2.1.

Biopsy Protocol

Human kidney samples were collected from transplant recipients with stable graft function $3\text{months}$ after engraftment of kidney from a deceased donor. Transplantations were performed at Hôpital Tenon, Paris, France. Needle-core biopsies were obtained using a biopsy gun (Monopty Bard, Covington, United Kingdom) with a 16-gauge needle.

2.2.

Models of Angiotensin-II-Induced Hypertension and Crescentic Glomerulonephritis

We studied male mice aged $8\text{to}14\text{weeks}$ with similar genetic background (C57BL/6J background). Baseline blood pressure measurements were performed on five consecutive days. After these baseline recordings, an osmotic minipump (Alzet Model 2004; Durect, California) infusing AngII (Sigma, Saint Louis, Missouri) at a rate of $1\mu \mathrm{g}∕\mathrm{kg}∕\min$ was implanted subcutaneous for $28\text{days}$ as described by Crowley ²⁸ Blood pressure was regularly monitored to verify continuous hypertension. In a subset of experiments, a second osmotic minipump was implanted on day 27 to ensure AngII-infusion up to day 49. The animals ingested a normal diet containing 5% sodium chloride (SDS-Dietex, France) to accentuate hypertension and kidney injury. We also used the accelerated antiglomerular basement membrane anti(GBM) crescentic glomerulonephritis (GN) model, as described by Lloyd ²⁹ Briefly, mice were given subcutaneous injections of $200\text{-}\mu \mathrm{g}$ normal sheep IgG [ $100\mu \mathrm{l}$ into each flank of normal sheep IgG (Sigma) diluted to $1\mathrm{mg}∕\mathrm{ml}$ in a solution of 50% Freund’s complete adjuvant and 50% saline]. Six days later (day 1), GN was induced by intravenous injection of sheep anti-GBM serum. Serum injections were repeated daily on days 2 and 3. The protocols followed the French Department of Agriculture and the National Institute of Health guidelines for animal care and protection.

2.3.

Histopathological Analysis

Human kidney biopsies specimens were fixed in formaldehyde. After fixation, tissues were dehydrated with a graded series of ethanol and xylene, embedded in paraffin, and cut into $10\text{-}\mu \mathrm{m}$ sections for SHG analysis.

Following 28 or $49\text{days}$ of AngII infusion or $17\text{days}$ after anti-GBM serum administration in C57BL6/J mice, murine kidneys were harvested and weighed. One half of a kidney was fixed in formalin, embedded in paraffin, sectioned $\left(3\mu \mathrm{m}\right)$ , and stained with Masson’s trichrome. All of the tissues were examined by a pathologist without knowledge of treatment groups. The pathological abnormalities were graded based on the presence and severity of component abnormalities, including glomerulosclerosis. Grading for glomerulosclerosis was performed using a semiquantitative scale as previously described.³⁰ Percent glomerulosclerosis was defined as the number of glomeruli with evidence of sclerosis divided by the total number of glomeruli in the section. For tubulointerstitial areas, individual scores adapted from Spurney ³¹ were obtained for the severity of interstitial inflammatory cell infiltrates and severity of tubular atrophy. The second half of the kidney was fixed in phosphate buffered saline (PBS) buffered 4% formaldehyde and used for multiphoton microscopy. $200\text{-}\mu \mathrm{m}$ -thick coronal slices were cut using a vibrating-blade microtome (VT1000S, Leica, Germany) at 25% of the kidney height.

2.4.

