Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging

Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman’s capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.


Introduction
Adult podocytes are terminally differentiated glomerular epithelial cells that form the outer layer of the glomerular filtration barrier and are unable to self-replicate [1]. As a result, a major limitation in their recovery and repair process in many disease states is their inability to restore their numbers following depletion [2,3]. Once total podocyte number decreases below a certain threshold in glomerular disease, glomerular scarring ensues [4][5][6]. For these reasons, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 with a combination of ketamine (100 mg per kg body weight) and xylazine (10 mg per kg body weight). The AIW consists of a re-usable titanium ring (kind gift from Ina Schiessl, University of Regensburg, Germany) with a 1-mm groove on the side and a 170 μm thick coverslip fixed on the top. The glass coverslip was secured using tissue glue. After preparation of the window, the AIW was surgically implanted into the flank incision on the dorsal skin and abdominal wall right above the left kidney, and held in position by a purse-string suture, which was concealed within the groove of the AIW ring to prevent mice from biting or pulling the sutures. To further reduce the risk of dislodgement of the AIW, a nonwoven non-resorbable suture (Prolene, Somerville, NJ, USA) was used, which can be tightened once the window is inserted and which can keep the window secured for >5 weeks. Postoperative care included animal monitoring to ensure that vital signs and body weight returned to preoperative status.

Inducing experimental FSGS typified by abrupt podocyte depletion
Experimental focal segmental glomerulosclerosis (FSGS) was induced in Ren1cCre /R26R-Con-fettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice strains with a cytotoxic sheep antipodocyte antibody, as previously described [22,27,28]. This cytotoxic antibody induces abrupt podocyte depletion, accompanied by glomerulosclerosis. Adult mice were given 2 doses of sheep anti-glomerular antibody at 12mg/20g body weight via IP injection, 24 hours apart. Thereafter, the experimental protocols differed by strain to address different questions as follows: Ren1cCre/R26R-ConfettiTG/WT mice were sacrificed randomly on day 14 (n = 6) and on day 28 (n = 7) of disease. Uninjected mice (n = 4) served as baselines. At sacrifice, mice were perfused with 10ml of ice cold PBS to remove excess red blood cells. Kidneys were split in half and one half was fixed overnight at 4˚C in 10% neutral buffered formalin (Globe Scientific, Paramus, NJ, USA), rinsed in 70% Ethanol, processed and embedded in paraffin. The other half was fixed for 45 minutes in 4% Paraformaldehyde Solution (PFA) in PBS (Affymetrix, Santa Clara, California, USA), washed in 30% sucrose overnight at 4˚C, patted dry, rinsed briefly in OCT, embedded in OCT, and frozen in a dry ice 100% ethanol bath. Sections were then cut from the paraffin or OCT blocks at 4μm thickness.
Ren1dCre/R26R-ConfettiTG/WT mice were used to assess the migration of CoRL from the juxta-glomerular compartment to the intra-glomerular compartment by intravital serial multiphoton microscopy (see below for details).

Intravital serial multiphoton microscopy (MPM)
Under continuous anesthesia (Isoflurane 1-4% inhalant via nose-cone), Ren1dCre /R26R-Con-fettiTG/WT mice with the AIW above were placed on the stage of the inverted microscope as described previously [29]. Body temperature was maintained with a homeothermic blanket system (Harvard Apparatus, Holliston, MA, USA). Alexa 594 bovine serum albumin was injected iv to label the vasculature. The images were acquired using a Leica TCS SP5 multiphoton confocal fluorescence imaging system with a 63× Leica glycerol-immersion objective (numerical aperture (NA) 1.3) powered by a Chameleon Ultra-II MP laser at 860 nm (Coherent) and a DMI 6000 inverted microscope's external nondescanned HyD detectors (Leica Microsystems, Heidelberg, Germany). Short-pass filters (680 nm for blue and red and 700 nm for green and yellow), dichroic mirrors (cut off at 515 nm for green and yellow and at 560 nm for blue and red) and bandpass filters were specific for detecting CFP, GFP, YFP and RFP emission (473, 514, 545 and 585 nm, respectively) (Chroma, Bellows Falls, VT, USA). The potential toxicity of laser excitation and fluorescence to the cells was minimized by using a low laser power and high scan speeds to keep total laser exposure as minimal as possible. The usual image acquisition consisted of only one z stack per glomerulus (<3 min), which resulted in no apparent cell injury. Serial imaging of the same glomerulus in the same animal/kidney was performed once every 3 days for up 12 days after the first imaging session.
BrdU/FDU labeling of mice to assess proliferation Amersham Cell Proliferation Labeling Reagent (GE Healthcare Life Sciences, Little Chalfont, UK) was administered to quantitate cell proliferation. Mice were given 3 consecutive IP injections of 10ul per gram body weight of 5-bromo-2-deoxyuridine and 5-fluoro-2'-deoxyuridine as recommended by the manufacturer, with the first dose given the day after the last dose of antibody. BrdU immunostaining was performed on paraffin embedded tissue that was processed as described above and incubated at 4˚C overnight with a primary anti-BrdU antibody (1:200, GE Healthcare Life Sciences, Little Chalfont, UK), and then with biotinylated antimouse IgG secondary (1:500, Vector Laboratories, Burlingame, CA, USA) followed by streptavidin-conjugated Alexa Fluor 594 (1:100, Molecular Probes, Eugene, OR, USA). FITC 488 conjugated anti-GFP antibody, which binds to all four of the confetti variants (Rockland Immunochemicals for Research, Gilbertsville, PA, USA) was used to detect all four confetti reporters at the 488nm wavelength channel (GFP).

