Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction?
Abstract
:1. Introduction
2. Results
2.1. Biopsies
2.2. Generation of CLCN5 Mutant Human Podocyte Cells through CRISPR/Cas9 Genome Editing
2.3. Qualitative Analysis of CLCN5 mRNA in Mutant Clones
2.4. Effects of CLCN5 Downregulation on Human Podocyte Cells
2.4.1. CLCN5 Downregulation Alters Nephrin Expression
2.4.2. CLCN5 Downregulation Alters Podocyte Actin Cytoskeleton
2.5. Effects of CLCN5 Downregulation on Human Podocyte Cells Treated with Albumin
2.5.1. Albumin Modulates ClC-5, Nephrin and Cubilin Expression
2.5.2. Albumin Alters Podocyte Actin Cytoskeleton
3. Discussion
4. Materials and Methods
4.1. Renal Biopsies
4.1.1. Immunohistochemistry
4.1.2. Immunofluorescence
4.1.3. Morphometric Analysis
4.2. Creation of CLCN5 Mutant Podocyte Cell Clones
4.2.1. Human Podocytes
4.2.2. CRISPR/Cas9-Mediated Genome Editing
4.2.3. CLCN5 Mutation Analysis
High Resolution Melting (HRM) Analysis
Sanger Sequencing
4.3. Albumin Treatment of Podocyte Cell Clones
4.4. Quantitative Analysis of Clones’ mRNA
4.4.1. Reverse Transcription
4.4.2. Real Time PCR
4.5. Qualitative Analysis of CLCN5 mRNA
4.6. Protein Expression in Podocyte Cell Clones
4.6.1. In-Cell Western (ICW)
4.6.2. F-Actin Cytoskeleton Staining
4.7. Cell Viability Assessment
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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DD1 | ClC-5 Mutation | Age at Biopsy (Years) | Indication for Biopsy | Proteinuria | Number of Glomeruli Examined | Number of Glomeruli with Global Sclerosis | Podocytes Structure (TEM) |
---|---|---|---|---|---|---|---|
a | p.(Q600*) | 6 | proteinuria | 1.2 g/day | 18 | 5 | foot process effacement |
b | p.(R34*) | 11 | Nephrotic syndrome | 1.6 g/day | 16 | 8 | foot process effacement |
c | p.(V308M) | 9 | proteinuria and hematuria | 0.5 g/day | 29 | 1 | normal |
CTRL (n = 4) | N/A | 55 (range 41–67) | N/A | N/A | 47 | 0 | N/A |
Target | Clone | Host | Manufacturer | Code | Conjugation | Dilution IHC | Dilution IF | Dilution ICW | |
---|---|---|---|---|---|---|---|---|---|
primary antibody | ClC-5 | - | rabbit | ATLAS ANTIBODY | HPA000401 | 1:200 | 1:200 | ||
ClC-3 | K-17 | goat | Santa Cruz Biotechnology | sc-17572 | 1:50 | ||||
ClC-4 | - | rabbit | ATLAS ANTIBODY | HPA063637 | 1:150 | ||||
Cubilin | - | sheep | R&D Systems | AF3700 | 1:200 | ||||
Nephrin | - | guinea pig | Progen | GP-N2 | 1:25 | 1:25 | |||
Podocalyxin | 4F10 | mouse | Santa Cruz Biothecnology | sc-23903 | 1:100 | ||||
IHC | anti-rabbit | - | - | Dako | K4002 | EnVision + System-HRP Labeled Polymer | Reedy to use | ||
IF secondary antibody | Anti-rabbit | - | donkey | Santa Cruz Biotechnology | sc-362291 | CFL 647 | 1:100 | ||
