# Decoupling shear stress and pressure effects in the biomechanics of autosomal dominant polycystic kidney disease using a perfused kidney-on-chip

Brice Lapin, Jessica Vandensteen, Giacomo Gropplero, Manal Mazloum, Frank Bienaimé, Stéphanie Descroix, Sylvie Coscoy

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## To cite this version:

Brice Lapin, Jessica Vandensteen, Giacomo Gropplero, Manal Mazloum, Frank Bienaimé, et al. Decoupling shear stress and pressure effects in the biomechanics of autosomal dominant polycystic kidney disease using a perfused kidney-on-chip. Acta Biomaterialia, 2025, 197, pp.326 - 338. 10.1016/j.actbio.2025.03.022. hal-05294384

HAL Id: hal-05294384

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Submitted on 2 Oct 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Decoupling shear stress and pressure effects in the biomechanics
of autosomal dominant polycystic kidney disease
using a perfused kidney-on-chip.

Brice Lapin¹, Jessica Vandensteen¹, Giacomo Groplero¹, Manal Mazloum², Frank Bienaimé²,³,
Stéphanie Descroix¹ *, Sylvie Coscoy¹ *

¹ Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physique des Cellules et Cancer, 75005 Paris, France.

² Université de Paris Cité, Institut Necker Enfants Malades-INEM, Département ‘Croissance et Signalisation’, INSERM UMR1151, CNRS UMR 8253 Paris, France

³ Service de Physiologie Hôpital Necker Enfants-Malades, Assistance Publique-Hôpitaux de Paris, 75015 Paris, France.

* Corresponding authors: Sylvie.Coscoy@curie.fr, Stephanie Descroix@curie.fr

Author version, manuscript accepted, Acta Biomater 2025 May 1:197:326-338. doi: 10.1016/j.actbio.2025.03.022. Epub 2025 Mar 13.
Abstract

Kidney tubular cells are submitted to two distinct mechanical forces generated by the
urine flow: shear stress and hydrostatic pressure. In addition, the mechanical properties of the
surrounding extracellular matrix modulate tubule deformation under constraints. These
mechanical factors likely play a role in the pathophysiology of kidney as exemplified by
autosomal dominant polycystic kidney disease, in which pressure, flow and matrix stiffness
have been proposed to modulate the cystic dilation of tubules with PKD1 mutations. The lack
of in vitro systems recapitulating the mechanical environment of kidney tubules impedes our
ability to dissect the role of these mechanical factors. Here we describe a perfused kidney-on-
chip with tunable extracellular matrix mechanical properties and hydrodynamic constraints,
that allows a decoupling of shear stress and flow. We used this system to dissect how these
mechanical cues affect Pkd1-/- tubule dilation. We investigated cell behavior for a flow shear
stress of 1 dyn/cm², combined or not with a 10-mbar intraluminal pressure. Our results
showed two distinct mechanisms leading to tubular dilation. For Pkd1-/- PCT cells (proximal
tubule), overproliferation mechanically leads to tubular dilation of 1.5-2-fold in 5 days,
regardless of the mechanical context. For mIMCD-3 cells (collecting duct), tube dilation was
associated with a squamous cell morphology but not with overproliferation and was highly
sensitive to extracellular matrix properties, with suppression of the dilation when switching
extracellular matrix composition from 6 to 9 mg/ml collagen. Contrary to PCT, mIMCD-3 tube
dilation was highly sensitive to the nature of hydrodynamic constraint. Surprisingly, flow alone
suppressed Pkd1-/- mIMCD-3 tubule dilation observed in static conditions, while the addition
of luminal pressure restored it. Our in vitro model emulating nephron geometrical and
mechanical organization sheds light on the roles of mechanical constraints in ADPKD and
demonstrates the importance of controlling intraluminal pressure in kidney tubule models.

**Keywords:** kidney-on-chip, pressure, shear stress, cyst formation, tube dilation, autosomal dominant polycystic kidney disease.

Introduction

Tubule flow in the kidney results in the application of flow shear stress (FSS), leading to tangential stress on cells; the effect of which has been abundantly described in renal epithelial cell studies¹⁻⁴. The flow rates in the nephron, which decrease in the different segments due to reabsorption events, result in shear stress in the order of 0.2-2 dyn/cm² in renal cells¹⁻³. On renal epithelial cells, flow has been reported to reinforce the apical domain of F-actincytoskeleton⁵, as well as *adherens* junctions and tight junctions between cells², to redistribute cytoskeletal tension and remodel focal adhesions⁶, and to trigger the expression of specific genes, in particular associated with endocytosis and reabsorption⁷. Flow is also intrinsically associated with intraluminal (transmural) pressure, which triggers cell distension⁸ and activates mechanotransduction pathways that are potentially different to those triggered by shear stress. Epithelial renal cells are thus constantly submitted apically to hydrodynamic constraints, flow shear stress, and luminal pressure⁹. In addition, at their basal side they respond to the geometrical and mechanical properties of their underlying basement membrane¹⁰,¹¹. It is now widely established that cell behavior is deeply influenced by 3D cues. More particularly, the high curvature experienced by cells in the tubule, of typical diameters of 50 µm in the proximal and distal parts, is also an important parameter governing the mechanical responses of renal cells¹²,¹³.
Tubular geometry and mechanical cues are deeply affected in autosomal dominant polycystic kidney disease (ADPKD). In this most common genetic kidney disease (incidence 1/1000-1/2500), mutations (mainly in *PKD1* or *PKD2* genes) inactivating a complex formed by polycystins 1 and 2, result in the development of thousands of kidney cysts in adulthood. This eventually leads to kidney failure, without curative therapies to date so that evolution largely leads to dialysis or transplantation¹⁴. Moreover, kidney tubules are characterized by their very dense packing, meaning that cyst expansion in ADPKD will lead to mechanical compression of the adjacent tubules. The nephron obstructions due to cyst formation result in altered flow shear stresses and luminal pressures in affected nephrons and their neighbors, which may account for the clustered and exponential propagation of cysts in the disease known as the snowball effect¹⁵.

Polycystins 1 and 2 are respectively a large orphan receptor and a Transient Receptor Potential cation channel, coupled to numerous signaling pathways including GPCRs, mTOR, JAK-STAT or Wnt pathways¹⁶,¹⁷. Although the mechanism underlying the initial local tube dilation at the origin of cyst formation is currently thought to rely on perturbations in proliferation, apoptosis and/or planar cell polarity¹⁸⁻²¹, it remains to date poorly understood. Many aspects, however, point to an important contribution of mechanical factors in cystogenesis. Cells integrate the mechanical stimuli through several organelles, including primary cilia and cell-substrate and cell-cell adhesions, with mechanosensitive properties deeply perturbed in ADPKD²²⁻²⁴. Polycystins are present in the primary cilia, a sensor of the luminal flow, although their mechanisms of action remain elusive²⁵,²⁶. However, primary cilia ablation reverts the cystic phenotype in ADPKD, pointing to the primary involvement of this organelle in the disease²⁷,²⁸. Moreover, transcriptomic assays comparing the effect of FSS on
WT or *Pkd1*/- proximal cells found that several genes were slightly more induced in the latter condition, suggesting that *Pkd1* here has a role in restraining FSS-regulated signaling<sup>29</sup>.

In addition, polycystins intervene in the organization of integrin-based basal complexes as well, with overexpression of several integrins and their extracellular matrix ligands in ADPKD<sup>30</sup>, along with a reversion of cystic phenotypes reported upon β1 integrin inactivation<sup>11</sup>. Polycystins are also strongly involved in acto-myosin organization<sup>10</sup> and actin-based mechanotransduction<sup>22</sup>, with RhoA-YAP-c-Myc axis identified as a key player in cystogenesis<sup>31</sup> that participates in stiffness sensing<sup>10</sup>.

Studying biomechanical aspects of cyst formation in a mice model of ADPKD, we recently discovered that *Pkd1* loss triggers the remodeling of the distal nephron basement membrane, leading to tubule distension, tubular cell stretching and increased tubule distensibility<sup>32</sup>. In this context, ureteral obstruction, which increases intraluminal pressure resulted in an excessive dilation of the distal nephron followed by explosive cystogenesis suggesting that increased luminal pressure could be a biomechanical driver of ADPKD<sup>32</sup>. Few studies have addressed the specific effects of pressure dependence on polycystin expression or function<sup>33-36</sup>, including mechanisms on renal cells relying on the inhibition of Piezo1-dependent stretch-activated channels<sup>34</sup> by polycystin-2. But this electrophysiology work was not performed in the frame of a realistic tubule geometry. The influence of decoupling these contributions for tube dilation and cyst formation in ADPKD remains unknown, despite the importance of identifying their respective role in the elucidation of the mechanosensitive pathways involved and for future therapeutic applications.

A variety of 3D models reproducing realistic features of the kidney tubules, as well as some relevant mechanical properties like perfusion or substrate elasticity, have been
developed in the past few years. These models involve organoids<sup>37,38</sup>, bioprinting tubules<sup>39,40</sup>, or organs-on-chips based on wire molding techniques<sup>41</sup>. However, the coupling of flow shear stress with hydraulic pressure in most experimental devices results in the fact that not only the relative contributions are not distinguished, but also that some effects observed can be attributed misleadingly to shear stress alone. In *in vitro* tubes, FSS and pressure contributions can however be decoupled by playing on the hydrodynamic properties of the system, like hydraulic resistance, diameter, or material mechanical properties, as described recently for endothelial cells<sup>42</sup>.

