptidase A3; TGF-β, transforming growth factor-beta; α-SMA, α-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole.

Secondary goat anti-mouse or anti-rabbit antibodies (AmpliStain anti-Mouse 1-Step HRP or AmpliStain anti-Rabbit 1-Step HRP [SDT GmbH, Baesweiler, Germany]) were applied for monoplex immunohistochemical detection of molecular targets with the DAB Peroxidase Substrate Kit (Vector Laboratories, Burlingame, California) according to the instructions (Table 2). Diplex immunohistochemical staining of tryptase and CPA3, as well as tryptase and chymase-bound primary antibodies, was visualized using secondary antibodies (Dianova, Hamburg, Germany, and Molecular Probes, Darmstadt, Germany) conjugated with Cy3 or Alexa Fluor-488, respectively (Tables 2 and 3). For tryptase and TGF-β simultaneous staining, OPAL 690 and OPAL 480 fluorochromes (Akoya Biosciences) were used to increase the range of excitation/emission maxima between the selected fluorophores and more accurately detect growth factor expression in MCs (Table 3). The final concentration of secondary antibodies was between 5 and 10 µg/mL of phosphate-buffered saline. Single and multiple immunofluorescence labeling was performed according to standard protocols. ³⁰

Sequential multiplex immunohistochemical staining for the simultaneous detection of tryptase, chymase, CPA3, and CD31 was performed according to the recommendations of Akoya Biosciences for the use of OPAL series fluorochromes in the Mantra 2 Quantitative Pathology Imaging System. The reagents used for this staining design were OPAL 480, 540, 620, and 690 (Tables 2 and 3). OPAL 480, 540, 570, 620, and 650 reagents were used to implement the immunohistochemical staining design with multiplex detection of chymase, CD38, CD163, CD68, and α-SMA, respectively (Tables 2 and 3). In addition, when using OPAL series fluorochromes for repeated retrieval, the EZ-Retriever System, MW015-IR (BioGenex, Fremont, CA, USA), was applied. Histochemical staining with toluidine blue, May–Grünwald Giemsa, Picro Mallory solutions, Heidenhain’s Azan trichrome, Alcian blue and periodic acid Schiff, Mayer’s hematoxylin and eosin, and silver impregnation was performed according to the manufacturer’s instructions (Table 2).

Controls

Control incubations were performed by omitting primary antibodies or substituting primary antibodies with the same IgG species (Dianova) at the same final concentration as the primary antibodies. The exclusion of either the primary or secondary antibody from the immunohistochemical reaction and the substitution of primary antibodies with the corresponding IgG at the same final concentration resulted in a lack of immunostaining. Specific and selective staining of different cells using primary antibodies from the same species on the same preparation is a sufficient control for immunostaining specificity.

Electron Microscopy

After fixation in solutions of glutaraldehyde and osmium tetroxide, the biomaterial was dehydrated in alcohol at an increasing concentration gradient (50%, 70%, 96%, and 100%) and then impregnated with a mixture of propylene oxide and Araldite resin. After impregnation, the material was placed in capsules, filled with Araldite resin, and placed in a thermostat at 60C for 2 days. Semi-thin sections (1.5–2 μm) were prepared from the obtained blocks. The sections were stained with toluidine blue. The images of light-optical preparations (section thickness 1.0–1.5 µm) were analyzed, and areas for ultramicrotomy were targeted. Ultrathin sections (100–200 nm) were prepared using an LKB Ultramicrotome (Stockholm, Sweden). The sections were then stained with uranyl acetate and lead citrate. ³⁰

Image Acquisition

Stained tissue sections were observed using a Zeiss Axio Imager.Z2 equipped with a Zeiss alpha Plan-Apochromat objective 100×/1.46 Oil DIC M27, a Zeiss Objective Plan-Apochromat 150×/1.35 Glyc DIC Corr M27, and a Zeiss Axiocam 712 color digital microscope camera. Captured images were processed with the software program “Zen 3.0 Light Microscopy Software Package,” “ZEN Module Bundle Intellesis & Analysis for Light Microscopy,” and “ZEN Module Z Stack Hardware” (Carl Zeiss Vision, Jena, Germany) and submitted with the final revision of the article at 300 DPI. Photomicrographs were captured using a Nikon D-Eclipse C1 Si confocal microscope based on Nikon Eclipse 90i.

