
==== Front
J Nephrol
J Nephrol
Journal of Nephrology
1121-8428
1724-6059
Springer International Publishing Cham

38300433
1886
10.1007/s40620-024-01886-y
Research Letter
Perihilar FSGS lesions originate from flat parietal epithelial cells
Chia-Gil Arnaldo 1
Floege Jürgen 1
http://orcid.org/0000-0001-7472-4907
Stamellou Eleni stamellou.eleni@gmail.com
estamellou@ukaachen.de

12
Moeller Marcus J. 1
1 https://ror.org/04xfq0f34 grid.1957.a 0000 0001 0728 696X Division of Nephrology and Clinical Immunology, RWTH Aachen University Hospital, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany
2 https://ror.org/03zww1h73 grid.411740.7 0000 0004 0622 9754 Department of Nephrology, University Hospital of Ioannina, Ioannina, Greece
1 2 2024
1 2 2024
2024
37 5 14051409
3 11 2023
1 1 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Graphical Abstract

Keywords

Focal segmental glomerulosclerosis (FSGS)
Perihilar lesion
Parietal epithelial cells (PECs)
Columbia classification
Secondary FSGS
BMBF01GM2202C Stamellou Eleni DFG45703531 Stamellou Eleni RWTH Aachen University (3131)Open Access funding enabled and organized by Projekt DEAL.

issue-copyright-statement© Italian Society of Nephrology 2024
==== Body
pmcFocal segmental glomerulosclerosis (FSGS) is not a specific disease entity but rather a histological pattern of glomerular injury. It is characterized by podocyte injury, which is considered a characteristic of, and potentially a prerequisite for, the development of FSGS. However, recent evidence suggests that parietal epithelial cells are the main effectors in the development of glomerulosclerotic lesions [1]. We have recently described a novel subgroup of parietal epithelial cells, known as cuboidal parietal epithelial cells, in addition to the classical flat parietal epithelial cells. These cuboidal parietal epithelial cells represent proximal tubular epithelial cells that colonize Bowman’s capsule to varying degrees from the tubular orifice [2].

Histologically, FSGS can be categorized into five morphologic variants using the Columbia classification based on light microscopy examination [3]. These variants include collapsing, tip, perihilar, and cellular variant and FSGS not otherwise specified [4]. Although several studies from large kidney biopsy centers have validated the clinical and prognostic usefulness of the morphologic subtypes, the histologic variants alone cannot reliably differentiate between the different clinical forms of FSGS [5]. Furthermore, the factors that drive formation of the different histologic variants remain incompletely understood.

Many animal models have been developed to gain insight into the complex pathophysiology of FSGS [6]. However, the prevalence of FSGS lesions and the contribution of different parietal epithelial cell subpopulations remain unclear. Since the pathophysiology of FSGS lesions can vary between different histological patterns, understanding the cells involved in these patterns may be crucial for developing targeted therapies.

In this study, we investigated the origin and prevalence of FSGS lesions in Munich Wistar-Fromter rats. Specifically, our aim was to explore the role of parietal epithelial cells and their subpopulations in glomerulosclerotic lesion development. Munich Wistar-Fromter rats carry an unknown genetic defect that likely impairs nephron formation, resulting in reduced nephron numbers and chronic pathological hyperfiltration [7]. At ten weeks old, they develop proteinuria, and by nine months, the kidneys exhibit significant glomerulosclerosis. Focal segmental glomerulosclerosis lesions form spontaneously more frequently in male rats than in females.

Munich Wistar-Fromter rats were sacrificed in groups at the following ages: 4–8, 13–17, 28–44, > 45 weeks old (n = 3–4 per group). Munich Wistar-Fromter rat kidneys were perfused with NaCl 0.9% solution with a pressure of 100 mmHg before tissue embedding. Kidneys were perfused with NaCl 0.9% solution with a pressure of 100 mmHg before extraction. Perfusion was based on Bunge and Schmiedeberg and adapted for rodents [11]. Kidneys were recovered, fixed overnight in 4% formaldehyde and then embedded in paraffin. Animals were held in rooms with constant temperature and humidity, 12 h/12 h light cycles, and had ad libitum access to drinking water (ozone-treated and acidified) and standard rat chow. The German Federal Authorities (Landesamt für Natur, Umwelt, und Verbraucherschutz (LANUV) Nordrhein-Westfalen) approved all animal procedures (Az 84–02.04.2015.A517).