In our past works, we developed a kidney-on-chip specifically dedicated to the study of ADPKD, by reproducing tightly packed tubes of physiological diameter in a biocompatible deformable collagen substrate, in which we observed a tube dilation specific for ADPKD proximal cells<sup>43</sup>. We recently implemented versatile possibilities of perfusion on our devices<sup>44</sup>. In this paper, we develop an original system to decouple flow and luminal pressure in our kidney-on-chips. This allows us to dissect the effects of flow alone, flow with luminal pressure, and matrix stiffness on tubule dilation, proliferation, and multicellular morphology. We find contrasting mechanisms of tube dilation, associated or not with proliferation, depending on cell lines that originate either from proximal or distal parts of the nephron. Importantly, our results highlight the prominent role of luminal pressure for tube dilation in collecting duct cells, which is of paramount importance as cysts form first in this nephron part in ADPKD.
# Methods

## Kidney-on-chips.

As previously described ⁴⁴, the chip consisted of a PDMS scaffold of 3 PDMS layers bonded to a glass slide. One part of the scaffold featured the microfluidic channels. For this part, a PDMS (Curing Agent to PDMS weight ratio of 1:10) was poured on a mold obtained with photolitography. This part laid on a flat PDMS membrane which elevated the channels from the glass slide. This flat PDMS layer was obtained by spincoating PDMS 1:10. On top, another flat 500 µm thick PDMS membrane closed the chip. The PDMS scaffold surface was activated by oxygen plasma and then treated with a 5% (3-aminopropyl) triethoxysilane (Sigma-Aldrich) solution in methanol for 45 min, rinsed thoroughly in methanol in an ultrasonic bath then in a 2.5% glutaraldehyde (Sigma-Aldrich) solution in ddH20 for 45min and rinsed thoroughly in ddH20 in an ultrasonic bath then at room temperature in ddH20 overnight. Then, 75 µm wide tungsten wires were inserted into the scaffold, which was sterilized with UV-Ozone. A 6mg/mL or 9 mg/mL collagen I gel was prepared on ice by neutralizing high concentration collagen gel from rat tail (Corning) with NaOH and diluted in PBS with 10% of total volume with PBS 10X. The gel was injected in the collagen chambers, still on ice. The collagen was set at 37°C for 2h. Chips were then stored in PBS at 4°C up to 2 weeks before use.

## Flow rate measurements.

5 µm fluorescent beads (Polysciences) in PBS were used. The flow of beads was imaged with a fluorescence microscope, and bead tracking was performed with ImageJ and the extension Trackmate. From this data, the average flow speed of beads within a channel was calculated: the maximum velocity of particles within their respective track was taken and the
average of all these velocities in each channel was computed to estimate an average fluid
velocity through a channel section. The corresponding flow rate in each channel was then
evaluated. We then estimated the wall shear stress in an 80µm micron wide tubule with the
same flow rate.

Cells.

Mouse PCT *Pkd1<sup>+/-</sup>* and *Pkd1<sup>-/-</sup>* cells (respectively PH2 and PN24 clones) were a kind gift of S. Somlo <sup>45,46</sup>. These cells, containing the Immortomouse transgene for the interferon-inducible expression of a thermolabile large tumor antigen, were amplified in proliferation conditions (33°C, with γ-interferon) and differentiated in differentiation conditions (37°C, without γ-interferon). Proliferation conditions were 33°C, 5% CO2, in DMEM/HamF12 supplemented with 3% FBS (Fetal Bovine Serum), 7.5 nM Na selenite, 1.9 nM triiodo-L-thyronine, 5 mg/ml insulin, 5 mg/ml transferrin, 100 UI/ml penicillin/streptomycin, 5 mg/ml nystatin (all from Sigma), and 10UI/ml γ-interferon (Millipore). Cells were differentiated in the same media without γ-interferon, and with 1% FBS instead of 3% FBS, at 37°C, 5% CO2.

Edited mIMCD-3 cell line (immortalized cells from murine inner medullary collecting duct, deleted for *Pkd1* gene using CRISPR-based genome editing or corresponding control (parental) <sup>47,48</sup>, were kindly provided by Michael Köttgen's lab and cultivated in DMEM/HamF12 supplemented with 10% FBS at 37°C, 5% CO2.

Tube seeding and perfusion.

The day before seeding, wires were partially removed to form the channels, an anti-adhesion solution was perfused in the tubes during a few seconds followed by a 1-hour incubation at room temperature, and a laminin 50µg/mL solution was injected in the chip
using a pressure controller and PTFE tubing followed by 1 hour-incubation at 37°C. Cells were
detached from flask upon confluency with trypsin, counted and resuspended at 10 million cells
/ml in complete medium. The cell suspension was then injected into collagen channels with a
pressure controller via PTFE tubing until the channels were filled with cells. Then tungsten
wires were inserted back into the channels to form a hollow tube. As described in our previous
work⁴⁴, it enabled seeding at confluency without lumen filling, together with high cell viability.
Chips were incubated in culture medium for 2 to 3 hours to let cells adhere, then tungsten
wires were removed. Before perfusion, the chip channel was flushed with a pressure
controller (Fluigent Flow E/Z) at 50 mbar with a 37°C PBS solution to remove unattached cells
and put back in the culture medium. Chips were perfused with 3 ml TERUMO syringes attached
to a microfluidic needle and maintained vertically by a homemade holder. Chips were then
cultured in 6-well plates in humid atmosphere at 37°C for up to 1 week, with medium changed
every two days.

Immunostaining.

**Immunostaining on whole chips.** This technique corresponds to the Results presented in the main text. Chips were washed with PBS twice before being fixed in a 4% paraformaldehyde solution in PBS for 45 min. Chips were then rinsed twice in PBS and stored in PBS at 4°C before immunostaining according to our previous described procedure⁴⁴. Cells were permeabilized by submerging the chip into PBS + 0.3% TritonX100 for 30 minutes and rinsed three times for 30 minutes with PBS-BSA 2%. The chip was submerged in PBS-BSA 2% with 0.1% Tween20 and 2% Goat Serum for saturation. A 100-150 µL drop of 1/200 antibody, 2% BSA, 1% Tween20 in PBS is delicately placed on the chip so that it does not fall. Incubation is carried out for 24 hours in a humid environment at room temperature. The chip was rinsed
3 times for 30 minutes in PBS-BSA 2%. A 100 µL drop of a solution containing 1 µg/ml Hoechst, 0.25 µg/ml fluorescent phalloidin, primary antibody (rabbit anti-Ki67, Abcam, ab16667, 1:100), 2% BSA, 1% Tween-20 in PBS was delicately placed to prevent spilling. Incubation was carried out for 24 hours in a humid environment at room temperature. The secondary antibody was a 641 nm anti-rabbit highly cross-adsorbed antibody. Imaging was performed by confocal microscopy.

**Collagen insert sectioning** (see SI). To obtain slices of the collagen matrix, the vibrating microtome (vibratome) was used to cut the collagen I matrix containing the tubules. The collagen insert was first removed from the chip and embedded in 4% agarose gel, which allowed better handling of the sample. The gel was hardened at 4°C and then placed in the vibratome water pool for cutting. Slices of 200 µm thick were obtained by inclining the razor blade perpendicular to the water surface. The cutting speed was put at 1 mm/s, and the amplitude at 1 mm. The slices were then stored in PBS at 4°C until staining.

## Fluorescence imaging.

After staining, the top half of the tubules was imaged within the chips with confocal microscopy (Leica DMi8) with a 25X immersion water objective (Fluotar VISIR 25x/§0.95 WATER). Acquisition parameters were optimized to highlight F-actin as a means of cell delimitation. Post-acquisition image processing consisted in contrast enhancement to further highlight F-actin.

## Quantifications.

Measurement and normalization of tube diameter was performed as previously described⁴³. Nuclei segmentation and identification of Ki67 positive nuclei was performed in
two steps, first presegmentation with the software Ilastik and construction of a training pool
with each pixel assigned to nuclei, actin, or background, second segmentation, with a home-
made Matlab algorithm. To compute the internuclear distance, for each nucleus, the 6 closest
nuclei were determined. To filter out outliers due to border localization or isolated nuclei, we
considered that the distances between nuclei and their 6 closest neighbors should follow a
Gaussian distribution, and only elements falling between the 1st and 3rd quartile of such
distribution was kept. Then distances between nuclei and their 6 closest neighbors were
averaged over each nucleus, and finally averaged over all the nuclei. To compute Ki67 positive
ratio, Ki67 positive nuclei were automatically identified and counted. Then total number of
Ki67 cells were divided by total number of nuclei.

Statistical analysis.

All statistical analyses were performed using the software Prism. For data following a
Gaussian distribution, we used parametric tests. Tubular dilation was analyzed with a repeated
measure two-way ANOVA with Sidak's multiple comparisons test comparing dilations between
genotypes within a given cell type and hydrodynamic stimulation normalized to different time
points. Internuclear distances were analyzed with an ordinary two-way ANOVA with Sidak's
multiple comparisons test, comparing chips fixed at different days after confluency within a
given genotype, cell type and hydrodynamic stimulation, but also between genotypes within
a cell type. Ki67 positive cell ratio, that were not following a distribution comparable to a
Gaussian distribution, were analyzed with multiple Mann-Whitney tests between cell
genotypes for each day using the Holm-Sidak method for multiple comparisons correction.
Mean values ± standard deviation are given in the text in the case of Gaussian distributions.
For the description of Ki67 distributions in the main text, not following a Gaussian distribution,
more meaningful median values were preferred, with dispersion that can be assessed by the
interquartile ranges shown in the graphs. For both Ki67 and internuclear distances analysis,
outliers were removed regarding possible errors due to the imaging and the automatic
analyses. This led to the removal of only one Ki67 channel with a null value and 3 channels for
internuclei distance as determined by ROUT method (integrated in PRISM) for outlier
detection, and then of few abnormally high internuclei distances or low proliferation levels.

COMSOL simulation.