The Mantra 2 Quantitative Pathology Imaging System (Akoya Biosciences), based on an Olympus BX43 microscope equipped with a scientific-grade multispectral 12-bit monochrome high-sensitivity CCD camera with a liquid crystal tunable spectral filter, was used to determine the profiles of specific MC proteases in multiple immunodetections of tryptase, chymase, CPA3, and CD31 using OPAL 690, 480, 620, and 540 fluorochromes, respectively (Table 3). In addition, multiplex staining results for chymase, CD38, CD163, CD68, and α-SMA were analyzed using OPAL 480, 540, 570, 620, and 650, respectively (Table 3).

Quantitative Analysis

A nephrofibrotic kidney was dissected into three to four fragments to increase the volume of the analyzed structural components, quantitative data, and, accordingly, the objectivity of the findings. Sections were prepared from each fragment and stained using the immunohistochemical methods described in Section “Immunohistochemistry and histochemistry”. Planimetric analysis to determine the number of MCs per unit area of kidney tissue, as well as the absolute number of MCs and other kidney cells, was performed using the open-source software QuPath for digital pathology image analysis. ³³ To identify the area of immunopositive structures on SMA and vimentin concerning the total size of the kidney tissues on histological sections and to determine the absolute number of tryptase-positive nuclei in relation to the total number of nuclei on sections, morphometric analysis was conducted using QuPath software ³³ with further calculation of the relative content (Table 4). The cortex and medulla of the kidney, as well as parts of the organ with pronounced manifestations of fibrotic changes, were analyzed separately. When determining SMA- and vimentin-positive cells and structures, kidney vessels were excluded from the analyzed areas to objectify the data obtained. To obtain the integral values of the studied MCs and fibrosis parameters, the average value was obtained from the analysis of each kidney fragment.

Table 4.

The Content of MCs in the Kidney Under Fibrosis.

Kidney area	Parameters	Tryptase+	CPA3+	Chymase+
Cortex of the kidney	Total number of cells in the analyzed area	8,410,320	8,585,787	8,384,222
	Absolute number of MCs in the analyzed area	12,598	10,302	4192
	Relative number of MCs in the analyzed area (в %)	0.15 ± 0.01	0.12 ± 0.02	0.05 ± 0.01
	The content of MCs/mm2	10.12 ± 0.95	7.14 ± 0.51	3.46 ± 0.32
Medulla of the kidney	Total number of cells in the analyzed area	3,483,864	3,586,712	3,412,747
	Absolute number of MCs in the analyzed area	4529	4304	1365
	Relative number of MCs in the analyzed area (в %)	0.13	0.12	0.04
	The content of MCs/mm2	8.34 ± 0.63	7.16 ± 0.52	3.12 ± 0.34
Fibrous-modified areas of the kidney	Total number of cells in the analyzed area	5,234,406	5,342,958	5,097,386
	Absolute number of MCs in the analyzed area	41,320	30,674	28,020
	Relative number of MCs in the analyzed area (в %)	0.78 ± 0.03	0.55 ± 0.02	0.56 ± 0.03
	The content of MCs/mm2	37.3 ± 1.9	25.5 ± 1.8	23.1 ± 2.0

Statistical Analysis

Statistical analysis was performed using the SPSS software package (v13.0, IBM, New York, USA). The results are presented as mean ± standard error of the mean.

Data Availability

The authors declare that all the data supporting the findings of this study are available within the article or from the corresponding author upon reasonable request.

Results

The fibrous-modified areas of the kidney contained the highest number of MCs, which was calculated per square millimeter and as the relative total number of cells in the tissue (Table 4, Fig. 1). In all observed microscopic fields, tryptase-positive MCs were the most abundant (Table 4, Fig. 2). The number of CPA3-positive MCs was lower (Table 4, Fig. 3), but in the renal medulla, the relative number was comparable to that of tryptase-positive MCs. Chymase-positive MCs were the least abundant (Table 4, Fig. 4) and were absent in the cortical and medullary areas of the kidney. However, it should be noted that in the fibrous foci of the kidney, the content of chymase-positive MCs was significantly higher, especially when determining the relative content among all cells of the fibrous tissue (Table 4, Fig. 4A).

Figure 1.

