Phosphoinositide 3 Kinase γ Plays a Critical Role in Acute Kidney Injury

doi:10.3390/cells11050772

. 2022 Feb 23;11(5):772.

doi: 10.3390/cells11050772.

Phosphoinositide 3 Kinase γ Plays a Critical Role in Acute Kidney Injury

Xiaogao Jin^{1

2

3

4}, Qinjun Chu¹, Liwei Sun¹, Melanie Tran⁵, Yanlin Wang^{4

5}

Affiliations

¹ Department of Anesthesiology and Perioperative Medicine, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, China.
² Research of Trauma Center, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, China.
³ Center for Advanced Medicine, College of Medicine, Zhengzhou University, Zhengzhou 450007, China.
⁴ Section of Nephrology, Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
⁵ Division of Nephrology, Department of Medicine and Department of Cell Biology, University of Connecticut School of Medicine, Farmington, CT 06030, USA.

PMID: 35269396
PMCID: PMC8909888
DOI: 10.3390/cells11050772

Phosphoinositide 3 Kinase γ Plays a Critical Role in Acute Kidney Injury

Xiaogao Jin et al. Cells. 2022.

. 2022 Feb 23;11(5):772.

doi: 10.3390/cells11050772.

Authors

Xiaogao Jin^{1

2

3

4}, Qinjun Chu¹, Liwei Sun¹, Melanie Tran⁵, Yanlin Wang^{4

5}

Affiliations

¹ Department of Anesthesiology and Perioperative Medicine, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, China.
² Research of Trauma Center, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, China.
³ Center for Advanced Medicine, College of Medicine, Zhengzhou University, Zhengzhou 450007, China.
⁴ Section of Nephrology, Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
⁵ Division of Nephrology, Department of Medicine and Department of Cell Biology, University of Connecticut School of Medicine, Farmington, CT 06030, USA.

PMID: 35269396
PMCID: PMC8909888
DOI: 10.3390/cells11050772

Abstract

Inflammatory cells contribute to the pathogenesis of renal ischemia-reperfusion injury (IRI). However, the signaling mechanisms underlying the infiltration of inflammatory cells into the kidney are not well understood. In this study, we examined the effects of phosphoinositide 3 kinase γ (PI3Kγ) on inflammatory cells infiltration into the kidney in response to ischemia-reperfusion injury. Compared with wild-type mice, PI3Kγ knockout mice displayed less IRI in the kidney with fewer tubular apoptotic cell. Furthermore, PI3Kγ deficiency decreased the number of infiltrated neutrophils, macrophages, and T cells in the kidney, which was accompanied by a decrease in the expression of pro-inflammatory cytokines in the kidney. Moreover, wild-type mice treated with AS-605240, a selective PI3Kγ inhibitor, displayed less tubular damage, accumulated fewer inflammatory cells, and expressed less proinflammatory molecules in the kidney following IRI. These results demonstrate that PI3Kγ has a critical role in the pathogenesis of kidney damage in IRI, indicating that PI3Kγ inhibition may serve as a potential therapeutic strategy for the prevention of ischemia-reperfusion-induced kidney injury.

Keywords: PI3Kγ; acute kidney injury; inflammation; ischemia-reperfusion injury.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
PI₃Kγ expressed in the kidney in the mice after IRI. (A) Representative photomicrographs of PI₃Kγ (red) expression in kidney slides, which were counterstained with hematoxylin (blue). (B) Quantitative analysis of PI3Kγ positive cells in the kidneys of PI3Kγ-KO and WT mice. ** p < 0.01 vs. WT sham. n = 6 per group. Mean ± SD is 9.83 ± 3.71 for Sham in WT, 37.50 ± 6.72 for IRI in WT, 0.33 ± 0.82 for Sham in PI3Kγ, and 0 ± 0 for IRI in PI3Kγ. HPF means high power field.

