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Nephrin and podocin mRNA detection in urine sediment of dogs with chronic kidney disease: preliminary observations

Camilla de Souza
Camilla de Souza
, 
Mariana Coelho
Mariana Coelho
, 
Daniel Silva Antonelo
Daniel Silva Antonelo
, 
Danielle Passarelli
Danielle Passarelli
, 
Arina Lázaro Rochetti
Arina Lázaro Rochetti
, 
Heidge Fukumasu
Heidge Fukumasu
 oraz 
Deise Carla Almeida Leite-Dellova
Deise Carla Almeida Leite-Dellova
   | 22 kwi 2022
Journal of Veterinary Research's Cover Image
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Data publikacji: 22 kwi 2022
Zakres stron: 281 - 288
Otrzymano: 27 paź 2021
Przyjęty: 29 mar 2022
DOI: https://doi.org/10.2478/jvetres-2022-0019
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© 2022 C. de Souza et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Electrophoresis of total RNA in agarose gel (2%). Column 1 – total RNA isolated from mouse lung cells; Columns 2 and 3 – total RNA isolated from urine sediment of subject dogs. Ribosomal RNA bands (28S and 18S) were observed in all samples, indicating adequate RNA isolation. The genomic DNA band appeared in samples 1 and 2 because DNAse had not yet been used in the reaction
Electrophoresis of total RNA in agarose gel (2%). Column 1 – total RNA isolated from mouse lung cells; Columns 2 and 3 – total RNA isolated from urine sediment of subject dogs. Ribosomal RNA bands (28S and 18S) were observed in all samples, indicating adequate RNA isolation. The genomic DNA band appeared in samples 1 and 2 because DNAse had not yet been used in the reaction

Fig. 2

cDNA product electrophoresis in 1.5% agarose gel. Neg. – negative control; Column 1 – sample from a dog with stage 3 chronic kidney disease; Column 2 – sample from a dog of the control group; Column 3 – sample from a dog with stage 1 chronic kidney disease. The length of 18S was 80 bp, that of NPHS1 was 156 bp, that of NPHS2 was 140 bp, and the DNA ladder was 100 bp long
cDNA product electrophoresis in 1.5% agarose gel. Neg. – negative control; Column 1 – sample from a dog with stage 3 chronic kidney disease; Column 2 – sample from a dog of the control group; Column 3 – sample from a dog with stage 1 chronic kidney disease. The length of 18S was 80 bp, that of NPHS1 was 156 bp, that of NPHS2 was 140 bp, and the DNA ladder was 100 bp long

Results for blood haematological parameters in healthy dogs and dogs with chronic kidney disease (CKD) at different stages

CKD dogs
Parameter (reference range) Healthy dogs (n = 10) Stage 1 or 2 (n = 5) Stage 3 or 4 (n = 9)
RBC (106/μL) (5.5–8.5) 6.7 ± 0.35 6.7 ± 0.49 4.7 ± 0.36**
HGB (g/dL) (12–18) 17.3 ± 0.83 16.7 ± 1.17 11.6 ± 0.88**
HCT (%) (37–55) 50.1 ± 2.49 49.7 ± 3.52 34.9 ± 2.63**
MCV (fL) (60–77) 75.2 ± 1.49 75.0 ± 2.11 75.2 ± 1.57
MCH (pg) (21–26) 26.0 ± 0.58 25.2 ± 0.82 25.0 ± 0.61
MCHC (%) (32–36) 34.6 ± 0.44 33.7 ± 0.62 33.2 ± 0.47
PLT (103/μL) (200–500) 307 ± 57.60 386 ± 39.7 378 ± 64.0
RBC morphology normal normal normal
WBC (103/μL) (6–17) 7.1 ± 0.63 8.8 ± 1.35 7.5 ± 1.02
WBC morphology normal normal normal

Results for blood biochemical parameters in healthy dogs and dogs with chronic kidney disease (CKD) at different stages

CKD dogs
Parameter (reference range) Healthy dogs (n = 10) Stage 1 or 2 (n = 5) Stage 3 or 4 (n = 9)
Albumin (g/dL) (2.3–3.8) 3.2 ± 0.16 2.8 ± 0.23 2.7 ± 0.20
Urea (mg/dL) (10–60) 39.4 ± 5.36 47.3 ± 13.48 259.4 ± 46.70*
Creatinine (mg/dL) (0.5–1.4) 1.1 ± 0.14 1.0 ± 0.17 7.4 ± 2.34*

Semi-quantitative analysis of detection of NPHS1 (nephrin mRNA) and NPHS2 (podocin mRNA) in urine sediment samples from healthy dogs and dogs with chronic kidney disease (CKD) at different stages

CKD dogs
Parameter Healthy dogs Stage 1 or 2 Stage 3 or 4 P value
NPHS1 detection 4/10 (40%) a 3/5 (60%) b 1/9 (11%) c 0.0009
NPHS2 detection 2/10 (20%) a 4/5 (80%) b 1/9 (11%) a 0.0188

Results for routine urinary parameters in healthy dogs and dogs with chronic kidney disease (CKD) at different stages

CKD dogs
Parameter (reference range) Healthy dogs (n = 10) Stage 1 or 2 (n = 5) Stage 3 or 4 (n = 9)
USG (1.015–1.045) 1.037 ± 0.0040 1.022 ± 0.0074* 1.014 ± 0.0009**
Protein (mg/dL) 29.4 ± 2.44 210.6 ± 11.67*** 124.0 ± 35.76*
Creatinine (mg/dL) 206.1 ± 24.00 94.4 ± 21.47** 70.0 ± 21.47**
UPC (< 0.5) 0.15 ± 0.01 2.25 ± 0.68* 1.76 ± 0.50*
Chemical analysis
(semiquantitative testing):
pH (6.0–7.5) 6.5 ± 0.37 6.4 ± 0.52 5.9 ± 0.39
Protein between 0 and 1+ between 1 and 3+ between 1 and 2+
Glucose negative negative negative
Ketones negative negative negative
Bilirubin negative negative negative
Urobilinogen normal normal normal
Occult blood negative 1+ in the urinary sediment of 1 dog 1+ in the urinary sediment of 2 dogs
Microscopic sediment
examination:
Epithelial cells/HPF (<3) 1.70 ± 0.26 1.00 ± 0.22 1.44 ± 0.43
Granular cast/HPF (<1) rare in the urinary sediment of 1 dog none rare in the urinary sediment of 1 dog
Triple phosphate crystal 1+ in the urinary sediment of 2 dogs 2+ in the urinary sediment of 1 dog none
Amorphous phosphate crystal none none 2+ in the urinary sediment of 1 dog
RBC/HPF (<5) 0.30 ± 0.13 0.30 ± 0.20 0.56 ± 0.49
WBC/HPF (<5) 0.50 ± 0.18 0.60 ± 0.25 0.83 ± 0.25
Bacteria none none none

Semi-quantitative analysis of renal ultrasonographic abnormalities in dogs with chronic kidney disease (CKD) at different stages

CKD dogs
Parameter Stage 1 or 2 Stage 3 or 4 P value
Irregular contour 5/5 (100%) 6/9 (67%) <0.0001
Loss of corticomedullary differentiation 4/5 (80%) 8/9 (89%) 0.1170
Increased cortical echogenicity 4/5 (80%) 8/9 (89%) 0.1170
Increased medullary echogenicity 0/5 5/9 (56%) 0.0197
eISSN:
2450-8608
Język:
Angielski
Częstotliwość wydawania:
4 razy w roku
Dziedziny czasopisma:
Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine
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