One of the major obstacles to prevent AKI-CKD transition is the lack of effective methods to follow and predict the ongoing kidney injury after an AKI episode. In the present study, we test the utility of urinary angiotensinogen (UAGT) for dynamically evaluating renal structural changes and predicting AKI-CKD progression by using both mild and severe bilateral renal ischemia/reperfusion injury mice. UAGT returns to pre-ischemic levels 14 days after mild AKI followed by kidney architecture restoration, whereas sustained increase in UAGT accompanies by ongoing renal fibrosis after severe AKI. UAGT at day 14–42 correlates with renal fibrosis 84 days after AKI. For predicting fibrosis at day 84, the area under receiver operating characteristics curve of UAGT at day 14 is 0.81. Persistent elevation in UAGT correlates with sustained activation of intrarenal renin–angiotensin system (RAS) during AKI-CKD transition. Abrogating RAS activation post AKI markedly reduced renal fibrosis, with early RAS intervention (from 14 days after IRI) more beneficial than late intervention (from 42 days after IRI) in alleviating fibrosis. Importantly, UAGT decreases after RAS intervention, and its level at day 14–28 correlates with the extent of renal fibrosis at day 42 post RAS blockade. A pilot study conducted in patients with acute tubular necrosis finds that compared with those recovered, patients with AKI-CKD progression exhibits elevated UAGT during the 3-month follow-up after biopsy. Our study suggests that UAGT enables the dynamical monitoring of renal structural recovery after an AKI episode and may serve as an early predictor for AKI-CKD progression and treatment response.

Introduction

Survivors of acute kidney injury (AKI) have a markedly increased risk for chronic kidney disease (CKD) [1]. However, not all AKI survivors are at equal risk of CKD transition [2–4]. After an AKI episode, some survivors experience successful renal repair leaving no evidence of damage, while others experience maladaptive repair resulting in progressive fibrotic lesions [5]. Investigations that can be used to dynamically detect renal structural changes would be helpful to facilitate timely and necessary interventions to prevent the AKI-CKD transition. Serum creatinine reflects late renal function changes, which do not necessarily parallel the structural injury in the kidney. Assessments of renal structural changes are only achievable with renal biopsies, which are obtained from patients via an invasive operation. In this setting, development of a noninvasive surrogate marker that closely parallels renal pathological changes is urgently needed.

Renin–angiotensin system (RAS) is a well-recognized important driver of fibrosis via promotion of inflammation and extracellular matrix production in multiple organs [6], including the kidney. Angiotensinogen (AGT) and angiotensin (Ang) II are significantly up-regulated in tubular cells following AKI [7,8]. RAS intervention in AKI mice preserved peritubular capillaries and prevented subsequent kidney inflammation and fibrosis [7,9,10]. Furthermore, tubular AGT, an important substrate of Ang II, can be secreted into the lumen and be detectable in the urine [6]. Urinary AGT reflects the intrarenal RAS status and has been proposed as a biomarker of renal injury severity in both human acute tubular necrosis (ATN) [8] and CKD [11]. However, the dynamics of urinary AGT and its relevance to kidney RAS status and renal histological changes post an AKI episode remain to be elucidated.

In the present study, our objective is to test and validate the hypothesis that urinary AGT may serve as an early predictive marker for renal structural recovery following an AKI episode. For this purpose, we characterized the profiles of urinary AGT and intrarenal RAS status after an episode of AKI in mild reversible ischemia reperfusion injury (IRI), in severe IRI with progressive renal fibrosis, and in human AKI.

Materials and methods

Mouse model of ischemic AKI

All animal experiments were approved by the Institutional Animal Ethics Committee. Six-week-old male C57BL/6J mice (20–24 g) were obtained from Institutional Animal Experiment Center.

To determine the impact of AKI severity on renal fibrosis, mice treated with 20- or 45-min ischemia were served as mild or severe AKI as previously described [12]. Sham-operated animals underwent the identical procedure except for clamping of the renal pedicles. The success of AKI model was confirmed by elevated serum creatinine levels and increased renal histological damage 24-h post ischemia [7]. All animals were followed up for 84 days. Mortality rate was approximately 20% in mice with 45-min ischemia.

To prepare IRI model with different AKI severity, mice were subjected to bilateral clamping of the renal pedicles for 20, 25, 30, 40, or 45 min as described previously [7]. All animals were followed up for 84 days.