Features of the mast cell (MC) association with elements of the kidney stroma in nephrofibrosis. Technique: (A, E–G) immunohistochemical staining of MC tryptase (A, G), α-smooth muscle actin (α-SMA; E), and vimentin (F); (B) silver impregnation; (C) Heidenhain’s Azan staining; (D) Alcian blue and periodic acid Schiff staining. (A) Selective localization of tryptase-positive MCs in the kidney with high protease secretion activity (arrowed). (A′) Enlarged fragment. (A, B–D) Formation of distinct tryptase-positive inductive fields, coinciding in localization with areas of high content of reticular fibers and collagen fibrillogenesis (B, B′, arrowed), as well as collagen fibers and an amorphous component of the extracellular matrix of the connective tissue (C, D, arrowed). (E, F) Colocalization of intraorganic expression of α-SMA (E) and vimentin (F) with MC histotopography (arrowed). (G) Participation of MCs in the formation of a profibrogenic niche with a tryptase-inductive zone in the unaltered kidney parenchyma (G′,G′′ arrowed). Bars A′, B′, G′ = 50 µm; bar G′′ = 5 µm; bars A–G = 500 µm.

Figure 2.

Histo- and cytotopography of mast cell tryptase under the formation of fibrotic changes in the kidney. (A) Close mast cell localization to the renal corpuscle in the cortex. (B–D) Target tryptase secretion by mast cells to the basement epithelial membrane of the proximal convoluted tubule of the nephron (arrowed) and interstitium cells (double arrowed). (E) Tryptase secretion to the basement membrane of the epithelium of the distal convoluted tubule of the nephron and the structures of the interstitium (arrowed). (F) Tryptase secretion into the extracellular matrix of the kidney interstitium in the cortex at the border of the distal and proximal convoluted tubules of the nephron (arrowed). (G) Interaction of mast cells with stromal cells in the renal cortex (arrowed), including fibroblast (double arrowed). (H–N) Options for the formation of profibrogenic niches by mast cells in the interstitium of the kidney with local tryptase accumulation in the extracellular matrix (arrowed). (O) Target tryptase secretion to the stromal cell with penetration into the nuclear structures (arrowed). (P–R) Tryptase-positive nuclei in the cells of a specific tissue microenvironment of profibrogenic renal niches. The positivity of the nucleus to tryptase, mainly in the cells of the interstitium (arrowed), is highlighted. O′ - enlarged fragment O. Q′ - enlarged fragment Q. Bars A–R = 10 µm.

Figure 3.

Carboxypeptidase A3 (CPA3)-positive mast cells (MCs) in the kidney in nephrofibrosis. (A, B) High content of MCs in the kidney area with fibrotic changes. (C) MCs form groups in the paracrine zone near the fibroblast (arrowed). (D) Colocalization of MCs with several stromal cells in the fibrosis site, active secretion of CPA3. (E–G) Secretion of CPA3 to the epithelium of the nephron (arrowed) in fibrous areas of the renal medulla. (H) Simultaneous effect of CPA3 on the nephron epithelium and interstitium cells (arrowed). (I) MC participation in the epithelial–mesenchymal transformation of nephron cells (presumably arrowed). (J) MC colocalization with myofibroblast of the interstitium of the kidney (presumably arrowed). (K–S) Morphological equivalents of MC participation in the formation of profibrogenic niches with CPA3 secretion (arrowed) to the targets of a specific tissue microenvironment, including the epithelium of the proximal and distal convoluted tubules (K–M), interstitium cells (N–R), and leaves of the Shumlyansky–Bowman’s capsule (S). Bars A and B = 50 µm; bars C–S and S′ = 5 µm.

Figure 4.

Mast cells (MCs) in the formation of profibrotic changes in the kidney. Techniques: (A–E) immunohistochemical staining of MC chymase; (F, G, K–N) staining with toluidine blue; (H–J) Giemsa staining; (O) Picro Mallory staining. (A) Accumulation of chymase in mature fibrous tissue (arrowed) at the border with the renal cortex. (B–D) Target chymase secretion to the basement membrane of the epithelium of the proximal (B, C, arrowed) and distal (D, arrowed) parts of the kidney nephron. (E–H) Directed MC degranulation to fibroblasts in the zone of fibrotic changes (arrowed). (I) MCs at the periphery of a profibrogenic niche with active secretory activity (arrowed). (J) Colocalization of an MC and eosinophil in the fibrous tissue. (K–M) Various variants of MC secretory activity through autonomous secretory granule excretion into the extracellular matrix (arrowed), resulting in a decreased number of granules in the cytoplasm (L) and the formation of areas of a specific tissue microenvironment filled with secretory granules (M). (N and O) Variants of MC colocalization with a fibrous component of the extracellular matrix in the kidney area with fibrotic changes. Bar A = 50 µm; bars B–O = 5 µm.

