Development of Human Renal Tubular Epithelial Cell Primary Cultures in Monolayers and Three-Dimensional Conditions

Summary

Here, we describe the protocols for obtaining primary cultures of human renal tubular epithelial cells (HRTEC) from the kidney cortex to develop monolayers and three-dimensional cultures on an extracellular matrix (3D-HRTEC).

Abstract

Human renal tubular epithelial cell (HRTEC) primary cultures are isolated from kidneys removed from pediatric patients undergoing nephrectomies indicated by the Pediatric Nephrology Unit of the Hospital General de Pediatría Pedro de Elizalde, Buenos Aires, Argentina. Macroscopically, the normal kidney cortex is dissected from the renal medulla and cut into fragments. Cortical fragments are then digested in Hank's solution supplemented with 0.1% collagenase type 1 and filtered through a 70 µm pore size mesh to separate the renal tubules from glomeruli. Renal tubules undergo a second digestion with collagenase to isolate epithelial cells. Finally, the enzyme digestion is stopped, and the epithelial cells are grown in flasks containing RPMI 1640 with supplements under 5% CO₂at 37 °C until confluence. HRTEC cells can be grown as monolayers on 96-well plates, glass coverslips into 24-well plates, or cell-culture holders coated with collagen. Also, three-dimensional human renal tubular epithelial cell (3D-HRTEC) cultures are obtained from HRTEC cells seeded on a basement membrane (BM) matrix. Cell aggregation and formation of tubular shape structures are monitored. We have used HRTEC primary cultures for many years to study the cytotoxic action caused by Shiga toxin in the pediatric kidney, which leads to pathophysiological alterations resulting in hemolytic uremic syndrome (HUS). Both HRTEC and 3D-HRTEC cultures can be used to evaluate the effects of different toxins, synthetic drugs, biological factors, hormones, etc, specifically on the human renal proximal tubule epithelium.

Introduction

In our group, primary cultures of human renal tubular epithelial cells (HRTEC) developed in monolayers and in three-dimensional (3D-HRTEC) conditions were used to study in the pediatric kidney the cytotoxic action of Shiga toxin type 2 (Stx2) caused after gastrointestinal infection with Shiga toxin-producing Escherichia coli (STEC).

Stx1 and Stx2 (Stxs) are the main virulence factors of STEC able to cause post-diarrheal hemolytic uremic syndrome (HUS)¹. HUS is a systemic complication that affects around 5-10% of STEC-infected children and consists of hemolytic anemia, thrombocytopenia, and pediatric acute kidney injury (AKI), requiring supportive treatment². In Argentina, STEC-HUS is an endemic disease with an annual incidence of 6.52 cases per 100,000 children under 5 years of age (the highest worldwide), representing one of the leading causes of acute kidney injury³. Although the mortality is less than 4%³, approximately 50% of patients develop renal sequels, constituting the second most frequent cause of kidney transplant in our country^4,5.

Prior to internalization and exerting its cytotoxic action, Stx2 binds specifically to the glycolipid globotriaosylceramide (Gb3) receptor expressed on the cell surface of target organs⁶. The kidney is the main organ damaged by Stxs due to the presence of Stx-sensitive cells, which express high amounts of biologically active Gb3 receptors in renal epithelial and endothelial cells^7,8.

Previous works carried out in HRTEC cultured as monolayers showed that Stx2 inhibited protein synthesis, decreased cell viability and cell proliferation, and induced apoptosis and necrosis^9,10,11,12. Moreover, the exposure of 3D-HRTEC to Stx2 inhibited cell migration and the development of tubular structures with characteristics of the renal proximal tubules^12,13.

The primary cultures of HRTEC and 3D-HRTEC cultures were also used to evaluate the effect of estradiol on cell proliferation by measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into the DNA of cells in the S-phase of the cell cycle¹⁴.

To isolate the epithelial cells, we start from renal fragments obtained from pediatric nephrectomies indicated for the correction of urological conditions.

Protocol

The Ethics Committee of the Hospital General de Niños Pedro de Elizalde approved the use of human renal tissues for the development of primary cell cultures for research purposes. Written informed consent from the next of kin or guardians on behalf of the children was obtained for the use of kidney samples for research.

NOTE: The primary cultures of human renal tubular epithelial cells (HRTEC) are derived from pediatric patients undergoing nephrectomies, indicated for the correction of urological conditions or tumor resections, at Hospital General de Pediatría Pedro de Elizalde in Buenos Aires, Argentina.

1. Development of primary cultures of human renal tubular epithelial cells (HRTEC)

NOTE: The cortical renal fragments are dissected from kidneys removed from pediatric patients undergoing nephrectomies at the hospital. The primary culture of the HRTEC is performed according to the method described previously¹¹.