To examine whether urinary AGT is responsive to treatment, we first established mice models at early or advanced stages of AKI-CKD transition by altering the reperfusion time (14, 28, or 42 days) post 45-min ischemia. Then, mice at different stages of AKI-CKD progression were randomly assigned to intragastric administration of losartan (150 mg/kg/day, Sigma, Saint Louis, MO) or phosphate-buffered saline (PBS, Sigma) for 42 days (illustrated in Figure 6A). Urinary AGT was monitored before and during the RAS/PBS treatment. Renal structural and functional changes were assessed at the end of the RAS/PBS treatment.

Measurement of renal function and mean arterial pressure

Serum creatinine concentration was measured using the Quantichrom Creatinine assay kit (BioAssay Systems, Hayward, CA). Urinary albumin excretion was measured with an ELISA kit (Bethyl Laboratories, Montgomery, TX). The glomerular filtration rate (GFR) was determined by inulin clearance [13]. Renal perfusion was determined by CEUS using a high-resolution ultrasonic imaging system (Sequoia 512, Acuson, Siemens, Malvern, PA) with a 15L8 HD probe (details are demonstrated in Supplementary Material) [12].

Mean blood pressure was measured in conscious mice via a catheter in the carotid artery by telemetry using TA11PA-C10 probes (Data Sciences International, St. Paul, MN), which were implanted 1 week before the renal ischemia–reperfusion injury or sham operation as previously described [14].

Renal histological analysis

Acute and chronic tubulointerstitial injury

The kidneys were dissected and processed for hematoxylin–eosin staining [15]. Acute tubular injury was assessed by determining the degree of renal tubular necrosis at the corticomedullary junction using a grading scale of 0 to 4 [15]. The index of chronic tubulointerstitial injury was determined according to the percentage of damaged (tubular atrophy or dilatation, interstitial macrophage infiltration or fibrosis) area in the renal cortex and corticomedullary junction [16]. Renal fibrosis was assessed by Masson’s trichrome staining. The extent of tubulointerstitial fibrosis was semiquantitated as previously described [17].

Tubulointerstitial inflammation was assessed by immunohistochemical staining using a monoclonal antibody recognizing macrophage marker F4/80 (1:50, Serotec, U.K.). Macrophage infiltration was quantitated by counting the F4/80-positive cells in ten randomly chosen (0.3 × 0.3 mm2) tubulointerstitial areas.

Extracellular matrix expression

Accumulation of extracellular matrix (collagen I) was evaluated by immunohistochemical staining using a rabbit anti-mouse collagen I (1:100, Millipore, Billerica, MA) antibody. Intrarenal expression of collagen I was semiquantitated as described previously [18]. The protein levels of collagen I in homogenates of renal cortex were determined by Western blot [19] using a rabbit anti-mouse collagen I (1:500, Millipore) and a rabbit anti-β-actin (1:1000, Cell Signaling Technology, Danvers, MA) antibodies.

All histological analyses were performed by two pathologists who were blinded to the treatment of the animals.

Intrarenal RAS expression

Immunohistochemistry analysis

To evaluate the expression of intrarenal RAS, 5-μm-thick sections were processed [20] using a rabbit anti-mouse AGT (1:200, IBL, Japan), a rabbit anti-Ang II (1:800, Peninsula Laboratories, San Carlos, CA), and a goat anti-mouse renin (1:50, Santa Cruz, Dallas, TA) antibodies. RAS expression in renal cortex was quantitated as previously described [21]; data were expressed as the ratio of integrated optical density (IOD) to observed area (IOD/area). Intrarenal renin expression was assessed and expressed as the renin-positive juxta-glomerular apparatus (JGA)/total JGA × 100 (%JGA) [22].

Localization of intrarenal AGT and Ang II was determined by double-staining with first primary antibody against AGT (1:200, IBL) or Ang II (1:800, Peninsula Laboratories) in separate sections, and second primary antibodies against the markers of tubular epithelial cells: anti-aquaporin 1 (AQP-1, a marker of proximal tubular epithelial cells; 1:100, Abcam, U.K.); anti-Tamm-Horsfall protein 1 (THP-1, a marker of ascending thick limbs of Henle’s loop; 1:100, Santa Cruz); anti-thiazide-sensitive NaCl cotransporter (NCCT, a marker of distal convoluted tubular epithelial cells; 1:500, Millipore); or anti-aquaporin 2 (AQP-2, a marker of collecting duct epithelial cells; 1:100, Novus Biologicals, Littleton, CO) antibody separately.

Western blot

The levels of AGT in renal homogenates were determined [23] using a rabbit anti-mouse AGT (1:400, IBL) and a rabbit anti-β-actin (1:1000, Cell Signaling Technology) antibodies.