Although the relative number of MCs in the fibrosis-modified area was low compared with the total number of other cells (Table 4), the high secretory activity of MCs has attracted attention. In this context, among the observed microscopic fields (zones), several were abundantly and heavily infiltrated by secretory granules or showed tryptase immunopositivity (Figs. 1, 2, and 5). The high content of secretory granules with autonomous activity potential and the gradual release of biologically active substances, including tryptase, chymase, and CPA3, could significantly enhance the biological role of MCs in individual loci of a specific tissue microenvironment.

Figure 5.

Profile of specific mast cell proteases during the formation of profibrogenic niches in the kidney stroma. (A) Active entry of tryptase and carboxypeptidase A3 (CPA3) to the targets of the extracellular matrix of the specific tissue microenvironment of the tissue niche in the composition of tunneling nanotubules (arrowed) and secretory granules (double arrowed). (B) Entry of tryptase and CPA3 into the profibrogenic niche as part of secretory granules (arrowed). (C, D) Dissemination of secretory granules with specific mast cell proteases in the interstitium (arrowed) adjacent to the stroma cells. (E) Contacting areas of the mast cell cytoplasm filled with large secretory granules, with predominant localization of tryptase and chymase along the periphery of the granules. (F, G) Mast cells in the state of secretion of specific proteases to the epitheliocytes of the nephron tubules (arrowed) of the renal medulla. (H, I) Active secretion of tryptase and CPA3 to interstitial cells of the fibrous part of the kidney (arrowed), including fibroblasts (presumably double arrowed). Bars A–I = 5 µm.

MCs forming protease-positive fields with unique inductive properties due to specific protease accumulation have been previously described in our studies related to other pathologies (Figs. 2 and 5).^34,35

In terms of cytotopography, specific proteases in certain MCs were packaged into secretory granules of various sizes that occupied peripheral positions (Fig. 5). Such intragranular localization of specific proteases is also typical of MCs located in other organs.^34–36 Tryptase, chymase, and CPA3 are often detected as progranules, and small immunopositive formations are distributed throughout the cytoplasm. This evidenced active processes of biogenesis, in which synthesis and post-translational rearrangements were accompanied by rapid secretion (Figs. 3 and 4). In addition, it is necessary to pay attention to the regulatory potential of MCs provided by the tunneling nanotubes, which are visible at certain loci of the specific tissue microenvironment of the kidney (Fig. 5). Occasionally, it was possible to observe the formation of elongated cytoplasmic outgrowths containing specific proteases directed to target cells.

Post-cellular structures were often found in fibrotic-modified areas of the kidneys and were autonomous fragments of the cytoplasm or freely located granules that are able to secrete biologically active substances for a certain time after separation from the cell.

Histotopographic features of MC distribution in the kidney interstitium revealed that, regardless of the specific protease expression, MCs in the cortical substance were most often located in the region of the proximal or distal convoluted tubules contacting the basement membrane of the nephron epithelium (Figs. 1G; 2A to H; 3K to S; 4B to D). The MCs were localized near the renal corpuscle and sometimes in contact with the parietal leaf of the Shumlyansky–Bowman capsule (Figs. 2A and 3S). The MCs in the medulla were most commonly localized in the interstitium near the loop of Henle (Fig. 6).

Figure 6.

Histotopography of mast cells (MCs) in the interstitium of the cortex and medulla of the kidney with fibrosis (in percentage of the total number of MCs, staining technique—monoplex immunohistochemical). Abbreviation: CPA3, carboxypeptidase A3.

MCs actively secreted specific proteases into the kidney stroma. The MCs actively interacted with the parietal leaf of the Shumlyansky–Bowman capsule (Fig. 3S), proximal convoluted tubules (Figs. 2B to D, G, and H; 3K to M, Q, and R; 4B and C), distal convoluted tubules, and loop of Henle (Figs. 2J and M; 3K and O; 4D). In the sclerotic zone, active MC interactions with other immunocompetent cells and, apparently, with stromal cells were noted (Figs. 2B to D, G, M, and O; 3C, D, G to J, and N to R; 4A, D to J, and L to N). MC interaction with other cells of the local tissue microenvironment was not accompanied by the classical pathways of mediator degranulation alone but also by the formation of tunneling nanotubes, which served as an effective way of targeted delivery of specific proteases to cellular targets (Fig. 5).