For light microscopy, the 4% buffered formalin-fixed kidney fragments were dehydrated and embedded in paraffin. Two-micrometer paraffin sections were stained with periodic-acid Schiff staining. At least 40 glomeruli per rat were evaluated for the presence of adhesion, sclerosis or hyalinosis. The proportion of cuboidal parietal epithelial cells in Bowman’s capsule was estimated only in glomeruli in which both the tubular and vascular pole were present.

For immunofluorescence, two-micrometer paraffin-embedded sections were stained with the following primary antibodies: CD44 (Cell Signaling Technology, Danvers, Massachusetts, USA), rabbit anti-aquaporin 1 polyclonal antibody (Abcam, Cambridge, UK). The following secondary antibodies were used: Alexa-Fluor-594-labeled donkey anti-mouse antibody (Dianova, Hamburg, Germany), Alexa-Fluor-488-labeled chicken anti-rabbit antibody (Invitrogen, Massachusetts, USA). The nuclei were stained using Hoechst 33,342 (Sigma-Aldrich, St. Louis, MO). Sections were evaluated with a Keyence BZ-9000 Microscope using BZ-II Analyzing software (Keyence Corporation, Osaka, Japan).

Levels of creatinine in serum and urine (enzymatic determination using the test kit Creatinine Plus Version 2; Roche Diagnostics) and urea nitrogen were analyzed using a Hitachi 9–17-E Autoanalyzer (Hitachi, Frankfurt am Main, Germany). Albumin level in urine was measured by a competitive two–step enzyme immunoassay using rabbit IgG to mouse albumin as first antibody (MP Biomedicals).

For assessing differences, an ANOVA or the Kruskal–Wallis test was used. Values of p < 0.05 were considered significant. All analyses were performed using Prism version 9.0 for Windows (GraphPad, San Diego, CA).

Animals aged without manipulation and were sacrificed for analysis at different time points, up to a maximum age of 64 weeks (Fig. 1A). Proteinuria began to increase at the age of 17 weeks and serum creatinine levels slowly increased throughout the observation period (Fig. 1B). Histological analysis revealed a steady increase in the frequency of FSGS lesions with age, starting around week 13 (2.1%) and eventually affecting almost all glomeruli (Fig. 1C). Perihilar FSGS lesions were the predominant lesion at all time points. This is noteworthy, as no other animal model or human disease is known to result in such a high frequency of perihilar lesions. Not otherwise specified FSGS lesions appeared at later time points, around week 28, and increased in frequency thereafter. Tip FSGS lesions arose at very low frequency at later time points (57 weeks). To investigate the role of cuboidal parietal epithelial cells in the formation of FSGS lesions, we assessed the presence of cuboidal parietal epithelial cells over time. In rats younger than 50 weeks, fewer than 11% of Bowman’s capsules were partially or completely colonized by cuboidal parietal epithelial cells. With higher age this colonization increased significantly (37.8 at 57 weeks) (Fig. 1D). Serial section analysis of affected glomeruli confirmed the lesion classification (Fig. 1E). De novo expression of CD44 is an established marker for parietal epithelial cell activation [8]. On confocal immunofluorescence, we found that CD44 + parietal epithelial cells in the lesions did not express aquaporin-1, which is known to be expressed only in proximal tubular epithelial cells and cuboidal parietal epithelial cells [2], thus confirming the exclusive involvement of flat parietal epithelial cells in the formation of perihilar lesions (Fig. 1F).Fig. 1 A Experimental set up. Rats were followed until the age of 64 weeks. B Renal functional parameters; serum creatinine, serum urea and urine protein/creatinine ratio (UPCR) in g/g creatinine (n = 4 per group). C. Frequency of FSGS-lesion type according to age. Note that perihilar FSGS represents the predominant lesion and increases substantially by the age of 28 weeks. D Frequency (%) of cuboidal parietal epithelial cells by age (20 glomeruli per rat, n = 4 per group). E Serial sections stained with periodic acid–Schiff (PAS). Higher magnification of picture 5 shows both tubular and vascular poles of the glomeruli. Sclerosis is depicted at the vascular pole, indicative of a perihilar lesion. Bar 50 μm. F Immunofluorescence staining of aquaporin 1 (green) and CD44 (red) confirmed that in a perihilar lesion (arrows) there is no connection to cuboidal parietal epithelial cells (arrow heads). CD44 was expressed by tubular cells in the scattered tubular cell phenotype and inflammatory cells. Additional autofluorescence staining comes from erythrocytes within the glomerular tuft