**Figure 2**
PI3Kγ deficiency or inhibition protects kidney from IRI. (A) Effect of PI3Kγ deficiency on blood urea nitrogen (BUN) in WT and PI3Kγ-KO mice after IRI or sham injury. ** p < 0.01 vs. WT sham; ⁺⁺ p < 0.01 vs. PI3Kγ-KO IRI; ^## p < 0.01 vs. WT IRI n = 6 per group. Mean ± SD is 41 ± 7.5 for Sham in WT, 160 ± 13 for IRI in WT, 41.7 ± 6.0 for Sham in PI3Kγ, and 102.8 ± 9.4 for IRI in PI3Kγ. n = 6 per group. (B) Effect of PI3Kγ deficiency on serum creatinine in WT and PI3Kγ-KO mice after IRI or sham injury. ^## p < 0.01 vs. WT IRI; ** p < 0.01 vs. WT sham; ⁺⁺ p < 0.01 vs. PI3Kγ-KO IRI. n = 6 per group. Mean ± SD is 0.35 ± 0.07 for Sham in WT, 2.05 ± 0.17 for IRI in WT, 0.33 ± 0.08 for Sham in PI3Kγ, and 0.92 ± 0.13 for IRI in PI3Kγ. n = 6 per group. (C) Representative photomicrographs of hematoxylin and eosin staining in kidney sections in WT and PI3Kγ-KO mice subjected to sham or IRI operation. (D) Quantitative histological assessment of tubular damage from IRI in WT and PI3Kγ-KO mice. ** p < 0.01 vs. WT IRI. Median with 25th and 75th percentiles is 0(0, 0) for Sham in WT, 3(2, 4) for IRI in WT, 0(0, 0) for Sham in PI3Kγ, and 1.5(1, 2) for IRI in PI3Kγ. n = 6 per group. (E) AS-605240 decrease serum BUN concentration in mice after IRI. ^## p < 0.05 vs. vehicle IRI; ** p < 0.01 vs. vehicle sham; ⁺⁺ p < 0.05 vs. AS-605240 IRI. n = 6 per group. Mean ± SD is 42.50 ± 9.35 for Sham in Vehicle group, 152.50 ± 14.40 for IRI in Vehicle group, 43.67 ± 7.50 for Sham in AS-605240 group, and 99.67 ± 7.12 for IRI in AS-605240 group. (F) AS-605240 reduced serum creatinine in mice after IRI. ^## p < 0.05 vs. vehicle IRI; ** p < 0.01 vs. vehicle sham; ⁺⁺ p < 0.05 vs. AS-605240 IRI. n = 6 per group. Mean ± SD is 0.40 ± 0.03 for Sham in Vehicle group, 2.38 ± 0.35 for IRI in Vehicle group, 0.41 ± 0.05 for Sham in AS-605240 group, and 0.83 ± 0.23 for IRI in AS-605240 group. (G) Representative photomicrographs of hematoxylin and eosin staining for kidney slides from the mice treated with IRI or sham injury in the presence of vehicle or AS-605240. (H) Tubular damage was quantitatively analyzed in the mice after IRI in the presence of vehicle or AS-605240. ** p < 0.05 vs. IRI. n = 6 in each group. Median with 25th and 75th percentiles is 0(0, 0) for Sham in Vehicle group, 3(2, 4) for IRI in Vehicle group, 0(0, 0) for Sham in AS-605240 group, and 1.5(1, 2) for IRI in AS-605240 group.

**Figure 3**
PI3Kγ deficiency protects tubular epithelial cells from apoptosis in IRI kidney. (A) Representative photomicrographs of kidney slides with apoptotic cells (brown) counterstaining with methyl green (green) in the IRI kidneys in WT and PI3Kγ-KO -treated mice (original magnification is 400×). (B) The apoptotic cells were quantitatively analyzed in the kidneys of WT and PI3Kγ-KO mice after sham injury or IRI. ^## p < 0.01 vs. WT IRI; ** p < 0.01 vs. WT sham; ⁺⁺ p < 0.01 vs. PI3Kγ-KO IRI. Mean ± SD is 3.50 ± 1.05 for Sham in WT, 32.50 ± 1.87 for IRI in WT, 3.67 ± 0.82 for Sham in PI3Kγ, and 12.00 ± 1.41 for IRI in PI3Kγ. n = 6 per group. HPF means high power field. (C) Representative Western blot shows the expression of active caspase 3 in the kidneys in WT and PI3Kγ KO mice. (D) The active caspase 3 expression was quantitatively analyzed in the kidneys in WT and PI3Kγ KO mice. ** p < 0.01 vs. WT sham. Mean ± SD is 1.00 ± 0.14 for Sham in WT, 3.60 ± 0.44 for IRI in WT, 1.05 ± 0.48 for Sham in PI3Kγ, and 1.79 ± 1.13 for IRI in PI3Kγ. n = 6 per group.

