Advertisement

Combined use of electron microscopy and intravital imaging captures morphological and functional features of podocyte detachment

  • James L. Burford
  • Georgina Gyarmati
  • Isao Shirato
  • Wilhelm Kriz
  • Kevin V. Lemley
  • János Peti-PeterdiEmail author
Invited Review

Abstract

The development of podocyte injury and albuminuria in various glomerular pathologies is still incompletely understood due to technical limitations in studying the glomerular filtration barrier (GFB) in real-time. We aimed to directly visualize the early morphological and functional changes of the GFB during the development of focal segmental glomerulosclerosis (FSGS) using a combination of transmission electron microscopy (TEM) and in vivo multiphoton microscopy (MPM) in the rat puromycin aminonucleoside (PAN) model. We hypothesized that this combined TEM + MPM experimental approach would provide a major technical improvement that would benefit our mechanistic understanding of podocyte detachment. Male Sprague-Dawley (for TEM) or Munich-Wistar-Frömter (for MPM) rats were given a single dose of 100–150 mg/kg body weight PAN i.p. and were either sacrificed and the kidneys processed for TEM or surgically instrumented for in vivo MPM imaging at various times 2–14 days after PAN administration. Both techniques demonstrated hypertrophy and cystic dilatations of the subpodocyte space that developed as early as 2–3 days after PAN. Adhesions of the visceral epithelium to the parietal Bowman’s capsule (synechiae) appeared at days 8–10. TEM provided unmatched resolution of podocyte foot process remodeling, while MPM revealed the rapid dynamics of pseudocyst filling, emptying, and rupture, as well as endothelial and podocyte injury, misdirected filtration, and podocyte shedding. Due to the complementary advantages of TEM and MPM, this combined approach can provide an unusally comprehensive and dynamic portrayal of the alterations in podocyte morphology and function during FSGS development. The results advance our understanding of the role and importance of the various cell types, hemodynamics, and mechanical forces in the development of glomerular pathology.

Keywords

Podocyte Glomerulus Puromycin Glomerulosclerosis Intravital microscopy 

Notes

Acknowledgments

This work was supported in part by the US National Institutes of Health grants DK064324 and DK100944 to J.P-P.

Authors’ contributions

JPP and WK designed the experiments. JLB, GG, and IS performed the experiments and analyzed the data. JPP, KL, and WK interpreted the results and wrote the manuscript.

Compliance with ethical standards

The experimental protocols for the in vivo experiments were approved by the Institutional Animal Care and Use Committee of the University of Southern California; the structural studies were conducted in accordance with the National Research Council guidlines for the Care and Use of laboratory animals of Juntendo University, Tokyo.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

ESM 1

(MP4 1836 kb)

ESM 2

(MP4 666 kb)

ESM 3

(MP4 1277 kb)

ESM 4

(MP4 1702 kb)

ESM 5

(MP4 4155 kb)

