cell culture normal rat kidney fibroblast cell line  (ATCC)

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Both mTORC1 and mTORC2 signaling are activated in kidney fibroblasts after IRI. a The graph showing the blood urea nitrogen (BUN) level in CD-1 mice at day 1 after UNx or IRI. *P < 0.05, n = 3 (left). Kidney histology as shown by periodic acid-Schiff (PAS) staining. Scale bar = 20 µm (right). b, c Western blot analyses showing the induction of p-Akt (Ser473), and p-S6 in the kidneys after ischemia-reperfusion injury (IRI). The numbers indicate each individual animal within the given group (b). The samples were pooled from three animals within each group (c). d Representative images showing the induction for p-Akt (Ser473) and p-S6 in Gli1- or Fsp1-positive fibroblasts from the IRI kidneys. White arrows indicate the co-staining positive cells. Scale bar = 20 µm

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Staining, Western Blot

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Ablation of fibroblast Rheb aggravates kidney ischemia/reperfusion injury in mice. a The strategy for inducing fibroblast Rheb gene ablation in Gli1-Cre+/−, Rhebfl/fl mice and renal IRI surgery. b Representative micrographs showing the ablation of Rheb in Gli1+ cells from Gli1+-Rheb−/− kidneys at day 1 after IRI. The kidney tissues were immune stained with Abs against Gli1 and Rheb. The asterisk indicates both Gli1- and Rheb-positive cells in Rheb+/+ kidneys. The white arrow indicates Gli1-positive but Rheb-negative cells in the knockout kidneys. Scale bar = 10 µm. c The graph showing the blood urea nitrogen (BUN) level in Gli1+-Rheb+/+ and Gli1+-Rheb−/− mice at 1 day after IRI. *P < 0.05, n = 11. d Kidney histology as shown by PAS staining. Representative immunofluorescent staining images for Ly6b and CD3 among groups as indicated. Scale bar = 50 µm. e Representative immunofluorescent staining images for cleaved caspase 3 and TUNEL staining for apoptotic cells among groups as indicated. Scale bar = 50 µm. f The graph showing the injury scores among groups. *P < 0.05 versus Rheb+/+ mice, n = 3–6; #P < 0.05 versus Rheb+/+ mice after IRI, n = 6. Each dot represents the average of five HPFs from each mouse. g, h Quantitative determination of Ly6b+ and CD3+ cells among groups as indicated. *P < 0.05 versus Rheb+/+ mice, n = 3–6; #P < 0.05 versus Rheb+/+ mice after IRI, n = 6. Each dot represents the average of five HPFs from each mouse. i Real-time qRT-PCR analysis showing the mRNA abundance for Rantes, Mcp-1, and Tnf-α in kidneys at day 1 after IRI. *P < 0.05 versus Rheb+/+ mice after IRI, n = 6. j, k Quantitative determination of cleaved caspase 3+ cells and TUNEL+ cells in kidneys among groups. *P < 0.05 versus Rheb+/+ mice, n = 3–6; #P < 0.05 versus Rheb+/+ mice after IRI, n = 5–6. Each dot represents the average of five HPFs from each mouse

