Protocol for induction of human kidney cell fibrosis

Summary

Here, we present a protocol for inducing fibrosis in human kidney-2 (HK2) cells followed by quantitative real-time PCR analysis of fibrosis-related genes. We describe steps for growing and expanding cells, inducing HK2 fibrosis, and collecting cells for downstream applications. Given the limited cell quantity in culture flasks and the challenges of cell collection, we utilized 10-cm Petri dishes for cell harvesting, with each experimental group comprising five replicate samples.

For complete details on the use and execution of this protocol, please refer to Zhang et al.¹

Graphical abstract

Highlights

• •Steps for inducing HK2 cell fibrosis
• •Harvest cells for further analysis
• •Extraction of high-quality RNA from HK2 cells and reverse transcription into cDNA

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for inducing fibrosis in human kidney-2 (HK2) cells followed by quantitative reverse-transcription PCR analysis of fibrosis-related genes. We describe steps for growing and expanding cells, inducing HK2 fibrosis, and collecting cells for downstream applications. Given the limited cell quantity in culture flasks and the challenges of cell collection, we utilized 10-cm Petri dishes for cell harvesting, with each experimental group comprising five replicate samples.

Before you begin

The following is a detailed description of how to perform a metabolomics study utilizing HK2 cells. Owing to the restricted quantity of cells within culture flasks and the challenge of collecting cells, in order to ensure that there were enough cells in the test samples, 10-cm Petri dishes were used to collect cells, and each group included five replicate samples. After collecting the cells, rapid freezing of the cells in liquid nitrogen for further analysis. The protocol below describes the specific steps for using HK2 cells. However, we have also used this protocol in NRK 52E cells and mRTEC cells.

Institutional permissions

The Ethics Committee of the Affiliated Huai’an Hospital of Xuzhou Medical University ensured that the research followed the principles outlined in the Declaration of Helsinki (1991) (HEYLL201883).

Resuscitate culture cell lines

Timing: approximately 3 days

• 1.Preheat the water bath to 37°C.
• 2.Replenish DMEM/F12 with 10% fetal bovine serum (FBS) in a sterile 50-mL centrifuge tube.
• 3.Rapidly transfer the HK2 cells in the cryopreservation tube from the liquid nitrogen to the water bath for 2 min.
• 4.Sterilize the tube by wiping it thoroughly with 75% alcohol.
• 5.Transfer the HK2 cells from the cryopreservation tube to a sterile 15-mL centrifuge tube.
• 6.Add 2 mL DMEM/F12 (10% FBS).

CRITICAL: The DMEM/F12 (10% FBS) need to be preheated at 37°C before use.

• 7.Centrifuge the tube at room temperature at 1000 × g for 5 min, retaining the precipitate.
• 8.Resuspend the precipitate in DMEM/F12 (10% FBS) and transfer it to the culture flask (25 cm²).
• 9.Add an additional 3 mL of DMEM/F12 (10% FBS) to the flask.
• 10.Incubate the culture flask for 24 h to 48 h.
• 11.Replace the medium in the culture flask.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Fetal bovine serum	Gibco Thermo Fisher Scientific	10099141C
DMEM/F12	Gibco Thermo Fisher Scientific	C11330500BT
0.25% Trypsin-EDTA (1X)	Gibco Thermo Fisher Scientific	25200056
Phosphate-buffered saline	Gibco Thermo Fisher Scientific	C10010500BT
rhTGF-β1 (10 μg)	R&D Systems	240-B-010
TRIzol	Takara	108-95-2
Bovine serum albumin	Beyotime	ST2249-5g
4 mM HCl	ECOTOP	ED-8460-500mL
Critical commercial assays
HiScript II Q RT SuperMix	Vazyme	R222-01
SYBR Green PCR mix	Vazyme	Q131-02
Experimental models: Cell lines
Human renal tubular cell line HK2	ATCC	CRL-2190
Oligonucleotides
Human XOR detection primers: forward primer 5′-GGACAGTTGTGGCTCTTGAGGT-3′ Reverse primer 5′-GGAAGGTTGGTTTTGCACAGCC-3′	PrimerBank	Figure 5
Human XDH detection primers: forward primer 5′-ACCCCGTGTTCATGGCCAGTG-3′ Reverse primer 5′-TCCGGGAGGCCTGCTTGAATG-3′	PrimerBank	Figure 5
Human ROMO1 detection primers: forward primer 5′-AAGCTGCTTCGACCGTGTC-3′ Reverse primer 5′-CCCGCATTCCGATCCTGAG-3′	PrimerBank	Figure 5
Human NOA1 detection primers: forward primer 5′-CCTTCCAGCACTCATCGAGTC-3′ Reverse primer 5′-TCCAGGATGTACTCCGGGAAC-3′	PrimerBank	Figure 5
Human COX-2 detection primers: forward primer 5′-TAAGTGCGATTGTACCCGGAC-3′ Reverse primer 5′-TTTGTAGCCATAGTCAGCATTGT-3′	PrimerBank	Figure 5
Human NF-κB detection primers: forward primer 5′-AACAGAGAGGATTTCGTTTCCG-3′ Reverse primer 5′-TTTGACCTGAGGGTAAGACTTCT-3′	PrimerBank	Figure 5
Human GAPDH detection primers: forward primer 5′-GGAGCGAGATCCCTCCAAAAT-3′ Reverse primer 5′-GGCTGTTGTCATACTTCTCATGG-3′	PrimerBank	Figure 5
Software and algorithms
GraphPad Prism 9 software	GraphPad Software	https://www.graphpad.com
Roche LightCycler 480II	LightCycler480_GeneScanning Software