We used the COMSOL Multiphysics® software (version 5.5, COMSOL AB, Stockholm, Sweden) to model fluid flow and luminal pressure in the kidney-on-chip. The simulations were conducted using a 3D model, which included the 5 rectangular serpentine channels followed by the round collagen channels. Each serpentine has a section with a width and height of 50 µm and were 6cm long in total. Each collagen channel had an 80µm diameter. A creeping flow was set along the channels with pressures in inlet 1 and 2 as boundary conditions. The inlet 1 pressure was set to 10 mbar in the flow configuration and 0 mbar in the flow and pressure configuration. The inlet 2 pressure was set to 0 mbar in the flow configuration and 10 mbar in the flow and pressure configuration. The computational mesh employed was the default fine physics-controlled mesh provided by COMSOL. For pressure, data were visualized with slice plots in the mid plan of the chip, extending to the total length of the microfluidic channels. For shear stress, data were visualized with slice plots showing the mid circular cross section of the tubes.
# Results

## 1. Decoupling flow and luminal pressure in kidney-on-chips.

To finely dissect the roles of flow shear stress and intraluminal pressure within the kidney tubule independently, we first optimized our previously developed kidney-on-chip system<sup>44</sup> (Figure 1.A). Briefly, a gel of collagen type I was polymerized in a polydimethylsiloxane (PDMS) microfluidic chip in a central open chamber; this gel featured parallel 80µm hollow tubules where kidney cells of two mouse cell lines were seeded, PCT and mIMCD-3 cells. The device is described and illustrated in depth in a recent methodology paper<sup>44</sup>. The chip not only integrated these collagen channels but also highly resistive serpentine microfluidic channels in the PDMS bulk perfusing the tubules, which allowed to decouple flow shear stress from intraluminal pressure. Flow and pressure in chips were generated by a home-made system, consisting in medium-filled syringes controlling the hydrostatic pressure; these simple devices allowed to perform the experiments in many chips in parallel. Channels were fluidically independent so that clogging in one channel would not affect the pressure gradient of the other channels. The chips could be perfused using two different inlets (Figure 1.A-B). A pressure difference between the “Pressure Offset” inlet and the “Set Pressure” inlet drove a flow within the collagen tubular channels (Figure 1.A). Due to a resistivity much higher than the rest of chip, the serpentine set the intensity of this flow. Furthermore, the intraluminal pressure in the collagen channels was effectively the pressure in the “Set Pressure” inlet as almost total pressure drop happens upstream of the serpentine. Hence, we had a mean to decorrelate the pressure within the collagen tubular channels and the wall shear stress by setting the pressure thanks to the “Set Pressure Inlet” and then by varying the “Pressure
Offset" inlet. We limited our study to a binary scenario: in one case there was physiological pressure and a flow shear stress in the tubules and in the other case there was only a flow shear stress in the tubules. By perfusing only one of the inlets at a time with the same pressure in both cases, the intraluminal pressure in tubules was either negligible when only perfusing the "Pressure Offset Inlet", or equivalent to the pressure in the "Set pressure" inlet with only this one perfused. In both cases, the resulting flow shear stress would then in theory be the same, as was verified by a finite element simulation (Figure 1.C). We chose to perfuse the chip with an input of 10 mbar pressure as a typical intraluminal pressure in the kidney nephron, 10 mbar being the typical physiological pressure in the proximal part ⁴⁹. The serpentine geometry was set so that this pressure generates a flow shear stress in the tubules in the 0.1-1 dyn/cm², as confirmed by a finite element simulation (Figure 1.C). It is worth mentioning that this system can also explore a wider range of pressure/shear stresses combinations by perfusing both inlets at the same time (Figure S1.A).

We experimentally measured the flow rate in tubules seeded with mIMCD-3 cells by particle tracking with 5µm fluorescent beads (Figure S1.B). From these measurements, we estimated the flow shear stress on cells lining an 80µm wide channel. We observed that 1 to 8 days after seeding, this flow shear stress remained within physiological range (Figure 1.D), although there was a drop in flow shear stress at long time point certainly due to cell clogging. For this reason, we restrained our study to less than 8 days after cell seeding and fixed an upper limit at day 5. Indeed, clogging was routinely assessed by evaluating level of syringes/debris flowing tubules, and most of the clogging was attributed to dead cells in the PDMS region, which was a rare event at day 5. Therefore, we expect the FSS at day 5 to be close to that at day1.
We also verified the tightness of the tubules obtained from mIMCD-3 cells and PCT cells. After perfusing confluent tubules with fluorescent dextran, looking at the fluorescence of the medium in and around the tubules, we demonstrated that mIMCD-3 cells formed a barrier for diffusion of molecules as small as 10 kDa. PCT tubules on the other hand appeared leaky for 10kDa dextran, but impermeable to 40 kDa dextran (Figure 1.E). This confirmed that the expected intraluminal pressure could build up within the tubules. Indeed, a perfusion in the flow and pressure configuration tubules lined with a confluent layer of kidney cells almost immediately led to a slight tubular dilation, not observed with naked collagen tubular channels (Figure S1.C). These observations confirmed the absence of leaks at the tubule edges.

## 2. Hydrodynamic constraints differentially affect the ability of *Pkd1*<sup>-/-</sup> mIMCD-3 and PCT cells to dilate kidney tubule-on-chip.

We used cell lines generated from the proximal tubule (*Pkd1*<sup>+/-</sup> PCT cells) and from the collecting tubule (*Pkd1*<sup>+/+</sup> mIMCD-3 cells), from which *Pkd1*<sup>-/-</sup> cells were generated. We previously reported the behavior of these cells in a similar tubular microenvironment but in static conditions<sup>43</sup>. In these conditions, both *Pkd1*<sup>-/-</sup> cell lines dilated the tubules significantly more than their parental cells<sup>32,43</sup> (Figure S2). After validation of our perfused system, we investigated the behavior of these cells submitted to a 1 dyn/cm<sup>2</sup> flow shear stress, combined or not with a 10-mbar intraluminal pressure.

### 2.1. Flow shear stress alone.
With PCT cells, a significant 1.38±0.21-fold dilation of *Pkd1*<sup>-/-</sup> tubules was observed 5 days after confluency when subjected to flow shear stress only, while parental PCT cells did not dilate tubules in these conditions (p<0.0001, Figure 2.A). This paralleled the *Pkd1*<sup>-/-</sup> specific dilation observed for these cells in static conditions. However, a very different situation was observed for mIMCD-3 cells. Surprisingly, with flow shear stress
the loss of *Pkd1* in mIMCD-3 cells was no longer sufficient to trigger the significant tubular dilation (Figure 2.B) that is observed in static conditions.

**2.2. Flow shear stress with pressure.** In the physiological conditions, flow shear stress is intrinsically associated to luminal pressure in small tubes. Combining FSS with a 10-mbar intraluminal pressure led to rapid tubular dilation of both cell types (Figure 2.C-2.D) and restored the ability of *Pkd1*⁻/⁻ mIMCD-3 cells to dilate tubes (Figure 2.D). We identified two phases in tube behavior: an initial non-*Pkd1*⁻/⁻-specific dilation occurring between day 0 and 1 in all conditions, of relative amplitudes on the order of 1.2-1.5 (PCT) to 1.7-1.8-fold (mIMCD-3 tubes) (Figure S3), and a subsequent smaller specific dilation after day 1. This latter is quantified by the D5/D1 dilation ratio between day 1 and day 5 and can be compared to the total dilation D5/D0 (Figure 2.C, 2.D). For **PCT cells**, after 5 days, the total D5/D0 tube dilation was clearly enhanced by the loss of *Pkd1*, with PCT tubules dilating by 1.62±0.40-fold for parental cells but more than 2-fold (2.05±0.53) for *Pkd1*⁻/⁻ cells (p<0.0001, Figure 2.C, left).

For **mIMCD-3 cells**, the addition of pressure to flow restored the excessive dilation of *Pkd1*⁻/⁻ tubes, with a total dilation between day0 and day5 reaching 2.25±0.41 fold – significantly different from parental dilation of 1.94±0.38 fold (p=0.0008, Figure 2.D). This total dilation encompassed an important unspecific dilation between D0 and D1, including a minor contribution of short-term tube deformation at the application of pressure, Figure S4. But while the parental tubes showed no significant dilation after day 1, mIMCD-3 *Pkd1*⁻/⁻ tubes exhibited an additional 1.29±0.13-fold further dilation between day 1 and day 5 (Figure 2.D).

At last, although this study is focused on elucidating the specific role of pressure in flow experiments corresponding to a physiological situation, one may wonder if pressure alone elicited a *Pkd1*⁻/⁻-specific dilation, which was indeed suggested by obstruction experiments
(Figure S5). In conclusion, our results show that for mIMCD-3 cells, the combination of FSS with intraluminal pressure restored the excessive dilation of mIMCD-3 *Pkd1*⁻/⁻ tubules which was lost upon shear stress only.

3. *Pkd1*-dependent tube dilation upon flow + pressure is prevented by increasing the collagen concentration for mIMCD-3 cells, but not for PCT cells.

As seen above, an intraluminal pressure of 10 mbar combined with FSS was sufficient to trigger tubular dilations, regardless of *Pkd1* status or cell type, in a 6mg/mL collagen I matrix. *Ex vivo* perfusion experiments demonstrated⁵⁰ that the matrix surrounding kidney tubules, more specifically the basement membrane, constitutes the principal barrier preventing tubular dilation when intraluminal pressure increases. To assess the importance of the surrounding matrix properties in *Pkd1*-dependent tubular dilation mechanisms, we grew mIMCD-3 and PCT cells in collagen hollow tubes made of increased collagen I concentration (9 mg/mL; Figure 3). We previously measured by tensile tests the Young modulus of 6 and 10 mg/mL collagen I gels employed in our study at 55±15 kPa to 86±47 kPa respectively (Bernheim-Dennery *et al.*, submitted). The relative increase in Young modulus thus remains moderate and is corroborated by a comprehensive review of existing literature, where we observe a quadratic relationship between the gel's Young's modulus and its concentration⁵¹⁻⁵⁴.

For **PCT cells** under flow+pressure, dilation after 5 days in 9 mg/mL collagen matrix remained close to 6 mg/mL conditions, with diameter at day 5 still increasing by 1.57±0.28-fold for control cells and significantly more for *Pkd1*⁻/⁻ cells, by 2.15±0.56-fold (p<0.0001, Figure 3.A). By contrast, for **mIMCD-3 cells** under flow+pressure, the tubule diameter increases at day 5 was no longer significantly different to parental tubules. Past the initial dilation of
approximately 1.5-fold between day 0 and day 1, the *Pkd1*⁻/⁻ mean tubule diameter appeared to stall, increasing only around 1.13±0.14-fold between day 1 and day 5, which was no more different from the parental tubes (Figure 3.B). Thus, increased collagen concentration limited tubular dilation under flow+pressure conditions for parental mIMCD-3 cells whereas it does not affect PCT cells.

4. Proliferation and morphology of the epithelium upon hydrodynamic stimulations suggest the existence of two dilation mechanisms due to loss of *Pkd1*.

The observed differences in tubular dilations as a function of hydrodynamic cues could be explained with differences either in net cell proliferation or/and with changes of cell morphology modulating the surface covered by the tissue. To unravel the cellular mechanisms involved in tubular dilation, we assessed levels of proliferation (KI67 labelling), as well as cell morphology. The mean internuclear distance within a tubule was used as a proxy for cell spreading.