In this study, we investigated the origin and prevalence of FSGS lesions in Munich Wistar-Fromter rats. Our first major finding revealed that the perihilar variant of FSGS was the most frequent lesion in Munich Wistar-Fromter rats. These results align with previous observations in humans, where perihilar lesion formation has been associated with secondary FSGS [4, 9, 10]. Notably, no other FSGS animal models exhibit such a predominance of perihilar lesions [11]. These results underscore the importance of the Munich Wistar-Fromter rat model in replicating the characteristics of secondary FSGS observed in humans.

Moreover, our study revealed that the perihilar variant of FSGS originates from flat parietal epithelial cells. Of note, we were not able to identify a marker that specifically labels flat parietal epithelial cells, so our findings are based on the exclusion of a role of cuboidal parietal epithelial cells in the formation of perihilar lesions. Interestingly, previous findings by Kuppe et al. demontrated the role of cuboidal cells in the development of tip lesions [2]. Taken together, these studies suggest that the two main subgroups of parietal epithelial cells, i.e., flat and cuboidal parietal epithelial cells, may become activated under different experimental or pathological conditions.

It is important to acknowledge the limitations of our study. Firstly, our findings in the Munich Wistar-Fromter rat model cannot be directly translated to human patients. We have yet to identify a marker that specifically labels flat parietal epithelial cells, which limits our ability to precisely characterize their role in FSGS. We attempted to minimize sampling bias by analyzing a sufficient number of glomeruli. Nevertheless, we believe that our findings provide substantial evidence to warrant further examination of our hypothesis in human patients.

Despite these limitations, our study contributes to the existing knowledge by providing novel insights into the pathogenesis of FSGS and the involvement of different parietal epithelial cell subpopulations. The identification of perihilar lesions as the predominant variant in the Munich Wistar-Fromter rat model, along with the exclusive involvement of flat parietal epithelial cells, fills an important gap in our understanding of FSGS pathology. These findings highlight the need for further research to elucidate the underlying mechanisms and identify potential therapeutic targets.

Acknowledgements

We thank Thiago Strieder and Astrid Fuß for their technical support.

Author contributions

ES and MJM designed and supervised research. ACG performed the experiments. ACG analyzed data. ACG and ES wrote the manuscript. ES, JF and MJM reviewed and edited the manuscript.

Funding

Open Access funding enabled and organized by Projekt DEAL. This research was supported by the consortium STOP-FSGS by the German Ministry for Science and Education (BMBF, 01GM1518A to M.J.M.a nd 01GM2202C to ES and MJM), the clinical research unit InteraKD consortium CRU 5011 (SP03 to E.S., AN 377/11–1 to M.J.M. KFO 5011/0, KFO5011/1) and by individual grants of the German Research Foundation (DFG, MO 1082/7–1, 8–1, 11–1 to M.M.). E.S. is supported by a START grant (19/21 to E.S.) and by a clinician scientist-program of the Faculty of Medicine of the RWTH Aachen University. E.S. has received a research grant of the German Society of Nephrology (DGfN).

Data availability

The authors confirm that the data supporting the findings of this study are available within the article. Further data are available from the corresponding author [ES] on request.

Declarations

Conflict of interest

The authors have no conflicts of interest to declare.

Ethical approval

The German Federal Authorities (Landesamt für Natur, Umwelt, und Verbraucherschutz (LANUV) Nordrhein-Westfalen) approved all animal procedures (Az 84-02.04.2015.A517).

Human and animal rights

All procedures performed were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This study adhered to the principles of the 3Rs (Replace, Reduce, Refine) and efforts were made to minimize animal suffering and to reduce the number of animals used.