**Figure 4**
PI3Kγ deficiency or inhibition decreases infiltration of inflammatory cells in the kidney after IRI. (A) Representative photomicrographs of kidney slides with MPO positive (a neutrophil marker) (brown) and counterstaining with hematoxylin (blue) in PI3Kγ-KO and WT mice after sham injury or IRI. (B) MPO⁺ neutrophils in the kidneys were quantitatively analyzed in WT and PI3Kγ-KO mice after sham injury or IRI. ^## p < 0.01 vs. WT IRI; ** p < 0.01 vs. WT sham, ⁺⁺ p < 0.01 vs. PI3Kγ-KO IRI. n = 6 per group. Mean ± SD is 4.83 ± 1.17 for Sham in WT, 45.17 ± 4.26 for IRI in WT, 4.67 ± 2.16 for Sham in PI3Kγ, and 25.17 ± 4.17 for IRI in PI3Kγ. (C) Representative photomicrographs of kidney slides with F4/80 (a macrophage marker) (brown) positive, nuclei were counterstained with hematoxylin (blue) in WT and PI3Kγ-KO mice after IRI or sham injury. (D) F4/80⁺ macrophages were quantitatively analyzed in the kidneys in WT and PI3Kγ-KO mice after IRI or sham injury. ^## p < 0.01 vs. WT IRI; ** p < 0.01 vs. WT sham; ⁺⁺ p < 0.01 vs. PI3Kγ-KO IRI. n = 6 per group. Mean ± SD is 1.67 ± 0.81 for Sham in WT, 32.00 ± 2.83 for IRI in WT, 1.50 ± 1.05 for Sham in PI3Kγ, and 7.83 ± 2.32 for IRI in PI3Kγ. (E) Representative photomicrographs of kidney slides with CD3 (a T-lymphocyte marker) (brown) positive in WT and PI3Kγ-KO mice after IRI or sham injury. Nuclei were counterstained with hematoxylin (blue) I. (F) CD3⁺ T cells were quantitatively analyzed in the kidneys of WT and PI3Kγ-KO mice after IRI or sham injury. ** p < 0.01 vs. WT sham; ^## p < 0.05 vs. WT IRI; ⁺⁺ p < 0.05 vs. PI3Kγ-KO IRI. n = 6 per group. Mean ± SD is 0.67 ± 0.82 for Sham in WT, 8.33 ± 1.21 for IRI in WT, 0.67 ± 0.52 for Sham in PI3Kγ, and 4.00 ± 0.89 for IRI in PI3Kγ. HPF means high power field. (G) Representative photomicrographs of kidney slices with MPO (brown) positive in the mice treated with sham or IRI in the presence of vehicle or AS-605240. Nuclei were counterstained with hematoxylin (blue). (H) MPO⁺ neutrophils were quantitatively analyzed in the kidneys of mice with sham injury or IRI in the presence of vehicle or AS-605240. ** p < 0.01 vs. vehicle sham, ^# p < 0.05 vs. vehicle IRI; ⁺ p < 0.05 vs. AS-605240 IRI. n = 6 per group. Mean ± SD is 1.17 ± 1.17 for Sham in Vehicle group, 35.83 ± 1.72 for IRI in Vehicle group, 1.17 ± 1.16 for Sham in AS-605,240 group, and 10.00 ± 1.41 for IRI in AS-605240 group. (I) Representative photomicrographs of kidney with F4/80 (brown) positive in the mice treated with sham or IRI in the presence of vehicle or AS-605240. Nuclei were counterstained with hematoxylin (blue). (J) F4/80+ macrophages were quantitatively analyzed in the kidneys of mice treated with sham injury or IRI in the presence of vehicle or AS-605240. ^## p < 0.05 vs. vehicle IRI; ** p < 0.01 vs. vehicle sham; ⁺⁺ p < 0.05 vs. AS-605240 IRI. n = 6 per group. Mean ± SD is 1.16 ± 1.16 for Sham in Vehicle group, 35.33 ± 3.56 for IRI in Vehicle group, 1.17 ± 1.10 for Sham in AS-605240 group, and 10.50 ± 1.87 for IRI in AS-605240 group. (K) Representative photomicrographs of kidney slides with CD3 (brown) expression. Nuclei were counterstained with hematoxylin (blue) in the kidneys of mice treated sham injury or IRI in the presence of vehicle or AS-605240. (L) CD3+ T cells were quantitatively analyzed in the kidneys of mice treated with sham injury or IRI in the presence of vehicle or AS-605240. ** p < 0.01 vs. vehicle sham, ^## p < 0.05 vs. vehicle IRI; ⁺⁺ p < 0.05 vs. AS-605240 IRI. n = 6 per group. Mean ± SD is 0.83 ± 0.75 for Sham in Vehicle group, 7.67 ± 1.63 for IRI in Vehicle group, 1.00 ± 0.89 for Sham in AS-605,240 group, and 3.33 ± 0.52 for IRI in AS-605240 group. HPF means high power field.