To this end, we fixed tubules 1 day after confluency, before any *Pkd1*⁻/⁻ specific dilation and others 5 days after confluency, at the end of the tubular dilation experiments. We compared cell spreading after 5 days of hydrodynamic stimulation with cell spreading of tubules fixed after 1 day to assess potential cell dilation concurrent with tubular dilation. On the same tubules, we assessed proliferation level differences between genotypes under different hydrodynamic conditions on the tubules fixed at early stages of stimulation but also on tubules fixed at the end of the experiment to evaluate the importance of proliferation before and after *Pkd1*⁻/⁻ specific dilation occurred (Figures 4, 5).
**4.1. PCT cells.** In PCT tubules subjected to flow only (Figure 4.A), the levels of proliferation at day 1 were similar on average between parental and *Pkd1*⁻/⁻ cells. But at day 5 *Pkd1*⁻/⁻ were more proliferative, with a median of 10% Ki-67 positive cells vs. 3% for parental cells (p<0.0001, Figure 4.B). Parental and *Pkd1*⁻/⁻ cells were hard to tell apart in terms of morphology and organization within the tissue at day 1, with no significant difference in internuclear distance. Sustained proliferation levels in *Pkd1*⁻/⁻ tubules resulted in denser tissues at day 5 as shown by cell morphology (Figure 4.A, S6-A) and was confirmed by a decrease of internuclear distance (Figure 4.C, statistically significant difference between day 1 and day 5 only for *Pkd1*⁻/⁻ tubes, with p<0.0001). Furthermore, in most instances we observed the formation of multilayers constituted of a few cells.

Applying intraluminal pressure initially increased PCT proliferation, irrespective of *Pkd1*
genotype, then proliferation levels dropped drastically 5 days after confluency (Figure 4.B, 4.E).
Nonetheless, *Pkd1*⁻/⁻ tubules sustained a significantly higher proliferation levels than parental
cells tubules, with medians for Ki67-positive cells of 3% vs 1.3% for parental tubules at day 5
(p<0.0001, Figure 4.E). Interestingly, this time, parental cells tubules became denser over time
in 6 mg/ml collagen, reflecting the acquisition of a more cuboidal shape than with flow only,
more reminiscent of *in vivo* proximal tubule cells (Figure 4.F, S6.B). *Pkd1*⁻/⁻ cell morphology and
density remained stable over time and comparable to parental cells, while still forming
multilayers of a few cells. At last, increasing matrix density from 6 to 9 mg/ml had no sizeable
impact either on cell density or on proliferation (Figure 4.E-F, S6.B).

**4.2. mIMCD-3 cells.** In mIMCD-3 tubules subjected to flow only (Figure 5.A), we observed an increase in proliferation at late stages (day 5) linked to the loss of *Pkd1*. Initially at day 1, the proportion of Ki-67 positive cells was in the order of 10-20% both for *Pkd1*⁻/⁻ and parental cells,
but it strongly increased with a median value of 25% for Pkd1⁻/⁻ cells at day 5, compared to 3%
only for parental cells (p<0.0001, Figure 5.B). This increased proliferation also translated into
an increased density for mIMCD-3 Pkd1⁻/⁻ cells, shown by a decrease in internuclear distance
(Figure 5.C, decrease between day1 and day5 with p<0.0001) and a more cuboidal shape
(Figure 5.A, S7-A). Contrary to PCT cells, this increased proliferation did not induce significant
tubular dilation, which may be in line with a high plasticity for Pkd1⁻/⁻ cells.

Contrary to PCT, addition of intraluminal pressure did not initially have a sizeable
impact on mIMCD-3 proliferation (Figure 5D, E). Interestingly however, at day 5 both parental
and Pkd1⁻/⁻ proliferation had decreased, still without a significant difference between them. In
contrast, the difference in cell morphology and density between Pkd1⁻/⁻ and parental cells was
striking (Figure 5.D, S7-B). Initially it was hard to distinguish between Pkd1⁻/⁻ and parental cells
based on appearance and by using the internuclear distance criteria (Figure 5.D-E, S7.B, day1).
At the beginning of tube dilation (day 2), both cell types exhibited a correct apicobasal
polarity and primary cilia expression, as well as secretion of their own basement
membrane (Heparan Sulfate, with MT1-MMP protease expression, Figure S8). At day 5,
Pkd1⁻/⁻ cells adopted a squamous morphology and the tissue became less dense, as evidenced
by the increase in internuclear distance between day1 and day5 (p=0.0075, Figure 5.F; Figure
S7.B, day5). Parental cells followed an opposite trend wherein the cells looked more cuboidal
keeping a similar cell density. These results suggest that, for mIMCD-3, the excessive dilation
of Pkd1⁻/⁻ tubules does not originate from enhanced proliferation but is correlated with the
acquisition of a squamous morphology in response to intraluminal pressure. Increasing the
matrix concentration favored the formation of denser tissues in Pkd1⁻/⁻ mIMCD-3 tubes, as
evidenced by the decrease in internuclear distance at day 5 between 6 and 9 mg/ml collagen,
with p=0.0002, Figure 5.F; Figure S7.B, day5, 9mg/mL); here Pkd1⁻/⁻ cells did not acquire a
flattened morphology over time, coincident with the absence of dilation in this condition of
concentrated collagen matrix. This further suggests that *Pkd1*⁻/⁻ dependent tubular dilation for
mIMCD-3 and an abnormally squamous cell morphology are two intertwined events.
Discussion

In a kidney-on-chip with deformable tubes of physiological dimensions, formed in a scaffold with tunable mechanical properties, we developed a method to decouple flow and luminal pressure. We present an innovative solution to decouple two generally intricate mechanical stimuli, pressure, and flow shear stress, relying on classic microfabrication and low-tech perfusion. The rationale for this decoupling comes from the fact that the effect of flow shear stress in the literature is generally studied without distinguishing it from the associated generating pressure. This pressure cannot be neglected in the case of small tubes and can even be, as strikingly reported here, the relevant constraint triggering the mechanism at play.

Our platform is modulable and allows to run in parallel a great number of different replicates for different mechanical conditions. This kidney-on-chip is also compatible with a wide range of biochemical studies, since we have developed associated protocols for immunofluorescence and mRNA extraction and tissue sectioning (Figure S8, S9). We applied this system to specifically investigate how these shear stress and pressure affect the shape and organization of tubules formed by kidney epithelial cell lines derived either from proximal (PCT) or distal (mIMCD-3) mouse nephron. In each of the cell lines, we further studied how these mechanical parameters affect the excessive tubule dilation caused by polycystin-1 inactivation. A tube diameter of 80 µm was chosen for mimicking proximal and distal segments because they have similar dimensions *in vivo*. While we focused here on the local biomechanical determinants of tube dilation, organ-on-chip technology would also make it possible to combine different devices mimicking different nephron segments including Henle loop ¹³ in order to study if interplays between them may play a role in tube dilation.
Strikingly, while *Pkd1* deletion in both PCT and mIMCD-3 led to excessive tubule dilation in static conditions, this phenotype was differentially affected by mechanical parameters across cell lines. Essentially, *Pkd1*-deficient PCT tubules underwent excessive dilation, increased proliferation and cell crowding, irrespective of pressure, flow or ECM stiffness conditions. This excessive proliferation of *Pkd1*⁻/⁻ PCT further was associated to cell crowding and epithelial multilayering, suggesting defective contact inhibition.

In contrast, the ability of *Pkd1*-deficient mIMCD-3 cells to deform their scaffold was drastically impacted by the mechanical conditions applied. *Pkd1*⁻/⁻ mIMCD-3 cells dilated the ECM scaffold more than the parental line in static conditions in a 6 mg/mL collagen scaffold (Figure S2, Mazloum *et al.* ³²). In the time frame of the experiment, applying shear stress alone prevented the excessive dilation of the tubule formed by *Pkd1*⁻/⁻ mIMCD-3 despite an increased proliferation. The absence of multilayering may suggest an enhanced regulation of the tissue homeostasis possibly through apical extrusion. Surprisingly, adding luminal pressure overcame the protective effect of flow shear stress and restored the ability of *Pkd1*⁻/⁻ to distend the ECM scaffold. In contrast, wild-type mIMCD-3 cells lacked in most cases this ability to distend the ECM past a rapid initial dilation.

Our results unveil two distinct mechanisms leading to tubular dilation due to the loss of *Pkd1*. For the PCT, over-proliferation was associated with tubular dilation regardless of mechanical constraints, hydrodynamic or from the extracellular matrix properties. For mIMCD-3 cells, a squamous cell phenotype favors tubular dilation through a mechanism both sensitive to the matrix properties and intraluminal hydrodynamic constraints but not associated with increased proliferation. The surprising results obtained with mIMCD-3 remarkably recapitulates our parallel *in-vivo* results³² regarding the initial dilation of *Pkd1*-deficient distal
tubule mice, which undergoes distension without a detectable increase in tubular cell proliferation index. The effect of pressure on tubule-on-chip dilation further echoes the effect of ureteral obstruction, which precipitates cyst formation specifically in the distended distal nephron. Importantly, the finding that the ability of *Pkd1*-deficient mIMCD-3 cells to dilate tube is not only impacted by flow shear stress but also by luminal pressure and matrix stiffness suggests that PC1 is connected to different mechanosensitive circuits responding to distinct physical parameters.

## Behavior of non-pathological cell models in the collagen kidney-on-chips

We used here two well-known mouse cell lines for proximal and distal kidney parts, proximal PCT control and *Pkd1*⁻/⁻ cells from transgenic mice – with a wide characterization of the effect of *Pkd1* inactivation for these cells⁴⁵,⁴⁶– and the classical mIMCD-3 collecting duct model, with CRISPR-Cas9 *Pkd1* inactivation³²,⁴⁷,⁴⁸. Note that the culture media for PCT and mIMCD-3 cells are different, which should be borne in mind when comparing growth of the two cell types. In addition, it is worth mentioning that PCT control cells are *Pkd1*⁺/⁻, while mIMCD-3 controls are *Pkd1*⁺/⁺ (parental). *Pkd1*⁺/⁻ cells have largely been considered as a model for control cells in the literature. In addition, we previously compared WT PCT (from a different genetic background) with *Pkd1*⁺/⁻ PCT and found the same behavior of absence of dilation in static conditions⁴³. However, we cannot exclude subtle differences in certain mechanical conditions, as cellular behavior could be sensitive to the precise dosage of *Pkd1*. It would be expected in that case that behaviors are closest between PCT *Pkd1*⁻/⁻ cells and control compared to the mIMCD-3 case – however, *Pkd1*⁻/⁻ and control PCT are already strongly different in all the conditions studied, so that we estimate that this element is not a confusing factor.
In the case of mIMCD-3 cells, we observed slight tubule dilation in the parental control. This may remind phenotypically the expansion of spherical cysts embedded in 3D hydrogels formed by basal to apical secretion⁵⁵⁻⁵⁹, although no tubule dilation occurred in our system with MDCK cells paradigmatic for this phenomenon⁴³. However, we can exclude such a mechanism in the presence of flow, which enables a fast luminal renewal with similar medium composition as the external media. Even in the absence of flow, we can exclude it for parental cells because the observed tube dilations were similar in static and flow conditions. We suggest instead that the slight mIMCD-3 parental dilation may result from an intrinsic proteolytic activity, possibly associated with proliferation. While we cannot exclude proliferation's role, IMCD cells have been reported to undergo spontaneous tubulogenesis in diluted collagen, and their tubulogenesis in synthetic hydrogels required MT1-MMP protease activity ⁶⁰⁻⁶². Although tubulogenesis is not observed in our concentrated collagen system, a proteolytic activity could contribute to mild enlargement of pre-formed tubes. Immunofluorescence experiments confirmed MT1-MMP secretion even for parental mIMCD-3 cells.