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Staining, Knock-Out, TUNEL Assay, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Ablation of fibroblast Tsc1 protects against kidney ischemia/reperfusion injury in mice. a The strategy for inducing fibroblast Tsc1 gene deletion in Gli1-Cre+/−, Tsc1fl/fl mice and renal IRI surgery. b Representative immunostaining images for Gli1 and Tsc1 in the kidneys at day 1 after IRI. The kidney tissue was stained with Abs against Gli1 and Tsc1. The asterisk indicates both Gli1- and Tsc1-positive cells in Tsc1+/+ kidneys. The arrow indicates Gli1-positive but Tsc1-negative cells in the knockout kidneys. Scale bar = 10 µm. c The graph showing the BUN level within groups at 1 day after IRI. *P < 0.05, n = 10. d PAS staining showing that the kidney injury was attenuated in the KO mice compared to their littermate controls at day 1 after IRI. Immunofluorescent staining revealing a decreased infiltration of Ly6b+ neutrophils and CD3+ T lymphocytes in the kidneys at day 1 after IRI. Scale bar = 50 µm. e Representative micrographs showing the apoptotic cells detected by immnuostaining for cleaved caspase 3 and TUNEL staining among groups. Scale bar = 50 µm. f The graph showing the injury scores among groups. *P < 0.05 versus Tsc1+/+ mice, n = 3–6; #P < 0.05 versus Tsc1+/+ mice after IRI, n = 6. Each dot represents the average of five HPFs from each mouse. g, h Quantitative determination of Ly6b+ and CD3+ cells among groups as indicated. *P < 0.05 versus Tsc1+/+ mice, n = 3–6; #P < 0.05 versus Tsc1+/+ mice after IRI, n = 5–6. Each dot represents the average of five HPFs from each mouse. i Real-time qRT-PCR analysis showing the mRNA abundance for Rantes, Mcp-1, and Tnf-α in Tsc1+/+ and knockout kidneys at day 1 after IRI. *P < 0.05 versus Tsc1+/+ mice after IRI, n = 6. j, k Quantitative determination of apoptotic cells among groups. Data are presented as the number of apoptotic cells per HPF (400×). *P < 0.05 versus Tsc1+/+ mice, n = 3–6; #P < 0.05 versus Tsc1+/+ mice after IRI, n = 6. Each dot represents the average of five HPFs from each mouse

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Immunostaining, Staining, Knock-Out, TUNEL Assay, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Ablation of fibroblast Rictor aggravates kidney ischemia/reperfusion injury in mice. a The strategy for inducing fibroblast Rictor gene deletion in Gli1-Cre+/−, Rictorfl/fl mice and renal IRI surgery. b Representative micrographs showing the ablation of Rictor in Gli1+ cells in IRI kidneys from Gli1+-Rictor−/− mice. The asterisk indicates both Gli1- and Rictor-positive cells in Rictor+/+ kidneys. The white arrow indicates Gli1-positive but Rictor-negative cells in the knockout kidneys. Scale bar = 10 µm. c The graph showing blood urea nitrogen (BUN) level in Gli1+-Rictor+/+ and Gli1+-Rictor−/− mice at 1 day after IRI. *P < 0.05, n = 10. d Kidney histology as shown by PAS staining. Representative immunofluorescent staining images for Ly6b and CD3 among groups as indicated. Scale bar = 50 µm. e Representative immunofluorescent staining images for cleaved caspase 3 and TUNEL staining for apoptotic cells among groups as indicated. f The graph showing the injury scores among groups. *P < 0.05 versus Rictor+/+ mice, n = 3–6; #P < 0.05 versus Rictor+/+ mice after IRI, n = 6. Each dot represents the average of five HPFs from each mouse. g, h Quantitative determination of Ly6b+ cells and CD3+ cells among groups as indicated. *P < 0.05 versus Rictor+/+ mice, n = 3–6; #P < 0.05 versus Rictor+/+ mice after IRI, n = 5–6. Each dot represents the average of four HPFs from each mouse. i Real-time qRT-PCR analysis showing the mRNA abundance for Rantes, Mcp-1, and Tnf-α in Rictor+/+ and the knockout kidneys at day 1 after IRI. *P < 0.05 versus Rictor+/+ mice after IRI, n = 6. j, k Quantitative determination of cleaved caspase 3+ cells and TUNEL+ cells. *P < 0.05 versus Rictor+/+ mice, n = 3–7; #P < 0.05 versus Rictor+/+ mice after IRI, n = 6–7. Each dot represents the average of five HPFs from each mouse