The protocol is described in the following steps:

1. Keep the kidney fragment refrigerated in sterile phosphate buffered saline (PBS) or Hanks' Balanced Salt solution (Hanks) until it is processed in the laboratory.
2. Dissect the renal capsule with forceps. Separate the renal cortex from the medulla and cut it into small fragments of around 1 mm.
3. To perform the digestion, place the kidney fragments in sterile Hanks solution without Ca²⁺ and Mg²⁺, supplemented with 0.1% collagenase type I.
   1. Incubate the renal fragments for 30 min at 37 °C with shaking.
4. To stop digestion, add one volume of PBS containing 10% fetal bovine serum (FBS) and wash the sample three times by centrifugation at 160 × g for 10 min each, in a centrifuge refrigerated at 4 °C. In each centrifugation, discard the supernatant and resuspend the pellet in the same solution.
5. Pass the product of digestion through a 70 µm pore mesh to filter the tubules and discard the glomeruli that remain retained in the filter.
6. Centrifuge the filtered tubules at 160 × g for 10 min at 4 °C, discard the supernatant, and resuspend the pellet in sterile Hanks solution, without Ca²⁺ and Mg²⁺, supplemented with 0.2% collagenase type I.
   1. Incubate the sample for 30 min at 37 °C with shaking.
7. To stop digestion, add one volume of PBS containing 10% fetal bovine serum (FBS).
   1. Wash the sample three times by centrifuging at 160 × g, 10 min each, in a centrifuge refrigerated at 4 °C. In each centrifugation, discard the supernatant and resuspend the pellet in the same solution.
8. After the last centrifugation, resuspend the cell pellet in 5 mL of complete culture medium (RPMI 1640 supplemented with 5% FBS, L-glutamine [2 mmol/L], penicillin [100 U/mL], streptomycin [100 µg/mL], and endothelial cell growth supplement [ECGS, 1%]).
   1. Seed the cell suspension in a 25 cm² culture flask with a vented lid.
   2. Incubate the cells at 37 °C in a humid atmosphere and 5% CO₂.
9. The next day, wash the cell culture 3 times, discarding the liquid from the flask, adding 5 mL of PBS each, and adding 5 mL of complete culture medium to the flask. These washes eliminate red blood cells and tubular fragments that were not fully digested and not attached to the culture flask.
10. Incubate the epithelial cells in the cell culture incubator at 37 °C, allowing them to grow in monolayer to confluence.
11. Change the medium with a fresh complete culture medium every 3 days and observe the cell culture daily under an inverted microscope.

2. Development of three-dimensional HRTEC cultures (3D-HRTEC)

NOTE: This section provides the steps to develop three-dimensional cultures (3D-HRTEC). The 3D-HRTEC cultures are performed according to the method described previously¹².

1. Use a BM matrix from Engelbreth-Holm-Swarm murine sarcoma. This BM matrix is delivered frozen, and in this state, it remains solid. Thaw the BM matrix solution overnight in an ice bath in the refrigerator at 4 °C, as indicated by the manufacturer.
2. Cool the tips in the freezer before placing the BM matrix and leave the multi-well plate on ice to prevent solidification of the matrix during the process.
3. Take an aliquot of liquid BM matrix (150 mL/cm²) and place it into each well of the plate to cover the bottom of the well. Incubate the plate in the cell incubator at 37 °C for 1 h to turn the BM matrix into a gel state.
4. Meanwhile, to detach confluent HRTEC primary cultures, discard the culture medium from the flask and add 1 mL of 0.25% trypsin and 0.02% EDTA solution.
   1. Spread the liquid over the entire surface of the flask and incubate for 5 min at 37 °C in the cell culture incubator.
   2. Observe under an inverted microscope whether all cells have detached.
5. Resuspend the cells in 10 mL of culture medium or PBS, pass them to a 15 mL tube, and centrifuge at 160 × g for 10 min at 4 °C.
   1. Discard the supernatant and resuspend the cell pellet in a complete culture medium containing the same supplements used for HRTEC primary cultures.
   2. Count the number of cells in a Neubauer chamber.
6. Seed 4 × 10⁴ cells/150 µL per well onto the BM matrix. Incubate the cell culture for several days in a cell culture incubator at 37 °C, in a humid atmosphere and 5% CO₂.
7. Change the medium every 2 days with fresh medium.
8. Monitor cell aggregation and formation of tubular shape structures under an inverted optical microscope.

Results

Confluent HRTEC primary cultures showed a similar morphology observed under light microscopy (Figure 1). Cells were characterized by immunofluorescence as described in our previous publications using antibodies against aquaporin 1 (AQP1) and PECAM CD31^9,12,13. Immunofluorescence studies showed that more than 90% of HRTEC were positive for AQP1, which demonstrates that their origin is in the human re...

Discussion

Primary cultures should be performed on the same day of nephrectomy. HRTEC primary cultures can be used between 1-5 passages to guarantee the morphogenetic stability of the cells. After the fifth passage, cells begin to change their morphology, showing fibroblast-like appearance or increase in vacuoles, and can become quiescent.

If a larger kidney fragment is used, the DNA released by cell lysis after digestion may interfere with the washing process. In this case, it is recommended to use a la...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase type I	Sigma Merck, St Louis, MO, USA	637958
Endothelial cell growth supplement	Sigma Merck, St Louis, MO, USA	E2759
Matrigel basement membrane matrix	BD Biosciences, USA; Sigma Merck, St Louis, MO, USA	356234
70 μm pore mesh filter	BD Bioscience, MA, USA	css010070
RPMI 1640 medium with L-glutamine	Sigma Merck, St Louis, MO, USA	R8758-500ml
Hanks’ Balanced salt Solution  Ca2+ and Mg2+ free	Gibco Invitrogen, NY, USA	14175046
Trypsin-EDTA solution	Sigma Merck, St Louis, MO, USA	T4049