**Characteristics and effects of the hydrodynamic and mechanical constraints.**

The application of hydrodynamic constraints had positive effects for maintaining long-term epithelial organization in tubes. Compared to the results that we obtained in static conditions ³²,⁴³ (Figure S2), applying flow shear stress with negligible pressure helped to maintain a stable hollow tubular geometry, limiting epithelial multilayering and cell accumulation in tubule lumen. Irrespective of the cell line considered, further applying pressure led to a progressive increase in tubule diameter, but also further limited multilayering, suggesting that all the studied cell lines respond to pressure. The initial pressure-induced dilation appeared to be initially independent of genotype and can be most likely
attributed to the mechanical stimuli induced by the elastic response of the confluent tubules.

This initial dilation upon pressure may occur because the system is in a transient state of initiation of pressure application and epithelial maturation at the same time. In contrast, in tubes *in vivo*, a steady state has been in place for months with long-term adaptations to hydrodynamic conditions. An experimental limitation of our system is that as the total observation time is limited, we had to apply pressure after the initial overnight cell colonization, when basement membrane or cell tissue may not have reached its final organization.

The initial response flow + pressure was then followed by a cell- and genotype-specific deformation of the tubules, indicating that different more complex mechanisms are at play. Overall, cell responses to these mechanical parameters were contrasted, depending not only on *Pkd1* status but also on the cell line considered. PCT expressing *Pkd1* adopted a more physiological shape when exposed to pressure, becoming more cuboidal and forming denser tissues only when adding intraluminal pressure, a behavior not observed in mIMCD-3 cells. This differential response may be linked to the physiologic exposure to pressure of the nephron segments from which those cell lines originate, as pressure drops from the proximal tubule to the distal nephron.

On a technical point of view for the analysis of flow + pressure experiments, we should also consider a progressive drop of flow shear stress in this condition as the tube diameter increases. At day 1 for *Pkd1*⁻/⁻ mIMCD-3 a dilation ranging from 1.6- to 1.9-fold (Figure S3) represented a drop in flow shear stress between 4-fold and 7-fold. Although still allowing a physiological flow shear stress between 0.1 and 1 dyn/cm², this represents an attenuation of this stress. However, this attenuation of the shear stress alone could not explain the dilation
attributed to the loss of Pkd1. Indeed, the Pkd1-/-specific dilation between day 1 and day 5, after the initial dilation, is greater under pressure + flow than in static condition used here as a control for the possible impact of a decrease of flow shear stress without pressure (Figure S2). We observed a small deformation of only 10% in absence of shear stress and pressure that can be compared to a 29% deformation with pressure and an attenuated shear stress (Figure 2.D). Even more strikingly, there was no longer significant difference in deformation compared to the wild type tubules in static condition.

At last, increasing ECM concentration prevented the excessive dilation of the tubules formed by Pkd1-/- mIMCD-3 under flow+pressure conditions. In addition, contrary to PCT cells, the excessive dilation of the tubule formed by Pkd1-deficient mIMCD-3 under pressure was not caused by increased cell proliferation but was associated with cell flattening, which was also prevented by an increase in ECM concentration. This suggests that cell flattening and tubule dilation for these cells are intertwined events in a tubule distension. Interestingly, a discrepancy between the effect of ECM concentration over this distension points to a nonlinear response of the tubule pertaining to an increase in matrix concentration. This nonlinear relationship between tissue mechanics and tubule response highlights how cell matrix interactions play a critical role in shaping Pkd1-/- mIMCD-3 tubule.

## Possible mechanisms of Pkd1-/- specific dilation.

A central question raised by our data is the biological mechanism of Pkd1-specific tube dilation. In general, three main factors have been identified as potential contributors to tube dilation<sup>63</sup>: increased basement membrane compliance, uncontrolled division of epithelial cells, and ion pumps disorders, e.g. causing excessive tube reabsorption or secretion. Forces exerted
by cells, actively flattening them and increasing the epithelial surface, may also be involved⁶⁴.

While deciphering these precise mechanisms goes beyond the scope of this paper, several hypotheses can be formulated.

With PCT, we found a correlation between *Pkd1*⁻/⁻ overproliferation and tube dilation, suggesting that PCT *Pkd1*⁻/⁻ tube dilation could be driven by this overproliferation ⁶³. Although the absolute values of PCT proliferation rate were not higher for PCT *Pkd1*⁻/⁻ tube than for mIMCD-3 *Pkd1*⁻/⁻ (not dilating; see Figure 4B, 5B), factors like extrusion and cell plasticity specific to each cell type can influence the global effect of proliferation. Altogether, we estimate likely a contribution of proliferation in PCT *Pkd1*⁻/⁻ dilation, possibly associated to a reduced contact inhibition. While overproliferation may be sufficient mechanistically to trigger dilation, we also observed previously an altered apicobasal polarity of *Pkd1*⁻/⁻ PCT cells ⁴³, which could further increase epithelial surface area by limiting extrusion mechanisms.

The mechanisms are more intriguing for mIMCD-3 tubes, models for nephron distal part where cysts form first in the disease. Indeed, mIMCD-3 cells did not proliferate more than parental cells under flow + pressure conditions, excluding a mechanism linked to overproliferation. Among the other possible mechanisms, it is excluded that the tube dilation is mainly driven by a basal to apical fluid secretion into the lumen for the experiments with flow constituting the core of the paper, for scaling reasons linked to the flow rates. Such a mechanism remains also unlikely in static conditions given the open configuration of this tubule-on-chip but cannot strictly be ruled it out, as reversion of the fluidic flux from basal to apical has been observed in the case of ADPKD⁶⁵. However, the most likely causal factors are linked with modifications of the basement membrane and altered cytoskeleton dynamics for *Pkd1*⁻/⁻ cells. In that respect, our study benefits from a companion *in vivo* study ³², with
observations in mice that early tubule dilation is associated with proliferation in the proximal part, but not in the distal part – paralleling the *in vitro* observations reported here. *In vivo* transcriptomic analysis showed an overexpression of genes coding for secreted proteins modifying basement membrane properties (proteases and “softeners” like heparan sulfate), and actin-related proteins which may govern mechanical forces exerted by cells. Preliminary experiments were performed in kidney-on-chips as a first characterization of epithelial organization, by labeling tubes 2 days after confluence, at the beginning of the *Pkd1*<sup>-/-</sup> specific dilation process when occurring (flow+pressure). In all conditions, apicobasal polarity was maintained, as demonstrated by Na-K ATPase, β1-integrin, ZO-1 and primary cilia staining (SI). We observed the adequate expression of primary cilia, an organelle centrally involved in mechanotransduction. MT1-MMP metalloprotease was expressed as soon as day 2, both in parental and *Pkd1*<sup>-/-</sup> tubes under flow+pressure. Accordingly, *in vivo* parallel experiments demonstrated that early distal dilation were accompanied by a thinning of the basement membrane and the secretion of matrix softeners<sup>32</sup>. Furthermore, it has already been observed that loss of *Pkd1* in other *in vitro* models with mIMCD-3 cells is accompanied by abnormal matrix deposition and cell-matrix interactions<sup>66</sup>. Further kinetic and pharmacological characterization would be required to study *in vitro* the contribution of proteases in the *Pkd1*<sup>-/-</sup> specific dilation process. Forces exerted by cells were not monitored here, but the general cytoskeleton dynamics is known to be dependent on the hydrodynamic condition. Future work will address several questions to explore the role of cytoskeleton dynamics, cellular forces and modifications of basement membrane as potential triggers of distal tube dilation.
Perspectives.

Our study deals with mouse ADPKD models, which enable a direct comparison to animal *in vivo* data³². However, it will be important as well to extent it to human cell models. Human tubular renal cell lines, particularly proximal cell lines, have been studied in related organ-on-chips for characterizing reabsorption or pharmacological assessments ⁶⁷,⁶⁸. Altogether the results obtained with PCT only partially coincide with the behavior of *Pkd1*-deficient proximal tubule *in vivo*. Increased cell proliferation and relative insensitivity to increased pressure are notable similarities. However, cell crowding as well as the striking formation of a multilayered epithelial layers are not observed in most orthologous ADPKD mice models³²,⁶⁹. This phenotype is nonetheless reminiscent of the micro-polyp and cord-like hyperplasia features found in a few percent of human ADPKD cysts⁷⁰. An important future development of our work will be to include humans, ideally derived from patients, to provide optimal translational applications of our tubule-on-chip model.

In conclusion, with this work, we show the importance of a specific control of intraluminal pressure in kidney tubule models, more particularly in the context of polycystic kidney disease. We demonstrate that even moderate levels of hydrostatic pressure have specific effects on kidney cells in pathological and non-pathological contexts, with mechanisms that may differ depending on the nephron segment. Although recent studies in different systems (blood microvessels ⁴²) have decoupled pressure from flow shear stress, tubular kidney models have yet to be separately dissected the role of these two mechanical stresses kidney function. Thus, with future *in vitro* kidney models, a specific control of the effects of flow and pressure could be pivotal to decipher the mechanisms underlying diseases such as ADPKD but also more generally, to improve the biomimicry of these systems.
**Disclosure statement.** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Essig, M., Terzi, F., Burtin, M. & Friedlander, G. Mechanical strains induced by tubular flow affect the phenotype of proximal tubular cells. *Am J Physiol Renal Physiol* **281**, F751-62 (2001).