**Figure 5**
Effect of PI3Kγ deficiency or inhibition on inflammatory molecules expression in the IRI kidney. (A) IL-6 mRNA expressions were shown by a dot plot. ^## p < 0.05 vs. WT IRI; ** p < 0.01 vs. WT sham; ⁺⁺ p < 0.05 vs. PI3Kγ-KO IRI. n = 6 per group. Mean ± SD is 1.00 ± 0.14 for Sham in WT, 5.13 ± 0.44 for IRI in WT, 0.98 ±0.12 for Sham in PI3Kγ, and 2.22 ± 0.28 for IRI in PI3Kγ. (B) MCP-1 mRNA expressions were shown by a dot plot. ^## p < 0.05 vs. WT IRI; ** p < 0.01 vs. WT sham; ⁺⁺ p < 0.05 vs. PI3Kγ-KO IRI. n = 6 per group. Mean ± SD is 1.00 ± 0.14 for Sham in WT, 9.83 ± 1.47 for IRI in WT, 0.99 ± 0.14 for Sham in PI3Kγ, and 3.90 ± 0.50 for IRI in PI3Kγ. (C). MIP-2 mRNA expressions were shown by a dot plot. ^## p < 0.05 vs. WT IRI; ** p < 0.01 vs. WT sham; ⁺⁺ p < 0.05 vs. PI3Kγ-KO IRI. n = 6 per group. Mean ± SD is 1.00 ± 0.14 for Sham in WT, 15.17 ± 1.29 for IRI in WT, 1.03 ± 0.13 for Sham in PI3Kγ, and 10.58 ± 1.02 for IRI in PI3Kγ. (D) TNFα mRNA expressions were shown by a dot plot. ^## p < 0.05 vs. WT IRI; ** p < 0.01 vs. WT sham; ⁺⁺ p < 0.05 vs. PI3Kγ-KO IRI. n = 6 per group. Mean ± SD is 1.00 ± 0.15 for Sham in WT, 5.05 ± 0.33 for IRI in WT, 0.99 ± 0.14 for Sham in PI3Kγ, and 2.05 ± 0.19 for IRI in PI3Kγ. Each dot represents the kidneys from WT and PI3Kγ-KO mice after IRI or sham injury. (E) IL-6 mRNA expressions were shown by a dot plot. Each dot represents the kidneys from the mice treated with sham injury or IRI in the presence of vehicle or AS-605240. ** p < 0.01 vs. vehicle sham, ^## p < 0.05 vs. vehicle IRI; ⁺⁺ p < 0.05 vs. AS-605240 IRI. n = 6 in each group. Mean ± SD is 1.00 ± 0.10for Sham in Vehicle group, 4.00 ± 0.35 for IRI in Vehicle group, 0.97 ± 0.13 for Sham in AS-605240 group, and 2.03 ± 0.11 for IRI in AS-605240 group. (F) MCP-1 mRNA expressions were shown by a dot plot. Each dot represents the kidneys from the mice treated with sham injury or IRI in the presence of vehicle or AS-605240. ** p < 0.01 vs. vehicle sham, ^## p < 0.05 vs. vehicle IRI; ⁺⁺ p < 0.05 vs. AS-605240 IRI. n = 6 in each group. Mean ± SD is 1.00 ± 0.14 for Sham in Vehicle group, 5.95 ± 0.19 for IRI in Vehicle group, 1.02 ± 0.14 for Sham in AS-605240 group, and 4.03 ± 0.38 for IRI in AS-605240 group. (G) MIP-2 mRNA expressions were shown by a dot plot. Each dot represents the kidneys from the mice treated with sham injury or IRI in the presence of vehicle or AS-605240. ** p < 0.01 vs vehicle sham, ^## p < 0.05 vs. vehicle IRI; ⁺⁺ p < 0.05 vs. AS-605240 IRI. n = 6 in each group. Mean ± SD is 1.00 ± 0.12 for Sham in Vehicle group, 16.78 ± 0.84 for IRI in Vehicle group, 1.02 ± 0.12 for Sham in AS-605240 group, and 7.83 ± 1.37 for IRI in AS-605240 group. (H) TNF-α mRNA expressions were shown by a dot plot. Each dot represents the kidneys from the mice treated with sham injury or IRI in the presence of vehicle or AS-605240. ** p < 0.01 vs vehicle sham, ^#^# p < 0.05 vs. vehicle IRI; ⁺⁺ p < 0.05 vs. AS-605240 IRI. n = 6 in each group. Mean ± SD is 1.00 ± 0.12 for Sham in Vehicle group, 2.76 ± 0.19 for IRI in Vehicle group, 1.03 ± 0.10 for Sham in AS-605240 group, and 1.50 ± 0.07 for IRI in AS-605240 group. GAPDH means glyceraldehyde-3-phosphate dehydrogenase.