Visualization of confetti reporter in Ren1cCre /R26R-ConfettiTG/WT mice
Immunostaining was examined on a Leica TCS SPE II laser scanning confocal microscope (Solms, Germany) with X40 (1.3 NA) or X60 (1.45 NA) oil objectives. In order to visualize the multicolor confetti reporter, four-μm cryo sections were rinsed in PBS (pH 7.4) to remove OCT compound (VWR, Radnor, PA, USA) and mounted with vectashield (Vector Labs, Burlingame, CA, USA). Confocal images were acquired in 1,024 X 1,024 pixel format with 8 bit intensity resolution. The acquisition was set in the green, red, yellow and cyan wavelengths as follows; CFP 458 nm excitation, 464-495 nm emission, GFP 488 nm excitation, 497-510 nm emission, YFP 514 nm excitation, 517-540 nm emission, and RFP 561 nm excitation, 575-654 nm emission.

Assessment of glomerular injury and podocyte density
In both reporter mouse strains, immunostaining was performed for p57 with Periodic Acid Schiff (PAS) counterstaining to measure podocyte number and assess glomerulosclerosis. In brief, paraffin sections were processed as described above, with antigen retrieval in 1 mM EDTA, pH 8.0. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide and sections were incubated overnight at 4˚C with a primary rabbit anti-p57 (1:800, Santa Cruz Biotechnology, Santa Cruz, CA,USA) followed by rabbit on rodent HRP-polymer (Biocare Medical, Concord, CA, USA). Visualization of immunostaining was by precipitation of diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO, USA). Counterstaining was performed with Periodic Acid Schiff (PAS) by washing slides in fresh 0.5% periodic acid (Sigma-Aldrich, St Louis, MO, USA) for 8 minutes, washed for 5 minutes in ddH2O, sections were incubated for 10 minutes at room temperature with Schiff's Reagent (Sigma-Aldrich, St Louis, MO, USA), washed 2x for 5 minutes in fresh 0.5% sodium metabisulfate (Sigma-Aldrich, St Louis, MO, USA), and washed for 5-10 minutes under running tap water. Slides were dehydrated in ethanol and mounted with Histomount. To measure podocyte density we used the correction factor (CF) method previously reported by Venkatareddy et al [30].

Identifying podocytes in Ren1cCre /R26R-ConfettiTG/WT mice reporter mice
Indirect immunofluorescence staining was performed on 4μm tissue sections from mouse renal biopsies fixed in 4%PFA as described above. Frozen sections were thawed from -80 o C storage and allowed to air-dry. All sections were equilibrated in PBS-buffered saline (pH7.4) then blocked with Background Buster (Accurate Chemical & Scientific Corporation, Westbury, NY, USA) for 30 minutes to minimize nonspecific protein interactions. Endogenous biotin activity was quenched with the Avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA). After blocking, tissue sections were incubated overnight at 4˚C with the appropriate primary antibodies: guinea pig antibody to nephrin (Fitzgerald Industries International. Inc., Concord, MA, USA), mouse antibody to synaptopodin (Fitzgerald Industries International. Inc., Concord, MA, USA), rabbit antibodies to podocin (Abcam, Cambridge, MA, USA) and rabbit antibody to p57 (1:800, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The appropriate biotinylated secondary antibody (Vector Laboratories) was applied followed by Streptavidin, AlexaFluor 647 conjugate (Life Technologies-Molecular Probes, Grand Island, NY, USA). All immunofluorescence samples were mounted using Vectashield mounting medium with DAPI (Vector Labs, Burlingame, CA, USA). A fluorescein isothiocyanate (FITC) conjugated anti-GFP antibody, which binds all four of the confetti variants (1:100, Rockland Immunochemicals for Research, Gilbertsville, PA, USA) was used to detect all four confetti reporters at the 488nm wavelength channel (FITC). As a negative control, all staining was performed without primary antibodies.