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Knock-Out, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Fibroblast mTORC1 and mTORC2 activation protects against staurosporine-induced tubular cell apoptosis. a The coculture system of tubular cells and kidney fibroblasts. b–d Western blot assay showing the abundance for Rheb, Tsc1, and Rictor in kidney fibroblasts. Primary cultured kidney fibroblasts generated from Rhebfl/fl, Tsc1fl/fl, and Rictorfl/fl mice were infected with adeno-Cre for 48 h to induce Rheb, Tsc1, and Rictor gene ablation, respectively. Fibroblasts infected with adeno-GFP were used as control fibroblasts. e–g Western blotting analyses showing the abundance of cleaved caspase 3 in primary cultured tubular cells incubated with CM from various primary cultured kidney fibroblasts as indicated. Tubular cells were treated with staurosporine (1 μM) for different duration. h–j Representative immunofluorescent staining images for cleaved caspase 3 among groups as indicated. Tubular cells were treated with staurosporine (1 μM) for 3 h. Scale bar = 20 µm. k–m Quantitative determination of cleaved caspase 3+ cells among groups as indicated. Data are presented as the percentage of the counted cells. *P < 0.05 versus vehicle-treated Rheb+/+ fibroblasts, n = 4; #P < 0.05 versus staurosporine-treated Rheb+/+ fibroblasts, n = 4. n = 4 refers to four independent repeats (k); *P < 0.05 versus vehicle-treated Tsc1+/+ fibroblasts, n = 3; #P < 0.05 versus staurosporine-treated Tsc1+/+ fibroblasts, n = 3. n = 3 refers to three independent repeats (l); *P < 0.05 versus vehicle-treated Rictor+/+ fibroblasts, n = 3; #P < 0.05 versus staurosporine-treated Rictor+/+ fibroblasts, n = 3. n = 3 refers to three independent repeats (m). CM conditioned media

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Activation Assay, Western Blot, Cell Culture, Generated, Infection, Control, Incubation, Staining

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Fibroblast mTOR/PPARγ dependent HGF production promotes tubular cell survival. a Western blot analyses showing the abundance of PPARγ in primary cultured Rheb−/−, Tsc1−/−, and Rictor−/− kidney fibroblasts, respectively. b Real-time qRT-PCR analysis showing the mRNA abundance for Hgf in primary cultured kidney fibroblasts. *P < 0.05 versus cells infected with adenovirus carrying GFP, n = 4. n = 4 refers to four independent repeats. c–e Western blotting analyses showing the abundance of p-c-met (Y1234/Y1235) and p-ERK1/2 (T202/Y204) as indicated. GAPDH was probed to show the equal loading. f Real-time qRT-PCR analysis showing the mRNA abundance for Hgf in NRK-49F cells. *P < 0.05 versus vehicle-treated cells, n = 3. n = 3 refers to three independent repeats. NRK-49F cells were pretreated with Pioglitazone for 24 h. g NRK-52E cells were treated with staurosporine (1 μM) for different duration as indicated. Western blot analyses showing the abundance of cleaved caspase 3. h Real-time qRT-PCR analysis showing the mRNA abundance Hgf in NRK-49F cells transfected with scramble or HGF siRNA as indicated. *P < 0.05 versus cells transfected with scramble siRNA, n = 3. n = 3 refers to three independent repeats. i, j NRK-52E cells were treated with staurosporine (1 μM) for different duration as indicated. Western blotting analysis showing that knocking down Hgf in NRK-49F cells (i) or blockade of tubular cell c-met signaling (j) could aggravate staurosporine-induced NRK-52E cell apoptosis. k, l NRK-52E cells were treated with staurosporine (1 μM) for 3 h. Representative immunofluorescent staining images for cleaved caspase 3 among groups as indicated. Scale bar = 20 µm. m, n Quantitative determination of cleaved caspase 3+ cells among groups as indicated. Data are presented as the percentage of the counted cells. *P < 0.05 versus vehicle-treated cells, n = 3; #P < 0.05 versus staurosporine-treated cells, n = 3. n = 3 refers to three independent repeats. CM conditioned media

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Western Blot, Cell Culture, Quantitative RT-PCR, Infection, Transfection, Staining

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Pharmacological blockade of fibroblast mTOR signaling represses HGF expression and aggravates tubular cell apoptosis. a Western blot analyses showing the reduction of PPARγ in NRK-49F cells treated with PP242. b Real-time qRT-PCR analysis showing the mRNA abundance for Hgf in NRK-49F cells. *P < 0.05 versus vehicle-treated cells, n = 4. n = 4 refers to four independent repeats. c Western blot analyses showing the abundance of p-c-met (Y1234/Y1235) and p-ERK1/2 (T202/Y204) as indicated. GAPDH was probed to show the equal loading. d NRK-52E cells were treated with staurosporine (1 μM) for different duration as indicated. Western blot analyses showing the abundance of cleaved caspase 3 in NRK-52E cells incubated with CM from PP242-treated NRK-49F cells. e Real-time qRT-PCR analysis showing that Pioglitazone could restore the mRNA abundance of Hgf suppressed by PP242 administration. *P < 0.05 versus vehicle-treated cells, n = 4; #P < 0.05 versus PP242-treated cells, n = 4. n = 4 refers to four independent repeats. f NRK-52E cells were treated with staurosporine (1 μM) for 3 h. Representative immunofluorescent staining images for cleaved caspase 3 among groups as indicated. Scale bar = 20 µm. g Quantitative determination of cleaved caspase 3+ cells among groups as indicated. Data are presented as the percentage of the counted cells. *P < 0.05 versus vehicle-treated cells, n = 3; #P < 0.05 versus staurosporine-treated cells that were incubated with CM from vehicle-treated fibroblasts, n = 3; $P < 0.05 versus staurosporine-treated cells that were incubated with CM from PP242-treated fibroblasts, n = 3. n = 3 refers to three independent repeats. CM conditioned media