2. Duan, Y., Gotoh, N., Yan, Q., Du, Z., Weinstein, A. M., Wang, T. & Weinbaum, S. Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes. *Proc Natl Acad Sci U S A* **105**, 11418–11423 (2008).

3. Verma, D., Bajpai, V. K., Ye, N., Maneshi, M. M., Jetta, D., Andreadis, S. T., Sachs, F. & Hua, S. Z. Flow induced adherens junction remodeling driven by cytoskeletal forces. *Exp Cell Res* **359**, (2017).

4. Kunnen, S. J., Malas, T. B., Semeins, C. M., Bakker, A. D. & Peters, D. J. M. Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells. *J Cell Physiol* (2018) doi:10.1002/jcp.26222.

5. Essig, M. & Friedlander, G. Tubular shear stress and phenotype of renal proximal tubular cells. *Journal of the American Society of Nephrology* **14**, (2003).

6. Verma, D., Meng, F., Sachs, F. & Hua, S. Z. Flow-induced focal adhesion remodeling mediated by local cytoskeletal stresses and reorganization. *Cell Adh Migr* **9**, 432–440 (2015).

7. Ross, E. J., Gordon, E. R., Sothers, H., Darji, R., Baron, O., Haithcock, D., Prabhakarpandian, B., Pant, K., Myers, R. M., Cooper, S. J. & Cox, N. J. Three dimensional modeling of biologically relevant fluid shear stress in human renal tubule cells mimics in vivo transcriptional profiles. *Sci Rep* **11**, (2021).
8. Della Corte, M. & Viggiano, D. Wall Tension and Tubular Resistance in Kidney Cystic Conditions. *Biomedicines* vol. 11 Preprint at https://doi.org/10.3390/biomedicines11061750 (2023).

9. Weimbs, T. Polycystic kidney disease and renal injury repair: common pathways, fluid flow, and the function of polycystin-1. *Am J Physiol Renal Physiol* **293**, F1423-32 (2007).

10. Nigro, E. A., Distefano, G., Chiaravalli, M., Matafora, V., Castelli, M., Pesenti Gritti, A., Bachi, A. & Boletta, A. Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness. *Sci Rep* (2019) doi:10.1038/s41598-019-53061-0.

11. Lee, K., Boctor, S., Barisoni, L. M. & Gusella, G. L. Inactivation of integrin-beta1 prevents the development of polycystic kidney disease after the loss of polycystin-1. *J Am Soc Nephrol* **26**, 888-895 (2015).

12. Xi, W., Sonam, S., Beng Saw, T., Ladoux, B. & Teck Lim, C. Emergent patterns of collective cell migration under tubular confinement. *Nat Commun* **8**, 1517 (2017).

13. Venzac, B., Madoun, R., Benarab, T., Monnier, S., Cayrac, F., Myram, S., Leconte, L., Amblard, F., Viovy, J. L., Descroix, S. & Coscoy, S. Engineering small tubes with changes in diameter for the study of kidney cell organization. *Biomicrofluidics* **12**, (2018).

14. Torres, V. E. & Harris, P. C. Mechanisms of Disease: Autosomal dominant and recessive polycystic kidney diseases. *Nat Clin Pract Nephrol* **2**, 40–55 (2006).

15. Leonhard, W. N., Zandbergen, M., Veraar, K., van den Berg, S., van der Weerd, L., Breuning, M., de Heer, E. & Peters, D. J. Scattered Deletion of PKD1 in Kidneys Causes a Cystic Snowball Effect and Recapitulates Polycystic Kidney Disease. *J Am Soc Nephrol* **26**, 1322–1333 (2015).

16. Cornec-Le Gall, E., Audrézet, M.-P., Meur, Y. Le, Chen, J.-M. & Férec, C. Genetics and pathogenesis of autosomal dominant polycystic kidney disease: 20 years on. *Hum Mutat* **35**, 1393-406 (2014).

17. Douguet, D., Patel, A. & Honoré, E. Structure and function of polycystins: insights into polycystic kidney disease. *Nature Reviews Nephrology* Preprint at https://doi.org/10.1038/s41581-019-0143-6 (2019).

18. Boca, M., Distefano, G., Qian, F., Bhunia, A. K., Germino, G. G. & Boletta, A. Polycystin-1 induces resistance to apoptosis through the phosphatidylinositol 3-kinase/Akt signaling pathway. *J Am Soc Nephrol* **17**, 637–647 (2006).

19. Castelli, M., Boca, M., Chiaravalli, M., Ramalingam, H., Rowe, I., Distefano, G., Carroll, T. & Boletta, A. Polycystin-1 binds Par3/aPKC and controls convergent extension during renal tubular morphogenesis. *Nat Commun* **4**, (2013).

20. Lee, E. J. Cell Proliferation and Apoptosis in ADPKD. in *Cystogenesis* vol. 933 25–34 (2016).
21. Fischer, E., Legue, E., Doyen, A., Nato, F., Nicolas, J. F., Torres, V., Yaniv, M. & Pontoglio, M. Defective planar cell polarity in polycystic kidney disease. *Nat Genet* **38**, 21–23 (2006).

22. Bhoonderowa, L., Hameurlaine, F., Arbabian, A., Faqir, F., Amblard, F. & Coscoy, S. Polycystins and intercellular mechanotransduction: A precise dosage of polycystin 2 is necessary for alpha-actinin reinforcement of junctions upon mechanical stimulation. *Exp Cell Res* **348**, 23–35 (2016).

23. Nigro, E. A., Distefano, G., Chiaravalli, M., Matafora, V., Castelli, M., Pesenti Gritti, A., Bachi, A. & Boletta, A. Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness. *Sci Rep* (2019) doi:10.1038/s41598-019-53061-0.

24. Flores, D., Battini, L., Gusella, G. L. & Rohatgi, R. Fluid shear stress induces renal epithelial gene expression through polycystin-2-dependent trafficking of extracellular regulated kinase. *Nephron Physiol* **117**, p27-36 (2011).

25. Nauli, S. M., Alenghat, F. J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A. E., Lu, W., Brown, E. M., Quinn, S. J., Ingber, D. E. & Zhou, J. Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. *Nat Genet* **33**, 129–137 (2003).

26. Delling, M., Indzhykulian, A. A., Liu, X., Li, Y., Xie, T., Corey, D. P. & Clapham, D. E. Primary cilia are not calcium-responsive mechanosensors. *Nature* **531**, 656–660 (2016).

27. Ma, M., Tian, X., Igarashi, P., Pazour, G. J. & Somlo, S. Loss of cilia suppresses cyst growth in genetic models of autosomal dominant polycystic kidney disease. *Nat Genet* (2013) doi:10.1038/ng.2715.

28. Viau, A. et al. Cilia-localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney. *EMBO J* **37**, (2018).

29. Kunnen, S. J., Malas, T. B., Formica, C., Leonhard, W. N., 't Hoen, P. A. C. & Peters, D. J. M. Comparative transcriptomics of shear stress treated Pkd1-/- cells and pre-cystic kidneys reveals pathways involved in early polycystic kidney disease. *Biomedicine & Pharmacotherapy* **108**, 1123–1134 (2018).

30. Zhang, Y., Reif, G. & Wallace, D. P. Extracellular matrix, integrins, and focal adhesion signaling in polycystic kidney disease. *Cell Signal* **7**, (2020).

31. Cai, J., Song, X., Wang, W., Watnick, T., Pei, Y., Qian, F. & Pan, D. A RhoA-YAP-c-Myc signaling axis promotes the development of polycystic kidney disease. *Genes Dev* (2018) doi:10.1101/gad.315127.118.

32. Mazloum, M. et al. Cilia to basement membrane signalling is a biomechanical driver of autosomal dominant polycystic kidney disease. *BioRxiv* (2024).

33. Sharif-Naeini, R., Folgering, J. H., Bichet, D., Duprat, F., Lauritzen, I., Arhatte, M., Jodar, M., Dedman, A., Chatelain, F. C., Schulte, U., Retailleau, K., Loufrani, L., Patel, A., Sachs, F.,
Delmas, P., Peters, D. J. & Honore, E. Polycystin-1 and -2 dosage regulates pressure sensing. *Cell* **139**, 587–596 (2009).

34. Peyronnet, R., Martins, J. R., Duprat, F., Demolombe, S., Arhatte, M., Jodar, M., Tauc, M., Duranton, C., Paulais, M., Teulon, J., Honore, E. & Patel, A. Piezo1-dependent stretch-activated channels are inhibited by Polycystin-2 in renal tubular epithelial cells. *EMBO Rep* **14**, 1143–1148 (2013).

35. Zoi, I., Gargalionis, A. N., Papavassiliou, K. A., Nasiri-Ansari, N., Piperi, C., Basdra, E. K. & Papavassiliou, A. G. Polycystin-1 and hydrostatic pressure are implicated in glioblastoma pathogenesis in vitro. *J Cell Mol Med* **26**, (2022).

36. Montalbetti, N., Li, Q., Gonzalez-Perrett, S., Semprine, J., Chen, X. Z. & Cantiello, H. F. Effect of hydro-osmotic pressure on polycystin-2 channel function in the human syncytiotrophoblast. *Pflugers Arch* **451**, 294–303 (2005).

37. Hiratsuka, K., Miyoshi, T., Kroll, K. T., Gupta, N. R., Valerius, M. T., Ferrante, T., Yamashita, M., Lewis, J. A. & Morizane, R. Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery. *Sci Adv* **8**, (2022).

38. Cruz, N. M., Song, X., Czerniecki, S. M., Gulieva, R. E., Churchill, A. J., Kim, Y. K., Winston, K., Tran, L. M., Diaz, M. A., Fu, H., Finn, L. S., Pei, Y., Himmelfarb, J. & Freedman, B. S. Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease. *Nature Materials* vol. 16 Preprint at https://doi.org/10.1038/NMAT4994 (2017).

39. Homan, K. A., Kolesky, D. B., Skylar-Scott, M. A., Herrmann, J., Obuobi, H., Moisan, A. & Lewis, J. A. Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips. *Sci Rep* **6**, (2016).

40. Lin, N. Y. C., Homan, K. A., Robinson, S. S., Kolesky, D. B., Duarte, N., Moisan, A. & Lewis, J. A. Renal reabsorption in 3D vascularized proximal tubule models. *Proceedings of the National Academy of Sciences* **116**, 5399–5404 (2019).