Statistical analysis
The results are expressed as mean ± SEM. Groups were compared using a one-way or two-way ANOVA for multiple comparisons with Bonferroni post hoc analysis with significant set at p<0.05.
Multiclonal cells of the renin lineage are detected in the JGC at baseline and after FSGS induction. Using mice harboring conditional or constitutive reporters, we showed that cells of renin lineage migrate from the juxta-to the intra-glomerular compartment following both acute [12] and chronic [34] podocyte depletion. In the current studies, we generated a CoRL/Cre/ confetti mouse (Ren1cCre/R26R-ConfettiTG/WT) to determine if the CoRL progenitors were of mono-or poly-clonal in origin. Results shown in Figs 2 and 3 indicated CoRL are multiclonal.
Renin expression accompanies the development of the entire pre-glomerular vascular tree but is transient except for cells in the juxta-glomerular complex (JGC). Reporter detection did not require the use of antibodies, as each reporter was readily detected using confocal microscopy on frozen tissue sections. At baseline prior to disease induction, typically JGC contained different cells with all four CoRL reporter colors (GFP, YFP, CFP RFP) along afferent arterioles (Figs 2A, 2B and 3A). The percentage of JGC containing cells with either one, two, three or all four of the different reporters is shown in Fig 2C. A higher percent of JGC contain cells expressing two reporter colors (21.3±6.0% vs. 10.0±1.0%, p = 0.038), three colors (16±3.6% vs. 10.0±1.0%, p = 0.386) or all four colors (45.2±5.0% vs 10.0±1.0%, p<0.0001) of the reporter colors versus a single color. This finding indicated that even if bi-colored cells (red/blue or green/yellow) were observed as a result of continuous renin expression, at least two reporter colors were present in the majority of JGC, indicating mostly non-clonal populations of CoRLs. In majority of glomeruli, there were no reporter labeled cells, nor renin stained cells in the intra-glomerular compartment (IGC). However, there was overlapping staining with renin in cells in the JGC, consistent with reporter labeled cells expressing renin at baseline in JGC ( Fig 2D). Following podocyte depletion in experimental FSGS, reporter labeled cells were detected in the IGC. The absence of renin staining in these IGC reporter positive cells (Fig 2E and 2F) suggests that CoRL appear to migrate from the JGC to the IGC (Fig 3B and 3C 3D).

CoRLs do not proliferate in the intra-glomerular compartment
BrdU staining was not detected in cells of JGC and IGC at baseline (Fig 3E). This was not a false negative, because BrdU was detected in occasional tubular epithelial cells as expected. At D14 of FSGS, reporter labeled cells in glomerular tuft did not stain for BrdU ( Fig 3F). Likewise, on D28 of FSGS, BrdU staining was not detected in the glomerular tuft ( Fig 3G).

Multi-clonal CoRL reporters in a glomerular location co-express podocyte proteins
To determine whether CoRL located in the intraglomerular compartment co-express podocyte proteins de novo in FSGS, double-staining was performed against the following four different podocyte proteins: podocin (Fig 4,  , all four reporter channels obtained on confocal microscopy were converted to appear green in color, and podocyte protein staining was converted to a red color, so that any co-localization created a yellow color (Figs 4A 7 , 4B 7 , 4C 7 & 5-7F).

Multi-Clonal CoRLs co-express PEC markers along Bowman's capsule in FSGS
We have reported that a subset of CoRL migrated to Bowman's capsule in disease co-express PEC markers [31,32]. In order to show that reporter labeled CoRL co-express PEC markers, staining for Claudin-1 ( Fig 8A 1 -8C   markers Claudin-1 ( Fig 8C 6 and 8C 7 ) and PAX8 (Fig 9C 6 and 9C 7 ). These results show that in Ren1cCre/R26R-ConfettiTG/WT mice with FSGS, a subset of multi-clonal CoRL were observed along Bowman's capsule and a subpopulation co-express PEC proteins.

