The Role of SETD2 and VHL in Promoting Renal Fibrosis

Yangtian Yan

Dulwich Zhuhai International High School, China

Keywords: Renal Fibrosis, SETD2, VHL, Inflammation-Related Genes, Tumour Suppressor Genes, Knockout.

Abstract: An excess accumulation of extracellular matrix is involved in renal fibrosis, which usually leads to a loss of

function when scar tissues replace normal tissues. This process is stimulated by multiple different

pathogenic factors that trigger the cascades of reparation converging in molecular signals responsible for

initiating and driving fibrosis. SETD2 and VHL are both tumour suppressor genes, the former acts as an

epigenetic modifier that is responsible for trimethylation of H3K36, and the latter works by controlling

extracellular matrix formation, apoptosis response, and the epithelial-to-mesenchymal transition. Whether

SETD2 and VHL play roles in promoting the occurrence of renal fibrosis is still unknown. Therefore, by

creating the mouse model with the specific knockout of VHL and SETD2 genes, our experiment analyses

their effects on the kidney tissue. The result indicates that there is an up-regulation for the expression level

of inflammation-related genes in the mouse kidney model with both VHL and SETD2 knocked out other

than with VHL-KO only, in combination with the HE, Masson, and Sirius Red staining images we

performed, we can get a conclusion that these two genes, SETD2 and VHL, can trigger the occurrence of

renal fibrosis. These results can provide a solid theoretical basis for the molecular mechanism of action and

prospects of relevant drug screening and clinical targeted therapy.

1 INTRODUCTION

Renal fibrosis is the final manifestation of chronic

kidney disease (CKD), which can be characterized

by tubulointerstitial fibrosis and glomerulosclerosis

(Cho, 2010). Even though a range of diseases related

to kidney such as glomerulonephritis, diabetes

mellitus, atherosclerosis and even polycystic kidney

disease, can be the main factors causing CKD, renal

fibrosis is always the common final result of CKD

(Cho, 2010). It appears to be a harmful process

leading to renal function deterioration inevitably,

independently of the previous renal diseases which

cause the original symptoms. Chronic Kidney

Disease and Renal Fibrosis affect 10% of the world's

population, and a significant proportion of people

progress to end-stage renal failure, requiring lifelong

dialysis and kidney transplants, placing a huge

financial burden on patients, families, and societies.

An excessive accumulation and deposition of

extracellular matrix (ECM) components are the main

characteristics of renal fibrosis that occur in almost

every type of CKD (Liu, 2006). It is worth knowing

that among many different fibrogenic factors that

regulate the process of renal fibrosis, transforming

growth factor-β (TGF-β) is the one that plays a

central role. The epithelial to mesenchymal

transition (EMT) of tubular epithelial cells means

they are transformed into mesenchymal fibroblasts

migrating to adjacent interstitial parenchyma, along

with local and circulating cells constitute the

principal mechanism of renal fibrosis (Humphreys,

2018). For the occurrence of renal fibrosis, although

many in vitro studies emphasize the significance of

one specific cellular event such as the activation of

fibroblast, it should be kept in mind that no single

type of isolated cell has the ability of initiating and

sustaining an entire scale of renal fibrosis (Liu,

2006). Many experimental studies have been carried

out to explain the specific pathway of renal fibrosis

and some significant progress has already been made

in the understanding of the cellular and molecular

mechanisms of renal fibrosis.

VHL is an important tumour suppressor gene, and

its mutation can promote the occurrence of renal

cancer. A person with VHL has nearly a 100%

chance of developing one or more VHL tumours in

their lifetime (Von Hippel-Lindau, 2022). Early

inactivation of VHL is commonly seen in clear-cell

renal cell carcinoma (ccRCC), and insights gained

386

Yan, Y.

The Role of SETD2 and VHL in Promoting Renal Fibrosis.

DOI: 10.5220/0012021400003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 386-391

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

from the functional analysis of pVHL have provided

the foundation for the routine treatment of advanced-

stage ccRCC with novel targeted therapies (Gossage,

2015). It is shown that VHL can influence the

content of the extracellular matrix, a proper

extracellular matrix cannot be organized in cells

lacking VHL. This in conjunction with enhanced

production of VEGF-A favours angiogenesis and

tumourigenesis (Patard, 2009). pVHL can also bind

with transcription factors, which will lead to a

decrease in the stability of certain mRNAs and

inhibit the transcription of genes such as VEGF-A

(Pal, 1997). The role of VHL in renal fibrosis is

undiscovered, but the variation of VHL and the

mechanism of renal fibrosis have some similarities,

such as the effect on extracellular matrix.