References

  1. 1.
    Burford JL, Villanueva K, Lam L, Riquier-Brison A, Hackl MJ, Pippin J, Shankland SJ, Peti-Peterdi J (2014) Intravital imaging of podocyte calcium in glomerular injury and disease. J Clin Invest 124:2050–2058. doi: 10.1172/jci71702 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Dunn KW, Sandoval RM, Kelly KJ, Dagher PC, Tanner GA, Atkinson SJ, Bacallao RL, Molitoris BA (2002) Functional studies of the kidney of living animals using multicolor two-photon microscopy. Am J Physiol—Cell Physiol 283:C905–C916. doi: 10.1152/ajpcell.00159.2002 CrossRefPubMedGoogle Scholar
  3. 3.
    Eyre J, Ioannou K, Grubb BD, Saleem MA, Mathieson PW, Brunskill NJ, Christensen EI, Topham PS (2007) Statin-sensitive endocytosis of albumin by glomerular podocytes. Am J Physiol Renal Physiol 292:F674–F681. doi: 10.1152/ajprenal.00272.2006 CrossRefPubMedGoogle Scholar
  4. 4.
    Garsen M, Lenoir O, Rops AL, Dijkman HB, Willemsen B, van Kuppevelt TH, Rabelink TJ, Berden JH, Tharaux PL, van der Vlag J (2016) Endothelin-1 induces proteinuria by heparanase-mediated disruption of the glomerular glycocalyx. J Am Soc Nephrol 27:3545–3551. doi: 10.1681/asn.2015091070 CrossRefPubMedGoogle Scholar
  5. 5.
    Hackl MJ, Burford JL, Villanueva K, Lam L, Susztak K, Schermer B, Benzing T, Peti-Peterdi J (2013) Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags. Nat Med 19:1661–1666. doi: 10.1038/nm.3405 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hall AM, Schuh CD, Haenni D (2017) New frontiers in intravital microscopy of the kidney. Curr Opin Nephrol Hypertens 26:172–178. doi: 10.1097/mnh.0000000000000313 CrossRefPubMedGoogle Scholar
  7. 7.
    Inokuchi S, Sakai T, Shirato I, Tomino Y, Koide H (1993) Ultrastructural changes in glomerular epithelial cells in acute puromycin aminonucleoside nephrosis: a study by high-resolution scanning electron microscopy. Virchows Arch A Pathol Anat Histopathol 423:111–119CrossRefPubMedGoogle Scholar
  8. 8.
    Inokuchi S, Shirato I, Kobayashi N, Koide H, Tomino Y, Sakai T (1996) Re-evaluation of foot process effacement in acute puromycin aminonucleoside nephrosis. Kidney Int 50:1278–1287CrossRefPubMedGoogle Scholar
  9. 9.
    Kang JJ, Toma I, Sipos A, McCulloch F, Peti-Peterdi J (2006) Quantitative imaging of basic functions in renal (patho)physiology. Am J Physiol Renal Physiol 291:F495–F502. doi: 10.1152/ajprenal.00521.2005 CrossRefPubMedGoogle Scholar
  10. 10.
    Kim YH, Goyal M, Kurnit D, Wharram B, Wiggins J, Holzman L, Kershaw D, Wiggins R (2001) Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat. Kidney Int 60:957–968. doi: 10.1046/j.1523-1755.2001.060003957.x CrossRefPubMedGoogle Scholar
  11. 11.
    Kretzler M, Koeppen-Hagemann I, Kriz W (1994) Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat. Virchows Arch 425:181–193CrossRefPubMedGoogle Scholar
  12. 12.
    Kriz W, Gretz N, Lemley KV (1998) Progression of glomerular diseases: is the podocyte the culprit? Kidney Int 54:687–697. doi: 10.1046/j.1523-1755.1998.00044.x CrossRefPubMedGoogle Scholar
  13. 13.
    Kriz W, Hahnel B, Hosser H, Rosener S, Waldherr R (2014) Structural analysis of how podocytes detach from the glomerular basement membrane under hypertrophic stress. Front Endocrinol (Lausanne) 5:207. doi: 10.3389/fendo.2014.00207 Google Scholar
  14. 14.
    Kriz W, LeHir M (2005) Pathways to nephron loss starting from glomerular diseases-insights from animal models. Kidney Int 67:404–419. doi: 10.1111/j.1523-1755.2005.67097.x CrossRefPubMedGoogle Scholar
  15. 15.
    Kriz W, Lemley KV (2015) A potential role for mechanical forces in the detachment of podocytes and the progression of CKD. J Am Soc Nephrol 26:258–269. doi: 10.1681/asn.2014030278 CrossRefPubMedGoogle Scholar
  16. 16.
    Kriz W, Lemley KV (2017) Mechanical challenges to the glomerular filtration barrier: adaptations and pathway to sclerosis. Pediatr Nephrol 32:405–417. doi: 10.1007/s00467-016-3358-9 CrossRefPubMedGoogle Scholar
  17. 17.
    Kriz W, Lemley KV (2017) Potential relevance of shear stress for slit diaphragm and podocyte function. Kidney Int 91:1283–1286. doi: 10.1016/j.kint.2017.02.032 CrossRefPubMedGoogle Scholar
  18. 18.
    Kriz W, Shirato I, Nagata M, LeHir M, Lemley KV (2013) The podocyte’s response to stress: the enigma of foot process effacement. Am J Physiol Renal Physiol 304:F333–F347. doi: 10.1152/ajprenal.00478.2012 CrossRefPubMedGoogle Scholar
  19. 19.
    Le Hir M, Keller C, Eschmann V, Hahnel B, Hosser H, Kriz W (2001) Podocyte bridges between the tuft and Bowman’s capsule: an early event in experimental crescentic glomerulonephritis. J Am Soc Nephrol 12:2060–2071PubMedGoogle Scholar
  20. 20.
    Nagata M, Kriz W (1992) Glomerular damage after uninephrectomy in young rats. II. Mechanical stress on podocytes as a pathway to sclerosis. Kidney Int 42:148–160CrossRefPubMedGoogle Scholar
