Renal mechanotransduction is an essential regulator of renin

The kidneys tightly control the composition of our internal environment to maintain homeostasis in the face of external variability. The regulation of blood volume begins in the kidneys and is essential for vertebrate life in terrestrial environments where salt and water availability are unpredictable1,2. Renin synthesis and release by juxtaglomerular granular cells of the kidney is the rate-limiting step in a hormonal cascade that modulates blood volume, filtration, and salt balance2. Renin is stimulated during hypovolemia and salt deprivation in response to chemical cues released from sympathetic efferent neurons and the macula densa onto the juxtaglomerular granular cells. Renin levels are also proposed to be modulated by mechanical forces elicited by changes in blood volume and/or pressure exerted upon juxtaglomerular cells2–4. However, the identity and significance of the juxtaglomerular mechanotransducer(s) was unknown. We found that the force-gated ion channel PIEZO2 is expressed in juxtaglomerular granular cells and in neighboring mesangial cells. Selective genetic ablation of PIEZO2 dysregulated the renin-angiotensin-aldosterone system by elevating renin, raising systemic blood pressure, inducing glomerular hyperfiltration, and exaggerating the hormonal response to volume depletion. These findings demonstrate that PIEZO2 contributes to renal blood volume sensing and kidney function in vivo.

assumed.Additionally, the in vivo consequences of loss of JG mechanosensitivity are unknown, particularly in light of parallel mechanisms to stimulate RAAS.In our study, we sought to elucidate the molecular identity and physiological significance of mechanotransduction within the JG apparatus (JGA).

PIEZO2 is expressed in juxtaglomerular and mesangial cells of the kidney
Given the link between intracellular calcium levels and renin, we hypothesized that mechanically activated nonselective cation channels might underlie the intrinsically mechanosensitive component of the renal baroreceptor.PIEZO1 and PIEZO2 comprise a family of ion channels that are exclusively gated by membrane stretch, and are both necessary and sufficient for cellular responses to a range of physiologically salient mechanical stimuli [23][24][25] .PIEZO1 is endogenously expressed in many tissue types including vascular endothelium and smooth muscle, and is important for vascular development and function 26,27 .PIEZO2 is mainly expressed in peripheral sensory neurons and specialized accessory sensory cells and is required for gentle touch sensation, proprioception, and gut and bladder function [28][29][30][31] .Together, PIEZO1 and PIEZO2 in vagal sensory neurons initiate the neuronal baroreflex through sensing of blood pressure in the aortic arch 5 .To establish the expression patterns of PIEZOs in the mouse kidney, we first examined the localization of mRNA transcripts of mechanosensory PIEZO channels using single molecule fluorescence in situ hybridization (smFISH).We found that Piezo2 and not Piezo1 was expressed in Ren1-expressing JG cells of the JGA and in Pdgfrb-expressing mesangial cells of the glomerulus 32,33 (Fig. 1a-c).Using a Piezo2 GFP-Cre fusion knock-in mouse 29 , we observed a pattern of fusion protein expression restricted to glomerular cells (Fig. 1d) as well as robust glomerular and JG cell labeling when crossed with the Ai9 fl/fl tdTomato reporter line 34 (Fig. 1e).By contrast, immunohistochemistry (IHC) of PIEZO1-tdTomato C-terminal fusion protein from a Piezo1 tdTomato knock-in mouse line showed expression largely restricted to basal aspects of a subset of tubular epithelial cells as previously reported 35 (Extended Data Fig. 1a).Our findings are in agreement with single-nucleus RNA-seq databases of mouse and human kidney tissues that support the expression of Piezo2 and not Piezo1 in mesangial and renin-expressing JG cells 36 .To further validate those results, we performed RNAscope for PIEZO2 and PDGFRB on kidney sections from a healthy human donor.We observed overlap of the two transcripts' expression patterns in kidney cortex (Extended Data Fig. 1b).
In order to target PIEZO2 for genetic ablation in the mouse kidney, we turned to an inducible pericyte Cre recombinase mouse line, Pdgfrb CreERT2 , that selectively targets specialized vascular smooth muscle cells that include mesangial and renin-expressing JG cells 37 after tamoxifen administration to adult mice to induce recombination (Fig. 1f, Extended Data Fig. 1c-d).Indeed, we found that after crossing this line with Piezo2 fl/fl mice, tamoxifen ablated Piezo2 expression in the kidney of Pdgfrb CreERT2 but not Pdgfrb WT animals, confirmed using an smFISH assay with probes against either the entire transcript (Piezo2) or the loxpflanked exons (Piezo2 E43-E45, Fig. 1g-h).We observed additional patterns of Cre recombination in Ren Cre and FoxD1 GFP-Cre mouse lines after crossing with the Ai9 fl/fl tdTomato reporter strain.In line with a published study, Ren Cre targeted renin-expressing putative JG cells of the JGA and renin-negative cells of the renin lineage along the glomerular arterioles of the kidney 38 , while largely sparing mesangial cells (Extended Data Fig. 1e-f).We observed that the stromal progenitor Cre line FoxD1 GFP-Cre targeted a broad population encompassing mesangial cells, renin-expressing cells, and fibroblasts, while sparing tubules and endothelial cells as previously reported 39 (Extended Data Fig. 1g-h).While our initial testing of a conditional Ren CreER reporter line used previously for lineage tracing 40 showed selective targeting of adult renin-expressing cells, we observed only partial recombination in these cells after tamoxifen administration, rendering it unsuitable for genetic loss-of-function studies in vivo (Extended Data Hill et al.