Liquid chromatography/Mass spectrometry

Ang II concentrations in renal homogenates were determined by liquid chromatography/mass spectrometry (LC/MS) [24]. Samples were extracted and separated with C18 Sep-Pak cartridges (Thermo Scientific, MA, U.S.A.), followed by detection with Q-ToF mass analyzer in the ESI+-MS ion mode.

Quantitation of AGT by ELISA

Concentrations of AGT in serum and urine samples were assessed by ELISA as dictated by the manufacturer’s protocol (IBL, Japan). AGT in urine samples were standardized for urinary creatinine and expressed as micrograms per milligram of creatinine.

Evaluation of urinary AGT in human acute tubular necrosis

The human study was approved by the Institutional Medical Ethics Committee (GZKYEC-201608-K1-02), and all study subjects gave written informed consent.

To further illustrate the feasibility of urinary AGT in human AKI-CKD progression, urinary AGT was monitored in patients with biopsy-proven ATN from 2015 to 2017. We excluded those with pre-existing CKD, those with comorbid diseases and those treated with RAS inhibitors at the time of sample collection. Baseline serum creatinine was defined as the lowest value in the last 6 months before AKI or the lowest value achieved during hospitalization in the absence of dialysis [8].

Urine and serum samples were collected in the morning in healthy volunteers and in ATN patients on the day of the biopsy and monthly for 3 months after biopsy. AGT were detected by an ELISA kit (IBL International). Creatinine was measured using an automatic biochemical analyzer (AU5831, Backman Coulter, CA). Urinary albumin excretion was measured using an automatic analyzer (BNPro Spec; Siemens, Germany).

Intrarenal expression of AGT and Ang II was determined [8] in renal biopsy samples obtained from ATN patients and in normal kidney tissue adjacent to renal carcinoma obtained from nephrectomy. The primary antibodies are anti-human AGT (1:200, R&D Systems, MN, U.S.A.) and anti-human Ang II (1:400, Peninsula Laboratories) antibodies. All histological analyses were performed by two pathologists who were blinded to the identity of the tissue.

Statistical data analysis

The continuous variables were expressed as mean ± SD or median (interquartile range). Categorical variables were expressed as percentages. For real-time PCR or measurement of renal function and AGT levels, each sample was determined in three replicates. Each value represents the mean of three replicates analyses. Differences between two groups were compared by the Student’s t-test or the Mann–Whitney U test. The categorical data were compared with Pearson chi-squared test. Correlations were assessed according to Pearson’s correlation analysis or the Spearman’s rank correlation analysis. To measure the sensitivity and specificity of urinary AGT at different cutoff values, a conventional receiver operating characteristics (ROC) curve was generated. In human study, correlations between urinary AGT and intrarenal expression of AGT or Ang II were determined using the Pearson’s correlation coefficient. In these correlation analyses, urinary AGT was logarithmically transformed to correct for dispersion of data. Statistical analyses were conducted with SPSS17.0 for Windows (SPSS Inc, Chicago, IL). A value of P<0.05 was considered statistically significant. Power analysis for the independent t-test was conducted to assess the number of animals necessary to obtain statistically significant data (alpha 0.05) for preselected powers 0.8 (nQuery version 8.2.1, Statistical Solutions Ltd., Ireland).

Results

Different severity of AKI associated with different fibrotic outcomes

We have previously shown that the AKI severity is a key determinant dictating the divergent outcomes of ischemic AKI, with 20-min IRI triggering near totally recovery, whereas over 30-min IRI resulting in progression to CKD 42 days after IRI [12]. In the present study, mice were subjected to 20 or 45 min of bilateral renal ischemia, followed by 84 days of reperfusion. Consistently, 20-min IRI induced reversible mild AKI followed by renal structural recovery. In contrast, 45-min IRI resulted in severe AKI. Although serum creatinine, urinary albumin excretion, and acute kidney injury score returned to baseline level within 14 days (Figure 1A,B and Supplementary Figure S1A), mice with severe AKI showed progressive chronic kidney damage through 84 days after IRI, characterized by progressive increases in tubulointerstitial inflammation and fibrosis (Figure 1D–F; Supplementary Figures S1B–F, S2, and S7A), and persistent decline in GFR and renal perfusion (Figure 1C and Supplementary Figure S3).

Different severity of AKI associated with different fibrotic outcomes

Figure 1
Different severity of AKI associated with different fibrotic outcomes

(A) Changes in serum creatinine in mice with mild (20-min IRI) and severe (45-min IRI) AKI. (B) Changes in urinary albumin excretion. (C) Changes in glomerular filtration rate measured by inulin clearance. (D) Representative photos of tubulointerstitial injury, presented by hematoxylin–eosin staining (HE) and Masson staining (MS). (E) Chronic tubulointerstitial injury index after severe AKI. (F) Tubulointerstitial fibrosis score after severe AKI; scale bar = 100 µm. Data are expressed as mean ± SD (n=8 in each study group). *P<0.05 versus sham mice at the same time point.

