Plasma and urine gelatinase-associated lipoxin (NGAL) with acute kidney injury or chronic kidney disease.

Gelatinase-associated lipokine (NGAL) is a protein used in human medicine as a real-time indicator of acute kidney injury (AKI). dogs and dogs with chronic kidney disease (Toil).
18 healthy control dogs, 17 dogs with chronic kidney disease, and 48 dogs with rheumatoid arthritis.
Methods
Over the course of one year, all dogs with renal blood nitrogenase were prospectively included. Urine and plasma samples were collected within the first 24 hours after presentation or after the development of renal azotemia. Plasma and urine NGAL concentrations were measured using a commercially available NGAL Elisa kit (Bioporto® Diagnostic) and UNCR was calculated. Plasma inulin clearance was performed with a single injection in healthy dogs.
The mean plasma concentration of NGAL (range) in healthy dogs, dogs with CKD and AKI was 10.7 ng/ml (2.5-21.2), 22.0 ng/ml (7.7-62.3 ) and 48.3 ng/ml (5.7-469.0), respectively. UNCR was 2 × 10 (-8) (0-46), 1424 × 10 (-8) (385-18,347), and 2366 × 10 (-8) (36-994,669), respectively. Affected dogs with renal blood nitrogen had significantly higher NGAL and UNCR concentrations than healthy dogs (P < 0.0001 for both). Plasma LAGN concentration was significantly higher in dogs with AKI than in dogs with chronic kidney disease (P = 0.027).

Plasma NGAL may be useful in differentiating between AKI and CKD in dogs with renal blood nitrogen.

Urinary gelatinase-associated lipocalin (NGAL) as a biomarker of acute kidney injury in dogs

Biomarkers for early prediction of canine acute kidney injury (AKI) are clinically important. Recently, neutrophil gelatinase-associated lipocalinase (NGAL) was found to be a sensitive biomarker for predicting very early-stage human AKI and progression of postoperative AKI. However, NGAL has not yet been studied in relation to kidney disease in dogs. In this study, the application of NGAL to dogs with AKI was investigated.

The canine NGAL gene has been successfully cloned and expressed. Polyclonal antibodies against NGAL for dogs were generated and used to develop an ELISA to measure NGAL protein in serum and urine samples collected from 39 dogs at different times after surgery.

AKI was defined by the standard method, that is, an increase in serum creatinine greater than or equal to 26.5 μmol/L from baseline within 48 hours. At 12 h after surgery, compared to the non-AKI group (12 dogs), the level of LAGN in the urine of seven dogs with AKI was significantly increased (mean 178.4 pg/mL vs. 88.0 pg/mL). /ml). /ml), and this difference persisted 72 hours

Because the increase in NGAL occurred long before the increase in serum creatinine, urinary NGAL appears to be able to serve as a sensitive and specific biomarker to predict AKI in dogs.

Acute kidney injury (AKI) shows a high mortality rate and occurs in patients undergoing cardiac or noncardiac surgery [1], and among those in the intensive care unit (ICU). However, due to the lack of a consensus definition of the severity of acute renal failure, early recognition of acute renal failure has been difficult. In the past, the Acute Dialysis Quality Initiative (ADQI) group published the RIFLE criteria in which the severity of acute renal failure was classified into risk, injury, and failure based on changes in blood creatinine and urine output; Using this approach, chronic renal failure has been classified into wasting and end-stage [2]. Recently, the Acute Kidney Injury Network (AKIN) modified these criteria to increase sensitivity and recommended that a small change in serum creatinine (greater than 26.5 μmol/L from baseline) within 48 hours be recognized as AKI in stage I [3]. Although there are no reference standards for AKI in veterinary medicine, the application of human standards has been validated as applicable to dogs with AKI [4, 5].

According to the consensus definition, the prediction of AKI is improved. Currently, rheumatoid arthritis is diagnosed primarily on the basis of an elevated serum creatinine level. However, serum creatinine can be affected by age, gender, muscle mass, and hydration status; Furthermore, it only increases after a loss of renal function of more than 50% [2]. Furthermore, an increase in serum creatinine may not be a real-time indicator of kidney damage, as it takes days to reach a steady state between serum creatinine production and decreased serum creatinine excretion [6, 7]. . Because AKI is important not only in humans but also in veterinary medicine, there is a need for other biomarkers that can be used to indicate AKI at an early stage.