MCs migrate into the intertubular matrix, and in some cases, protease secretion directly to the basement epithelial membrane of various parts of the nephron causes an obvious loss of contact between the epithelium and basement membrane. This evidence can be associated with the phenomenon of epithelial–mesenchymal transition, in which the cellular functional potential changes significantly, leading to a pronounced ability to synthesize ECM components.

Tryptase-positive areas were formed in isolated loci of the tissue microenvironment; they had pronounced immunopositivity to the protease and induced the development of inflammation (Figs. 1A and G and 2H to N). When determining α-SMA and vimentin expression in various areas of the kidney, it was found that the highest values of the cell and structure immunopositivity were detected in the fibrotic zone (Figs. 1E and F and 7). Concurrently, the values of the content of α-SMA and vimentin correlated with each other, a fact supporting an increased representation of mesenchymal structures due to a potential intensification of the epithelial–mesenchymal transformation of the kidney parenchyma. Much lower values of the content of α-SMA and vimentin-positive structures were obtained in the cortical and medulla of the kidney (Figs. 1E and F and 7).

Figure 7.

The content of α-smooth muscle actin (α-SMA)- and vimentin-positive cells and structures in the kidney (in percentage of the total number on the analysis area, staining technique—monoplex immunohistochemical).

MCs appear to initiate primary changes in the kidney stroma at sites of subsequent inflammatory processes and fibrosis development, creating conditions for the formation of a profibrogenic niche (Fig. 1G). A limited zone with a large number of tryptase-positive nuclei was detected at the border of the unaltered kidney parenchyma in contact with the fibrous tissue (Fig. 2P). In the loci of profibrogenic niches, tryptase-positive nuclei were present; these were combined with an almost complete lack of tryptase expression in the cell nuclei in both the parenchyma and interstitium of the cortex and medulla of the kidney (Figs. 1G and 2O to R). The epigenetic effects of tryptase were involved in creating the profibrotic phenotype of a specific tissue microenvironment by reprogramming and modifying the functional activity of other cells, leading to further progression of fibrotic changes (Fig. 2O). The frequency of tryptase-containing nuclei in kidney cells was the highest in areas with fibrotic changes or in the border areas of the normal parenchyma with pathologically altered areas. Planimetric analysis revealed a significantly excessive number of cell nuclei with tryptase immunopositivity in the fibrous foci of the kidney compared with similar parameters in the cortex and medulla, reaching differences of 10 or more times (Table 5).

Table 5.

Tryptase Expression in the Cell Nuclei of Various Kidney Areas.

Immunopositivity of Nuclear Structures to Tryptase	Cortex	Medulla	Zone of Fibrosis
	Percent of the Total Number of Nuclei	Percent of the Total Number of Nuclei	Percent of the Total Number of Nuclei
Moderate (+)	0.0284 ± 0.0012	0.0312 ± 0.0021	0.3814 ± 0.031
Pronounced (++)	0.0081 ± 0.0004	0.0061 ± 0.0004	0.1118 ± 0.0131

MCs were often colocalized with cells expressing α-SMA such as smooth myocytes (in the wall of the vascular bed of the kidney) and myofibroblasts, because their number increased in areas with fibrotic changes.

Epifluorescence and confocal microscopy allowed the evaluation of the spatial orientation of the MC secretome effect, in particular, tryptase and CPA3, on the structural components of the epithelium in various parts of the nephron. The secretion of protease-containing components could occur in a fairly large area of the basement membrane in both the proximal and distal convoluted tubules of the nephron. Undoubtedly, the effect of specific proteases was accompanied by significant changes in nephron epithelium activity. However, MCs could simultaneously influence several targets of the local tissue microenvironment, synchronizing the induced changes in the corresponding signaling systems in multiple cells (Fig. 5). The formation of a large contact area between MCs and other cells potentiated the biological effects of tryptase, CPA3, and chymase (Fig. 5A, B, and F). In the area of fibrosis, MCs had a simultaneous effect on a greater number of other cells in the local tissue microenvironment than in the cortex or medulla of the kidney. In some cases, an MC could directly secrete proteases simultaneously into eight or more cells, depending on the thickness of the histological section (Fig. 5).