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Fig. 1i).It is worth noting that while none of our Cre lines (Pdgfrb CreERT2 , Ren Cre , and FoxD1 GFP- Cre ) on their own are selective for renin-expressing JG cells, PIEZO2, unlike PIEZO1, is expressed in only handful of cell types in the body and mainly found within the peripheral nervous system 5,28,41 .Although PIEZO2 expression has been sporadically observed in vascular endothelial cells 42 (its function in these cells is unknown), none of our Cre lines target this particular cell type and we were unable detect Piezo2 transcript expression in glomerular capillary endothelial cells.As such, by using Pdgfrb CreERT2 , Ren Cre , and FoxD1 GFP-Cre in combination we can infer the function of PIEZO2 in JG cells, which are uniquely targeted by all three of the genetic strategies.

Renal PIEZO2 is an essential regulator of plasma renin
We next sought to investigate the consequences of loss of functional PIEZO2 to renin levels and RAAS.Since the primary role of the JG cells is to synthesize and release renin, we measured renin in plasma harvested from naïve mice.Initially, to avoid potential confounds due to developmental phenotypes that could be present in Ren Cre or FoxD1 GFP-Cre conditional mutant mice, and to target all PIEZO2-expressing renal cells (both mesangial and putative JG cells), we examined the inducible Piezo2 fl/fl ; Pdgfrb CreERT2 conditional knockout mice.We observed a significant increase in plasma renin levels in the Piezo2 fl/fl ; Pdgfrb CreERT2 mice compared to Piezo2 fl/fl ; Pdgfrb WT wild-type littermate controls that also received tamoxifen (Fig. 2a).This effect was not exacerbated in Piezo1 fl/fl ; Piezo2 fl/fl ; Pdgfrb CreERT2 mice lacking both ion channels (Extended Data Fig. 2a), consistent with our earlier observation that PIEZO2 and not PIEZO1 is expressed in mesangial and JG cells.The elevated renin levels were phenocopied in Piezo2 fl/fl ; FoxD1 GFP-Cre animals, suggesting that loss of PIEZO2 in stromal cells was sufficient to reproduce the phenotype and was not likely due to PIEZO2 in non-stromal populations including tubular and endothelial cells (Extended Data Fig. 2b).Notably, we did not observe elevations in Hill et al.
downstream RAAS hormones aldosterone and angiotensin II (Fig. 2b-c) in Piezo2 fl/fl ; Pdgfrb CreERT2 mice, suggesting that the elevated renin levels were insufficient to trigger induction of downstream RAAS hormones under baseline conditions.While hyperreninemia is sometimes associated with hypertension, this is typically attributed to a systemic elevation in angiotensin II 43,44 , which was not observed in plasma from the conditional knockout mice (Fig. 2c).We measured the systemic blood pressure Piezo2 fl/fl ; Pdgfrb WT and Pdgfrb CreERT2 mice using the radiotelemetry-validated volume pressure recording (VPR) method 45 and found a significant elevation in Pdgfrb CreERT2 mice that was not indicative of hypertension (Fig. 2d, Extended Data Figs.3a-b, 4a-c).The effect was less than that reported in transgenic rodent models with elevated RAAS signaling 44 , and may reflect the lack of significantly elevated angiotensin II in the naïve PIEZO2 conditional knockout mice.However, it is possible that the elevated baseline renin observed in the conditional knockout mice contributes to sporadic or mild elevations in angiotensin II not captured by our enzyme-based measurement methods, but which are still capable of triggering mildly elevated blood pressure.
To determine whether mesangial and/or renin-lineage cells contribute to the elevated renin in PIEZO2 conditional mutants, we measured plasma renin and blood pressure in Piezo2 fl/fl ; Ren Cre conditional knockouts and littermate controls.Our findings in this strain phenocopied those observed with Pdgfrb CreERT2 -mediated deletion of PIEZO2 (Figs. 2e-f, Extended Data Figs.5a-b, 6a-c).As Ren Cre targets JG cells and other cells of renin lineage within the kidney but spares the majority of mesangial cells 38,40 , we conclude from these experiments that the phenotype is primarily driven by PIEZO2 in renin+ JG cells rather than in mesangial cells, which are known to play key roles in the pathogenesis of glomerular disease and are relatively unstudied in the context of RAAS 46 , though we cannot completely rule out their involvement.

