Divergent effects of AKI to CKD models on inflammation and fibrosis

Abstract

Chronic kidney disease (CKD) is a condition with significant morbidity and mortality that affects 15% of adults in the United States. One cause of CKD is acute kidney injury (AKI), which commonly occurs secondary to sepsis, ischemic events, and drug-induced nephrotoxicity. Unilateral ischemia-reperfusion injury (UIRI) without contralateral nephrectomy (CLN) and repeated low-dose cisplatin (RLDC) models of AKI to CKD demonstrate responses characteristic of the transition; however, previous studies have not effectively compared the pathogenesis. We demonstrate both models instigate renal dysfunction, inflammatory cytokine responses, and fibrosis. However, the models exhibit differences in urinary excretory function, inflammatory cell infiltration, and degree of fibrotic response. UIRI without CLN demonstrated worsening perfusion and function, measured with ^99mTc-mercaptoacetyltriglycine-3 imaging, and physiologic compensation in the contralateral kidney. Furthermore, UIRI without CLN elicited a robust inflammatory response that was characterized by a prolonged polymorphonuclear cell and natural killer cell infiltrate and an early expansion of kidney resident macrophages, followed by T-cell infiltration. Symmetrical diminished function occurred in RLDC kidneys and progressively worsened until day 17 of the study. Surprisingly, RLDC mice demonstrated a decrease in inflammatory cell numbers relative to controls. However, RLDC kidneys expressed increased levels of kidney injury molecule-1 (KIM-1), high mobility group box-1 ( HMGB1), and colony stimulating factor-1 ( CSF-1), which likely recruits inflammatory cells in response to injury. These data emphasize how the divergent etiologies of AKI to CKD models affect the kidney microenvironment and outcomes. This study provides support for subtyping AKI by etiology in human studies, aiding in the elucidation of injury-specific pathophysiologic mechanisms of the AKI to CKD transition.

Keywords: chronic kidney disease; cisplatin; fibrosis; glomerular filtration rate; inflammation; ischemia-reperfusion.

Fig. 1.

Renal function in unilateral ischemia-reperfusion injury without contralateral nephrectomy (UIRI without CLN) and repeated low-dose cisplatin models of acute kidney injury to chronic kidney disease. A and B: experimental design for UIRI without CLN (A) and cisplatin administration (9 mg/kg; B). Male C57BL/6 mice were used in all experiments. C: serum creatinine levels were measured and expressed as milligrams per deciliter as a time course from UIRI without CLN and cisplatin mice and their respective controls. D: glomerular filtration rate (GFR) was measured using transcutaneous FITC-sinistrin clearance over the course of the study in UIRI without CLN and cisplatin mice and their respective controls. Data represented as means ± SE; n = 3–8 per group. Statistical significance was determined by 2-way ANOVA followed by Sidak’s multiple comparisons test * P < 0.05; n = 3–8 per group.

Fig. 2.

^99mTc-mercaptoacetyltriglycine-3 (MAG3) renal function percent injected dose curves at varying time points during acute kidney injury to chronic kidney disease. A and B: ^99mTc-MAG3 imaging was performed to analyze renal function over the course of the study for unilateral ischemia-reperfusion injury (UIRI) without contralateral nephrectomy (CLN) (A) and repeated low-dose cisplatin (RLDC) (B). Background-corrected percent injected dose within individual kidneys are plotted as a function of time after IV administration of radioisotope. Varying time points after UIRI and cisplatin injury induction were tested. Representative still images, shown adjacent to graphs, were obtained from the first frame before signal saturation in the bladder (within 15–60 s of intravenous injection of ^99mTc-MAG3). If present, curvilinear opacities represent mouse tails at the site of injection. C and D: area under the curve (AUC) measurements for elimination curves fit to a 1-compartment model for UIRI without CLN (C) and RLDC (D). Two-way ANOVA with Tukey’s posttest: *P < 0.05 within time points; #P < 0.05 across time points; n = 4–5 kidneys for IR; n = 3 kidneys for sham, quantifying each kidney independently; n = 6–14 kidneys for cisplatin (3–7 mice quantifying both kidneys); n = 6–8 kidneys for saline (3–4 mice quantifying both kidneys).

Fig. 3.