Serial intravital multiphoton microscopy imaging of Ren1d-Confetti mouse kidneys
To understand the pattern and dynamics of glomerular cell remodeling by CoRL after IgGinduced podocyte injury, we performed serial multiphoton microscopy (MPM) of the same glomerulus in the same Ren1d-Confetti mouse kidneys in vivo. Dorsal abdominal imaging windows were implanted before the start of experiments, which allowed non-invasive optical z- Multi-clonal cells of renin lineage sectioning of the same intact kidney every 3 rd day for the first two weeks after cytotoxic IgG injection. Individual CoRLs were identified at the single cell level using the unique Confetti color (either membrane-targeted CFP, nuclear GFP, or cytosolic YFP or RFP). Changes in CoRL cell distribution within glomeruli was determined by comparing z-stacks acquired at each time point (S1-S5 Movies). Because of the slow cell migration and remodeling process, serial MPM imaging of the same glomerulus was performed intermittently (every 3 rd day) rather than continuously. Therefore, it couldn't be established with absolute certainty that the exact same cell was followed throughout the experiment. However, the number and relative position of pre-existing single cells (e.g. single yellow cell between blue cells, S1-S5 Movies), and the overall CoRL cell distribution (registered at each time point based on the unique single cell identifier color) appeared to be identical between time points, strongly suggesting that indeed the same cells were captured and followed over time. Also, we never observed any single cell within the glomerulus switching colors between consecutive imaging sessions (S1-S5 Movies), again strongly suggesting that renin/Cre were inactive in intra-glomerular CoRL. In addition, intravital MPM provided visual confirmation of the classic signs of podocyte injury and FSGS development after IgG treatment, including glomerular albumin leakage and increased proximal tubular albumin uptake (Fig 10D and 10E).

CoRL-mediated remodeling of the PEC layer of Bowman's capsule
We evaluated changes in CoRL distribution along the parietal epithelial cells (PEC) of Bowman's capsule. We found that the position of select individual, CoRL-derived PECs was continuously shifting laterally away from the Bowman's capsule into the early proximal tubule segment (Fig 10). The original position of PECs was taken by other CoRL that showed identical color with those PECs closer to the glomerular vascular pole and in the terminal afferent arteriole (blue, CFP-expressing in Fig 10), suggesting that the afferent arteriole provides a continuous supply of cells that differentiate into and migrate along the PEC layer. In the case of CoRL-derived PECs, the speed of lateral cell migration was very slow, approx. 20 μm over 3 days. This was measured by the change in the relative position of a single cell between imaging sessions that was assumed to be the same cell (Fig 10). Also, evidence was found for some CoRL disappearing from the PEC layer of Bowman's capsule (Fig 10A-10C) suggesting that cell types other than the CoRL may also act as PEC precursors.

Migration of CoRL to the visceral layer of the Bowman's capsule
Serial intravital MPM imaging was able to track the migration of individually labeled single CoRL into the visceral layer of the Bowman's capsule in intact living kidneys. Fig 11A-11C illustrates that cells which belong to the CoRL appear to detach from the terminal afferent arteriole compartment and migrate into the glomerular tuft, including locations in the mesangium . Bar is 20 μm. Note the 1-2 yellow-labeled (cytosolic YFP expressing) CoRL (arrows) localized exactly at the glomerulo-tubular junction (dashed line). (B-E) Three-totwelve days after IgG injection, the same YFP+ cells in the same glomerulus (G 1 ) moved continuously away from the glomerulo-tubular junction and deeper into the proximal tubule. During the same time, CFP+ cells along the parietal Bowman's capsule of the other glomerulus (G 2 ) disappeared from Bowman's capsule (arrowhead) between D3-6 (B-C). Plasma was labeled red using Alexa594-albumin. Note the significantly increased albumin content in G and PT fluid and within PT cells in Day 9-12 (intense red). and outside the glomerular capillary. This pattern and direction of CoRL migration may take several days (Fig 11A-11C). Importantly, the overall number of CoRL observed in the glomerular tuft occupying either mesangial and visceral Bowman's capsule positions increased approximately 5-fold in response to podocyte injury compared to both baseline and timed controls over the first two weeks after IgG treatment (6.0±2.7 vs. 1.0± 1.5, p = 0.005 vs. control D12 FSGS) (Fig 11D).