Renin regulates the GFR via Ang(1-7)/Mas in a PIEZO2-dependent manner
Another important function of renin is modulating the glomerular filtration rate (GFR) in response to changes in renal blood flow and macula densa signaling 4,47,48 .We observed that Piezo2 fl/fl ; Pdgfrb CreERT2 mice exhibited glomerular hyperfiltration in an assay in which FITCsinistrin (a freely filterable molecule that is neither reabsorbed into the blood nor secreted through the peritublar capillaries) fluorescence signal decay is observed by continuous transdermal measurement 49 (Fig. 3a-b), consistent with a dysregulation of renin.The GFR values of the conditional knockout mice were similar to those observed in genetic models of hyperfiltration 50 .Whereas kidney disease is typically associated with a low GFR, early in its pathogenesis certain diseases such as diabetes trigger glomerular hyperfiltration 51,52 .As such, we also explored the possibility that the Piezo2 fl/fl ; Pdgfrb CreERT2 mice had renal disease by remeasuring GFR in the same adult mice eight months after the initial experiments.We found that the GFR of the Piezo2 fl/fl ; Pdgfrb CreERT2 mice declined as expected with age [53][54][55] , comparable to the decline observed in wild-type littermates (Extended Data Fig. 7a).The conditional knockout mice maintained elevated GFR compared to controls, suggesting that the observed phenotype could be due to elevated renin rather than kidney disease (Extended Data Fig. 7a-b).
Additionally, the conditional knockout mice did not exhibit elevated urinary albumin, a hallmark of kidney glomerular disease (Extended Data Fig. 7c).We also observed that the elevated GFR was phenocopied in Piezo2 fl/fl ; Ren Cre conditional knockouts, suggesting the phenotype was mediated by renin-expressing cells rather than glomerular mesangial cells (Fig. 3c).Importantly, loss of PIEZO1 and PIEZO2 in peripheral neuronal baroreceptors using Piezo1 fl/fl ; Piezo2 fl/fl ; SNS Cre 56 did not induce a GFR phenotype (Extended Data Fig. 7d), suggesting that these findings are independent of any indirect effects of neural baroreception on kidney function 3,5,57 , for example, due to modulation of sympathetic norepinephrine release onto the JGA.
Examination of PAS-and H&E-stained sections showed that neither Piezo2 fl/fl ; Pdgfrb CreERT2 nor Piezo2 fl/fl ; Ren Cre kidneys exhibited consistent histological abnormalities compared to controls that were indicative of glomerular disease or dysfunction (Extended Data Fig. 7e-h), further supporting the idea that the elevated GFR was not due to overt glomerular disease.
We postulated that the elevated GFR, as it did not arise from overt kidney disease nor from loss of PIEZO2 in mesangial cells, could be caused by aberrant signaling downstream of renin.Elevated renin is a consequence of administration of captopril, an angiotensin converting enzyme (ACE) inhibitor commonly prescribed to treat hypertension that disrupts feedback inhibition of angiotensin II on renin production 58 .The effects of this drug on GFR are unclear 59- 62 .We reasoned that if captopril causes elevated plasma renin, then we would expect captopril to enhance the GFR of wild-type mice to a level comparable to that of the conditional knockouts (Fig. 2a).Indeed, we observed that six days of captopril administration (Fig. 3d) elevated the GFR of wild-type mice such that it was not significantly different from the Piezo2 fl/fl ; Pdgfrb CreERT2 littermates also receiving captopril (Fig. 3e).Consistent with captopril's known effects, we observed elevated plasma renin (Fig. 3f) and decreased aldosterone (Extended Data Fig. 8a) in mice of both genotypes on day seven 58 .It was of note that the renin levels observed in treated Piezo2 fl/fl ; Pdgfrb CreERT2 animals were higher than those observed earlier in untreated animals (Fig. 2a), however, their GFR values were within a similar range (Figs.3b,e).Taken together, our results suggest that elevation of plasma renin in the absence of elevated angiotensin II is sufficient to elevate the GFR.Our observation that the higher renin levels elicited by captopril in treated animals did not raise the GFR beyond that of untreated conditional mutant mice additionally suggests that there is a limit to renin's ability to increase the GFR.
How does elevated renin increase the GFR?The relationship between RAAS activation and GFR is complex.For example, angiotensin II produced by RAAS activation can have opposing effects on GFR through differential effects on glomerular arteriole constriction and mesangial cell contractility [61][62][63][64] .As our conditional knockout mice had normal angiotensin II Hill et al. 10 levels, we did not suspect this molecule was playing a role in elevating the GFR of these animals.Notably, the related peptide Ang(1-7) is produced downstream of renin's cleavage of angiotensinogen to angiotensin I and its subsequent conversion to Ang(1-7) by specialized proteases 65,66 (Fig. 3g).Ang(1-7) acts via the G-protein coupled receptor (GPCR) Mas within in the glomerular vasculature to increase the GFR 67,68 .Intriguingly, this regulation by the Ang(1-7)/Mas axis is reported to occur under salt-depleted conditions 68 , as elevated renin drives Ang(1-7) production, and can occur after treatment with ACE inhibitors such as captopril that similarly drive elevated Ang(1-7) through loss of angiotensin I conversion to angiotensin II 66 (Fig. 3g).We examined whether the elevated GFR could be a direct consequence of elevated renin and subsequent Ang(1-7)/Mas signaling in the Piezo2 fl/fl ; Pdgfrb CreERT2 mice.We first tested whether Mas signaling via Ang(1-7) contributed to elevated GFR in the cKO mice.We measured the GFR before and after selectively blocking Mas signaling via daily administration of the pharmacological antagonist A779 [67][68][69][70] (Fig. 3h).Strikingly, Mas blockade fully rescued normal GFR in conditional knockout mice and had no effect on the GFR of littermate controls (Fig. 3i), demonstrating that Mas signaling underlies the elevation in GFR caused by loss of PIEZO2.Our findings with the captopril and A779 experiments establish a molecular pathway by which PIEZO2-dependent renin regulation is required for maintenance of a GFR within the normal range via Ang(1-7)/Mas signaling.