Step-by-step method details

Cell culture

Timing: approximately 1 week

In this step, we elucidate the procedure for passaging and culturing HK2 cells before inducing fibrosis.

• 1.
  Culture eight culture flasks of HK2 cells.

  • a.
    After aspirating the medium, cells are gently washed twice with PBS.
  • b.
    Add two milliliters of 0.25% trypsin-EDTA in the flasks.
  • c.
    Incubate culture flasks for 2 min in incubator (37°C with 5% CO2) to make cells loosely adherent.
  • d.
    Aspirate the trypsin from cell culture flasks.
  • e.
    Add 1.5 mL DMEM/F12 medium (including 10% FBS) into the culture flasks.
  • f.
    Utilize a pipette tip to rinse the sample up and down several times to disperse the cells.
  • g.
    Collect all the cell suspension in a 15-mL centrifuge tube.
  • h.
    Gently add one milliliter of the HK2 cell suspension to ten 10-cm Petri dishes, and discard the remaining.
  • i.
    Introduce another 7 mL of DMEM/F12 medium (including 10% FBS), bringing the total medium volume to 8 mL.

    CRITICAL: Rock the petri dishes gently in all directions to ensure uniform cells distribution.

  • j.
    Incubate dishes at 37°C for 24 h–48 h, until they reach approximately 70% confluence.

Induction of HK2 cell fibrosis by TGF-β1 and harvest cells for further analysis

Timing: approximately 1 week

In this step, we provide details of how to induce fibrosis successfully.¹

• 2.
  Induce five randomly selected Petri dishes HK2 cells fibrosis for 24 h.

  • a.
    Dissolve 10 μg TGF-β1 lyophilized powder in sterile 4 mM HCl containing 0.1% bovine serum albumin and stock in −20°C (Table 1).
  • b.
    Add 1 μL TGF-β1 stock solution into 1 mL DMEM/F12 medium as working medium, resulting in a working concentration of 10 ng/mL (Table 2).
  • c.
    Add 1 μL 4 mM HCl containing 0.1% bovine serum albumin into 1 mL DMEM/F12 medium as control medium (Table 3).
  • d.
    Choose five Petri dishes randomly.
  • e.
    Aspirate supernatants and refill 8 mL working medium (10 ng/mL TGF-β1).
  • f.
    Aspirate supernatants and refill 8 mL Control medium in another five Petri dishes.
• 3.
  Collect all cells for further analysis.

  • a.
    Aspirate the supernatant from all Petri dishes and gently washes twice with PBS.
  • b.
    Add one milliliter of PBS to each dish, and scraped off the cells carefully by cell scraper (China, LABSELECT, 1601).

    CRITICAL: Make efforts to ensure minimal cell adherence to the dishes.

