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

Souza, Camilla de; Coelho, Mariana; Antonelo, Daniel Silva; Passarelli, Danielle; Rochetti, Arina Lázaro; Fukumasu, Heidge; Leite-Dellova, Deise Carla Almeida

doi:10.2478/jvetres-2022-0019

Abstract

Introduction

Dogs with chronic kidney disease (CKD) may have alterations in the glomerular filtration barrier, including podocyte loss. Detection of podocyte mRNA in urine could be useful for assessing podocyturia in dogs with kidney disease. The objective of this study was to evaluate the presence of nephrin mRNA (NPHS1) and podocin mRNA (NPHS2) in urine sediments of dogs with naturally occurring CKD and healthy dogs.

Material and Methods

Twenty-four dogs, 14 with CKD and 10 as healthy controls, underwent clinical evaluation. The dogs with CKD were divided into two groups, according to the International Renal Interest Society criteria: stage 1 or 2 CKD (n = 5) and stage 3 or 4 CKD (n = 9). Urine was collected by catheterisation or free catch and RNA isolation from the urine sediments was optimised using glycogen as a co-precipitant. Detection of NPHS1 and NPHS2 in the sediment samples was performed using quantitative real-time PCR.

Results

Both types of mRNA were detected in samples from all groups, but the percentages of detection were higher in the group of dogs with stage 1 or 2 CKD and lower in the group of dogs with stage 3 or 4 disease.

Conclusion

Physiological podocyturia was observed in healthy dogs, and the results suggest differential podocyturia in dogs with CKD, according to the stage of the disease, i.e. an increase in podocyturia in dogs at stage 1 or 2 and a reduction in podocyturia in dogs at stage 3 or 4.