41. Weber, E. J., Chapron, A., Chapron, B. D., Voellinger, J. L., Lidberg, K. A., Yeung, C. K., Wang, Z., Yamaura, Y., Hailey, D. W., Neumann, T., Shen, D. D., Thummel, K. E., Muczynski, K. A., Himmelfarb, J. & Kelly, E. J. Development of a microphysiological model of human kidney proximal tubule function. *Kidney Int* (2016) doi:10.1016/j.kint.2016.06.011.

42. Dessalles, C. A., Ramón-Lozano, C., Babataheri, A. & Barakat, A. I. Luminal flow actuation generates coupled shear and strain in a microvessel-on-chip. *Biofabrication* **14**, (2022).

43. Myram, S., Venzac, B., Lapin, B., Battistella, A., Cayrac, F., Cinquin, B., Cavaniol, C., Gropplero, G., Bonnet, I., Demolombe, S., Descroix, S. & Coscoy, S. A Multitubular Kidney-on-Chip to Decipher Pathophysiological Mechanisms in Renal Cystic Diseases. *Front Bioeng Biotechnol* **9**, 283 (2021).
44. Lapin, B., Myram, S., Nguyen, M. L., Gropplero, G., Coscoy, S. & Descroix, S. Construction of a Multitubular Perfusable Kidney-on-Chip for the Study of Renal Diseases. *Methods Mol Biol* **2664**, 85–106 (2023).

45. Joly, D., Ishibe, S., Nickel, C., Yu, Z., Somlo, S. & Cantley, L. G. The polycystin 1-C-terminal fragment stimulates ERK-dependent spreading of renal epithelial cells. *J Biol Chem* **281**, 26329–26339 (2006).

46. Wei, F., Karihaloo, A., Yu, Z., Marlier, A., Seth, P., Shibazaki, S., Wang, T., Sukhatme, V. P., Somlo, S. & Cantley, L. G. Neutrophil gelatinase-associated lipocalin suppresses cyst growth by Pkd1 null cells in vitro and in vivo. *Kidney Int* **74**, 1310–1318 (2008).

47. Hofherr, A., Busch, T., Huber, N., Nold, A., Bohn, A., Viau, A., Bienaimé, F., Kuehn, E. W., Arnold, S. J. & Köttgen, M. Efficient genome editing of differentiated renal epithelial cells. *Pflugers Arch* **469**, 303–311 (2017).

48. Westermann, L., Li, Y., Göcmen, B., Niedermoser, M., Rhein, K., Jahn, J., Cascante, I., Schöler, F., Moser, N., Neubauer, B., Hofherr, A., Behrens, Y. L., Göhring, G., Köttgen, A., Köttgen, M. & Busch, T. Wildtype heterogeneity contributes to clonal variability in genome edited cells. *Sci Rep* **12**, (2022).

49. Gilmer, G. G., Deshpande, V. G., Chou, C. L. & Knepper, M. Flow resistance along the rat renal tubule. *Am J Physiol Renal Physiol* **315**, F1398–F1405 (2018).

50. Welling, L. W. & Grantham, J. J. *Physical Properties of Isolated Perfused Renal Tubules and Tubular Basement Membranes*. Clin. Res vol. 19 (1970).

51. Verhulsel, M., Shivokhin, M. E., Simon, A., Descroix, S., Frétigny, C. & Talini, L. High bandwidth noninvasive measurements of the linear viscoelasticity of collagen gels. *J Rheol (N Y N Y)* (2016) doi:10.1122/1.4965039.

52. Raub, C. B., Putnam, A. J., Tromberg, B. J. & George, S. C. Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy. *Acta Biomater* **6**, 4657–4665 (2010).

53. Lama, M., Raveendranathan, B., Brun, J., Fernandes, F. M., Boissière, C., Nassif, N. & Marcellan, A. Biomimetic Tough Gels with Weak Bonds Unravel the Role of Collagen from Fibril to Suprafibrillar Self-Assembly. *Macromol Biosci* **21**, (2021).

54. Vidmar, C. S., Bazzi, M. & Lai, V. K. Computational and experimental comparison on the effects of flow-induced compression on the permeability of collagen gels. *J Mech Behav Biomed Mater* **128**, (2022).

55. Engelberg, J. A., Datta, A., Mostov, K. E. & Hunt, C. A. MDCK cystogenesis driven by cell stabilization within computational analogues. *PLoS Comput Biol* **7**, e1002030 (2011).
56. Gin, E., Tanaka, E. M. & Brusch, L. A model for cyst lumen expansion and size regulation via fluid secretion. *J Theor Biol* **264**, 1077–1088 (2010).

57. Torres-Sánchez, A., Kerr Winter, M. & Salbreux, G. Tissue hydraulics: Physics of lumen formation and interaction. *Cells and Development* vol. 168 Preprint at https://doi.org/10.1016/j.cdev.2021.203724 (2021).

58. McAteer, J. A., Evan, A. P., Vance, E. E. & Gardner, K. D. MDCK cysts: An in vitro model of epithelial cyst formation and growth. *Journal of Tissue Culture Methods* **10**, (1986).

59. Capra, J. P. & Eskelinen, S. M. MDCK cells are capable of water secretion and reabsorption in response to changes in the ionic environment. *Can J Physiol Pharmacol* **95**, (2017).

60. Barros, E. J. G., Santos, O. F. P., Matsumoto, K., Nakamura, T. & Nigam, S. K. Differential tubulogenic and branching morphogenetic activities of growth factors: Implications for epithelial tissue development. *Proc Natl Acad Sci U S A* **92**, (1995).

61. Chen, D., Roberts, R., Pohl, M., Nigam, S., Kreidberg, J., Wang, Z., Heino, J., Ivaska, J., Coffa, S., Harris, R. C., Pozzi, A. & Zent, R. Differential expression of collagen- and laminin-binding integrins mediates ureteric bud and inner medullary collecting duct cell tubulogenesis. *Am J Physiol Renal Physiol* **287**, (2004).

62. Cruz-Acuña, R., Mulero-Russe, A., Clark, A. Y., Zent, R. & García, A. J. Identification of matrix physicochemical properties required for renal epithelial cell tubulogenesis by using synthetic hydrogels. *J Cell Sci* **132**, (2019).

63. Hannezo, E., Prost, J. & Joanny, J. F. Mechanical instabilities of biological tubes. *Phys Rev Lett* **109**, 18101 (2012).

64. Kela, S. A., Sethi, K., Tan, P. Y., Suresh, D., Ong, H. T., Castaneda, P. G., Amin, M. R., Laviv, T., Cram, E. J., Faix, J. & Zaidel-Bar, R. Tension-dependent RHGF-1 recruitment to stress fibers drives robust spermathecal tissue contraction. *J Cell Biol* **222**, (2023).

65. Choudhury, M. I., Li, Y., Mistriotis, P., Vasconcelos, A. C. N., Dixon, E. E., Yang, J., Benson, M., Maity, D., Walker, R., Martin, L., Koroma, F., Qian, F., Konstantopoulos, K., Woodward, O. M. & Sun, S. X. Kidney epithelial cells are active mechano-biological fluid pumps. *Nature Communications* **2022** **13:1** **13**, 1–13 (2022).

66. Subramanian, B., Ko, W. C., Yadav, V., DesRochers, T. M., Perrone, R. D., Zhou, J. & Kaplan, D. L. The Regulation of Cystogenesis in a Tissue Engineered Kidney Disease System by Abnormal Matrix Interactions. *Biomaterials* **33**, 8383 (2012).

67. Weber, E. J., Chapron, A., Chapron, B. D., Voellinger, J. L., Lidberg, K. A., Yeung, C. K., Wang, Z., Yamaura, Y., Hailey, D. W., Neumann, T., Shen, D. D., Thummel, K. E., Muczynski, K. A., Himmelfarb, J. & Kelly, E. J. Development of a microphysiological model of human kidney proximal tubule function. *Kidney Int* (2016) doi:10.1016/j.kint.2016.06.011.
68. Sakolish, C., Chen, Z., Dalaijamts, C., Mitra, K., Liu, Y., Fulton, T., Wade, T. L., Kelly, E. J., Rusyn, I. & Chiu, W. A. Predicting tubular reabsorption with a human kidney proximal tubule tissue-on-a-chip and physiologically-based modeling. *Toxicology in Vitro* **63**, (2020).

69. Dong, K., Zhang, C., Tian, X., Coman, D., Hyder, F., Ma, M. & Somlo, S. Renal plasticity revealed through reversal of polycystic kidney disease in mice. *Nat Genet* **53**, (2021).

70. Grantham, J. J., Geiser, J. L. & Evan, A. P. Cyst formation and growth in autosomal dominant polycystic kidney disease. *Kidney Int* **31**, 1145-1152 (1987).

**Acknowledgements.** The authors acknowledge all the members of the MMBM and PBME groups for their precious support in the process development and in microfabrication. We are grateful to Sarah Myram, Sophie Demolombe, Eric Honoré, Bénédicte Delaval, Abdul Barakat, Jacques Fattaccioli, Pierre Ucla, Isabelle Bonnet, Pascal Silberzan and Axel Buguin for insightful discussions, and to Polina Rapoport and Lorraine Couteau for their participation in the experiments. We thank George M. O’Brien Kidney Center at Yale (NIH P30 DK079310) and Stefan Somlo for the kind gift of PCT *Pkd1<sup>+/-</sup>* and PCT *Pkd1<sup>-/-</sup>* cell lines and Michael Köttgen, Lukas Westermann and Tilman Busch, Freiburg University, for the kind gift of mIMCD-3 control and *Pkd1*-KO (*Pkd1<sup>-/-</sup>* ) cell lines. This work benefited from the technical contribution of the joint service unit CNRS UAR 3750. The authors would like to thank the engineers of this unit (in particular Bertrand Cinquin) for their advice during the development of the experiments. The authors greatly acknowledge the Cell and Tissue Imaging core facility (PICT-IBiSA), Institut Curie, member of the French National Research Infrastructure France-Biolmaging (ANR10-INBS-04).

**Funding.** This work was supported by a doctoral grant from AMX ("Ministère de la Recherche et de l'Enseignement supérieur", France) (recipient B. Lapin), and the 'Fondation pour la
Recherche Médicale' (FRM, recipient B. Lapin, program FDT; Fondation Durlach). It was supported by the European Commission grant FET Open program (FETOPEN-01-2016-2017). This work has received the support of 'Association Polykystose France', provided by the 'Société Francophone de Néphrologie, Dialyse et Transplantation' (SFNDT). This work has received the support of "Institut Pierre-Gilles de Gennes" laboratoire d'excellence, "Investissements d'avenir" program ANR-10-IDEX-0001-02. This work received support from the grants ANR-11-LABX-0038, ANR-10-IDEX-0001-02 (LabEx CelTisPhyBio - Cell(n)Scale). This work was supported by the Centre National de la Recherche Scientifique (CNRS), Sorbonne Université and Institut Curie.