Figure 1
Different severity of AKI associated with different fibrotic outcomes

(A) Changes in serum creatinine in mice with mild (20-min IRI) and severe (45-min IRI) AKI. (B) Changes in urinary albumin excretion. (C) Changes in glomerular filtration rate measured by inulin clearance. (D) Representative photos of tubulointerstitial injury, presented by hematoxylin–eosin staining (HE) and Masson staining (MS). (E) Chronic tubulointerstitial injury index after severe AKI. (F) Tubulointerstitial fibrosis score after severe AKI; scale bar = 100 µm. Data are expressed as mean ± SD (n=8 in each study group). *P<0.05 versus sham mice at the same time point.

Sustained increase in urinary AGT after AKI predicts progressive renal fibrosis

We next investigated the dynamics of urinary AGT in mice with mild (20-min IRI) and severe (45-min IRI) AKI at various time points after the acute insult. In mild IRI where fibrosis did not occur, urinary AGT level fell to baseline within 2 weeks after IRI, whereas in the severe AKI characterized by progressive fibrosis, elevated urinary AGT was sustained for 84 days (Figure 2A). There was no difference in urinary AGT level among sham groups at different time points after the surgery (data not shown). In contrast with urinalysis, AGT level in serum samples did not change significantly after IRI (Figure 2B). There is no correlation between serum and urinary AGT level (Figure 2C, r = 0.17, P=0.094).

Dynamics of urinary AGT in mice with mild and severe IRI

Figure 2
Dynamics of urinary AGT in mice with mild and severe IRI

(A) Urinary AGT levels in mice with mild (20-min IRI) and severe (45-min IRI) AKI. (B) Serum AGT levels in mice with mild (20-min IRI) and severe (45-min IRI) AKI. (C) Correlation between urinary and serum AGT. Data are expressed as mean ± SD (n=8 in each study group). *P<0.05 versus sham mice at the same time point.

Figure 2
Dynamics of urinary AGT in mice with mild and severe IRI

(A) Urinary AGT levels in mice with mild (20-min IRI) and severe (45-min IRI) AKI. (B) Serum AGT levels in mice with mild (20-min IRI) and severe (45-min IRI) AKI. (C) Correlation between urinary and serum AGT. Data are expressed as mean ± SD (n=8 in each study group). *P<0.05 versus sham mice at the same time point.

To further examine the correlation between urinary AGT and subsequent chronic kidney injury, mice were challenged with incremental periods (20, 25, 30, 40, and 45 min) of bilateral ischemia. Urinary AGT level was determined before IRI and 14–84 days post IRI. Animals were killed at day 84 for histological assessment. As shown in Figure 3A–D, ischemic strike for 30 min or longer resulted in sustained increase in urinary AGT and progressive tubulointerstitial fibrosis. Level of urinary AGT at day 14 after IRI positively correlated with the accumulation of collagen I and tubulointerstitial fibrosis score at day 84 (Figure 3E,F). Significant correlation was observed between the urinary AGT level at day 14-42 and tubulointerstitial fibrosis score at day 84 (Figure 3G). To evaluate the performance of urinary AGT for predicting renal fibrosis at day 84, a ROC curve was generated. The areas under the ROC curve at 14–42 days for predicting renal fibrosis were 0.81–0.90 (Figure 3H,I).

Sustained increase in urinary AGT after AKI predicts progressive renal fibrosis

Figure 3
Sustained increase in urinary AGT after AKI predicts progressive renal fibrosis

Mice treated with IRI for different ischemia time. Urinary AGT was measured at different time point post ischemia. The animals were killed at day 84 after IRI. (A) Changes of urinary AGT levels. (B) Quantitative data of tubulointerstitial Co I staining at day 84 after IRI. (C) Tubulointerstitial fibrosis score at day 84 after IRI. (D) Representative photos of tubulointerstitial fibrosis showed by Masson staining (MS) and immunohistochemical staining of collagen I (Co I) at day 84 after IRI. (E and F) Urinary AGT level at day 14 after IRI correlated with accumulation of collagen I (E) and tubulointerstitial fibrosis score (F) at day 84 after IRI. (G) Urinary AGT level at day 14–42 after IRI correlated with tubulointerstitial fibrosis score at day 84 after IRI. (H and I) Receive-operating characteristic curve analysis to determine the performance of urinary AGT at day 14–42 for predicting renal fibrosis at day 84; scale bar = 100 µm. Data are expressed as mean ± SD (n = 8 in each study group). *P<0.05 versus pre-ischemic levels in (A). *P<0.05 versus sham mice in (B) and (C).

