Chemokine Therapy in Cats With Experimental Renal Fibrosis and in a Kidney Disease Pilot Study

Abstract

Background: Chronic tubulointerstitial fibrosis is a common final pathway leading to end stage kidney disease in cats and has no effective treatment. The use of cell-based molecules to treat kidney fibrosis may be a promising approach. The objectives were to test the effects of intra-renal chemokine CXCL12 injection in a pre-clinical cat model of unilateral ischemia/reperfusion (I/R)-induced kidney fibrosis and then, within a clinical pilot study, test the safety/feasibility of CXCL12 injection in cats that might have early chronic kidney disease (CKD). Methods: Pre-clinical: Thirty cats received intra-renal injection of 100, 200, or 400 ng of recombinant human CXCL12, or sterile saline, into the I/R kidney 70 days post-injury, or were non-injured, non-injected controls (n = 6/group). Kidney collagen content was quantified 4 months post-treatment using Masson's Trichrome and Picrosirius Red (PSR) stained tissues. In a separate study (n = 2) exploring short-term effects of CXCL12, 200 ng CXCL12 was injected into I/R kidneys and then harvested either 30 min (n = 1) or 1 month (n = 1) post-injection. Kidney concentrations of CXCL12, matrix metalloproteinase 1 (MMP-1), and lysyl oxidase-like enzyme 2 (LOXL-2) were quantified via ELISA. Clinical Pilot: 14 client-owned cats with potential early kidney disease received a single-treatment, bilateral intra-renal injection of 200 ng CXCL12 (n = 7), or received no injection (n = 7). Blood/urine samples were collected monthly for 9 months to assess renal function and CKD staging. Results: Pre-clinical: I/R increased the affected kidney collagen content, which both mid and high doses of CXCL12 restored to normal (ps < 0.05 vs. untreated). I/R increased collagen fiber width, which both mid and high doses of CXCL12 restored to normal (p < 0.001 vs. untreated). Early changes in kidney MMP-1, associated with collagen breakdown, and subsequent decreases in LOXL-2, associated with collagen cross-linking, in response to CXCL12 treatment may contribute to these findings. Clinical Pilot: Bilateral intra-renal injection of CXCL12 using ultrasound guidance in cats with CKD was feasible and safe in a general practice clinical setting with no obvious side effects noted during the 9-month follow-up period. Conclusions: Intra-renal injection of CXCL12 may prove to be an effective treatment for kidney fibrosis in cats with CKD. Additional mechanistic and clinical evaluations are needed.

Keywords: chemokine CXCL12; histomorphometry of collagen fibers; ischemia/reperfusion injury; local intra-renal injection; stromal cell-derived factor (SDF)-1α; tubulo- interstitial fibrosis.

Figure 1

Visual Abstract Summary Diagram.

Figure 2

(A) Schematic of pre-clinical study design and timeline using 5 groups of cats to test the effects of intra-renal injection of 100, 200, or 400 ng (n = 6/dose) of CXCL12, or sterile saline carrier (n = 6), administered 70 days after I/R-induced renal fibrosis. Six cats in a control group were uninjured and not injected. Baseline blood/urine renal function tests and ultrasounds were performed at the time of I/R Injury, Day 42, Day 70 (Treatment injection), and then monthly for 4 months post-injection. (B) Intraoperative images during I/R injury surgery with vascular clamps occluding left renal artery and left renal vein and purple discoloration of the kidney. Clamps remained in place for 65 min and then were released restoring renal blood flow that was confirmed using a Doppler probe to detect renal artery pulsation.

Figure 3

(A) Gross necropsy of left kidney for Control, I/R injured, and CXCL12 treated. I/R resulted in renal fibrosis (white discoloration) that was restored with treatment. (B) Histology of left kidney CMJ stained with Masson's Trichrome under 200× magnification for each study group. I/R (Carrier) increased collagen accumulation, stained blue. CXCL12 Treatment restored I/R-induced changes in collagen in a dose-dependent manner. (C) Masson's Trichrome stain image and cellSens Imaging Software-generated image used to calculate Area % of collagen (yellow). (D) One-way ANOVA for Area % of positive staining for collagen for each group on Masson's Trichrome stain showed a significant group effect, F_(4,25) = 8.408, p < 0.01. n = 6/group. Values are individual dot plot and mean ± SD. *p < 0.05.

Figure 4

(A) Histology of left kidney CMJ stained with PSR and PSR-POL under 100× magnification for each study group. I/R (Carrier) increased birefringent collagen fiber signal. CXCL12 Treatment restored I/R-induced changes in birefringent collagen fiber signal in a dose-dependent manner. (B) PSR-POL image used to calculate total collagen in pixels and segmentation software CT-FIRE-generated image used to quantify collagen fiber length and width. (C) One-way ANOVA for collagen fiber width for each group on PSR-POL showed a significant group effect, F_(4,25) = 36.634, p < 0.001; and (D) One-way ANOVA for total birefringent collagen signal in pixels for each group on PSR-POL showed a significant group effect, F_(4,25) = 15.165, p < 0.001. n = 6/group. Values are individual dot plot and mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Pre-clinical study clinical pathology results of renal function over time (Baseline, Day 70, Month 1, Month 2, Month 3, Month 4) for each group depicted as dot plot and mean ± SD showing: USG (A), serum creatinine (B), body weight (C), SDMA (D), serum BUN (E), and serum phosphorus (F). Average % change in body weight (kg) (G) and USG (H) over time (Month 1, Month 2, Month 3, Month 4) for each group when compared to day 70 post-injury (i.e., maximum injury), just prior to treatment injection. n = 6/group. Values are mean ± SD.