Multiphoton Microscopy

Multiphoton images were recorded using a custom-built upright laser scanning microscope, as previously described.²² Briefly, SHG and 2PEF signals were simultaneously excited by a femtosecond titanium-sapphire laser adjusted to $860\mathrm{nm}$ (Tsunami, Spectra-Physics, Irvine, California), and dispatched to two photon-counting epidetection channels using a dichroic mirror (FF458-Di01, Semrock, Rochester, New York) and appropriate spectral filters (E700SP and GG455 in the 2PEF channel, E700SP and HQ430/20 in the SHG channel, Chroma, Rockingham, Vermont). Alternatively, the SHG signal from thin biopsies was detected in the forward direction using the same filters. We used circular polarization to minimize orientation effects in the image plane, similarly to circularly polarized Picrosirius imaging.⁹

Visualization of micrometer or submicrometer collagen fibers that were heterogeneously distributed in the kidney tissue at millimeter scale required both a large field of view and a good spatial resolution. We therefore used a $20\times$ 0.95-NA objective lens (Olympus, Japan) with $512\times 512\text{-}{\mu \mathrm{m}}^2$ field of view and 0.40- $\mu \mathrm{m}\left(\text{lateral}\right)\times 1.6\text{-}\mu \mathrm{m}$ (axial) resolution near the sample surface. Furthermore, we scanned the tissue using a motorized stage with $12\text{-}\mathrm{mm}$ travel range. We always scanned $2.5\times 5\text{-}{\mathrm{mm}}^2$ areas with $0.8\times 0.8\text{-}\mu {\mathrm{m}}^2$ pixel size, at $100\text{-}\mathrm{kHz}$ pixel rate and $50\text{-}\mathrm{mW}$ excitation, and we acquired eight images spaced out $5\mu \mathrm{m}$ apart within the depth of the tissue. Voxel size was set to a slightly greater value than the optical resolution near the sample surface to restrain imaging time to typically $90\min$ per sample. Nevertheless, images acquired for 3-D reconstructions were recorded with $0.4\times 0.4\text{-}\mu {\mathrm{m}}^2$ pixel size and were spaced out $0.5\mu \mathrm{m}$ apart within the tissue.

2.5.

Second Harmonic Generation Image Processing and Segmentation

Stacks of SHG and 2PEF images were combined and processed using an algorithm developed to enhance the fiber contrast, as previously described.²² 3-D reconstructions were performed using Amira (Mercury Computer Systems, Chelmsford, Massachusetts). Fibrosis scores were obtained from SHG data using a phenomenological approach as previously published:²² they were calculated as the volume density of voxels exhibiting significant SHG signal in the image stack.

To take into account the specific morphology of every murine kidney, that is the variability in kidney shape and in radial location of the arteries, we developed a segmentation algorithm and calculated fibrosis scores on distinct morphological regions of the kidney. We first manually outlined the capsule and the papilla in every SHG/2PEF image, and selected the centers of the arcuate arteries in the cortico-medullary area. Our algorithm then yielded three curves: Ca, along the capsule, Ar, through the arterial centers, and PM, at the edge of medulla and papilla [see Fig. 1a ]. These curves were fitted to ellipses, enabling their dilation or contraction toward the center of the ellipse, as shown in Fig. 1b. Three regions were defined by our algorithm [Fig. 1c]: the outer cortical region [C] between $\mathrm{Ca}-40\mu \mathrm{m}$ and $\mathrm{Ar}+160\mu \mathrm{m}$ , the medulla region [M] between $\mathrm{Ar}-160\mu \mathrm{m}$ and $\mathrm{PM}+40\mu \mathrm{m}$ , and the papilla region [P] limited by $\mathrm{PM}-40\mu \mathrm{m}$ . The contraction of Ca by $40\mu \mathrm{m}$ ensures that large SHG signals from the capsule do not contribute to the cortical SHG score. The $\pm 160\text{-}\mu \mathrm{m}$ shift of the Ar curve apart from the artery centers aims at excluding the arteries from any SHG scoring, because their quantity and size in every kidney sample show large fluctuations (see results). The $\pm 40\text{-}\mu \mathrm{m}$ replicas of MP minimizes the uncertainty of the borderline position between the medulla and the papilla. Our algorithm finally provided SHG scores for all these regions, which were calculated as the percentage of nonzero SHG voxels averaged over the $40\text{-}\mu \mathrm{m}$ $z$ stack.