Figure 3
Sustained increase in urinary AGT after AKI predicts progressive renal fibrosis

Mice treated with IRI for different ischemia time. Urinary AGT was measured at different time point post ischemia. The animals were killed at day 84 after IRI. (A) Changes of urinary AGT levels. (B) Quantitative data of tubulointerstitial Co I staining at day 84 after IRI. (C) Tubulointerstitial fibrosis score at day 84 after IRI. (D) Representative photos of tubulointerstitial fibrosis showed by Masson staining (MS) and immunohistochemical staining of collagen I (Co I) at day 84 after IRI. (E and F) Urinary AGT level at day 14 after IRI correlated with accumulation of collagen I (E) and tubulointerstitial fibrosis score (F) at day 84 after IRI. (G) Urinary AGT level at day 14–42 after IRI correlated with tubulointerstitial fibrosis score at day 84 after IRI. (H and I) Receive-operating characteristic curve analysis to determine the performance of urinary AGT at day 14–42 for predicting renal fibrosis at day 84; scale bar = 100 µm. Data are expressed as mean ± SD (n = 8 in each study group). *P<0.05 versus pre-ischemic levels in (A). *P<0.05 versus sham mice in (B) and (C).

Persistent elevation in urinary AGT reflects sustained activation of intrarenal RAS

To investigate the relationship between urinary AGT and intrarenal RAS, intrarenal expression of AGT and Ang II was determined by immunohistochemistry staining in mice with mild (20-min IRI) and severe (45-min IRI) AKI at various time points after the acute phase of AKI. The trend of changes in intrarenal expression of AGT and Ang II was similar to that in urinary AGT. Up-regulation of AGT and Ang II in the kidney had returned to baseline at day 14 after mild IRI, but they remained elevated until day 84 after severe injury (Figure 4A–C). Overexpression of AGT in the kidney was observed predominantly in proximal tubules (Figure 5A), while staining of Ang II was detected mainly in ascending thick limbs of Henle’s loop, distal convoluted tubules, and collecting ducts (Figure 5B). This contrasts with immunodetectable renin expression that was confined to the juxtaglomerular apparatus and decreased at day 42 and 84 after severe IRI (Supplementary Figure S4A–C). The expression pattern of intrarenal RAS after IRI was further confirmed by the changes of AGT and Ang II levels in kidney homogenates (Figure 4D,E and Supplementary Figure S7B). Staining score of intrarenal AGT and Ang II closely correlated with level of urinary AGT (Figure 4F,G).

Persistent elevation in urinary AGT reflects sustained activation of intrarenal RAS

Figure 4
Persistent elevation in urinary AGT reflects sustained activation of intrarenal RAS

(A) Representative photos of intrarenal RAS expression showed by immunohistochemical staining of angiotensinogen (AGT) and angiotensin II (Ang II) in mice with mild and severe AKI. (B) Quantitative data of AGT staining. (C) Quantitative data of Ang II staining. (D) Expression of intrarenal AGT in homogenates of renal cortex measured by Western blot. Full annotated Westerns are provided in Supplementary Figure S7B. (E) The concentration of Ang II in homogenates of renal cortex. (F) Correlation between intrarenal AGT expression and urinary AGT. (G) Correlation between intrarenal Ang II expression and urinary AGT; scale bar = 100 µm. Data are expressed as mean ± SD (n = 8 in each study group). *P<0.05 versus sham mice at the same time point.

Figure 4
Persistent elevation in urinary AGT reflects sustained activation of intrarenal RAS

(A) Representative photos of intrarenal RAS expression showed by immunohistochemical staining of angiotensinogen (AGT) and angiotensin II (Ang II) in mice with mild and severe AKI. (B) Quantitative data of AGT staining. (C) Quantitative data of Ang II staining. (D) Expression of intrarenal AGT in homogenates of renal cortex measured by Western blot. Full annotated Westerns are provided in Supplementary Figure S7B. (E) The concentration of Ang II in homogenates of renal cortex. (F) Correlation between intrarenal AGT expression and urinary AGT. (G) Correlation between intrarenal Ang II expression and urinary AGT; scale bar = 100 µm. Data are expressed as mean ± SD (n = 8 in each study group). *P<0.05 versus sham mice at the same time point.