  21. 21.
    Nakano D, Kobori H, Burford JL, Gevorgyan H, Seidel S, Hitomi H, Nishiyama A, Peti-Peterdi J (2012) Multiphoton imaging of the glomerular permeability of angiotensinogen. J Am Soc Nephrol 23:1847–1856. doi: 10.1681/asn.2012010078 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Neal CR, Crook H, Bell E, Harper SJ, Bates DO (2005) Three-dimensional reconstruction of glomeruli by electron microscopy reveals a distinct restrictive urinary subpodocyte space. J Am Soc Nephrol 16:1223–1235. doi: 10.1681/asn.2004100822 CrossRefPubMedGoogle Scholar
  23. 23.
    Pavenstadt H, Kriz W, Kretzler M (2003) Cell biology of the glomerular podocyte. Physiol Rev 83:253–307. doi: 10.1152/physrev.00020.2002 CrossRefPubMedGoogle Scholar
  24. 24.
    Peti-Peterdi J, Burford JL, Hackl MJ (2012) The first decade of using multiphoton microscopy for high-power kidney imaging. Am J Physiol Renal Physiol 302:F227–F233. doi: 10.1152/ajprenal.00561.2011 CrossRefPubMedGoogle Scholar
  25. 25.
    Peti-Peterdi J, Kidokoro K, Riquier-Brison A (2015) Novel in vivo techniques to visualize kidney anatomy and function. Kidney Int 88:44–51. doi: 10.1038/ki.2015.65 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Peti-Peterdi J, Kidokoro K, Riquier-Brison A (2016) Intravital imaging in the kidney. Curr Opin Nephrol Hypertens 25:168–173. doi: 10.1097/mnh.0000000000000219 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Peti-Peterdi J, Sipos A (2010) A high-powered view of the filtration barrier. J Am Soc Nephrol 21:1835–1841. doi: 10.1681/asn.2010040378 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Qi H, Casalena G, Shi S, Yu L, Ebefors K, Sun Y, Zhang W, D'Agati V, Schlondorff D, Haraldsson B, Bottinger E, Daehn I (2017) Glomerular endothelial mitochondrial dysfunction is essential and characteristic of diabetic kidney disease susceptibility. Diabetes 66:763–778. doi: 10.2337/db16-0695 CrossRefPubMedGoogle Scholar
  29. 29.
    Rosivall L, Mirzahosseini S, Toma I, Sipos A, Peti-Peterdi J (2006) Fluid flow in the juxtaglomerular interstitium visualized in vivo. Am J Physiol Renal Physiol 291:F1241–F1247. doi: 10.1152/ajprenal.00203.2006 CrossRefPubMedGoogle Scholar
  30. 30.
    Salmon AH, Ferguson JK, Burford JL, Gevorgyan H, Nakano D, Harper SJ, Bates DO, Peti-Peterdi J (2012) Loss of the endothelial glycocalyx links albuminuria and vascular dysfunction. J Am Soc Nephrol : JASN 23:1339–1350. doi: 10.1681/asn.2012010017 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Salmon AH, Toma I, Sipos A, Muston PR, Harper SJ, Bates DO, Neal CR, Peti-Peterdi J (2007) Evidence for restriction of fluid and solute movement across the glomerular capillary wall by the subpodocyte space. Am J Physiol Renal Physiol 293:F1777–F1786. doi: 10.1152/ajprenal.00187.2007 CrossRefPubMedGoogle Scholar
  32. 32.
    Schiessl IM, Hammer A, Kattler V, Gess B, Theilig F, Witzgall R, Castrop H (2016) Intravital imaging reveals angiotensin II-induced transcytosis of albumin by podocytes. J Am Soc Nephrol 27:731–744. doi: 10.1681/asn.2014111125 CrossRefPubMedGoogle Scholar
  33. 33.
    Shirato I, Hosser H, Kimura K, Sakai T, Tomino Y, Kriz W (1996) The development of focal segmental glomerulosclerosis in masugi nephritis is based on progressive podocyte damage. Virchows Arch 429:255–273PubMedGoogle Scholar
  34. 34.
    Siegerist F, Zhou W, Endlich K, Endlich N (2017) 4D in vivo imaging of glomerular barrier function in a zebrafish podocyte injury model. Acta Physiol (Oxf) 220:167–173. doi: 10.1111/apha.12754 CrossRefGoogle Scholar
  35. 35.
    Sipos A, Toma I, Kang JJ, Rosivall L, Peti-Peterdi J (2007) Advances in renal (patho)physiology using multiphoton microscopy. Kidney Int 72:1188–1191. doi: 10.1038/sj.ki.5002461 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Smeets B, Uhlig S, Fuss A, Mooren F, Wetzels JF, Floege J, Moeller MJ (2009) Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells. J Am Soc Nephrol : JASN 20:2604–2615. doi: 10.1681/ASN.2009010122 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Sun YB, Qu X, Zhang X, Caruana G, Bertram JF, Li J (2013) Glomerular endothelial cell injury and damage precedes that of podocytes in adriamycin-induced nephropathy. PLoS One 8:e55027. doi: 10.1371/journal.pone.0055027 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Physiology and Biophysics, Zilkha Neurogenetic InstituteUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.Division of Nephrology, Department of Internal Medicine, School of MedicineJuntendo UniversityTokyoJapan
  3. 3.Centre for Biomedicine and Medical Technology Mannheim (CBTM), Neuroanatomy, Medical Faculty MannheimHeidelberg UniversityMannheimGermany
  4. 4.Division of NephrologyChildren’s Hospital Los AngelesCAUSA

Personalised recommendations