  • c.
    Collect cell suspensions in separate microtubes.
  • d.
    Centrifuge tubes at 1000 × g for 5 min at 4°C.
  • e.
    Carefully aspirate the supernatant from the microtubes.
  • f.
    Snap-freeze the samples in liquid nitrogen for downstream applications such as metabolomics analysis.

Table 1.

10 μg/mL TGF-β1 stock solution

Steps	Reagent	Amount
Prepare the solvent	4 mM HCl	1000 μL
	bovine serum albumin	1 mg
Stock solution	TGF-β1	10 μg

Stock solution.

Table 2.

Working medium

Reagent	Amount
stock solution	1 μL
DMEM/F12 medium	1 mL

Table 3.

Control medium

Reagent	Amount
4 mM HCl	1 μL
bovine serum albumin	1 μg
DMEM/F12 medium	1 mL

Real-time fluorescence quantitative PCR

Timing: approximately 2 weeks

In this step, we elucidate the procedure for passaging and culturing HK2 cells before RT-qPCR. Moreover, we provide Synthesis of cDNA in detail.

Note: qRT-PCR primers sequences are shown in the key resources table, and once ordered dissolved in nuclease-free water to a final concentration of 10 μM for future analyses.

• 4.
  Selection of primers.

  • a.
    Select primers from PrimerBank, which are detailed in the key resources table.
  • b.
    Synthesize Primers by Tsingke Biotechnology Co.3 (Beijing, China).
• 5.
  Preparations before HK2 cell induction.

  • a.
    Obtain one culture flask of HK2 cells.
  • b.
    Aspirate the medium.
  • c.
    Gently wash the cells twice with PBS.
  • d.
    Add two milliliters of 0.25% trypsin-EDTA.
  • e.
    Incubate culture flasks for 2 min in incubator (37°C with 5% CO2) to make cells loosely adherent.
  • f.
    Aspirate trypsin.
  • g.
    Add 1.5 mL of DMEM/F12 medium (including 10% FBS) into the culture flasks.
  • h.
    Utilize a pipette tip to rinse sample up and down several times to disperse the cells.
  • i.
    Collect the cell suspension in a 2-mL EP tube.
  • j.
    Slowly pipette 100 μL cell suspension into each well of a 12-well plate.
  • k.
    Add another 1 mL DMEM/F12 medium (including 10% FBS).

    CRITICAL: Slide the plate gently in both forward and reverse directions to achieve uniform cell dispersions.

  • l.
    Place the plate in the incubator and culture at 37°C with 5% CO2 for 24 h.
• 6.
  Induce HK2 cell fibrosis for 24 h.

  • a.
    Carefully aspirate 6 well’s supernatant and replace it with 1 mL Working medium (10 ng/mL TGF-β1).
  • b.
    Aspirate another 6 well’s supernatant and replace it with 1 mL Control medium.
  • c.
    Place the plate in the incubator and culture at 37°C with 5% CO2 for 24 h.
• 7.
  Collect all cells.

  • a.
    Discard the medium and wash the cells with 2 mL of PBS twice.
• 8.
  RNA isolation.

  Note: The aforementioned procedures must be performed within a biosafety cabinet, and the samples must always be kept on ice.

  • a.
    Add 1 mL of Trizol to each cell sample.
  • b.
    Use a pipette tip to pipette the sample up and down several times to disperse and lyse the cells.
  • c.
    Transfer the sample to a 1.5-mL EP tube.
  • d.
    Add 200 μL chloroform to each EP tube.
  • e.
    Invert the sample 10 times to ensure a complete mixing.
  • f.
    Centrifugate the sample at 12000 × g for 20 min at 4°C.
  • g.
    Carefully pipette 400 μL of the top transparent liquid (aqueous phase) and transfer it to the EP tube.

    CRITICAL: Take care not to aspirate the middle interphase and lower organic phase.