Localization of activated RAS in renal tubules in IRI mice with AKI-CKD transition

Figure 5
Localization of activated RAS in renal tubules in IRI mice with AKI-CKD transition

(A) Representative photographs of angiotensinogen (AGT) localization determined with double staining of antibody against AGT, and antibodies against markers of renal tubular segments. (B) Representative photographs of angiotensin II (Ang II) localization determined with double staining of antibody against Ang II, and antibodies against markers of renal tubular segments; scale bar = 100 µm. AQP-1, aquaporin 1 (proximal tubule); AQP-2, aquaporin 2 (collecting duct); NCCT, thiazide-sensitive NaCl cotransporter (distal tubule); THP-1, Tamm–Horsfall protein 1 (thick ascending limb).

Figure 5
Localization of activated RAS in renal tubules in IRI mice with AKI-CKD transition

(A) Representative photographs of angiotensinogen (AGT) localization determined with double staining of antibody against AGT, and antibodies against markers of renal tubular segments. (B) Representative photographs of angiotensin II (Ang II) localization determined with double staining of antibody against Ang II, and antibodies against markers of renal tubular segments; scale bar = 100 µm. AQP-1, aquaporin 1 (proximal tubule); AQP-2, aquaporin 2 (collecting duct); NCCT, thiazide-sensitive NaCl cotransporter (distal tubule); THP-1, Tamm–Horsfall protein 1 (thick ascending limb).

Urinary AGT serves as an indicator for predicting treatment response

To examine whether urinary AGT is responsive to treatment, urinary AGT was monitored in 45-min IRI mice with losartan treatment starting at different stages of AKI-CKD transition (illustrated in Figure 6A). Animals were killed after losartan treatment for histological assessment.

Urinary AGT serves as an indicator for predicting treatment response

Figure 6
Urinary AGT serves as an indicator for predicting treatment response

(A) Adult mice were treated with PBS or losartan (LOS, 150 mg/kg per day) for 42 days, starting from 14, 28, or 42 days post 45-min ischemia, respectively. (B) Representative photos of Masson staining (MS) and immunohistochemical staining of collagen I (Co I) at day 42 post treatment. (C) Tubulointerstitial fibrosis score at day 42 post treatment. (D) Quantitative data of Co I staining at day 42 post treatment. (E) Changes of urinary AGT post treatment. (F and G) Urinary AGT level at day 14 post LOS treatment correlated with tubulointerstitial fibrosis score (F) and accumulation of collagen I (G) at day 42 post LOS treatment. (H) Urinary AGT level at day 14–28 post LOS treatment correlated with tubulointerstitial fibrosis score at day 42 post LOS treatment; scale bar = 100 µm. Data are expressed as mean ± SD (n = 10 in each study group). *P<0.05 versus 45-min IRI mice treated with vehicle (PBS).

Figure 6
Urinary AGT serves as an indicator for predicting treatment response

(A) Adult mice were treated with PBS or losartan (LOS, 150 mg/kg per day) for 42 days, starting from 14, 28, or 42 days post 45-min ischemia, respectively. (B) Representative photos of Masson staining (MS) and immunohistochemical staining of collagen I (Co I) at day 42 post treatment. (C) Tubulointerstitial fibrosis score at day 42 post treatment. (D) Quantitative data of Co I staining at day 42 post treatment. (E) Changes of urinary AGT post treatment. (F and G) Urinary AGT level at day 14 post LOS treatment correlated with tubulointerstitial fibrosis score (F) and accumulation of collagen I (G) at day 42 post LOS treatment. (H) Urinary AGT level at day 14–28 post LOS treatment correlated with tubulointerstitial fibrosis score at day 42 post LOS treatment; scale bar = 100 µm. Data are expressed as mean ± SD (n = 10 in each study group). *P<0.05 versus 45-min IRI mice treated with vehicle (PBS).

Renal fibrosis was improved in all groups after losartan treatment (Figure 6B,C), with a more pronounced improvement in groups with RAS intervention initiation at early compared with advanced stages of AKI-CKD transition. This coincided with less collagen I accumulation and higher GFR in groups with early compared with delayed onset of losartan treatment (Figure 6B,D; Supplementary Figure S5A,B and S7C).

More interestingly, urinary AGT generally reduced after RAS intervention (Figure 6E and Supplementary Figure S5C), and its level at day 14 post losartan treatment correlated with the extent of renal fibrosis and accumulation of Co I at day 42 post losartan treatment (Figure 6F,G; P<0.001). Significant correlation was observed between the urinary AGT at day 14–28 and tubulointerstitial fibrosis score at day 42 post losartan treatment (Figure 6H).