Fig. 1

Segmentation procedure. (a) Plot of three curves underlining morphological key features of the kidney: Ca, along the capsule, Ar, through the arteries centers, and PM at the edge of medulla and papilla. (b) Dilation or contraction of these curves, using elliptical fitting to define a reference center, to exclude the region of the arcuate arteries and the borderline regions. (c) Location of the three regions for SHG scoring: the cortex region [C] between $\mathrm{Ca}\text{-}40\mu \mathrm{m}$ and $\mathrm{Ar}+160\mu \mathrm{m}$ , the medulla region [M] between $\mathrm{Ar}\text{-}160\mu \mathrm{m}$ and $\mathrm{PM}+40\mu \mathrm{m}$ , and the papilla region [P] limited by PM $+40\mu \mathrm{m}$ .

2.6.

Quantitative Analysis of the Arcuate Artery Hypertensive Remodeling

We performed a quantitative analysis of the morphology of arcuate arteries. For every mouse, we selected one to five arteries. Their section was approximately elliptical because their orientation in the tissue was not absolutely perpendicular to the image plane. We considered only the small axis of the ellipse, which was not distorted by the 2-D sectioning, and measured: 1. the lumen radius L, as delineated by 2PEF from the internal elastic lamina; 2. the thickness of the media (region exhibiting 2PEF) M; and 3. the thickness of the adventitia (region exhibiting SHG) A. The measured artery radii $(\mathrm{L}+\mathrm{M}+\mathrm{A})$ ranged from $21\text{to}84\mu \mathrm{m}$ (average: $40\mu \mathrm{m}$ ). The mean cross sectional area (CSA) was calculated for each experimental condition. We normalized the data with respect to the media outer radius $(\mathrm{L}+\mathrm{M})$ for every artery to compare the morphology of arteries having different sizes. We then calculated mean values of the raw data (in $\mu \mathrm{m}$ ) and of the normalized dimensions (in %) for every mouse. The results were averaged within the three groups: the control mice $(n=7)$ , the $28\text{days}$ -infused mice $(n=7)$ , and the $49\text{days}$ -infused mice $(n=7)$ . The same method was used for the calculation of the media to lumen (M/L) and wall to lumen $[(\mathrm{A}+\mathrm{M})∕\mathrm{L}]$ ratios.

2.7.

Statistical Methods

The values for each parameter within a group were expressed as the $\text{mean}\pm \text{the}$ standard error of the mean (SEM). For comparisons between groups, a one-way nonparametric Kruskal-Wallis test was employed followed by Dunn’s Multiple Comparison test (Prism 5, GraphPad Software Incorporated, San Diego, California). For comparison of different measurements performed on the same group, we used the Kendall tau coefficient.

3.1.

Multiphoton Images of Human and Murine Renal Sections

We first studied human renal biopsies removed three months after renal transplant to assess interstitial fibrosis. Figure 2a shows a multiphoton image of an unstained formalin-fixed paraffin-embedded section: SHG and 2PEF contrasts were recorded simultaneously and displayed with green and red colors, respectively, in the combined image. 2PEF from endogenous cellular chromophores revealed tubules, glomeruli, and arterioles. SHG revealed collagen fibers in the Bowman's capsule and in the tubular interstitium. We also observed small fluorescent structures surrounded by collagen fibers in the interstitium, consistent with capillary morphology.

Fig. 2

SHG microscopy versus histological staining of kidney cortex. (a) Multiphoton image of an unstained human renal implantation biopsy: SHG (green color) reveals collagen fibers in the Bowman's capsule and the tubular interstitium, and endogenous 2PEF (red color) underlines tubules, glomeruli, and arterioles. PCT: proximal convoluted tubules, DT: distal tubules, G: glomerulus, white arrows: arterioles. (b) and (c) Histological versus (d) and (e) multiphoton images [same color code as for (a)] of serial renal sections of control mouse (b) and (d) and hypertensive mouse infused with AngII for $28\text{days}$ (c) and (e). Note that hypertension promoted the accumulation of nonfibrillar extracellular matrix within the glomerular tuft stained with Masson’s trichrome (c) [SHG silent in (f)] and the accumulation of fibrillar collagen in the Bowman's capsule and the surrounding interstitium, similarly to the observation in the human biopsy in (a). Scale bars: (a) $100\mu \mathrm{m}$ , (b), (c), (e), and (f) $20\mu \mathrm{m}$ and (d) and (g) $50\mu \mathrm{m}$ .