Ultrastructural analysis of MCs in fibrous niches demonstrated an active secretome effect on the fibrous ECM, primarily on collagen fibers (Fig. 8). Particularly, a mesophase state formed around the MCs, which created favorable conditions for the initiation of MC molecule polymerization with the formation of collagen microfibrils (Fig. 8A). MC degranulation was accompanied by specific positioning of the secretome components (Fig. 8B). In addition, MC secretome exposure resulted in the thickening of microfibrils and their spatial distribution in the peripheral region of collagen fibers (Fig. 8C and D).

Figure 8.

Mast cells (MCs) in the extracellular matrix (ECM) remodeling during the formation of fibrogenic niches in the tissue microenvironment of the kidneys. Electronic micrographs. (A) MC in the ECM mesophase with tactoid formation (arrowed) and the initial stages of collagen microfibril formation (double arrowed). A′—magnified fragment of A. (B) MC degranulation with specific positioning of secretome components (arrowed) and collagen fibrillogenesis initiation with microfibril formation (double arrowed). (C) Ultrastructural signs of the MC granule secretory material involved in collagen fibrillogenesis with ECM tactoid nucleation, specific positioning of collagen protofibrils (arrowed), and thickening of microfibrils (double arrowed). (D) Grouping of collagen microfibrils into target-specific bundles with developing features of the microfibril spatial distribution in the peripheral region of the collagen fiber (arrowed). Bars A–D = 1 µm. Abbreviation: CF, collagen fibers; MF, microfibril; SG, secretory granule.

Multiplex immunohistochemistry data demonstrated that MCs with a high level of TGF-β were detected in the renal parenchyma intact up to that time (Fig. 9A and B). MCs in the area of kidney fibrosis had the highest degree of TGF-β expression.

Figure 9.

Spatial mapping and profile of specific mast cell (MC) proteases in the stromal and immune landscapes of fibrogenic niches. Technique: sequential multiplex immunohistochemical detection of molecular targets. (A, B) Sequential staining of tryptase and transforming growth factor-beta (TGF-β). High TGF-β expression in MCs adjacent to the nephron epithelium (arrowed) in the presence of MCs with moderate TGF-β expression. (C, D) Sequential staining of tryptase, chymase, carboxypeptidase A3 (CPA3), and CD31. The predominant phenotype of MCs, Tryptase^High, Chymase^high, and CPA3^High, contact with CD31+ cells (arrowed). (E) Sequential staining of chymase, α-smooth muscle actin (α-SMA), CD163, CD68, CD38, and chymase. The most frequent MC colocalization with α-SMA-positive stromal cells of fibrous niches. Bars A–E = 50 µm.

In addition, MCs were found to have a protease content of Try+, Chy+, and CPA3+ and were often localized around the microvasculature or angiogenesis sites (Fig. 9C and D). Mapping MCs in the fibrotic area revealed their close colocalization in the stromal landscape with α-SMA-positive cells, whereas in the immune landscape, they were colocalized with CD38+ cells (Fig. 9E).

Discussion

In terms of pathobiology, fibrosis is viewed as a protective reaction aimed at isolating the focus of inflammation from the surrounding tissues and systemic blood flow. Fibrous tissue replacement results in a gradual loss of their specific functions when a certain extent of the integral dysfunction of the organ is reached. The reasons for such changes include radiation, trauma, infection, allergy, autoimmunity, and inflammation. Damage to the kidney can provide a special status of the integrative-buffer metabolic environment of the tissue microenvironment, which undergoes dynamic changes in its composition and content under regeneration and remodeling of the ECM and spatial architectonics of the organ. To structurally specify these changes in the tissue microenvironment, the term “fibrogenic niche promoting fibroblast activation and myofibroblast formation in organ fibrosis” was previously appropriately proposed. ³⁷ One of the mechanisms of fibrosis formation is the epithelial–mesenchymal transition, in which epithelial cells acquire the phenotypic properties of mesenchymal cells. Our findings involving the kidney of a patient with advanced nephrofibrosis showed direct MC involvement in the formation of fibrogenic niches and an active contribution to fibrosis through several mechanisms.

