Zhengwei Ma ∗,∗, Xiaoru Hu ∗,†, Han-Fei Ding ‡, Ming Zhang ∗, Yuqing Huo ∗,§, Zheng Dong ∗,¶,∗
Posted: April 19, 2022
Abstract
Cisplatin induces both acute and chronic nephrotoxicity during chemotherapy in patients with cancer. Presented here is the first study of single-nucleus RNA sequencing (snRNA-seq) of cisplatin-induced nephrotoxicity. Repeated low-dose cisplatin treatment (RLDC) led to decreases in renal function and kidney weight in mice at 9 weeks. The kidneys of these mice showed tubular degeneration and dilation. snRNA-seq identified 16 cell types and 17 cell clusters in these kidneys. Cluster-by-cluster comparison demonstrated cell type–specific changes in gene expression and identified a unique proximal tubule (PT) injury/repair cluster that co-expressed the injury marker kidney injury molecule-1 (Kim1) and the proliferation marker Ki-67. Compared with control, post-RLDC kidneys had 424 differentially expressed genes in PT cells, including tubular transporters and cytochrome P450 enzymes involved in lipid metabolism. snRNA-seq also revealed transcriptional changes in potential PT injury markers (Krt222, Eda2r, Ltbp2, and Masp1) and repair marker (Bex4). RLDC induced inflammation and proinflammatory cytokines (RelB, TNF-α, Il7, Ccl2, and Cxcl2) and the expression of fibrosis markers (fibronectin, collagen I, connective tissue growth factor, vimentin, and α-smooth muscle actin). Together, these results provide new insights into RLDC-induced transcriptional changes at the single-cell level that may contribute to the development of chronic kidney problems in patients with cancer after cisplatin chemotherapy.
Single-cell or single-nucleus RNA sequencing (snRNA-seq) is a powerful, unbiased technology for comprehensive analysis of cell subtypes, cell-specific gene expression, and their changes in physiological and pathologic conditions. Using this technology, recent studies have begun to unveil the single-cell transcriptional profiles in kidneys under physiological and disease conditions,1, 2, 3, 4 including kidney development,5, 6, 7 diabetic kidney disease,8, 9, 10 kidney transplantation,11, 12, 13 ischemic and endotoxic acute kidney injury,14 and maladaptive kidney repair and renal fibrosis.15,16
Cisplatin is a potent and widely used chemotherapy drug for cancer treatment, but is notorious for adverse effects in normal tissues and organs, especially in the kidneys. Over a quarter of patients with cancer develop kidney problems during or after cisplatin chemotherapy. Acute nephrotoxicity of cisplatin has the typical feature of acute kidney injury (AKI), characterized by abrupt loss of renal function and damage to renal tubules. Chronic kidney problems after cisplatin exposure exhibit some key features of chronic kidney disease (CKD), including a gradual decline of renal function and tubule-interstitial histopathologies, such as tubular atrophy, atubular glomeruli, and tubulointerstitial fibrosis. Although cisplatin-induced AKI was a focus of research for years, recent studies have turned interest to chronic kidney problems after cisplatin exposure. This is facilitated by the establishment of the animal and cellular models of repeated low-dose cisplatin treatment (RLDC), which permits the investigation of the chronic changes in kidneys after cisplatin exposure.17, 18, 19, 20, 21, 22, 23
The present study was designed to analyze the changes of gene transcriptional profile at the single-cell resolution during the development of chronic kidney problems after RLDC treatment. Specifically, the kidney tissues were analyzed in a well-characterized mouse model of RLDC by snRNA-seq. The analysis led to the identification of a unique proximal tubule (PT) cell population co-expressing the cell proliferation marker Ki-67 and the PT injury marker Kim1. In addition, keratin 222 (Krt222), ectodysplasin A2 receptor (Eda2r), latent transforming growth factor beta binding protein 2 (Ltbp2), and MBL associated serine protease 1 (Masp1) were identified as the potential new markers of PT injury, whereas brain expressed, X-linked 4 (Bex4) was identified as a potential PT repair marker in the RLDC model. RLDC also induced PT-specific transcription changes in genes important for transport, inflammation, and fibrosis. Together, these results provide new insights into RLDC-induced transcriptional changes at the single-cell level that may contribute to the development of chronic kidney problems in patients with cancer after cisplatin chemotherapy.
NeoBiotechnologies’ products were used in this study:
Kidney tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline, embedded in paraffin, and sectioned at 5 μmol/L. Sirius red staining was performed according to a standard protocol from the manufacturer (Chondrex Inc., Woodinville, WA). Periodic acid–Schiff staining was performed according to a standard protocol from Sigma. For immunohistochemical staining, tissue sections were heated in the antigen retrieval buffer. Then, the sections were incubated with 3% H2O2 and then blocking buffer. The slides were then exposed to the primary antibody at 4°C overnight. After washing, the slides were incubated with ImmPRESS HRP Horse Anti-Rabbit IgG (MP-7401) or ImmPRESS-AP Horse Anti-Goat IgG (MP-5405) (both from Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. After washing, color was developed with a DAB kit ImmPACT DAB Substrate, Peroxidase (HRP) (SK-4105) and ImmPACT Vector Red Substrate, Alkaline Phosphatase (SK-5105) (both from Vector Laboratories). The primary antibodies for immunostaining included anti–Ki-67 (9129; Cell Signaling), anti-Kim1 (AF1817; R&D Systems, Minneapolis, MN), anti-Vcam1 (ab134047; Abcam), and anti-Krt20 (provided by Dr. Venkatachalam at the University of Texas Health Science Center at San Antonio as a gift from NeoBiotechnologies, Union City, CA).
Publication History:
Nucleic Acids Res. 2025 Mar 4;53(5):gkaf135. doi: 10.1093/nar/gkaf135
