P3H2-Knockout Ölümsüzleştirilmiş İnsan Podosit Hücre Hattında Endoplazmik Retikulum Stresin Araştırılması

Abstract

Prolil 3-hidroksilaz 2 (P3H2), podositlerde glomerüler bazal membranın (GBM) temel yapısal bileşeni olan kolajen IV'ün post-translasyonal modifikasyonunu katalize eden bir enzimdir. P3H2 kaybının, bazal membran kusurlarıyla karakterize böbrek hastalıklarıyla ilişkili olduğu bilinmesine rağmen, hücresel etkileri hala net değildir. Bu çalışmada, P3H2 eksikliğinin ölümsüzleştirilmiş insan podosit hücre hattında endoplazmik retikulum (ER) stresine neden olup olmadığını değerlendirmek amaçlandı. Daha önce oluşturulmuş P3H2-Knockout (KO) podositleri ve vahşi tip (WT) podositler kontrol grubu olarak kullanıldı ve sonuçlar iki grup arasında karşılaştırıldı. ER stresi, kantitatif PCR (qPCR) ve Western blot yöntemleriyle ER stres belirteçlerinin mRNA ve protein seviyeleri ölçülerek değerlendirildi. Ayrıca immünofloresan boyama ile kolajen IV’ün hücre içindeki lokalizasyonu incelendi. Sonuçlar, WT ve KO hücreler arasında ER stres belirteçlerinin RNA ve protein düzeylerinde anlamlı bir fark olmadığını göstermiştir. Kolajen IV lokalizasyonu her iki grupta da benzer şekilde plazma membranı ve sitoplazmada gözlemlendi. Bu bulgular, P3H2 kaybının bazal koşullar altında podositlerde ER stresine neden olmadığını göstermektedir; ancak, P3H2 eksikliğinin podosit fonksiyonu üzerindeki etkilerini daha iyi anlamak için ileri düzeyde araştırmalara ihtiyaç vardır.

Keywords

• podosit
• prolil 3-hidroksilaz 2 (P3H2)
• ER stresi
• kolajen IV

Investigation of Endoplasmic Reticulum Stress in P3H2-Knockout Immortalized Human Podocyte Cell Line

Abstract

Prolyl 3-hydroxylase 2 (P3H2) is an enzyme that catalyzes the post-translational modification of collagen IV, a key structural component of the glomerular basement membrane (GBM) in podocytes. While P3H2 loss has been linked to kidney diseases characterized by basement membrane defects, its cellular effects remain unclear. This study aimed to evaluate whether P3H2 deficiency induces endoplasmic reticulum (ER) stress in an immortalized human podocyte cell line. Previously generated P3H2-Knockout (KO) podocytes were used and compared to wild-type (WT) controls. ER stress was assessed by measuring mRNA and protein levels of ER stress markers via quantitative PCR (qPCR) and Western blot. Immunofluorescence staining was performed to examine collagen IV localization. Results showed no significant differences in ER stress marker expression at the RNA and protein levels between WT and KO cells. Collagen IV localization was similarly observed in the plasma membrane and cytoplasm of both groups. These findings indicate that P3H2 loss does not induce ER stress in podocytes under basal conditions; however, further investigation is required to better understand the impact of P3H2 deficiency on podocyte function.

Keywords

• podocytes
• prolyl 3 hydroxylase 2 (P3H2)
• ER stress

References

1. 1. Grahammer F. New structural insights into podocyte biology. Cell Tissue Res. 2017;369(1):5-10. doi:10.1007/s00441-017-2590-3
2. 2. Kocylowski MK, Aypek H, Bildl W, et al. A slit-diaphragm-associated protein network for dynamic control of renal filtration. Nat Commun. 2022;13(1). doi:10.1038/s41467-022-33748-1
3. 3. Kopp JB, Anders HJ, Susztak K, et al. Podocytopathies. Nat Rev Dis Primers. 2020;6(1). doi:10.1038/s41572-020-0196-7
4. 4. Loreth D, Sachs W, Meyer‐Schwesinger C. The Life of a Kidney Podocyte. Acta Physiologica. 2025;241(8):e70081. doi:10.1111/apha.70081
5. 5. Järnum S, Kjellman C, Darabi A, Nilsson I, Edvardsen K, Åman P. LEPREL1, a novel ER and Golgi resident member of the Leprecan family. Biochemical and Biophysical Research Communications. 2004;317(2):342-351. doi:10.1016/j.bbrc.2004.03.060
6. 6. Pokidysheva E, Boudko S, Vranka J, et al. Biological role of prolyl 3-hydroxylation in type IV collagen. Proc Natl Acad Sci USA. 2014;111(1):161-166. doi:10.1073/pnas.1307597111
7. 7. Tiainen P, Pasanen A, Sormunen R, Myllyharju J. Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV. Journal of Biological Chemistry. 2008;283(28):19432-19439. doi:10.1074/jbc.m802973200
8. 8. Hudson DM, Joeng KS, Werther R, et al. Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations. Journal of Biological Chemistry. 2015;290(13):8613-8622. doi:10.1074/jbc.m114.634915