Neutrophil gelatinase-associated lipocalin (NGAL, also called lipocalin 2 or 24p3), a small 25-kDa protein belonging to the lupuscalin family, is highly expressed during ischemic kidney injury in animal models [8]. Recently, several studies have indicated that abnormal cerebrospinal fluid in blood and urine are sensitive and specific biomarkers for predicting AKI [9-11], as well as the development of acute renal failure in human patients after surgery, such as cardiac surgery [ 12 ].- 15], non-cardiac surgery [16], kidney transplant [17, 18] and in trauma patients [19], as well as when there is IgA nephropathy [20].

However, the role of NGAL has not been studied in dogs. Therefore, the aim of this study was to determine whether NGAL could be used for early prediction of AKI in dogs. In this work, we first generated an ELISA that allows the measurement of NGAL in dogs. In addition, the validity of serum NGAL and urine NGAL for early identification of AKI in postoperative dogs was determined.

Amplification of LAGN (cDNA)

Mammary gland tumors (MGTs) with a high degree of NGAL have been reported, therefore, to clone the NGAL gene sequence, total RNA from MGT was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. . . First strand cDNA was synthesized by SuperScript III reverse transcriptase (Invitrogen) using RNA (1 µg) and primers (50 µM). NGAL was then amplified by polymerase chain reaction (PCR) using a primer designed according to GenBank entry no. XM_548441 (NGAL-F: 5′-ATGACCCAAGTTCTCTCTGTG; NGAL-R: 5′-TCACTCATCAATGCACTGGTC). Thermal cycling conditions began with 94 °C for 5 min, followed by 35 cycles of thermal denaturation at 94 °C for 30 s, primer annealing at 60 °C for 30 s, DNA stretching at 72 °C for 30 s, and final extension at 72 °C for 5 min. The resulting PCR product was isolated at the expected size and cloned into the TOPO 2.1 PCR vector (Invitrogen), designated c-NGAL/TOPO. The identity of NGAL was verified by automated sequencing (Mission Biotech, Taipei, Taiwan).

Construction of a plasmid expressing recombinant NGAL
Initially, the canine NGAL gene was amplified by PCR from the plasmid c-NGAL/TOPO using the primer set F-BamH: 5′-aggatccaatgacccaagttctcctg-3′ and R-Xho: 5′-ttctcgagctcatcaatgcactggtc-3′. PCR conditions were similar to those used for LAGN amplification from tissues, except for primer annealing at 49°C. The PCR product was then digested with BamHI/XhoI and cloned into pET32b. The resulting plasmid was confirmed by bidirectional sequencing.

Preparation of the recombinant NGAL dog
Protein expression was performed in Escherichia coli. strain BL21 AI (Invitrogen) following the procedures described previously [22]. Briefly, protein expression was induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG, 0.8 mM) and 0.2% L-arabinose (CALBIOCHEM) at 16 °C for 24 h. Bacterial pellets were suspended in lysis buffer (500 mM NaCl, 500 mM HCl, 20 mM imidazole, and 8 M urea; pH 7.4) followed by three cycles of 30-second sonication (sonic deuterator 550; Fisher Scientific)). . After a second centrifugation, the cell supernatant containing the recombinant proteins was recovered and purified using a Sepharose Fast Flow clutch (GE Healthcare) following the manufacturer’s instructions. Finally, bound protein was filtered into 4 mL of elution buffer (0.05 M Tris-HCl, 0.5 M NaCl, 400 mM imidazole, and 8 M urea, pH 7.4) and then washed with 1x PBS at 4 °C to remove excess imidazole. and urea.

western transfer
The purified proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoresed on a nitrocellulose membrane. Western blot analysis procedures followed those of a previous report [23]. Briefly, after a blocking step in PBS containing 0.1% Tween-20 (PBST) and 5% skim milk powder for 1 h at room temperature, membranes were incubated with 1:5000 diluted anti-label antibody (AbD Serotec) or 1:500 diluted rabbits immunized with recombinant NGAL in PBST-5% milk powder at 4°C overnight. The filter was then washed in PBST, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody in PBST-2% dry milk at room temperature for 1 hour. After extensive washing with PBST, the filter was developed using an enzyme-linked chemiluminescence system (ECL, Amersham, GE Healthcare) and exposed to X-ray film.

Production of antibodies against NGAL
Three eight week old BALB/c mice and one three month old New Zealand white rabbit were vaccinated with 50 µg NGAL/mouse (200 µg/rabbit) mixed with complete Freund’s adjuvant (Sigma). After the initial immunization, two boosters of the same dose were given every 2 weeks. Blood was collected from the rat submandibular vein by needle puncture and from the rabbit ear vein. After centrifugation, the plasma was transferred to a fresh tube and stored at -20°C until use. The study was approved by the Institutional Animal Care and Use Committee of Chongqing National University, Permit Number: 100-66.