PIEZO2 acts as a brake on the hormonal response to hypovolemia
A primary function of renin is to activate RAAS to conserve bodily salt and water during hypovolemia when blood volume is depleted.To test whether renal PIEZO2-dependent mechanotransduction plays a role in hypovolemia-evoked RAAS, we subjected Piezo2 fl/fl ; Pdgfrb CreERT2 and littermate controls to the polyethylene glycol (PEG)-evoked hypovolemia model 71 (Fig. 4a), where PEG draws bodily fluid into the subcutaneous space to cause a volume Hill et al.

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depletion from the blood into a secondary compartment without directly affecting salt balance, as with administration of the loop diuretic furosemide 8 .We found that the PEG-injected Piezo2 fl/fl ; Pdgfrb CreERT2 mice exhibited an exaggerated hormonal response to hypovolemia, with elevated renin, angiotensin II, and aldosterone compared to littermate controls (Figs.4b-d).The renin levels we measured were greater than that seen under normovolemic conditions (Fig. 2), and might explain the stronger induction of angiotensin II and aldosterone in conditional knockouts that were not seen in normovolemic Piezo2 fl/fl ; Pdgfrb CreERT2 mice.Enhanced hypovolemia-evoked renin and aldosterone levels were phenocopied with the Piezo2 fl/fl ; Ren Cre and FoxD1 Cre strains affecting the renin lineage and stromal progenitors (Figs.4e-h), suggesting that loss of functional PIEZO2 in the renin-lineage alone was sufficient to drive the effect.Similar to our findings under naïve conditions, loss of both PIEZOs using Piezo1 fl/fl ; Piezo2 fl/fl ; Pdgfrb CreERT2 had no further effect beyond loss of PIEZO2 alone (Extended Data Fig. 9a).No difference was observed between conditional knockout and controls with dual loss of PIEZO1 and PIEZO2 in peripheral neuronal baroreceptors (Extended Data Fig. 9b).We conclude from these experiments that PIEZO2 in renin-expressing cells blunts the RAAS response to hypovolemia.