Persistence of CoRL-derived glomerular cell clusters
Next, we addressed the persistence of CoRL recruitment to the glomerular tuft. Several clusters of CoRL were observed both along the parietal Bowman's capsule (Fig 10) and surrounding glomerular capillaries (Fig 12). The cells on the outside of glomerular capillaries featured large, canopy-shaped cell bodies and several long cell processes wrapping around the capillaries, suggesting that they were differentiated podocytes (Fig 12A). Further support was that the same cells persisted at the same anatomical location for days (Fig 12A and 12B). what is likely the same RFP+ cell in the same glomerulus (G) appears detached from the AA and localizes in the glomerular tuft (arrow). (C) Six days after IgG injection the RFP+ cell appears localized around a glomerular capillary (arrow). In contrast to the migrating RFP+ cell, a YFP+ CoRL (yellow, cytosolic YFP expressing) appears stationary in the intraglomerular mesangium (arrowhead). Plasma was labeled red using Alexa594-albumin. (D) Analysis of the total number of Confetti+ CoRL in the glomerular tuft in time-control (Ctrl, n = 5) and IgG-injected (IgG, n = 4) mice during the first two weeks of podocyte injury. https://doi.org/10.1371/journal.pone.0173891.g011

Discussion
Replacement of glomerular cells, in particular terminally differentiated podocytes, is essential for the return of normal glomerular function following disease. Although studies are ongoing to determine the extent of progenitor involvement in these processes, the ability to study the clonality and movement of these populations has recently been made possible with the use of cell fate mapping strategies using multi-colored reporters and serial imaging of live cells. In the current study we show that following podocyte injury, cells of the renin lineage (CoRL) populate the glomeruli, and adopt phenotypic characteristics of both glomerular epithelial cell types namely podocytes and parietal epithelial cells (PECs).
These findings agree with previous work from several groups including ours and indicate that CoRL have remarkable plasticity, with the ability to change fate towards at least three glomerular cell types in disease: podocytes, PECs and mesangial cells [11,[15][16][17]. Yet, and until now, it was not known whether the CoRL originated from one cell in particular, or from multiple cells. To answer this fundamental question, we used reporter mice whereby cells of the renin lineage can be fate mapped stochastically by random selection of one of four colors. We found that CoRL which populated the glomerulus following abrupt podocyte depletion presented a mosaic pattern displaying 4 distinct colors. The results strongly indicate that during glomerular injury, cells from the renin progeny do not originate from a single dividing cell or clone but are instead multiclonal in origin. Although an inducible reporter system was not used, renin staining in both health and disease was limited to cells in the juxta-glomerular compartment, and was not detected in the glomerulus in disease in the current studies. This is similar to what we have reported for renin protein and mRNA expression in inducible renin reporter mice in this model [15]. These findings suggest that once in the glomerular compartment, cells of the renin lineage stop making renin and differentiate into other cell types as previously shown during normal development and glomerular disease [11,17]. Therefore, despite studies not being in an inducible reporter system, the cessation of renin expression in the cells Multi-clonal cells of renin lineage that migrate into the glomerular compartment would preclude de novo reporter expression resulting in the four distinct colors that were found within the glomerulus.
Using multiple injections of BrdU, we next asked if the presence of multiple clones of CoRL in diseased glomeruli was due to their proliferation within the glomerulus. The results showed that there were BrdU labeled cells in the tubules as expected. However, in the few BrdU labeled cells near the glomerulus, staining was restricted to labeled (and unlabeled) CoRL in the juxtaglomerular compartment, with barely any BRDU labeled cells detected within the glomeruli at the time points studied. This is consistent with our previous report [15]. We interpret these findings to indicate that the presence of multiple clones of CoRL in glomeruli is not the consequence of cell proliferation but likely from the movement of multiple individual cells from the juxta-glomerular compartment. This interpretation does not exclude the possibility, however, that local intraglomerular or glomerular parietal epithelial cells that derive from cells that transiently expressed renin in the early embryo may have also contributed to the regeneration of injured glomeruli. To be sure, similar studies would have to be performed using lineage tracing of podocytes and/or PECs. In fact, the relative contribution of each cell type to the regeneration of the injured glomerulus remains to be discerned, as multiple cell lineages may contribute to glomerular repair.
We next asked if all clones had the ability to co-express podocyte proteins. Indeed, the results showed that CoRL labeled with YFP, GFP, RFP and CFP all had the ability to co-express the podocyte markers podocin, nephrin, p57 and WT1. These results also support recently published work that CoRL serve an adult podocyte progenitor niche [13,15,28]. Similarly, multiple clones of labeled CoRL co-express the PEC markers claudin-1 and PAX8, consistent with the notion that they too serve as adult PEC progenitors in disease. The latter has also been shown during normal glomerular development [22,35]. Taken together, both glomerular epithelial cell types could derive from multiple clones of CoRL following abrupt podocyte depletion. Nevertheless, as mentioned above, additional fate mapping with specific inducible cre lines will need to be performed to discern whether, and to what extent if any, PECs and/or remaining resident podocytes themselves contribute to repopulate the injured glomeruli.