Set domain containing 2 (SETD2) Set domain

containing 2 (SETD2) is a histone modifier that is

generally known as the single human gene

responsible for trimethylation of lysine 36 of histone

H3 (H3K36). H3K36me3 readers recruit protein

complexes to carry out specific processes, such as

transcription elongation, RNA processing, and DNA

repair, to determine the impact of this histone

modification. Histone H3K36 trimethylation is a

highly conserved chromatin mark related to

transcriptional elongation, and it accumulates mainly

across the body of genes that are actively

transcribed. Cells tend to become significantly

vulnerable and sensitive to DNA-damaging agents

after loss of the H3K36me3 mark through SETD2

mutation or loss (Li, 2016). SETD2 mutation can

promote the occurrence of ccRCC, its mutation rate

in clinical patients is as high as 12%, ranking third,

which can lead to structural abnormalities in renal

tubules. A series of researches have revealed that

SETD2 is mutated or its function is lost in a range of

solid cancers (Hu, 2020), lung cancer,

gastrointestinal cancer, renal cancer, pancreatic

cancer, osteosarcoma, and so on. Mutation, or

functional loss, of the SETD2 gene produces

dysfunction in corresponding tissue proteins so that

a series of adverse functions will be leaded

(Molenaar, 2022).

Now, little is known about the roles of SETD2 or

VHL in renal fibrosis, so by analyzing the mouse

renal tubular tissue with the specific knockout of

VHL and SETD2 genes, the specific possible

molecular mechanism of action can be elucidated,

which provides a solid theoretical basis and

development prospect for corresponding drug

screening and clinical targeted therapy.

2 METHODS

2.1 Mice Preparation

Setd2fl/fl mice were generated as described [ref].

The Ksp-Cre mice (B6.CgTg (Cdh16-cre) 91Igr/J)

and VHLfl/fl mice were purchased from The Jackson

Laboratory.

Setd2fl/fl mice were mated with Ksp-Cre mice to

generate Ksp-Cre; Setd2flox/flox (Setd2–KO) mice

in C57BL/6 background. SETD2–KO mice were

mated with VHLfl/fl mice to generate Ksp-Cre;

VHLfl/fl&Setd2fl/fl mice (Setd2-VHL-KO) housing

under the same condition.

2.2 DNA Extraction from Mouse Tail

for Genotyping

Cutting the tip of the mouse tail and place it into an

Eppendorf tube and then add 100ul to 150ul reagent

A depending on the size of your tail sample. Boil the

sample at 95 to 98°C for 1 hour or so till the tail is

“melted”. Cool down to room temperature. Adding

equal volume of reagent B and then mixing well.

Centrifuging the Eppendorf tube at full speed for 5

minutes. Finally, take 1ul to 2ul supernatant for PCR.

2.3 Buffer Recipe

Reagent A: 25Mm NaOH/0.2Mm EDTA

Note: 5x stock solution which can be kept at

room temp

Reagen B: 40Mm Tris HCl (pH 5.5)

2.4 RNA Isolation and Quantitative

Qrt-PCR

Total RNA was isolated from fresh kidney samples.

cDNA (complementary DNA) was made using the

Prime Script RT reagent kit and subjected to

quantitative RT-PCR. Calculating the relative

abundance of mRNA by normalization to actin-beta

or GAPDH mRNA.

The primer sequences are as follows: Il1rap-

Forward: 5’-TGCCTGGGGGAATTGTCAC-3’,

Il1rap-Reverse: 5’-

CTTAGCCCGCTTCAGCTCTTT-3’; Il18r1-

Forward: 5’-TCACCGATCACAAATTCATGTGG-

3’, Il18r1-Reverse: 5’-

TGGTGGCTGTTTCATTCCTGT-3’; Il7r-Forward:

5’-GCGGACGATCACTCCTTCTG-3’, Il7r-

Reverse: 5’-AGCCCCACATATTTGAAATTCCA-

3’; Il1r1-Forward: 5’-

GTGCTACTGGGGCTCATTTGT-3’, Il1r1-

The Role of SETD2 and VHL in Promoting Renal Fibrosis

387

Reverse: 5’-GTGCTACTGGGGCTCATTTGT-3’

(Fig.2).