First, it should be noted that MCs form a pro-inflammatory secretory profile in the local tissue microenvironment of the kidney with the help of specific proteases. Temporal and spatial patterns of tryptase secretion in the local tissue microenvironment of the kidney during MC activation are the most significant pathogenetic events in the inflammatory process and the further formation of a profibrogenic niche. Chronic MC activation can induce hypersensitivity to several components of a specific tissue microenvironment owing to the restructuring of its proper receptor apparatus, thus provoking higher secretion of biogenic amines, cytokines, and specific proteases.^38–40

Tryptase is characterized by high biofunction that affects numerous cellular and non-cellular components of the tissue microenvironment.^34,41–43 Several studies have demonstrated that tryptase is closely involved in angiogenesis,^44,45 which is associated with connective tissue remodeling and the secretion of growth factors, cytokines, chemokines, and matrix metalloproteinases. ⁴⁶

Kidney fibrosis is accompanied by an increased number of myofibroblasts, more than one third of which originate from the tubular epithelial cells.^47,48 Tryptase affects fibroblast differentiation, causing active migration, mitotic division, and collagen synthesis. Therefore, tryptase may contribute to the development of renal fibrosis. The increased expression of α-SMA demonstrated in our study supports the increased number of myofibroblasts at the sites with fibrotic changes and the high intensity of epithelial–mesenchymal transition under the influence of specific MC proteases.

In addition, tryptase has a high affinity for protease-activated receptor 2 in various cells in a specific tissue microenvironment, potentiating the development of inflammation. ⁴⁶ A crucial tryptase regulatory mechanism that potentiates inflammation is the persistently increased expression of protease-activated receptor 2 in target cells.^44,49–51

Chymase plays an important role in signaling molecular–cellular integrative mechanisms in a specific tissue microenvironment.^35,43,52 There is evidence of an increased accumulation of various types of inflammatory cells, including eosinophils, under the action of chymase, as recorded in our study (Fig. 4K).^52–54 Chymase has a direct effect on the activity of various ECM components and has greater destructive potential than tryptase.^46,52,55

As previously reported, chymase actively correlates with the progression of inflammatory diseases in various organs, including the kidneys.^52,56 Pro-inflammatory effects of chymase are associated with the activation of cytokines and growth factors, including IL-1β, IL-8, IL-18, TRF-β, endothelin-1 and -2, and neutrophil-activating protein-2, which leads to the recruitment of granulocytes, lymphocytes, and monocytes into the tissue microenvironment. ⁵² Chymase is likely to cause the degradation of contacts and structures that ensure the strength of attachment of epithelial cells to each other and to the basement membrane, ⁵⁷ which can indirectly contribute to epithelial–mesenchymal transformation.

Chymase increases the mitotic activity and biosynthetic potential of fibroblasts. In addition, chymase is involved in the modification of procollagen molecules, thereby inducing collagen fibril formation.^46,58 Studies have shown that active MCs participate in the mechanisms of fibrillogenesis, which is manifested by an inductive effect on the formation of the fibrous component of the tissue microenvironment, primarily in the pericellular space of fibroblastic differentiation cells. Reticular fibers or points of fibrillogenesis initiation have also been detected near the MC plasmalemma. ²⁷ The results of the electron microscopic analysis performed in our study indicated the direct involvement of MCs in collagen fiber formation (Fig. 8). MCs most likely form the required qualitative composition of the ECM to initiate collagen fibril polymerization. ²⁷ However, indirect effects, including those caused by TGF-β, should also be taken into account. It is obvious that in the zone of fibrous niche formation, MCs become one of the main producers of TGF-β, a fact that should be properly regarded when developing translational biomarkers of kidney fibrosis. Taking into account MC colocalization with α-SMA-positive cells, it can be assumed that there is a cascade increase in the production of collagens and ECM proteins by myofibroblasts.

Despite the abundance of CPA3 in MCs, there is currently a lack of knowledge regarding its biological effects compared with other specific MC proteases, such as tryptase and chymase.^43,59,60 CPA3 has a crucial role in the biogenesis of the fibrous components of the ECM and ECM remodeling. On the one hand, the CPA3–chymase complex can induce increased mitotic activity of fibroblasts, along with their biosynthetic potential. This complex or protease participates solely in procollagen molecule modification, inducing the formation of collagen fibrils.^46,58

Post-translational histone modifications result in the activation or suppression of target genes by modulating the binding of transcription factors to their respective nuclear promoter elements. ⁶¹ Several studies have reported that histone modifications are critical in the development and progression of renal fibrosis. ⁶²

Histone modifications stimulate the epithelial–mesenchymal transition, which promotes the transformation of the epithelium of the nephron tubules into stromal cells. This mechanism causes dysfunction of all parts of the nephron during chronic renal failure.^63,64 Activated myofibroblasts formed during epithelial–mesenchymal transition arising from the epithelium of the nephron can play a key role in the development of kidney fibrosis, leading to excessive ECM formation.^13,65 The increased expression of α-SMA, demonstrated in our study, supports the increased number of myofibroblasts at the sites with fibrotic changes and the high intensity of the epithelial–mesenchymal transition.

