Secreted Cytokines within the Urine of AKI Patients Modulate TP53 and SIRT1 Levels in a Human Podocyte Cell Model

Abstract

Acute kidney injury (AKI) is a major kidney disease with a poor clinical outcome. It is a common complication, with an incidence of 10-15% of patients admitted to hospital. This rate even increases for patients who are admitted to the intensive care unit, with an incidence of >50%. AKI is characterized by a rapid increase in serum creatinine, decrease in urine output, or both. The associated symptoms include feeling sick or being sick, diarrhoea, dehydration, decreased urine output (although occasionally the urine output remains normal), fluid retention causing swelling in the legs or ankles, shortness of breath, fatigue and nausea. However, sometimes acute kidney injury causes no signs or symptoms and is detected by lab tests. Therefore, the identification of cytokines for the early detection and diagnosis of AKI is highly desirable, as their application might enable the prevention of the progression from AKI to chronic kidney disease (CKD). In this study, we analysed the secretome of the urine of an AKI patient cohort by employing a kidney-biomarker cytokine assay. Based on these results, we suggest ADIPOQ, EGF and SERPIN3A as potential cytokines that might be able to detect AKI as early as 24 h post-surgery. For the later stages, as common cytokines for the detection of AKI in both male and female patients, we suggest VEGF, SERPIN3A, TNFSF12, ANPEP, CXCL1, REN, CLU and PLAU. These cytokines in combination might present a robust strategy for identifying the development of AKI as early as 24 h or 72 h post-surgery. Furthermore, we evaluated the effect of patient and healthy urine on human podocyte cells. We conclude that cytokines abundant in the urine of AKI patients trigger processes that are needed to repair the damaged nephron and activate TP53 and SIRT1 to maintain the balance between proliferation, angiogenesis, and cell cycle arrest.

Keywords: SIRT1; TP53; acute kidney injury; cytokines.

Figure 1

AKI cytokines identified in patients 24 h post-surgery. (A) Experiments clustered into CKD (n = 6) and healthy control (n = 6) based on the global kidney cytokine expression. (B) Heatmap and (C) barplot of markers in females and barplot of markers in males (D). The histograms represent means and the error bars standard errors of the mean (n = 3). The cytokines depicted here were differentially upregulated with a p-value (test of the R package limma (Smyth 2004 [28])) p < 0.05 and a ratio > 1.2, or downregulated with a p < 0.05, FDR < 0.25 and ratio < 0.8333.

Figure 2

AKI cytokines identified in patients 72 h post-surgery. (A) Experiments clustered into CKD (n = 6) and healthy control (n = 6) based on the global kidney cytokine expression. (B) Heatmap and (C) barplot of markers in females and barplot of markers in males (D). The histograms represent means and the error bars standard errors of the mean of technical duplicates (n = 2). The cytokines depicted here were differentially upregulated with a p-value (test of the R package limma) p < 0.05 and a ratio > 1.2, or downregulated with a p < 0.05, FDR < 0.25 and ratio < 0.8333.

Figure 3

Upregulation of TP53 and SIRT1 induced by AKI stage 2/3 urine in podocytes. The podocytes were incubated with 72 h post-surgery healthy (n = 6) and AKI stage 2/3 urine (n = 6) for 5 days. The relative protein expression normalized to GAPDH for SIRT1, TP53 and H2A.X phosphorylation was detected by Western blot (A). The mRNA expression of TP53 and SIRT1 was determined by quantitative real-time PCR (B). The TP53 expression in the podocytes exposed to AKI 2/3 urine was detected by immunofluorescence-based staining (C). Scale bars indicate 100 µm.

Figure 4

Proposed AKI signalling cascade. Urine-containing cytokines for the detection of AKI, which are male-enriched (AHSG), female-enriched (CCN1, IL6, CCL2, THBS1, IL1RN) and common in both (VEGF, SERPIN3A, TNFSF12, ANPEP, CXCL1, REN, CLU and PLAU). Furthermore, mechanistically, the cytokines ADIPOQ, EGF, EGFR, REN and VEGF present in the urine of AKI 2/3 patients trigger processes such as angiogenesis that are needed to repair the damaged nephron and the activation of TP53 and SIRT1 to maintain the balance between proliferation, angiogenesis, repair and cell-cycle arrest. Upregulated Cyokines and Proteins are indicated by a red arrow, while downregulated ones are indicated by a green arrow. Created with BioRender.com.