  • h.
    Add 400 μL pre-chilled isopropanol and store on ice.
  • i.
    Mix completely by repeated inversion for 15 s, place it on ice, and incubate for 10 min.
  • j.
    Centrifugate at 12000 × g for 20 min at 4°C.
  • k.
    Discard the supernatant and be cautious that not discard the precipitate while disposing of the supernatant.
  • l.
    Prepared the 75% ethanol and pre-cooled on ice.
  • m.
    Add 750 μL of 75% ethanol to the precipitate. And invert the mixture several times to thoroughly wash the precipitate.
  • n.
    Centrifugate at 7500 × g for 15 min at 4°C, then discard the supernatant.
  • o.
    Incorporate 750 μL of 75% ethanol, centrifugate at 7500 × g for 15 min at 4°C, and then dispose the supernatant.
  • p.
    Carefully and thoroughly aspirated off lingering ethanol by the micropipette
  • q.
    Allow the precipitate to air-dry for 10 min at 4°C.
  • r.
    Dissolve the precipitate with RNase-free water.
  • s.
    Test the purity of the extracted RNA by UV-Vis Spectrophotometer (Thermo Fisher Scientific, America, NanoDrop1000).

    Note: A260/A280 can characterize the purity of the RNA sample, and the ratio of A260/A280 is greater than 2.0. A ratio of less than 2.0 indicates the presence of a protein or phenolic influence. A260/A230 is commonly used to assess the presence of contaminants in RNA, the ratio of A260/A230 less than 1.8 indicates the presence of pollutants absorbed wavelength at 230 nm. And reverse-transcript the RNA into cDNA immediately. Then the remaining RNA can be stored at −80°C for several months.

• 9.
  Synthesis of cDNA.

  • a.
    Synthesize cDNA using the HiScript II Q RT SuperMix for qPCR kit, following manufacturer’s instructions (https://www.vazyme.com/product/98.html) (Table 4).
  • b.
    After carefully combining the reactants, perform the reverse-transcription procedure. Initiate the cDNA synthesis program in accordance with Table 5.
• 10.
  Examine the HK2 cell fibrosis and XDH related gene expression using qRT-PCR.

  Note: Before preparation of the qRT-PCR mixture, dilute the synthesized cDNA to a concentration of 1/5 for use (add 80 μL RNase-free H₂O).

  • a.
    Perform qPCR experiments in accordance with the manufacturer’s instructions (https://www.vazyme.com/product/68.html), by use Vazyme’s “AceQ qPCR SYBR Green Master Mix (Low ROX Premixed)” kit.
  • b.
    Prepare the total mix (Table 6) as shown in the tables below.
  • c.
    In a 96-well white plate, pipette 10 μL of the qRT-PCR mixture into each well.
  • d.
    Perform instantaneous centrifugation by inserting the 96-well plate in a centrifuge fitted with the appropriate rotor.
  • e.
    Prepare the real-time PCR detection system (Roche LightCycler 480II) by inserting the 96-well plate.
  • f.
    Apply the PCR program outlined in Table 7.

Table 4.

cDNA synthesis reaction

Reagent	Amount
Total RNA	1,000 ng
5×HiScript II qRT SuperMix	4 μL
RNase-free H2O	Up to 20 μL
Total	20 μL

Table 5.

cDNA synthesis program

Temperature	Time	Cycles
50°C	15 min	1
85°C	5 s	1
4°C	Forever	1

Table 6.

PCR reaction master mix

Reagent	Amount
2 × ChamQ Universal SYBR qPCR Master Mix	5 μL
Primer Forward	0.2 μL
Primer Reverse	0.2 μL
dd H2O	0.6 μL
cDNA	4 μL
Total	10 μL

Table 7.

qPCR program

Steps	Temperature	Time	Cycles
Holding Stage	Increase to 50°C	2 min	1
Holding Stage	Increase to 95°C	10 min	2
Cycling Stage	95°C	15 s	2–40 cycles
	60°C	1 min
Melt Curve Stage	Increase to 95°C	15 s	——
	60°C	1 min	——
	95°C	30 s	——
	60°C	15 s	——
Hold	4°C	Forever

Expected outcomes

The present protocol facilitates the initiation of HK2 cell fibrosis, resulting in the manifestation of unique cellular morphology and the increased expression of certain marker genes, such as fibronectin. In this experiment, we aim to further validate the expression levels of XDH, KOX-1, ROMO1, NOA1, NF-kB, and COX-2. Treatment with TGF-β led to considerable upregulation of XDH, KOX-1, and ROMO1 when compared with the control groups (Figure 1) but had no discernible effect on the expression levels of NOA1, NF-kB, and COX-2. Furthermore, this approach may be used to study the effects of TGF-β on the fibrosis of mouse and rat renal tubular epithelial cells and renal tubular duct epithelial cells.