To study the progression of interstitial fibrosis and to compare the features of mouse renal fibrosis with human renal fibrosis, we used a well-characterized animal model of hypertensive renal fibrosis.²⁸ C57B16 mice were infused for $28\text{days}$ with AngII, and they developed increased systolic blood pressure, albuminuria (data not shown), and glomerulosclerosis compared with normotensive counterparts, as evidenced by Masson’s trichrome staining [Figs. 2b, 2c, 2d]. Serial sections of kidney cortex from the same control and hypertensive mice were visualized using multiphoton microscopy [Figs. 2e, 2f, 2g]. Multiphoton images of representative sclerosed glomeruli [Figs. 2f and 2g] showed numerous collagen fibers in the Bowman's capsule and in the tubular interstitium, whereas the control unstained section [Fig. 2e] showed no SHG signal, thus no fibrillar collagen accumulation, except for a few thin fibers in the tubular interstitium. Notably, as in human kidneys [Fig. 2a], collagen fibers were never observed within the glomerular tuft despite patent glomerulosclerosis after chronic and severe hypertension [Figs. 2f and 2g].

3.2.

Three-Dimensional Distribution of Collagen Fibers in Murine Fibrotic Kidney Tissue

To characterize the collagen distribution on a large scale, we imaged renal cortex and medulla in thick coronal slices [Figs. 3a, 3b, 3c ]. Figure 3a shows a combined SHG/2PEF image from a $49\text{day}$ -infused mouse obtained by stitching many images acquired using an objective with a high numerical aperture. We observed collagen fibers in the adventitia of arcuate arteries and arterioles and in the tubular interstitium.

Fig. 3

Multiphoton images of fibrotic murine kidney tissue. (a) Multiphoton image of a representative coronal renal section from a $49\text{days}$ -AngII infused mouse showing renal papilla (P), medulla (M), outer cortex (C), and arcuate arteries (A) along the boundary between cortex and medulla. SHG (green color) reveals collagen fibers in the tubular interstitium and in the arterial adventitia, and 2PEF (red color) underlines tubules. This $4.8\times 2.4\text{-}{\mathrm{mm}}^2$ image is obtained by stitching $10\times 20$ laser scanned images (dimension: $270\times 270\mu {\mathrm{m}}^2$ , pixel size: $0.8\times 0.8\mu {\mathrm{m}}^2$ ) acquired sequentially by moving the kidney sample with a motorized microscope stage. Scale bar: $500\mu \mathrm{m}$ . The dotted white lines underline segmentation used for SHG scoring (excluding the artery region and the borders). (b) Scheme of the laser scanning multiphoton microscope showing epidetection of $z$ stacks of SHG/2PEF images. (c) Scheme of the kidney coronal slicing for multiphoton microscopy. (d) and (e) 3-D reconstructions of underlined areas in (a) ( $270\times 270\times 40\mu {\mathrm{m}}^3$ with $0.4\times 0.4\times 1\text{-}{\mu \mathrm{m}}^3$ voxel size), showing (d) an arcuate artery and (e) interstitial fibrosis. The artery morphology in (d) is revealed by 2PEF (red color) obtained from elastin in the internal lamina and from cellular chromophores in the media, and by SHG (green color) from collagen fibers in the adventitia. Enhanced SHG contrast in (e) underlines the 3-D distribution of collagen fibers around arterioles and tubules (no 2PEF displayed here).