Changes of urinary AGT in human acute tubular necrosis

To further illustrate the utility of urinary AGT in human AKI-CKD progression, 20 patients with biopsy-proven ATN and 20 age-and gender-matched healthy volunteers were included. The patient characteristics were presented in Supplementary Table S1.

Patients with ATN exhibited significant higher level of urinary AGT and albumin at the day of biopsy compared with healthy controls (Supplementary Figure S6A and B). Supplementary Figure S6D and E displayed serial measures of urinary AGT and albumin during the 90-day follow-up after biopsy. We found a significant higher level of urinary AGT at almost all-time points during the follow-up in the group with CKD progression, compared with those without CKD progression (P<0.05, Supplementary Figure S6D). Urinary albumin fell and did not differ significantly at any time point in patients with or without CKD progression (Supplementary Figure S6E). In addition, serum AGT levels were comparable between patients with and without AKI-CKD progression (Supplementary Figure S6C,F). While increased immunoreactivity of AGT and Ang II was noted in renal biopsies from ATN patients, and the extent of their expression correlated with urinary AGT level at the day of biopsy (Supplementary Figure S6G–I).

Discussion

One of the major obstacles to prevent AKI-CKD transition is the lack of effective method to follow and predict the ongoing kidney injury in AKI survivors. Currently, evaluation of kidney injury can be only obtained in invasive renal biopsies. Therefore, it is difficult to dynamically monitor the renal histological changes, which may allow for identification of progressive kidney injury and facilitation of personalized post-AKI treatment in clinical circumstances. In the present study, we demonstrate that the level of urinary AGT paralleled the status of kidney RAS and correlated with the degree of renal architectural restoration post an AKI episode. Urinary AGT decreased after RAS intervention, and its level determined early after RAS intervention correlated with the extent of renal structural recovery post RAS intervention. Therefore, tracking urinary AGT after the acute insult predicts the ensuing development of progressive renal fibrosis and could be used to evaluate treatment response. A pilot study conducted in patients with ATN indicates the utility of urinary AGT for predicting AKI-CKD progression in human AKI.

We first demonstrate that sustained elevation in urinary AGT post an AKI episode predicts subsequent CKD progression before rises in serum creatinine and urinary albumin excretion. We previously reported urinary AGT as an indicator of severity of renal histology in AKI [8]. However, the relationship between urinary AGT and renal histological changes following the acute insult has not been established. Here, we characterized the dynamics of urinary AGT in vivo in mice with different severity of AKI. Through this analysis, we found that changes in urinary AGT positively correlated with the likelihood of fibrotic outcome. A transient increase in urinary AGT post mild AKI associated with complete recovery in renal function and structure. While a sustained increase in urinary AGT level after severe AKI associated with incomplete repair indicated by progressive renal fibrosis. The elevated level of urinary AGT at day 14–42 was significantly related to the extent of renal fibrosis 84 days after AKI. Therefore, urinary AGT, determined at early stage of AKI-CKD transition, predicts maladaptive repair, thereby a fibrotic outcome. Early measuring and tracking urinary AGT post AKI may provide clinicians an essential time window to halt or reverse ongoing renal injury.

One interesting finding is that persistent elevated level of urinary AGT indicated lasting activation of intrarenal RAS in mice with CKD progression after AKI. In the present study, we examined the AGT level in the urine, serum, and kidney simultaneously. Only intrarenal expression of AGT and Ang II strongly correlated with urinary AGT level, suggesting that the increase in urinary AGT excretion may potentially originate from the enhanced production of AGT in the kidney. Consistently, intrarenal AGT, an important substrate of Ang II, has been mainly detected in proximal tubular cells, which makes its secretion to the urine possible [25]. This increased synthesis of AGT in the kidney under pathological conditions can be mediated through a vicious circle from Ang II stimulation [26,27]. As such, urinary AGT can be served as a marker of intrarenal RAS status during AKI-CKD transition. Similar findings are also observed in subjects with CKD [6,28], AKI [8], polycystic disease [29,30], and type 2 diabetes [31,32].

