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

Öz

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.

Anahtar Kelimeler

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

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

Öz

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.

Anahtar Kelimeler

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

Kaynakça

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