PIEZO2 contributes to RAAS independently of sympathetic and macula densa function
We next sought to determine how the contribution of PIEZO2 to RAAS is weighted against that of the other pathways regulating renin synthesis and release from JG cells, namely efferent sympathetic and macula densa signaling.To address this question, we designed an experiment in which we ablated or blocked these two respective arms through simultaneous chemical sympathectomy using the drug 6-hydroxydopamine 72,73 (6-OHDA) and acute blockade of synthesis of prostaglandins, the primary macula densa-derived chemical signal stimulating renin, using the cyclooxygenase-1 and -2 inhibitor indomethacin 12,13 .After chemical sympathectomy and indomethacin injection, Piezo2 fl/fl ; Ren Cre mice and littermate controls were subjected to PEG-evoked hypovolemia or saline control treatment and blood plasma was isolated for measurement of renin (Fig. 5a).We found that plasma renin levels were strongly suppressed, in some cases to below the assay's limit of detection, with blockade of both sympathetic and macula densa prostaglandin signaling in wild-type mice treated with saline (Fig. 5a), suggestive of a successful inhibition of renin-stimulating pathways.We also observed a substantial loss of TH+ sympathetic nerve fibers in the kidney, indicating the 6-OHDA treatment was efficacious (Extended Data Fig. 10a,b).Plasma renin was still elevated in response to PEG hypovolemia despite loss of these two important pathways, suggesting that a third (i.e.PIEZO2-dependent) pathway regulates renin levels after pharmacological blockade (Fig. 5b).Values observed in our wild-type littermate controls were lower than we had observed in our other hypovolemia experiments, indicating that the pharmacological manipulations did lower plasma renin levels (compare Fig. 5b to Fig. 4e).In Piezo2 fl/fl ; Ren Cre conditional knockout mice injected with saline, renin levels were largely unaffected by the blockade and similar to mice with intact sympathetic and macula densa signaling (compare to Fig. 2e).In PEG-injected conditional knockout mice, renin levels were substantially elevated beyond that of littermate controls and similar to hypovolemic Piezo2 fl/fl ; Ren Cre mice with intact sympathetic and macula densa signaling, suggesting that loss of mechanotransduction in renin-expressing cells dysregulates RAAS independently of the sympathetic and macular densa prostaglandin pathways (Fig. 5b).Although we cannot completely rule out remnant function of the macula densa after indomethacin treatment, the stark differences observed between the control and conditional knockout mice support our conclusions regardless of this possibility.Aldosterone levels were similarly exacerbated by hypovolemia in the knockouts, demonstrating that the changes in plasma renin translated into downstream RAAS signaling (Fig. 5c).