References

Bartges J.W.: Chronic kidney disease in dogs and cats. Vet Clin North Am Small Anim Pract 2012, 42, 669–692, doi: 10.1016/j.cvsm.2012.04.008.
Open DOI Search in Google Scholar Back to article
Bradley M.S., Boudreau M.H., Grenier C., Huang Z., Murphy S.K., Siddiqui N.Y.: Urine RNA Processing in a Clinical Setting: Comparison of Three Protocols. Female Pelvic Med Reconstr Surg 2019, 25, 247–251, doi: 10.1097/SPV.0000000000000525.
Open DOI Search in Google Scholar Back to article
Bragato N., Borges N.C., Fioravanti M.C.S.: B-mode and Doppler ultrasound of chronic kidney disease in dogs and cats. Vet Res Commun 2017, 41, 307–315, doi: 10.1007/s11259-017-9694-9.
Open DOI Search in Google Scholar Back to article
Chagnac A., Zingerman B., Rozen-Zvi B., Herman-Edelstein M.: Consequences of glomerular hyperfiltration: the role of physical forces in the pathogenesis of chronic kidney disease in diabetes and obesity. Nephron 2019, 143, 38–42, doi: 10.1159/000499486.
Open DOI Search in Google Scholar Back to article
Coady M., Fletcher D.J., Goggs R.: Severity of ionized hypercalcemia and hypocalcemia is associated with etiology in dogs and cats. Front Vet Sci 2019, 6, e276, doi: 10.3389/fvets.2019.00276.
Open DOI Search in Google Scholar Back to article
Crivellenti L.Z., Borin‐Crivellenti S., Fertal K.L., Contin C.M., Miranda C.M., Santana A.E.: Occult gastrointestinal bleeding is a common finding in dogs with chronic kidney disease. Vet Clin Pathol 2017, 46, 132–137, doi: 10.1111/vcp.12461.
Open DOI Search in Google Scholar Back to article
De Medeiros Torres M., Chitarra C.S., Nakazato L., de Almeida A.B.P.F., Sousa V.R.F.: Nephrin gene expression in chronic kidney disease of dogs with Leishmania (Leishmania) infantum chagasi. Braz J Infect Dis 2016, 20, 516–518, doi: 10.1016/j.bjid.2016.07.008.
Open DOI Search in Google Scholar Back to article
Dunaevich A., Chen H., Musseri D., Kuzi S., Mazaki‐Tovi M., Aroch I., Segev G.: Acute on chronic kidney disease in dogs: Etiology, clinical and clinicopathologic findings, prognostic markers, and survival. J Vet Intern Med 2020, 34, 2507–2515, doi: 10.1111/jvim.15931.
Open DOI Search in Google Scholar Back to article
Endlich N., Lange T., Kuhn J., Klemm P., Kotb A.M., Siegerist F., Kindt F., Lindenmeyer M.T., Cohen C.D., Kuss A.W., Nath N., Retting R., Lendeckel U., Zimmermann U., Amann K., Stracke S., Endlich K.: BDNF: mRNA expression in urine cells of patients with chronic kidney disease and its role in kidney function. J Cell Mol Med 2018, 22, 5265–5277, doi: 10.1111/jcmm.13762.
Open DOI Search in Google Scholar Back to article
Fukuda A., Minakawa A., Kikuchi M., Sato Y., Nagatomo M., Nakamura S., Mizoguchi T., Fukunaga N., Shibata H., Naik A.S., Wiggins R.C., Fujimoto S.: Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy. Sci Rep 2020, 10, 1–11, doi: 10.1038/s41598-020-75320-1.
Open DOI Search in Google Scholar Back to article
Green M.R., Sambrook J.: Precipitation of RNA with Ethanol. Cold Spring Harb Protoc 2020, doi: 10.1101/pdb.prot101717.
Open DOI Search in Google Scholar Back to article
Guo Y., Pace J., Li Z., Ma’ayan A., Wang Z., Revelo M.P., Chen E., Gu X., Attalah A., Yang Y., Estrada C., Yang V.W., He J.C., Mallipattu S.K.: Podocyte-specific induction of Krüppel-like factor 15 restores differentiation markers and attenuates kidney injury in proteinuric kidney disease. J Am Soc Nephrol 2018, 29, 2529–2545, doi: 10.1681/ASN.2018030324.
Open DOI Search in Google Scholar Back to article
Helal I., Fick-Brosnahan G.M., Reed-Gitomer B., Schrier R.W.: Glomerular hyperfiltration: definitions, mechanisms and clinical implications. Nat Rev Nephrol 2012, 8, 293–300, doi: 10.1038/nrneph.2012.19.
Open DOI Search in Google Scholar Back to article
Ichii O., Yabuki A., Sasaki N., Otsuka S., Ohta H., Yamasaki M., Takiguchi M., Nami Y., Hashimoto Y., Endoh D., Kon Y.: Pathological correlations between podocyte injuries and renal functions in canine and feline chronic kidney diseases. Histol Histopathol 2011, 26, 1243–1255, doi: 10.14670/HH-26.1243.
Open DOI Search in Google Scholar Back to article
International Renal Interest Society: IRIS Staging of CKD (modified 2019). International Renal Interest Society, Cambridge, 2019. http://www.iris-kidney.com/pdf/IRIS_Staging_of_CKD_modified_2019.pdf
Search in Google Scholar Back to article
Khan T.M., Khan K.N.M.: Acute kidney injury and chronic kidney disease. Vet Pathol 2015, 52, 441–444, doi: 10.1177/0300985814568358.
Open DOI Search in Google Scholar Back to article