**Author Contributions.** B.L., F.B., S.D. and S.C. designed the study; B.L., J.V., G.G. and M.M. performed experiments; B.L., J.V., S.D. and S.C. analyzed data; S.D. and S.C. supervised the study and wrote the manuscript; B.L., F.B., S.D. and S.C. drafted and revised the manuscript; all authors approved the final version of the manuscript.
<figure><img src="image_3.png" /><figcaption>**Figure 1:** A perfused kidney-on chip to decouple flow shear stress from intraluminal pressure. A 3D model of the chip pointing at its key features is shown in (A) with a zoom on the collagen microtubules (see additional schemes of the device in Lapin et al.⁴⁴). A large opening in the PDMS cover enables free exchanges between the collagen around the tubes and the external medium. Depending on where the chip is perfused (B), either in the Pressure Offset inlet (top) or in the Set Pressure inlet (bottom), we show with a finite element simulation (C) (COMSOL Multiphysics) that a pressure of 10 mbar will result in a negligible intraluminal pressure or in a 10-mbar intraluminal pressure respectively with the same flow shear stress in the tubules around 1 dyn/cm². (D) Estimation of the intraluminal shear stress for 80µm wide tubules, calculated from measurements of the flow rates in confluent tubules of mIMCD-3 cells 1 day (n=27) and 8 days (n=20) after seeding. (Boxplots showing are 1st quartile, median and 3rd quartile, with whiskers showing extrema. Each dot represents a tubule.) (E) Impermeability of m-IMCD-3 (top) and PCT (bottom) tubules to FITC Dextran 10 kDa and 40 kDa respectively. Pictures show the temporal evolutions of one mIMCD-3 and one PCT tubule at the initial time point (top of each image pair) and after 180 min. Scale bars 100 µm.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2:** Under flow shear stress, loss of *Pkd1* leads to increased enlargement of PCT tubules, but intraluminal pressure is necessary for increased enlargement of mIMCD-3 tubules. PCT (A,C) or mIMCD-3 (B, D) tubules were perfused after seeding without intraluminal pressure (A,B) or with intraluminal pressure (C,D). Representative tubules are shown in these configurations, on the day of confluency and one or several days after confluency. Mean diameters of the tubules 5 days after confluency normalized by their mean diameters at the day of confluency (D5/D0) are indicated. In the flow+pressure condition, characterized by *Pkd1*⁻/⁻ dilation for both cell types, the evolution following the initial dilation is also quantified, with diameters at day 5 normalized by diameter at day 1 (D5/D1, right). Parental PCT cells (blue, A, flow N=8 chips/n=29 tubules, C, flow+pressure N=8 chips/n=39 tubules), PCT *Pkd1*⁻/⁻ cells (green, A, flow N=11 chips/ n=41 tubules, C, flow+pressure N=8 chips/n=30 tubules), parental mIMCD-3 cells (blue, B, flow N=9 chips/ n=41 tubules, D, flow+pressure N=5 chips /n=25 tubules) and mIMCD-3 *Pkd1*⁻/⁻ cells (green, B, flow N=10 chips/n=47 tubules, D, flow+pressure N=5 chips/n=24 tubules). Each dot represents a tubule. The data presented in this figure correspond to three different independent cell seedings. (ns: p-value>0.05, *: p-</figcaption></figure>
value≤0.05, **: p-value≤0.01, ****: p-value≤0.0001, actual p-values are indicated below the symbols). Scale bars: 100µm.

<figure><img src="image_5.png" /><figcaption>**Figure 3:** Increase in pressure-driven tubular dilation due to loss of *Pkd1* is mitigated by denser extracellular matrix in *mIMCD-3* tubules but not in *PCT* tubules. To study the impact of matrix mechanical properties on pressure-driven dilation, PCT and *mIMCD-3* cells were cultured under flow with intraluminal pressure in a denser 9 mg/mL collagen I matrix instead of 6 mg/mL Here representative PCT (A) and *mIMCD-3* (B) tubules are shown in this configuration. On the right of these images are plotted the mean diameters of the tubules 5</figcaption></figure>
days after confluency normalized by their mean diameters at the day of confluency (left, D5/D0) and 1 day after confluency (right, D5/D1). As reference, median and 95% confidence interval of analogous experiment in a 6mg/mL collagen gel are indicated by black superimposed lines. (A) PCT tubules still display higher levels of dilation for *Pkd1*⁻/⁻ cells (green, N=6 chips/n=26 tubules) than parental ones (blue, N=6 chips/n=28 tubules). (B) On contrary, for mIMCD-3 tubes there is no longer significant difference between the *Pkd1*⁻/⁻ (green, N=5 chips/n=25 tubules) and parental cells (blue, N=5 chips/n=24 tubules). Boxplots shown are 1st quartile, median and 3rd quartile, with whiskers showing extrema. Each dot represents a tubule. The data presented in this figure correspond to three independent cell seedings. (ns: p-value>0.05, *: p-value≤0.05, ****: p-value≤0.0001, actual p-values are indicated below the symbols). Scale bars: 100µm.

Flow
PCT
Pkd1⁻/⁻

A

Day 1, 6mg/mL

<figure><img src="image_6.png" /></figure>

Day 5, 6mg/mL

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

B

<figure><img src="image_10.png" /></figure>

C

<figure><img src="image_11.png" /></figure>

Flow+Pressure
PCT
Pkd1⁻/⁻

D

Day 1, 6mg/mL

<figure><img src="image_12.png" /></figure>

Day 5, 6mg/mL

<figure><img src="image_13.png" /></figure>

Day 5, 9 mg/mL

<figure><img src="image_14.png" /></figure>

Nuclei F-actin Ki67

E

<figure><img src="image_15.png" /></figure>

F

<figure><img src="image_16.png" /></figure>
<caption>Figure 4: In PCT tubules, loss of *Pkd1* leads to higher proliferation levels and denser tissues.</caption>

PCT tubules left under flow without pressure (A) or under flow with pressure (D) were fixed 1 day and 5 days after confluency in 6 mg/mL collagen I matrix, 5 days after confluency in 9 mg/mL collagen, and stained for nuclei (blue), F-actin (green), and Ki-67 (red). Cell proliferation was quantified by computing the ratio of Ki67 positive nuclei over the total number of nuclei (B, E); comparisons between parental (blue) and *Pkd1*⁻/⁻ cells (green) are shown. Cell morphology was assessed by measuring the average internuclear distance within each tubule (C, F) as a proxy for cell spreading. For internuclear distance, either parental (blue) or *Pkd1*⁻/⁻ (green) values are shown side to side, to focus on the relative evolution in cell spreading related to time or matrix properties. (B-C) PCT tubes under flow, control *Pkd1*⁺/⁻ at day 1 and day 5 (N=5 chips /n=19 tubules and N=10 chips/n=39 tubules), and *Pkd1*⁻/⁻ at day 1 and day 5 (N=5/n=17 and N=12/n=44), (E-F) PCT tubes under flow+pressure, control *Pkd1*⁺/⁻ at day 1, day 5 and day 5 in 9mg/mL collagen (N=8/n=27, N=11/n=51 and N=4/n=19 respectively) and *Pkd1*⁻/⁻ at day 1, day 5 and day 5 in 9mg/mL collagen (N=5/n=24, N=12/n=57 and N=5/n=23 respectively). Boxplots shown are 1st quartile, median and 3rd quartile, with whiskers showing extrema. Each dot represents a tubule. The data presented in this figure correspond to two independent cell seedings. (ns: p-value>0.05, *: p-value≤0.05, ****: p-value≤0.0001, actual p-values are indicated below the symbols). Scale bars: 50µm. Overall, *Pkd1*⁻/⁻ cells exhibit higher proliferation levels than the parental cells. This correlates with *Pkd1*⁻/⁻ tubules that become denser over time under flow and often form multilayer in all conditions.
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

**Figure 5:** In mMCD-3 tubules, increased tubular dilation is not correlated to higher proliferation levels but to increased cell spreading. mMCD-3 tubules left under flow without pressure (A) or under flow with pressure (D) were fixed 1 day and 5 days after confluency in 6 mg/mL collagen I matrix, 5 days after confluency in 9 mg/mL collagen, and stained for nuclei (blue), F-actin (green), and Ki-67 (red). Cell proliferation was quantified by computing the ratio of Ki67 positive nuclei over the total number of nuclei (B, E); comparisons between parental (blue) and *Pkd1*⁻/⁻ cells (green) are shown. Cell morphology was assessed by measuring the average internuclear distance within each tubule (C, F) as a proxy for cell spreading. For internuclear distance, either parental (blue) or *Pkd1*⁻/⁻ (green) values are shown side to side, to focus on the relative evolution in cell spreading related to time or matrix properties. (B-C) mMCD-3 tubes under flow, control parental cells at day 1 and day 5 (N=3 chips/n=14 tubules and N=3 chips/n=15 tubules), and *Pkd1*⁻/⁻ cells at day 1 and day 5 (N=3/n=11 and N=4/n=18),
(E-F) mIMCD-3 tubes under flow+pressure, control parental cells at day 1, day 5 and day 5 in 9mg/mL collagen (N=6/n=28, N=8/n=24, N=6/n=25 respectively) and *Pkd1*⁻/⁻ cells at day 1, day 5 and day 5 in 9mg/mL collagen (N=7/n=28, N=6/n=26, N=5/n=25 respectively). Boxplots shown are 1st quartile, median and 3rd quartile, with whiskers showing extrema. Each dot represents a tubule. The data presented in this figure correspond to two independent cell seedings. (ns: p-value>0.05, *: p-value≤0.05, **: p-value≤0.01, **: p-value≤0.001, ***: p-value≤0.0001, actual p-values are indicated below the symbols). Scale bars: 50µm. Contrary to PCT tubules, higher proliferation levels are associated with *Pkd1*⁻/⁻ tubules only under flow without intraluminal pressure that become denser over time. Under flow with luminal pressure, however, if parental cells get significantly denser over time, *Pkd1*⁻/⁻ cells become significantly more spread. This intraluminal pressure effect is mitigated by a denser matrix.