Discussion
This work demonstrates that PIEZO2 in renin-expressing JG cells of the kidney regulates RAAS in response to changes in blood volume in vivo (Fig. 5d).Isolating the intrinsically mechanosensitive component of the renal baroreceptor is complicated due to the numerous intersecting feedback loops that encompass several organ systems, as well as the intrarenal chemical signaling pathways that modulate the activity of JG cells 64 .Our findings show that selectively perturbing the mechanosensory capability of renin-expressing cells through conditional deletion of Piezo2 is sufficient to increase plasma renin in healthy mice.We demonstrate that elevated renin has downstream consequences for glomerular filtration rate, systemic blood pressure, and the hormonal response to hypovolemia.It can be challenging to reconcile the microscale changes in renal hemodynamics downstream of renin and TG feedback that were observed using micropuncture techniques in the rat 61,74,75 with the wholeorganism effects on RAAS we observed with selective gene perturbation in the mouse.Our findings suggest that altered JGA mechanotransduction has specific and profound outcomes for renal physiology in vivo and align with previous studies 61,74,75 .For example, we identified Ang(1-7)/Mas signaling as a major effector of PIEZO2 and renin's effects on the GFR in the absence of elevated angiotensin II.Our data additionally support the conclusion that that PIEZO2 in reninexpressing cells acts independently of the parallel mechanisms (i.e.macula densa prostaglandin signaling and/or sympathetic activity) controlling renin levels during hypovolemia.
Taken together, our study establishes a cellular and molecular mechanism for the response of the JGA to hypovolemia, underscores the therapeutic potential for targeting JG cell mechanotransduction pathways to modulate renal RAAS, and highlights the role of PIEZO proteins as general effectors of baroreception throughout the body.
Hill et al.Piezo2 fl/fl ; Ren WT , and n = 4 Piezo2 fl/fl ; Ren Cre mice were analyzed.Representative images of kidney cortex were acquired using a Keyence BZ-X710 microscope using brightfield imaging with a 40x objective and the supplied color camera.
Captopril administration: Water valves were removed from cages and mice were supplied with 400mg/L captopril (Sigma-Aldrich, C4042) in the drinking water, prepared fresh each day in a water bottle.Mice had access to standard chow.After six days of treatment, GFR was measured.On the seventh day, blood was harvested for ELISA.
A779 administration: Mice were injected i.p. once daily for seven consecutive days with 0.5 mg/kg A779 (Cayman Chemical, 23396) dissolved in 0.9% NaCl in water.
Polyethylene glycol (PEG) mouse model of hypovolemia: Mice were lightly and briefly (< 5 min) anesthetized with 2% isoflurane and injected with 40% w/v PEG-8000 (Sigma-Aldrich, 89510) in sterile 0.9% NaCl subcutaneously using a 28G insulin syringe.Food and water were removed from the cage.After six hours, mice were euthanized and blood was collected.For the 6-OHDA and indomethacin experiments, 15 minutes after indomethacin injection, mice received either sterile saline or 40% PEG.Blood was collected six hours following PEG or saline injection.

Figure 1 .
Figure 1.PIEZO2 is expressed in JG and mesangial cells of the kidney.a, smFISH of sectioned mouse kidney for Piezo2, Ren1, and counterstained with DAPI.b, smFISH with IHC of sectioned mouse kidney for Piezo2, Pdgfrb, anti-NPHS2, and counterstained with DAPI.c, smFISH with IHC of sectioned mouse kidney for Piezo1, Pdgfrb, anti-NPHS2, and counterstained with DAPI.d, Sectioned mouse kidney stained with anti-GFP antibody and counterstained with TO-PRO-3.e, Sectioned mouse kidney with native tdTomato fluorescence and counterstained with DAPI.f, Sectioned mouse kidney with native tdTomato fluorescence, stained with anti-Renin antibody, and counterstained with DAPI.Asterisk (*) indicates extraglomerular expression at putative Renin+ vascular pole.g-h, smFISH of sectioned mouse kidney for Piezo2 (left), floxed exon-specific probe Piezo2 E43-45 (center), and merged image counterstained with DAPI (right).Scale bars = 100 µm.Dotted circles indicate renal corpuscles.Each experiment was repeated on at least N=2 mice.
Sectioned mouse kidney stained with anti-tdTomato AlexaFluor 647-conjugated nanobody.Asterisk (*) indicates distal convoluted tubule.b, smFISH of sectioned human kidney for PIEZO2, PDGFRB, and counterstained with DAPI.c, Sectioned mouse kidney with native tdTomato fluorescence, stained with anti-Renin and anti-PECAM1 antibodies.d, Sectioned mouse kidney with native tdTomato fluorescence, stained with anti-NPHS2 and anti-PECAM1 antibodies.e, Sectioned mouse kidney with native tdTomato fluorescence, stained with anti-Renin and anti-PECAM1 antibodies.f, Sectioned mouse kidney with native tdTomato fluorescence, stained with anti-NPHS2 and anti-PECAM1 antibodies.g, Sectioned mouse kidney with native tdTomato fluorescence, stained with anti-NPHS2 and anti-PECAM1 antibodies.h, Sectioned mouse kidney with native tdTomato fluorescence, stained with anti-Renin and anti-PECAM1 antibodies.Dotted circles outline renal corpuscles.i, Sectioned mouse kidney with native tdTomato fluorescence, stained with anti-Renin antibodies.Asterisks (*) are placed to the immediate left of JGA.Scale bars = 100 µm.Each experiment was repeated on N=2 mice.