Although fate mapping is used as proof for the migration of a labeled cell from one kidney location to another, an important question remained regarding whether CoRL migrate from the juxta-to the intra-glomerular compartment. Accordingly in these experiments we used a powerful complementary strategy, a recently developed direct visual approach to track the fate of fluorescently labeled glomerular cells in the intact living kidney using serial MPM imaging [22,26]. The use of the multi-color Confetti reporter allowed us to label and identify individual CoRL and to perform genetic fate tracing in combination with serial MPM. It should be noted that although our Cre/lox-based experimental approach used a constitutively active Cre model (Ren1d-Cre), which labels cells that expressed renin in the early embryonic kidney and their descendants (even if actual renin expression has ceased), the combination with serial MPM imaging alleviated the potential technical issues.
Direct visualization of the CoRL cell population in the same glomerulus over time provided direct visual clues for the first time, suggesting the migration of individual CoRL from the terminal afferent arteriole to the parietal Bowman's capsule and early proximal tubule, as well as to the glomerular tuft including the mesangium and the visceral layer of Bowman's capsule. It should be emphasized that serial MPM imaging of the same glomeruli was performed intermittently rather than continuously over two weeks, therefore the identification of the same cell cannot be established with absolute certainty. This technical limitation was due to the rather slow process of CoRL migration and glomerular cellular remodeling. However, this experimental design was necessary to avoid cumbersome technical issues with continuous anesthesia, intravenous fluids and feeding, and significant cell damage due to laser exposure that would have been necessary and unavoidable with continuous MPM imaging. We believe that our serial MPM imaging approach provided strong visual clues regarding CoRL migration, and the new results represent a significant step forward in understanding the novel functions of CoRL. We feel confident that due to the advantages of the multi-color Confetti reporter and serial MPM approach, we indeed tracked the fate and migration of the same CoRL. Although it cannot be ruled out, we find it highly unlikely that another cell of the exact same Confetti-color belonging to the same CoRL lineage appeared in the exact same anatomical position at the exact same time.
We recognize limitations in the current study. First is the use of a constitutive reporter, which opens the possibility of cre-mediated recombination within podocytes and PECs following injury, due to activation of the Ren1c or Ren1d promotors in the two mouse strains used. We believe this is unlikely in the absence of any renin staining in podocytes or PECs. Furthermore, we have previously shown no Ren1c promotor activity in podocytes following injury using Ren1cGFP reporter mice [36]. Although not immediately feasible, it would be ideal to genetically pulse-label cells once, using an inducible system or perform single cell injection of intravital dyes before the injury and then track the cell movements along the chosen nephrovascular units. While those experiments are being contemplated, the results presented here in aggregate represent a significant advance and are consistent with the hypothesis that regeneration/repopulation of injured glomeruli may occur with the participation of CoRL that may reach the intraglomerular compartment from the juxta-glomerular compartment. Second, as stated above, the relative participation of PECs or local intraglomerular podocytes to the increase in podocyte mass after glomerular injury remains to be determined. Third, the experimental model used has partial podocyte replacement, but does not fully recover podocyte number to baseline levels following depletion.
Select CoRL appeared to differentiate into podocytes and remained at the same position around the glomerular capillary for several days. The increased number of CoRL recruited to the glomerular tuft after cytotoxic IgG injection suggests that podocyte injury triggers or augments glomerular remodeling by CoRL. These results are consistent with the previously established distribution of CoRL, which includes the glomerular afferent arterioles, mesangium, the parietal layer of Bowman's capsule as well as various tubule segments and interstitium [10,11,19,20]. Also, the relatively low number of CoRL observed in the present study in the glomerular tuft after podocyte injury is in good agreement with our previous work [13,31].
In summary, we have used molecular genetic fate map reporting and live imaging approaches to better understand CoRL clonality during podocyte replacement and to monitor the fate of CoRL over time in the intact living kidney in experimental FSGS. Our data suggest that multi-clonal CoRL may migrate from the juxta-glomerular compartment and replace a subset of podocytes and PECs in experimental FSGS. Further work will be needed to determine whether the participation of CoRL and/or other cell types to the morphological repair results in lasting functional improvement of affected glomeruli.