Persistent activation of intrarenal RAS during AKI-CKD progression suggests a potential benefit of RAS blockade in AKI survivors who are at higher risk of progressive CKD. RAS blockers, however, have been considered to exacerbate AKI by interfering with Ang II-dependent efferent glomerular arteriole vasoconstriction [33,34], and RAS inhibition currently is not recommended in this acute setting. Here, by altering the reperfusion time (14–42 days) post 45-min ischemia, we established models at different stages of AKI-CKD progression. RAS intervention was performed at least 14 days after ischemia, thus the initial acute kidney injury was not affected by this treatment (by serum creatinine, urinary albumin excretion, and acute kidney injury score indicated in Figure 1A,B and Supplementary Figure S1A). Blockade of RAS post the acute phase of AKI significantly slowed down the ensuing progressive CKD, with early RAS intervention is more beneficial than late intervention in delaying CKD progression. These results are consistent with recent observational studies [35,36] that RAS intervention post an AKI episode associated with a lower 1-year mortality and a decreased risk of CKD progression, and suggest that RAS blockade should be initiated early after the acute insult in high-risk subjects to avoid the involvement of Ang II in the maladaptive renal reparation [9,37] in long term.

More importantly, we demonstrate that urinary AGT is response to treatment. Since we monitored urinary AGT before and during losartan treatment, urinary AGT level and its relationship with renal recovery after treatment could be evaluated. Urinary AGT generally reduced after RAS intervention, and its level determined early after RAS intervention correlated with the extent of renal structural recovery post RAS intervention. Therefore, it is conceivable to speculate that urinary AGT is predictive of treatment response and may be helpful to guide the initiation, monitoring, and titration of novel AKI-CKD therapies.

Monitoring of urinary AGT could be beneficial in clinical practice. In the present study, we evaluated the usefulness of urinary AGT in predicting AKI-CKD progression in patients with ATN. The increase in urinary AGT level at the time of biopsy directly reflected enhanced activity of intrarenal RAS. Importantly, compared with those who recovered, AKI survivors with CKD progression showed elevated urinary AGT in advance of urinary albumin during the first 3-month follow-up after biopsy. In clinical reality, identifying patients at risk of AKI is difficult [38,39]. Most patients, when visiting the outpatient department, have presented with the established disease [40,41]. Furthermore, not all AKI survivors are at equal risk of CKD progression. Therefore, there is a critical need to discover biomarkers able to identify AKI survivors likely to develop progressive CKD. Our present study, including the pilot study in a relatively small sample size, shows the potential use of urinary AGT in predicting renal structural recovery over time after AKI. Dynamically detection of urinary AGT may be useful to early risk-stratify patients and predict renal outcome, which would provide an opportunity for earlier intervention and would be helpful to develop renal-preserving managements. Future studies in a larger cohort of population are required to extend our observations.

In sum, urinary AGT, determined at early stage of AKI-CKD progression, correlates with the degree of renal architectural restoration. Urinary AGT generally reduced after RAS intervention. The level of urinary AGT determined early after RAS intervention correlated with the extent of renal structural recovery post RAS intervention. Tracking urinary AGT following an AKI episode predicts the subsequent development of CKD with excellent performance and could be helpful to evaluate treatment response. The results of our study justify the future clinical studies for the transition of AKI to CKD.

Clinical perspectives

  • One of the major obstacles to prevent AKI-CKD transition is the lack of effective method to follow and predict the renal structural changes following an episode of AKI.

  • Here, our study suggests that urinary AGT enables the evaluation of renal structural recovery over time in IRI mice, tracking urinary AGT following an AKI episode predicts the subsequent development of CKD with excellent performance and could be helpful to evaluate treatment response.

  • Since not all AKI survivors are at equal risk of CKD progression, monitoring urinary AGT may be useful to early risk-stratify patients and predict renal outcome, which would provide an opportunity for earlier intervention and would be helpful to develop renal-preserving managements.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China [81570619 and 81870473 (to W.C.)].

Author Contribution

S.C. and L.L.W. performed the animal experiments and analyzed the data. X.D.F., H.J.S., Z.M.Z., W.H.L., M.S., and Z.C.Y. performed histology and biochemical experiments. X.D.F., C.L.S., and Y.J.L. conducted the clinical study and collected human samples. W.C. designed and financed the study, wrote and edited the manuscript.

Abbreviations

     
  • AGT

    angiotensinogen

  •  
  • AKI

    acute kidney injury

  •  
  • Ang

    angiotensin

  •  
  • ATN

    acute tubular necrosis

  •  
  • CKD

    chronic kidney disease

  •  
  • GFR

    glomerular filtration rate

  •  
  • IOD

    integrated optical density

  •  
  • IRI

    ischemia reperfusion injury

  •  
  • JGA

    juxta-glomerular apparatus

  •  
  • LC/MS

    liquid chromatography/mass spectrometry

  •  
  • RAS

    renin–angiotensin system

  •  
  • ROC

    receiver operating characteristics

  •  
  • uAGT

    urinary angiotensinogen

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Author notes

*

These authors contributed equally to this work.