We also took advantage of the 3-D imaging capability of multiphoton microscopy to visualize the 3-D distribution of collagen fibers in the adventitia from arcuate arteries [Fig. 3d] and in the tubular interstitium [Fig. 3e]. For this purpose, we reconstructed 3-D images from $z$ stacks of combined SHG/2PEF images. Strong 2PEF signals arose from elastic lamina in the artery media as previously observed^{19, 20} [Fig. 3d]. As expected, SHG revealed collagen fibers in the vessel adventitia [Fig. 3d] and in the tubular interstitium [Fig. 3e], where they appeared to form a quasicontinuous network.

3.3.

Quantitative Analysis of Arcuate Artery Hypertensive Remodeling

Characterization of the remodeling of resistive arteries in end organs is of major importance in evaluating tissue perfusion, since the peripheral resistance is mainly determined by the distal part of the arterial vasculature (the resistance vessels), consisting of small arteries (diameter $<300\mu \mathrm{m}$ ) and arterioles.^{32, 33} Arcuate arteries are less than $50\mu \mathrm{m}$ in diameter in the mouse kidney, well below the usual threshold of $300\mu \mathrm{m}$ . We focused on these arteries and measured the overall vascular size, the adventitia thickness, the media thickness, and the lumen radius, as shown in Fig. 4a . Table 1 displays raw and normalized results for control and hypertensive mice. Mean raw data did not present significant changes with hypertension, because of the size heterogeneity of the arteries. However, normalization, taking into account artery radius, revealed a decrease in the lumen radius in hypertensive animals and a concomitant increase of the media thickness. By contrast, we observed no significant change in the adventitia normalized thickness [Fig. 4b].

Fig. 4

Quantitative analysis of arcuate artery remodeling in hypertensive animals. (a) Multiphoton 2-D image of the same artery as in Fig. 2d. Measurements performed along the small nondistorted axis are schematized by double arrows: $\mathrm{A}=\text{adventitia}$ thickness, $\mathrm{M}=\text{media}$ thickness, $\mathrm{L}=\text{lumen}$ radius, and $\mathrm{A}+\mathrm{M}=\text{wall}$ thickness. Scale bar: $50\mu \mathrm{m}$ . (b) Stability of adventitia surface normalized to cross sectional area in 28 and 49-day AngII-infused mice versus control mice. (c) Increase in media to lumen ratio (M/L) in 28 and 49-day AngII-infused mice versus control mice. (d) Increase in wall to lumen ratio $[(\mathrm{A}+\mathrm{M})∕\mathrm{L}]$ in 28 and $49\text{-}\text{day}$ AngII-infused mice versus control mice. ( ${}^{*}\mathrm{p}<0.05$ versus control, ${}^{**}\mathrm{p}<0.01$ versus control, $\mathrm{n}=7$ per condition.)

Table 1

Dimensions of arcuate arteries in control and hypertensive mice. Mean values and standard errors of the mean of the arcuate artery dimensions for control mice versus 28- and 49-days AngII-infused mice. Raw values are directly obtained from length measurements, as schematized in Fig. 4a. Normalized values are obtained as the ratio of the raw data to the media outer radius (L+M) in every artery, as explained in the methods. ( p*<0.05 versus control value, n=7 ).

These structural properties of arteries helped determine the physiologically important media to lumen (M/L) and wall to lumen ratios $[(\mathrm{M}+\mathrm{A})∕\mathrm{L}]$ [Figs. 4c and 4d]. Hypertension significantly increased media to lumen and wall to lumen ratios in these renal resistance arteries. This altered morphology was not associated with any significant increase in the media cross sectional area (Table 1), suggesting rearrangement of the media around a smaller diameter, so-called eutrophic remodeling.³²

3.4.