Anti-guinea pig | donkey | Jackson ImmunoResearch laboratory | 706-545-148 | Alexa 488 | 1:100 | ||||
Anti-mouse | goat | ThermoFisher Scientific | A-11001 | Alexa 488 | 1:1000 | ||||
ICW secondary antibody | Anti-rabbit | - | donkey | LI-COR | 926-32213 | IRDye 800CW | 1:800 | ||
Anti-sheep | - | donkey | ThermoFisher Scientific | A-21102 | Alexa Fluor 680 | 1:1000 | |||
Anti-goat | - | donkey | LI-COR | 926-32214 | IRDye 800CW | 1:800 | |||
Anti-guinea pig | - | donkey | LI-COR | 925-32411 | IRDye 800CW | 1:600 |
Name | NCBI Reference Sequence | Sequence (5′-3′) | [PRIMER] µM | Ta (°C) | Size (bp) | Efficiency (%) |
---|---|---|---|---|---|---|
GAPDH For | NM_17851.1 | GAAGGTGAAGGTCGGAGT | 0.4 | 62 | 92 | 96 |
GAPDH Rev | TGGCAACAATATCCACTTTACCA | 0.4 | ||||
HPRT1 For | NM_000194.2 | CCTGGCGTCGTGATTAGTGA | 0.4 | 62 | 140 | 86 |
HPRT1 Rev | TCTCGAGCAAGACGTTCAGT | 0.4 | ||||
CLCN5 For | NM_000084.4 | TGCTGGAACTCTGAGCATGT | 0.2 | 64 | 162 | 99 |
CLCN5 Rev | TACGGCAAGGAAGGCAAATA | 0.2 | ||||
CLCN3 For | NM_001829.4 | TGGAGCAGGTGTTATTATGGAC | 0.4 | 62 | 105 | 94 |
CLCN3 Rev | ATGCTGCCTCCATTTGTCAT | 0.4 | ||||
CLCN4 For | NM_001830.4 | GTCGCGCTGAAGAAAGGAT | 0.4 | 62 | 122 | 89 |
CLCN4 Rev | TCAGGTTTCCAGAGCCACTC | 0.4 | ||||
CUBN For | NM_001081.3 | GCCGTGAGAAAGGATTTCAG | 0.4 | 62 | 118 | 85 |
CUBN Rev | TCCTTGTTTGGTGGATACCTG | 0.4 | ||||
NPHS1 For | NM_004646.3 | CAACTGGGAGAGACTGGGAGAA | 0.2 | 64 | 189 | 87 |
NPHS1 Rev | AATCTGACAACAAGACGGAGCA | 0.2 |
Name | NCBI Reference Sequence | Sequence (5′-3′) | [PRIMER] µM | Ta (°C) | Size (bp) | |
---|---|---|---|---|---|---|
Canonical isoform CLCN5-204 | 1F | NM_000084.4 | AAGCTCCCCAACCTGAATGA | 20 | 56 | 360 |
4R | TGTCTATCAAACCAGCTAACGA | 20 | ||||
Isoform CLCN5-206 | 8F | CAGCCATCACTGCCATCCTG | 20 | 64 | 630 | |
10R | CATCTATGATGCCCACATCCGT | 20 | ||||
nested 206F | GGCAGCTGGTTTAACACTC | 20 | 60 | 110 | ||
nested 206R | AGCCTGAACTCTCCAGACCA | 20 |
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Priante, G.; Ceol, M.; Gianesello, L.; Bizzotto, D.; Braghetta, P.; Calò, L.A.; Del Prete, D.; Anglani, F. Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction? Int. J. Mol. Sci. 2023, 24, 1313. https://doi.org/10.3390/ijms24021313
Priante G, Ceol M, Gianesello L, Bizzotto D, Braghetta P, Calò LA, Del Prete D, Anglani F. Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction? International Journal of Molecular Sciences. 2023; 24(2):1313. https://doi.org/10.3390/ijms24021313
Chicago/Turabian StylePriante, Giovanna, Monica Ceol, Lisa Gianesello, Dario Bizzotto, Paola Braghetta, Lorenzo Arcangelo Calò, Dorella Del Prete, and Franca Anglani. 2023. "Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction?" International Journal of Molecular Sciences 24, no. 2: 1313. https://doi.org/10.3390/ijms24021313