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References

1. Malek M., Nematbakhsh M. Renal ischemia/reperfusion injury; from pathophysiology to treatment. J. Ren. Inj. Prev. 2015;4:20–27. - PMC - PubMed
1. Shiva N., Sharma N., Kulkarni Y.A., Mulay S.R., Gaikwad A.B. Renal ischemia/reperfusion injury: An insight on in vitro and in vivo models. Life Sci. 2020;256:117860. doi: 10.1016/j.lfs.2020.117860. - DOI - PubMed
1. Jin X., Chen J., Hu Z., Chan L., Wang Y. Genetic deficiency of adiponectin protects against acute kidney injury. Kidney Int. 2013;83:604–614. doi: 10.1038/ki.2012.408. - DOI - PMC - PubMed
1. Tammaro A., Kers J., Scantlebery A.M.L., Florquin S. Metabolic Flexibility and Innate Immunity in Renal Ischemia Reperfusion Injury: The Fine Balance Between Adaptive Repair and Tissue Degeneration. Front. Immunol. 2020;11:1346. doi: 10.3389/fimmu.2020.01346. - DOI - PMC - PubMed
1. Zhao M., Wang Y., Li L., Liu S., Wang C., Yuan Y., Yang G., Chen Y., Cheng J., Lu Y., et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics. 2021;11:1845–1863. doi: 10.7150/thno.50905. - DOI - PMC - PubMed

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[1] Malek M., Nematbakhsh M. Renal ischemia/reperfusion injury; from pathophysiology to treatment. J. Ren. Inj. Prev. 2015;4:20–27. - PMC - PubMed

[2] Malek M., Nematbakhsh M. Renal ischemia/reperfusion injury; from pathophysiology to treatment. J. Ren. Inj. Prev. 2015;4:20–27. - PMC - PubMed

[3] Shiva N., Sharma N., Kulkarni Y.A., Mulay S.R., Gaikwad A.B. Renal ischemia/reperfusion injury: An insight on in vitro and in vivo models. Life Sci. 2020;256:117860. doi: 10.1016/j.lfs.2020.117860. - DOI - PubMed

[4] Shiva N., Sharma N., Kulkarni Y.A., Mulay S.R., Gaikwad A.B. Renal ischemia/reperfusion injury: An insight on in vitro and in vivo models. Life Sci. 2020;256:117860. doi: 10.1016/j.lfs.2020.117860. - DOI - PubMed

[5] Jin X., Chen J., Hu Z., Chan L., Wang Y. Genetic deficiency of adiponectin protects against acute kidney injury. Kidney Int. 2013;83:604–614. doi: 10.1038/ki.2012.408. - DOI - PMC - PubMed

[6] Jin X., Chen J., Hu Z., Chan L., Wang Y. Genetic deficiency of adiponectin protects against acute kidney injury. Kidney Int. 2013;83:604–614. doi: 10.1038/ki.2012.408. - DOI - PMC - PubMed

[7] Tammaro A., Kers J., Scantlebery A.M.L., Florquin S. Metabolic Flexibility and Innate Immunity in Renal Ischemia Reperfusion Injury: The Fine Balance Between Adaptive Repair and Tissue Degeneration. Front. Immunol. 2020;11:1346. doi: 10.3389/fimmu.2020.01346. - DOI - PMC - PubMed

[8] Tammaro A., Kers J., Scantlebery A.M.L., Florquin S. Metabolic Flexibility and Innate Immunity in Renal Ischemia Reperfusion Injury: The Fine Balance Between Adaptive Repair and Tissue Degeneration. Front. Immunol. 2020;11:1346. doi: 10.3389/fimmu.2020.01346. - DOI - PMC - PubMed

[9] Zhao M., Wang Y., Li L., Liu S., Wang C., Yuan Y., Yang G., Chen Y., Cheng J., Lu Y., et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics. 2021;11:1845–1863. doi: 10.7150/thno.50905. - DOI - PMC - PubMed

[10] Zhao M., Wang Y., Li L., Liu S., Wang C., Yuan Y., Yang G., Chen Y., Cheng J., Lu Y., et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics. 2021;11:1845–1863. doi: 10.7150/thno.50905. - DOI - PMC - PubMed

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Phosphoinositide 3 Kinase γ Plays a Critical Role in Acute Kidney Injury

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Phosphoinositide 3 Kinase γ Plays a Critical Role in Acute Kidney Injury

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Abstract

Conflict of interest statement

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Abstract

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