Figure 1.

Comparison of gene expression levels of six marker genes between the controls and TGFβ samples via qRT-PCR

qRT-PCR was used to determine the mRNA levels of Fibronectin, XDH, KOX-1, ROMO1, NOA1, COX-2, and NF-κB in TGF-β-treated HK2 cells and control cells. Variations were determined by contrasting gene expression levels to those in HK2 cells and utilizing the 2^−ΔΔCt technique. Student’s t test was used to analyze all data. Asterisks and ns indicate differences between the categories that are statistically significant and insignificant, respectively. n = 6, ∗∗P < 0.01, ∗∗∗∗P < 0.0001. Figure reprinted and adapted with permission from Zhang et al., 2023¹

Limitations

The major limitation is that only one time point and one dosage of TGF-β1 has been utilized.

It was difficult to ensure uniform cell distribution if the Petri dishes were moved into a 37°C incubator immediately after being gently rocked. The Petri dishes were therefore outside the incubator for 10 min to allow the cells to sink to the bottom.

The DMEM/F12 medium contained no FBS when inducing HK2 cell fibrosis for 24 h, which may have resulted in modest changes to cellular states.

Troubleshooting

Problem 1

RNA degrades during the process of isolation (related to step 8).

Potential solution

RNA is a molecule that exhibits inherent instability and is susceptible to degradation as a result of its presence of RNase, which can be encountered in multiple environmental contexts, including on skin, hair, saliva, and microorganisms. If RNA degradation is observed during the isolation process, the following recommendations can be implemented to enhance the quality of the RNA.

• •Cells must be lysed as quickly as possible after collection by adding Trizol and pipette vigorously, followed by rapid cDNA synthesis.
• •An RNase-free area must be allocated in the lab specifically for RNA extraction and associated investigations.
• •Use RNase-free water to prepare 75% ethanol.
• •After touching skin, door handles, and common object surfaces, change gloves.

Problem 2

RNA samples were contaminated during the process of isolation (related to step 8 to step 9).

Potential solution

RNA samples are easily contaminated by the surrounding environment like respiratory droplets, which may lead to inaccurate experimental results. So, the following steps can be implemented to enhance the quality of RNA.

• •All the pipette tips, EP tubes that may be used in this experiment should be sterilized by autoclaving in advance.
• •All steps were performed in a dedicated biosafety cabinet, and put on a new mask and gloves during the process.

Problem 3

The gene could not be detected by using the primers selected from PrimerBank. (related to step 4).

Potential solution

The gene may most be detectable by using the primers in PrimerBank, because the primers included in PrimerBank have not been verified. In this situation, new primers can be designed from NCBI Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).

Problem 4

The poor effect of TGF-β1 inducing HK2 fibrosis (related to step 6).

Potential solution

TGF-β1 is the primary factor that drives fibrosis in almost forms of CKD, so TGF-β1 is commonly used to induce cell fibrosis. If the degree of cells fibrosis does not meet the needs of the experiment, the following recommendations can be implemented to enhance the effect of TGF-β1.

• •Carefully aspirate the supernatant from 12-well plate and replace it with 1 mL DMEM/F12 medium without FBS and culture the 12-well plate for 12 h before inducing cell fibrosis.
• •In order to determine the best induction protocol, different incubation times, concentrations and serum contents should be explored again.

Problem 5

The cells are non-uniform within the 12-well plate (related to step 5).

Potential solution

Non-uniform cells may generate a failed experiment results, so if cells are non-uniform within the 12-well plate or petri dishes, the following recommendations can be implemented to enhance the uniformity of cells.

• •Add enough cells culture medium in 12-well plate (more than 0.8 mL) and petri dishes (more than 6 mL).
• •Gently shake the 12-well plate with a small amplitude(“十” shaking) and gently shake the petri dishes with a large amplitude("8″ shaking).

Resource availability

Lead contact

Further information, requests, and inquiries should be directed to and will be fulfilled by the lead contact, Donghui Zheng (zddwjj@126.com).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Yiyuan Zhang (358861513@qq.com).

Materials availability

No novel reagents or cell lines were developed in this investigation. The full list of reagents is available in Key resources table.

Data and code availability

The key resources table includes the appropriate accession numbers. No original code was used in this analysis.