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Incubation, Staining

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: MTOR regulates PPARγ/HGF expression in kidney fibroblasts in mice. a, c, e Western blot analyses showing the PPARγ abundance in the sham and IRI kidneys from the knockouts and littermate controls. Numbers indicate each individual animal within each group (left). The graphs showing the quantification of the PPARγ in the IRI and sham kidneys. *P < 0.05 versus littermate controls, #P < 0.05 versus littermate controls after IRI, n = 5. b, d, f Real-time qRT-PCR analysis showing the mRNA abundance for Hgf in the knockout and littermate control kidneys. *P < 0.05 versus WT mice, n = 5–6. g–i Immunostaining showing PPARγ in the interstitial cells among groups as indicated. The asterisk indicates both Gli1- and PPARγ-positive cells in the Rheb+/+, Rictor+/+, and Tsc1−/− kidneys. The arrow indicates Gli1-positive but PPARγ-negative cells in the Rheb−/−, Rictor−/−, and Tsc1+/+ kidneys. Scale bar = 20 µm

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Knock-Out, Control, Immunostaining

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Tsc1/Rheb/mTORC1 does not affect mTORC2 signaling activation in kidney fibroblasts. a, b The primary cultured kidney fibroblasts generated from Rhebfl/fl, or Tsc1fl/fl mice were infected with adeno-Cre virus to induce Rheb, or Tsc1 gene deletion. Western blotting assay showing that ablation of Rheb or Tsc1 could not affect the p-Akt (Ser473) abundance. c, d Western blot analyses showing p-Akt (Ser473) abundance in the Sham and IRI kidneys. Numbers indicate each individual animal within each group. e Real-time qRT-PCR analysis showing Raptor mRNA abundance in NRK-49F cells transfected with scramble or Raptor siRNA as indicated. *P < 0.05 versus cells transfected with scramble siRNA, n = 3. n = 3 refers to three independent repeats. f Western blotting analyses showing that knocking down Raptor expression inhibited S6 phosphorylation but not Akt phosphorylation at Ser473. g Western blot analyses showing the abundance of p-S6 and p-Akt (Ser473) in NRK-49F cells after rapamycin treatment

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Activation Assay, Cell Culture, Generated, Infection, Virus, Western Blot, Quantitative RT-PCR, Transfection, Expressing, Phospho-proteomics

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: mTORC2 is indispensable for mTORC1 signaling activation in kidney fibroblasts. a Western blot assay showing that ablation of Rictor reduced p-S6 abundance in kidney fibroblasts. The primary cultured fibroblasts generated from the kidneys of Rictorfl/fl mice were infected with adeno-Cre virus to induce Rictor gene deletion. b Western blot analyses showing p-S6 abundance in the Sham and IRI kidneys. Numbers indicate each individual animal within each group. c Representative images showing the less induction of p-S6 in the kidney fibroblasts from Gli1+-Rictor−/− mice at day 1 after IRI. The asterisk indicates both PDGFRβ-and p-S6-positive cells in littermate control kidneys. The arrow indicates PDGFRβ-positive but p-S6-negative cells in the knockout kidneys. Scale bar = 20 μm

Article Snippet: Cell culture Normal rat kidney fibroblasts (NRK-49F) and Normal rat kidney tubular epithelial cells (NRK-52E) were ordered from ATCC (Manassas, VA).

Techniques: Activation Assay, Western Blot, Cell Culture, Generated, Infection, Virus, Control, Knock-Out