Informed consent

For this type of study, formal consent was not necessary.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Smeets B Kuppe C Sicking EM Fuss A Jirak P van Kuppevelt TH Parietal epithelial cells participate in the formation of sclerotic lesions in focal segmental glomerulosclerosis J Am Soc Nephrol 2011 22 7 1262 1274 21719782
Smeets B, Kuppe C, Sicking EM, Fuss A, Jirak P, van Kuppevelt TH et al (2011) Parietal epithelial cells participate in the formation of sclerotic lesions in focal segmental glomerulosclerosis. J Am Soc Nephrol 22(7):1262–127421719782
2. Kuppe C Leuchtle K Wagner A Kabgani N Saritas T Puelles VG Novel parietal epithelial cell subpopulations contribute to focal segmental glomerulosclerosis and glomerular tip lesions Kidney Int 2019 96 1 80 93 31029503
Kuppe C, Leuchtle K, Wagner A, Kabgani N, Saritas T, Puelles VG et al (2019) Novel parietal epithelial cell subpopulations contribute to focal segmental glomerulosclerosis and glomerular tip lesions. Kidney Int 96(1):80–9331029503
3. D'Agati VD Kaskel FJ Falk RJ Focal segmental glomerulosclerosis N Engl J Med 2011 365 25 2398 2411 22187987
D’Agati VD, Kaskel FJ, Falk RJ (2011) Focal segmental glomerulosclerosis. N Engl J Med 365(25):2398–241122187987
4. D'Agati VD Fogo AB Bruijn JA Jennette JC Pathologic classification of focal segmental glomerulosclerosis: a working proposal Am J Kidney Dis 2004 43 2 368 382 14750104
D’Agati VD, Fogo AB, Bruijn JA, Jennette JC (2004) Pathologic classification of focal segmental glomerulosclerosis: a working proposal. Am J Kidney Dis 43(2):368–38214750104
5. Sethi S Zand L Nasr SH Glassock RJ Fervenza FC Focal and segmental glomerulosclerosis: clinical and kidney biopsy correlations Clin Kidney J 2014 7 6 531 537 25503953
Sethi S, Zand L, Nasr SH, Glassock RJ, Fervenza FC (2014) Focal and segmental glomerulosclerosis: clinical and kidney biopsy correlations. Clin Kidney J 7(6):531–53725503953
6. Yang JW Dettmar AK Kronbichler A Gee HY Saleem M Kim SH Recent advances of animal model of focal segmental glomerulosclerosis Clin Exp Nephrol 2018 22 4 752 763 29556761
Yang JW, Dettmar AK, Kronbichler A, Gee HY, Saleem M, Kim SH et al (2018) Recent advances of animal model of focal segmental glomerulosclerosis. Clin Exp Nephrol 22(4):752–76329556761
7. Fassi A Sangalli F Maffi R Colombi F Mohamed EI Brenner BM Progressive glomerular injury in the MWF rat is predicted by inborn nephron deficit J Am Soc Nephrol 1998 9 8 1399 1406 9697661
Fassi A, Sangalli F, Maffi R, Colombi F, Mohamed EI, Brenner BM et al (1998) Progressive glomerular injury in the MWF rat is predicted by inborn nephron deficit. J Am Soc Nephrol 9(8):1399–14069697661
8. Smeets B Uhlig S Fuss A Mooren F Wetzels JF Floege J Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells J Am Soc Nephrol 2009 20 12 2604 2615 19917779
Smeets B, Uhlig S, Fuss A, Mooren F, Wetzels JF, Floege J et al (2009) Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells. J Am Soc Nephrol 20(12):2604–261519917779
9. Kambham N Markowitz GS Valeri AM Lin J D'Agati VD Obesity-related glomerulopathy: an emerging epidemic Kidney Int 2001 59 4 1498 1509 11260414
Kambham N, Markowitz GS, Valeri AM, Lin J, D’Agati VD (2001) Obesity-related glomerulopathy: an emerging epidemic. Kidney Int 59(4):1498–150911260414
10. Harvey JM Howie AJ Lee SJ Newbold KM Adu D Michael J Renal biopsy findings in hypertensive patients with proteinuria Lancet 1992 340 8833 1435 1436 1360561
Harvey JM, Howie AJ, Lee SJ, Newbold KM, Adu D, Michael J et al (1992) Renal biopsy findings in hypertensive patients with proteinuria. Lancet 340(8833):1435–14361360561
11. Hayashi A Okamoto T Yamazaki T Sato Y Takahashi T Ariga T CD44-positive glomerular parietal epithelial cells in a mouse model of calcineurin inhibitors-induced nephrotoxicity Nephron 2019 142 1 71 81 30799414
Hayashi A, Okamoto T, Yamazaki T, Sato Y, Takahashi T, Ariga T (2019) CD44-positive glomerular parietal epithelial cells in a mouse model of calcineurin inhibitors-induced nephrotoxicity. Nephron 142(1):71–8130799414