2.5 Histology and IHC Staining

Fixing mouse kidneys in 10% formaldehyde, then

embedding them in paraffin, and staining for

Masson’s trichrome (Sigma-Aldrich) and Picrosirius

red (Abcam) separately after cutting them in 5μm

thickness. Measurement of the tissue fibrotic area

could be identified with contrastive images.

Fixing tissues in 10% buffered formalin and then

sectioning them for hematoxylin and eosin staining.

RNA sequencing and analyses

For IHC staining, treating paraffin-embedded

tissues with 0.01 mol/L sodium citrate (pH 6.0) to

deparaffinized, rehydrate, and subject them to a

heat-induced epitope retrieval step. 0.3% (v/v)

hydrogen peroxide in distilled water was used to

block the activity of endogenous peroxidase. Then

incubating the sections with 0.3% Triton X-100 in

PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 10

mmol/L Na2HPO4, 2 mmol/L KH2PO4, pH 7.4) for

15 minutes, followed by 1 hour’s 10% goat serum in

PBS.

3 RESULTS

To ensure that the mouse gene model is needed for

the experiment, we carried out PCR tests to identify

the genotype. It can be seen that the fourth column is

the model of SETD2 and VHL double knockout. At

this time, the band size of SETD2 is 130bp and that

of VHL is 500bp. The third column is the model of

only knocking out the VHL gene; the band sizes of

SETD2 and VHL are 75bp and 500bp respectively.

The second column represents a model with only

SETD2 knockout. (Fig.1).

Figure 1: Results of mouse DNA PCR test.

QueryID forward reverse

Il1rap TGCCTGGGGGAATTGTC

CTTAGCCCGCTTCAGCT

CTTT

Il18r1 TCACCGATCACAAATTC

ATGTGG

TGGTGGCTGTTTCATTC

CTGT

Il7r GCGGACGATCACTCCTT

CTG

AGCCCCACATATTTGAA

ATTCCA

Il1r1 GTGCTACTGGGGCTCAT

TTGT

GTGCTACTGGGGCTCAT

TTGT

Figure 2: Inflammation related genes are up-regulated in SETD2 knockout mice.

Figure 3: Results of qPCR test of inflammation related genes.

Many studies have shown that inflammation

shares some of the same biological mechanisms as a

range of other conditions, such as fibrosis

(Inflammation, 2022). Increased expression levels of

inflammation-related genes suggest the presence of

diseases with the same mechanism. The gray column

of each graph in Fig. 3 represents the expression

level of inflammation-related genes in the VHL-KO

model, and the red column represents the expression

level in the VHL-KO and SETD2-KO models. It can

be seen that Il1r1 and Il7r have significant rises from

the gray column to the red column, and the before

and after comparisons of Il18r1 and Il1rap are more

obvious. The results of the qPCR test for four

inflammation-related genes indicate that the

Knockout of SETD2 in VHL knockout mouse

ICBB 2022 - International Conference on Biotechnology and Biomedicine

388

kidneys results in renal fibrosis in mice because we

can see that the expression level of these

inflammation-related genes all experience an up-

regulation in the mouse kidney model with both VHL

and SETD2 knocked out other than VHL-KO only

(Fig.3).

Figure 4: Knockout of SETD2 in VHL knockout mouse kidneys results in renal fibrosis in mouse(scale bars, 80um).

Knockout of SETD2 in VHL knockout mouse

kidneys results in renal fibrosis in mice (scale bars,

80um)

By staining the renal tubular tissue of the mouse

kidney model with VHL knockout only and the

mouse kidney model with both SETD2 and VHL

knockout at the same time, we can see that there is

more cellular fibrosis in the double knockout mouse

model (Fig.4). In HE staining, the blue stained part

represents the nucleus. In the double knockout model

experiment, we can find a significant increase in the

number of nuclei in the tissue. In Masson's staining,

blue represents fibers, and Sirius Red staining is on a

yellow background, in which the red part is the

fibrotic part. Both of these two staining images show

a vivid upward trend in the occurrence of fibrosis

from VHL-KO only to SETD2-KO and VHL-KO.

