Osteopontin deficiency ameliorates Alport pathology by preventing tubular metabolic deficits
Wen Ding, Keyvan Yousefi, Stefania Goncalves, Bradley J. Goldstein, Alfonso L. Sabater, Amy Kloosterboer, Portia Ritter, Guerline Lambert, Armando J. Mendez, Lina A. Shehadeh
Wen Ding, Keyvan Yousefi, Stefania Goncalves, Bradley J. Goldstein, Alfonso L. Sabater, Amy Kloosterboer, Portia Ritter, Guerline Lambert, Armando J. Mendez, Lina A. Shehadeh
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Research Article Metabolism Nephrology

Osteopontin deficiency ameliorates Alport pathology by preventing tubular metabolic deficits

Abstract

Alport syndrome is a rare hereditary renal disorder with no etiologic therapy. We found that osteopontin (OPN) is highly expressed in the renal tubules of the Alport mouse and plays a causative pathological role. OPN genetic deletion ameliorated albuminuria, hypertension, tubulointerstitial proliferation, renal apoptosis, and hearing and visual deficits in the Alport mouse. In Alport renal tubules we found extensive cholesterol accumulation and increased protein expression of dynamin-3 (DNM3) and LDL receptor (LDLR) in addition to dysmorphic mitochondria with defective bioenergetics. Increased pathological cholesterol influx was confirmed by a remarkably increased uptake of injected DiI-LDL cholesterol by Alport renal tubules, and by the improved lifespan of the Alport mice when crossed with the Ldlr–/– mice with defective cholesterol influx. Moreover, OPN-deficient Alport mice demonstrated significant reduction of DNM3 and LDLR expression. In human renal epithelial cells, overexpressing DNM3 resulted in elevated LDLR protein expression and defective mitochondrial respiration. Our results suggest a potentially new pathway in Alport pathology where tubular OPN causes DNM3- and LDLR-mediated enhanced cholesterol influx and impaired mitochondrial respiration.

Authors

Wen Ding, Keyvan Yousefi, Stefania Goncalves, Bradley J. Goldstein, Alfonso L. Sabater, Amy Kloosterboer, Portia Ritter, Guerline Lambert, Armando J. Mendez, Lina A. Shehadeh

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Figure 6

OPN deficiency reduces DNM3-mediated cholesterol influx.

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OPN deficiency reduces DNM3-mediated cholesterol influx. (A and B) Osteo...
(A and B) Osteopontin (OPN) recombinant protein treatment causes increase of dynamin-3 (DNM3) protein expression in HK-2 cells. In panel B, n = 3–4 per group. Statistics based on ANOVA with Tukey’s post hoc test. (C and D) DNM3 was overexpressed by lentivirus in 293T and HK-2 cells and validated by qPCR. SCR, scrambled lentivirus. (E) DNM3 overexpression in HK-2 cells causes overexpression of LDL receptor (LDLR) protein as shown by Western blot. (F) Inhibition of dynamins by dynasore treatment in 30-minute TGF-β–stimulated HK-2 cells causes upregulation of PAI-1 and downregulation of LDLR protein levels 24 hours after treatment. (G) Inhibition of dynamins by dynasore treatment in 30-minute TGF-β–stimulated primary tubular epithelial cells (TECs) isolated from wild-type (WT) or Alport mice causes a decrease in LDLR protein levels 24 hours after treatment. In panels C–G, n = 2–4 per group. Statistics based on Student’s t test. n.d.u., normalized densitometry units. (H and I) LDLR protein expression is increased in Alport kidneys and normalized in Col4a3–/– Opn–/– kidneys. In panel I, n = 4–6 mice per group, and 4 images were quantified per mouse. Statistics based on ANOVA with Tukey’s post hoc test. Scale bars: 20 μm. (J) Infusion of 150 μg DiI-LDL cholesterol in Alport mice shows a significant 45.9-fold increase in LDL cholesterol uptake by Alport renal tubules after 2 hours. In panel J, n = 3 mice per group, and 3 images were quantified per mouse. Statistics based on Student’s t test. Scale bars: 20 μm. (K) Heterozygous deletion of LDLR in Alport mice tends to improve survival as shown by Kaplan-Meier curve. n = 8 Col4a3–/– mice and n = 12 Col4a3–/– Ldlr–/– mice. All mice are littermates. For all, data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.