9. 9. Mordechai S, Gradstein L, Pasanen A, et al. High Myopia Caused by a Mutation in LEPREL1, Encoding Prolyl 3-Hydroxylase 2. The American Journal of Human Genetics. 2011;89(3):438-445. doi:10.1016/j.ajhg.2011.08.003
10. 10. Guo H, Tong P, Peng Y, et al. Homozygous loss‐of‐function mutation of the LEPREL1 gene causes severe non‐syndromic high myopia with early‐onset cataract. Clinical Genetics. 2014;86(6):575-579. doi:10.1111/cge.12309
11. 11. Aypek H, Krisp C, Lu S, et al. Loss of the collagen IV modifier prolyl 3-hydroxylase 2 causes thin basement membrane nephropathy. Journal of Clinical Investigation. 2022;132(9). doi:10.1172/jci147253
12. 12. Inagi R. Endoplasmic Reticulum Stress in the Kidney as a Novel Mediator of Kidney Injury. Nephron Exp Nephrol. 2009;112(1):e1-e9. doi:10.1159/000210573
13. 13. Av C. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases. Nature reviews Nephrology. 2017;13(11). doi:10.1038/nrneph.2017.129
14. 14. Saleem MA, O’Hare MJ, Reiser J, et al. A Conditionally Immortalized Human Podocyte Cell Line Demonstrating Nephrin and Podocin Expression. Journal of the American Society of Nephrology. 2002;13(3):630-638. doi:10.1681/asn.v133630
15. 15. Lin Q, Banu K, Ni Z, Leventhal JS, Menon MC. Podocyte Autophagy in Homeostasis and Disease. JCM. 2021;10(6):1184. doi:10.3390/jcm10061184
16. 16. Hartleben B, Gödel M, Meyer-Schwesinger C, et al. Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest. 2010;120(4):1084-1096. doi:10.1172/jci39492
17. 17. Bork T, Liang ,Wei, Yamahara ,Kosuke, et al. Podocytes maintain high basal levels of autophagy independent of mtor signaling. Autophagy. 2020;16(11):1932-1948. doi:10.1080/15548627.2019.1705007
18. 18. Besio R, Garibaldi N, Leoni L, et al. Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate. Disease Models & Mechanisms. 2019;12(6):dmm038521. doi:10.1242/dmm.038521
19. 19. Pincus D, Chevalier MW, Aragón T, et al. BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response. Kelly JW, ed. PLoS Biol. 2010;8(7):e1000415. doi:10.1371/journal.pbio.1000415
20. 20. Pokidysheva E, Boudko S, Vranka J, et al. Biological role of prolyl 3-hydroxylation in type IV collagen. Proc Natl Acad Sci USA. 2014;111(1):161-166. doi:10.1073/pnas.1307597111
21. 21. Montgomery NT, Zientek KD, Pokidysheva EN, Bächinger HP. Post-translational modification of type IV collagen with 3-hydroxyproline affects its interactions with glycoprotein VI and nidogens 1 and 2. Journal of Biological Chemistry. 2018;293(16):5987-5999. doi:10.1074/jbc.RA117.000406
22. 22. Chittiprol S, Chen P, Petrovic-Djergovic D, Eichler T, Ransom RF. Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes. American Journal of Physiology-Renal Physiology. 2011;301(3):F660-F671. doi:10.1152/ajprenal.00234.2011
23. 23. Agarwal S, Sudhini YR, Reiser J, Altintas MM. From Infancy to Fancy: A Glimpse into the Evolutionary Journey of Podocytes in Culture. Kidney360. 2021;2(2):385-397. doi:10.34067/kid.0006492020