Therefore, we can conclude that simultaneous

mutations of SETD2 and VHL can induce the

occurrence of renal fibrosis.

4 CONCLUSION

By creating the mouse model with the specific

knockout of VHL and SETD2 genes, we analyze its

effects on the kidney tissue. From the up-regulation

for the expression level of inflammation-related

genes (including Il1rap, Il18r1, Il7r, and Il1r1) in the

mouse kidney model with both VHL and SETD2

knocked out other than with VHL-KO only,

combined with the HE, Masson and Sirius Red

staining images we performed, we can get a

conclusion that these two genes of SETD2 and VHL

can also trigger the occurrence of renal fibrosis.

These inflammation-related genes also play a role

in the study of fibrosis. Pro-inflammatory proteins

can be synthesized by the induction of Interleukin 1

(IL-1) during tissue damage or infection, IL-1 does

this by forming a complex with an interleukin 1

receptor and an accessory protein at the cell

membrane (Rouillard, 2016). This gene is

responsible for encoding the interleukin-1 receptor

accessory protein (Il1rap). Il1rap can recognize IL-1

and it is the co-receptor for signaling pathways.

Interleukin 18 receptor 1 (Il18r1) is a protein-coding

gene that encodes a cytokine receptor that belongs to

the interleukin-1 receptor group. Interleukin 18 (Il18)

is specifically bound to this receptor, which is also a

pro-inflammatory cytokine (IL18R1, 2022).

Instructions for making the interleukin-7 (Il-7)

receptor alpha chain are provided by the Interleukin-

7 receptor subunit alpha (Il7r) gene. These Il-7

receptors can be embedded in the cell membrane of

cells in the immune system (New11., 2008). They

are usually found in B cells, T cells, and also the

early blood-forming cells that give rise to them.

Interleukin-7 (Il-7) is a protein that can interact with

the Il-7 receptor at the cell surface to regulate the

activity of immune system cells (Plumb, 2017).

Signaling across the Il-7 receptor helps mature B

cells and T cells to develop properly and it also

stimulates the later proliferation of these cells (Corfe,

2012). Similarly, the interleukin-1 receptor type 1

(Il1r1) gene encodes a cytokine receptor which also

belongs to the IL-1 receptor family. It is a significant

mediator involved in many cytokine-induced

immune and inflammatory responses (IL1R1, 2022).

Some studies suggested that pro-inflammatory

stimuli can induce this receptor and it may be

involved in the function of helper T cells (IL1R1

protein overview, 2022). These series of responses

caused by the cytokine interleukin released by

inflammatory-related molecules are proof of the

persistence of inflammation, and also directly or

indirectly reflect the development of fibrosis.

The results indicate that there is an up-regulation

for the expression level of inflammation-related

The Role of SETD2 and VHL in Promoting Renal Fibrosis

389

genes in the mouse kidney model with both VHL and

SETD2 knocked out other than with VHL-KO only.

It is known that renal fibrosis is a kind of

pathophysiological change, which is a gradual

process of renal function from health to injury, then

to loss of function. Due to the stimulation of trauma,

infection, inflammation, blood circulation disorders,

immune response, and other pathogenic factors, cells

of the kidney are damaged, and a large amount of

collagen deposition and accumulation appear in the

later stage of development [2], causing the renal

parenchyma to gradually harden and form scars until

the kidney completely loses organ function [3]. The

process of hardening in the kidney is also the process

of renal fibrosis. In the three staining methods we

used, HE, Masson, and Sirius Red staining, the image

results indicate an increasing tendency of tissue

fibrosis from the mouse model with VHL-KO only to

that with both SETD2-KO and VHL-KO. Therefore,

we can get the conclusion that the knockout of

SETD2 in VHL knockout mouse kidneys results in

renal fibrosis in mice.

However, SETD2 and VHL are well known as

tumour suppressor genes and their effects of mutation

are mainly found and studied in clinical patients with

renal clear cell carcinoma (ccRCC). In this

experiment, we did not study carcinogenic effects, so

some potential limitations about the specific

knockout of these two genes may also be present

throughout the process. More experiments need to be

done and these results have to be repeatedly

deliberated to give out a more reliable conclusion.

Taken together, these results obtained can provide

a solid theoretical basis for the mechanism of

molecular action and prospects for corresponding

drug screening and clinical target therapy.

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