^99mTc-mercaptoacetyltriglycine-3 physiologic parameters for kidney function related to perfusion and excretion. A–D: time to peak (A), peak percent injected dose (B), percent injected dose (%ID; C) during 15- to 45-s intervals after radioisotope injection, and peak:10-min ratio (D) were measured. Data represented as means ± SE. Two-way ANOVA with Tukey’s posttest: *P < 0.05 within time points; #P < 0.05 across time points. n = 4–5 for IR; n = 3 for sham; n = 6–14 for cisplatin (3–7 mice quantifying both kidneys); n = 6–8 for saline (3–4 mice quantifying both kidneys).

Fig. 4.

Intrarenal leukocytic populations in unilateral ischemia-reperfusion injury without contralateral nephrectomy and repeated low-dose cisplatin. Flow cytometry from whole kidney single cell suspensions normalized to mass of tissue. A: gating scheme. B: CD45⁺ leukocytes and neutrophils [polymorphonuclear cell (PMNs)]. C: F4/80^Hi resident macrophages and F4/80^Low infiltrating macrophages and dendritic cells. D: CD4 and CD8 T cells. E: B cells and natural killer (NK) cells. Black dots: sham controls collapsed from 7, 14, and 21 days; red dots: IR; blue dots: vehicle; orange dots: cisplatin. MHCII, major histocompatibility complex class II. Data represented as means ± SE. *P < 0.05 by one-way ANOVA and Dunnett’s posttest, comparing each time point vs. sham or vehicle controls; n = 5–9 for IR; n = 10 for sham; n = 3 for cisplatin and vehicle groups.

Fig. 5.

Proinflammatory cytokines and damage patterns are exacerbated in acute kidney injury to chronic kidney disease models. A–H: total RNA was isolated from whole kidney tissue at end points [ischemia-reperfusion (IR), 21 days; cisplatin (Cp), 24 days] and was analyzed for expression of interleukin-6 (IL-6; A), high mobility group box-1 (HMGB1; B), colony stimulating factor-1 (CSF-1; C), monocyte chemoattractant protein-1 (MCP-1; D), IL-1β (E), tumor necrosis factor-α (TNF-α; F), neutrophil gelatinase associated lipocalin (NGAL; G), and nitric oxide synthase-2 (NOS2; H) by real-time PCR. Data are normalized to GAPDH and expressed as fold change compared with sham or vehicle (veh) controls. Data are represented as means ± SE; n = 3–8 per group. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparisons test: *P < 0.05. I: protein lysates from whole kidney tissue were analyzed for kidney injury molecule-1 (KIM-1) and NGAL expression. Anti-GAPDH was used as a loading control. n = 2–5 per group. J and K: densitometric values were normalized to GAPDH and represented as arbitrary units (AU) for KIM-1 (J) and NGAL (K) using ImageJ. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparisons test: *P < 0.05; n = 2–5 per group.

Fig. 6.

Unilateral ischemia-reperfusion injury (UIRI) without contralateral nephrectomy (CLN) and repeated low-dose cisplatin (RLDC) demonstrate histopathologic signs of interstitial fibrosis at study end points. A: Picrosirius red (PSR) and periodic acid Schiff-hematoxylin (PASH) stained kidney transverse sections at low and high power. C, cyst-like space; arrow: perivascular and periglomerular fibrosis; arrowhead: anucleated epithelium; red arrowhead: cellular swelling. B: percent area above threshold for PSR. C: intensity of PSR stain encompassing positive staining areas above threshold. Statistical significance was determined by unpaired t-test: *P < 0.05; n = 2–8 per group.

Fig. 7.

Unilateral ischemia-reperfusion injury (UIRI) induces a more severe fibrotic response compared with cisplatin (Cp). Total RNA was isolated from whole kidney tissue at end points was analyzed for expression of fibronectin (Fn; A), transforming growth factor-β (TGF-β; B), collagen I (C), and α-smooth muscle actin (α-SMA; D) by real-time PCR. Data were normalized to GAPDH and expressed as fold change compared with sham or vehicle (veh) controls; n = 3–8 per group. E: protein lysates from whole kidney tissue were analyzed for Fn and α-SMA expression. Anti-GAPDH was used as a loading control; n = 2–5 per group. F and G: densitometric values were normalized to GAPDH and represented as arbitrary units (AU) for Fn (F) and α-SMA (G) using ImageJ. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparisons test: *P < 0.05; n = 2–5 per group.