These results indirectly support the involvement of specific MC proteases in the epithelial–mesenchymal process. The nuclear histone state is epigenetically regulated by tryptase.^66–68 Enzymatic tryptase activity in the nucleus is stabilized by DNA molecules, thus potentiating long-term regulation of the state of histones.^66,68,69 Regulation of core histone epigenetic modifications is a specific function of human MC tryptase. ⁶⁸ The close colocalization of CPA3 with tryptase, which we have identified in our studies (unpublished findings), creates prerequisites for CPA3 participation in particular epigenetic effects. ³⁶

The results of our research demonstrated the formation of certain areas of cells containing tryptase, which supports potential mechanisms for protease participation in epithelial–mesenchymal transformation. Concurrently, the undulating-like process affects a limited number of cells in the first stage; these cells are likely to become active cellular foci for increased synthesis of ECM components.

Strong evidence for CPA3 participation in the regulation of fibrous niches suggests unknown mechanisms. Based on the known biological effects of CPA3 in cooperation with chymases, one can assume the importance of this protease in the progression of fibrotic changes. Thus, the results of this analysis emphasize the role of MCs in fibrotic changes in the kidneys. The potential to develop a pro-inflammatory profile of a specific tissue microenvironment and provide epigenetic modification of histones using tryptase invents a new mechanism of kidney tubulointerstitial fibrosis progression, which has important diagnostic criteria and allows the determination of future pharmacological targets for the prevention and treatment of the disease. In essence, the formation of local areas of the stroma with high tryptase content should be limited by additional structural elements that regulate the diffusion rate and their final concentration in a strictly limited tissue volume. This regulation can be performed by telocytes, which have a significant function in the organization and regulation of kidney stroma^70,71; however, this issue requires further detailed analysis.

Our data indicate a sharp increase in the number of MCs containing specific proteases in pathological lesions of the kidneys. The greatest severity was observed in chymase-positive MCs, the content of which in areas with fibrotic changes increased more than 10 times (relative values, Table 4) compared with the number of MCs in other areas of the cortex and medulla. To a lesser extent, the number of tryptase-positive and CPA3-positive cells increased in the fibrotic-changed kidney loci; their relative values increased by 5 and 4.3 times, respectively, compared with unchanged kidney loci. Thus, as for the quantification of the fibrous component of the ECM, the level of chymase is the most significant cause of fibrotic changes in the kidneys. However, considering the onset of pathogenic transformations during kidney fibrosis and the fundamental mechanisms of the initiated development of subsequent irreversible processes of stromal reorganization, we considered tryptase to be the most essential among other specific MC proteases. Tryptase is the key tool for creating a pro-inflammatory background and, as a result, induces further multistage changes, resulting in excessive formation of connective tissue. Therefore, we propose tryptase as the most promising pharmacological target, and its blockade can help inhibit the creation of conditions for profibrotic niche formation. The results of this experiment cannot provide comprehensive answers to determine the most significant MC protease involved in tubulointerstitial fibrosis formation. Moreover, the relationship among the effects of chymase, CPA3, and tryptase can potentiate their isolated effects on inflammation and fibrotic changes. However, further experiments with the widespread use of multiplex immunohistochemical staining will help uncover the patterns of interaction of diverse MC phenotypes with other immune and stromal cells in the kidney interstitium. This will bring new issues to the forefront of personalized medicine and the development of targeted solutions for both the prevention and treatment of fibrotic changes in the kidneys.

The results showed that along with pronounced fibrotic changes in the pathogenic kidney, a multilocular process was present, and new profibrogenic niches were formed with active MC participation through tryptase, chymase, and CPA3 secretion. MCs can develop inducing effects of the local tissue microenvironment on fibroblast activation, inflammation, epithelial–mesenchymal transition activity, and vascular homeostasis in profibrogenic niches. Identified direct effects of collagen fibrillogenesis initiation by MCs are enhanced by mediated profibrotic effects with the help of specific proteases and TGF-β. Identification of the molecular biological parameters of MCs, primarily those associated with inflammatory loci, is an innovative approach to identifying the fundamental mechanisms of nephrofibrosis. The biological relevance of MCs in the formation of fibrogenic niches can be used not only to make timely and correct diagnoses and administer effective therapy but also to prevent tubulointerstitial fibrosis of various etiologies.