Quantitative Analysis of Segmented Second Harmonic Generation Images Reveals Angiotensin-2-Induced Interstitial Fibrosis in the Cortex

We compared the progression of fibrosis in normal and hypertensive mice infused for 28 or $49\text{days}$ . Interstitial fibers were heterogeneously distributed in a thin network that accumulated mostly in the inner cortex, close to arcuate arteries, and extended radially in the medulla and in the outer cortex [Figs. 5a, 5b, 5c ]. Surprisingly, we observed physical connections (or at least very close proximity below the resolution of the microscope) between perivascular, periglomerular, and peritubular fibers. For comparison, we also visualized kidneys from a mouse model of crescentic glomerulonephritis, and we observed a similar distribution of collagen fibers [see Fig. 5d].

Fig. 5

SHG imaging of renal fibrosis. SHG density images of coronal renal slices from representative (a) 0, (b) 28, and (c) $49\text{day}$ -AngII-infused mice and (d) GN mouse, giving the percentage of voxels above the threshold over the $40\text{-}\mu \mathrm{m}$ $z$ stack ( $\text{black}=0\%$ to $\text{white}=100\%$ ). Scale bar: $500\mu \mathrm{m}$ . The white lines underline segmentation used for SHG scoring (excluding the artery region and the borders): (C) (cortex), (M) medulla, and (P) papilla. Mean value of the SHG scores calculated on these regions are the following: (a) D0 mouse: (C) 0.31%, (M) 0.85%, (P) 0.49%; (b) D28 mouse: (C) 0.42%, (M) 2.13%, (P) 0.11%; (c) D49 mouse: (C) 0.74%, (M) 1.83%, (P) 0.20%; and (d) GN model: (C) 0.41%, (M) 2.79%, (P) 0.29%.

To discriminate between de novo interstitial fibrosis and constitutive adventitial collagen around arcuate arteries, we performed a radial segmentation of SHG images excluding arcuate arteries as well as border regions, and selected outer-cortical, medullar, and papillary regions [see white dotted line in Figs. 3c and 5]. We then obtained fibrosis scores in various anatomical regions from the kidney tissue by measuring the density of voxels exhibiting nonzero SHG signal.

Averaged values of the cortical scores and of the total scores are shown in Fig. 6 for control and hypertensive mice. Both scores increased for infused mice, and with the infusion duration. Cortical scores showed the larger relative increase in hypertensive kidneys. Medullar scores were highly heterogeneous in control kidneys and showed no significant change with hypertension (data not shown). Papillary SHG signals were scarce, with low signal over noise ratios and unreliable SHG scores.

Fig. 6

Quantitative scoring of renal fibrosis. (a) Mean values of SHG scores calculated in the whole SHG images and in cortical areas for six control mice (white triangles) versus AngII-infused mice for $28\text{days}$ (gray diamond) and $49\text{days}$ (black circle). Errors bars correspond to standard errors of the mean. The cortical score shows smaller absolute values but larger differences on AngII-induced hypertension. ( ${}^{*}\mathrm{p}<0.05$ versus control; ${}^{**}\mathrm{p}<0.01$ versus control; $\mathrm{n}=6$ to 8 per condition). (b) Automated cortical SHG score plotted as a function of a semiquantitative estimate of interstitial damage for control and hypertensive mice $(\tau =0.68,p<0.01)$ . (c) Glomerulosclerosis index versus cortical SHG score for control and hypertensive mice $(\tau =0.55,p<0.01)$ .

Finally, we confronted cortical SHG scores with usual histopathological methods that evaluate kidney damages in Masson’s trichrome stained sections. We plotted cortical SHG scores as a function of histopathological evaluation of interstitial damage and inflammation [Fig. 6b] and glomerulosclerosis scores [Fig. 6c] for all mice in the three groups (control, $28-\text{day}$ and $49-\text{day}$ AngII-infused mice, $n=21$ mice). Semiquantitative histopathological evaluation of interstitial damage and SHG cortical score were found to be correlated ( $\tau =0.68$ , $p<0.01$ ) [Fig. 6b]. Glomerulosclerosis correlated better with cortical SHG scores ( $\tau =0.55$ , $p<0.01$ ) than with global SHG scores ( $\tau =0.32$ , $p<0.05$ ).