Medyńska A., Chrzanowska J., Kościelska-Kasprzak K., Bartoszek D., Żabińska M., Zwolińska D.: Alpha-1 Acid Glycoprotein and Podocin mRNA as Novel Biomarkers for Early Glomerular Injury in Obese Children. J Clin Med 2021, 10, 4129, doi: 10.3390/jcm10184129.
Open DOI Search in Google Scholar Back to article
Monteiro M.B., Santos-Bezerra D.P., Thieme K., Passarelli M., Machado U.F., Lin C.J., Corrêa-Giannella M.L.: Optimization of total RNA isolation from human urinary sediment. Clin Chim Acta 2016, 462, 158–161, doi: 10.1016/j.cca.2016.09.018.
Open DOI Search in Google Scholar Back to article
Nagata D., Hishida E., Masuda T.: Practical Strategy for Treating Chronic Kidney Disease (CKD)-Associated with Hypertension. Int J Nephrol Renovasc Dis 2020, 13, 171–178, doi: 10.2147/IJNRD.S259931.
Open DOI Search in Google Scholar Back to article
Neal C.R.: Podocytes… What’s under yours? (Podocytes and foot processes and how they change in nephropathy). Front Endocrinol 2015, 6, e9, doi: 10.3389/fendo.2015.00009.
Open DOI Search in Google Scholar Back to article
O’Neill D.G., Elliott J., Church D.B., McGreevy P.D., Thomson P.C., Brodbelt D.C.: Chronic kidney disease in dogs in UK veterinary practices: prevalence, risk factors, and survival. J Vet Int Med 2013, 27, 814–821, doi: 10.1111/jvim.12090.
Open DOI Search in Google Scholar Back to article
O’Neill W.C.: Targeting serum calcium in chronic kidney disease and end-stage renal disease: is normal too high? Kidney Int 2016, 89, 40–45, doi: 10.1016/j.kint.2015.10.001.
Open DOI Search in Google Scholar Back to article
Perondi F., Lippi I., Marchetti V., Bruno B., Borrelli A., Citi S.: How Ultrasound Can Be Useful for Staging Chronic Kidney Disease in Dogs: Ultrasound Findings in 855 Cases. Vet Sci 2020, 7, e147, doi: 10.3390/vetsci7040147.
Open DOI Search in Google Scholar Back to article
Rudinsky A.J., Harjes L.M., Byron J., Chew D.J., Toribio R.E., Langston C., Parker V.J.: Factors associated with survival in dogs with chronic kidney disease. J Vet Int Med 2018, 32, 1977– 1982, doi: 10.1111/jvim.15322.
Open DOI Search in Google Scholar Back to article
Sato Y., Wharram B.L., Lee S.K., Wickman L., Goyal M., Venkatareddy M., Chang J.W., Wiggins J.E., Lienczewski C., Kretzler M., Wiggins R.C.: Urine podocyte mRNAs mark progression of renal disease. J Am Soc Nephrol 2009, 20, 1041– 1052, doi: 10.1681/ASN.2007121328.
Open DOI Search in Google Scholar Back to article
Siwińska N., Pasławska U., Bąchor R., Szczepankiewicz B., Żak A., Grocholska P., Szewczuk Z.: Evaluation of podocin in urine in horses using qualitative and quantitative methods. PLOS One 2020, 15, e0240586, doi: 10.1371/journal.pone.0240586.
Open DOI Search in Google Scholar Back to article
Szczepankiewicz B., Bąchor R., Pasławski R., Siwińska N., Pasławska U., Konieczny A., Szewczuk Z.: Evaluation of tryptic podocin peptide in urine sediment using LC-MS-MRM method as a potential biomarker of glomerular injury in dogs with clinical signs of renal and cardiac disorders. Molecules 2019, 24, e3088, doi: 10.3390/molecules24173088.
Open DOI Search in Google Scholar Back to article
Szczepankiewicz B., Pasławska U., Pasławski R., Gębarowski T., Zasada W., Michałek M., Noszczyk-Nowak A.: The urine podocin/creatinine ratio as a novel biomarker of cardiorenal syndrome in dogs due to degenerative mitral valve disease. J Physiol Pharmacol 2019, 70, 2, 229–238, doi: 10.26402/jpp.2019.2.06.
Open DOI Search in Google Scholar Back to article
Trimarchi H.: Podocyturia: What is in a name? J Transl Intern Med 2015, 3, 51–56, doi: 10.1515/jtim-2015-0003.
Open DOI Search in Google Scholar Back to article
Untergasser A., Nijveen H., Rao X., Bisseling T., Geurts R., Leunissen J.A.M.: Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 2007, 35, W71–W74, doi: 10.1093/nar/gkm306.
Open DOI Search in Google Scholar Back to article
Vogelmann S.U., Nelson W.J., Myers B.D., Lemley K.V.: Urinary excretion of viable podocytes in health and renal disease. Am J Physiol Renal Physiol 2003, 285, F40–F48, doi: 10.1152/ajprenal.00404.2002.
Open DOI Search in Google Scholar Back to article
Watson A.D.J., Lefebvre H.P., Elliott J.: Using urine specific gravity (revised 2015). International Renal Interest Society, Cambridge, 2015. http://www.iris-kidney.com/education/urine_specific_gravity.html
Search in Google Scholar Back to article
Wickman L., Afshinnia F., Wang S.Q., Yang Y., Wang F., Chowdhury M., Graham D., Hawkins J., Nishizono R., Tanzer M., Wiggins J.E., Escobar G.A., Rovin B., Song P., Gipson D., Kershaw D., Wiggins R.C.: Urine podocyte mRNAs, proteinuria, and progression in human glomerular diseases. J Am Soc Nephrol 2013, 24, 2081–2095, doi: 10.1681/ASN.2013020173.
Open DOI Search in Google Scholar Back to article