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Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, the NetherlandsDepartment of Neurosurgery, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany
Address correspondence to Jan-Luuk Hillebrands, Ph.D., Department of Pathology and Medical Biology – Pathology, University of Groningen, University Medical Center Groningen, HPC EA10, Post Office Box 30.001, 9700 RB Groningen, the Netherlands.
Hyalinosis is a vascular lesion affecting the renal vasculature and contributing to aging-related renal function decline. We assessed whether arteriolar hyalinosis is caused by Klotho deficiency, a state known to induce both renal and vascular phenotypes associated with aging. Histochemistry was used to assess hyalinosis in Klotho−/− kidneys, compared with Klotho+/− and wild-type littermates. Immunohistochemistry was used to investigate vascular lesion composition and the different layers of the vascular wall. Finally, spironolactone was used to inhibit calcification in kl/kl mice, and vascular lesions were characterized in the kidney. Arteriolar hyalinosis was detected in Klotho−/− mice, which was present up to the afferent arterioles. Hyalinosis was accompanied by loss of α-smooth muscle actin expression, whereas the endothelial lining was mostly intact. Hyalinous lesions were positive for IgM and iC3b/c/d, indicating subendothelial leakage of plasma proteins. The presence of extracellular matrix proteins suggested increased production by smooth muscle cells (SMCs). Finally, in Klotho−/− mice with marked vascular calcification, treatment with spironolactone allowed for replacement of calcification by hyalinosis. Klotho deficiency potentiates both endothelial hyperpermeability and SMC dedifferentiation. In the absence of a calcification-inducing stimulus, SMCs assume a synthetic phenotype in response to subendothelial leakage of plasma proteins. In the kidney, this results in arteriolar hyalinosis, which contributes to the decline in renal function. Klotho may play a role in preventing aging-related arteriolar hyalinosis.
Arteriolar hyalinosis, or hyaline arteriolosclerosis, is a vascular lesion often found in the kidney in aging and hypertension,
In the kidney, the afferent arterioles are the most commonly affected vessels, likely because of their role of major resistance arterioles, placing great local stress on the endothelium. It is thought that hyalinosis of the afferent arterioles leads to loss of autoregulation, which, in turn, leads to glomerular damage and the decline of renal function.
We determined whether murine Klotho deficiency, as a model for premature aging that also leads to a spontaneous decrease in renal function and develops sequelae of chronic renal failure, is affected by hyalinosis as well. Klotho is a renal antiaging protein that is predominantly expressed in the distal convoluted tubule.
Given the profound protective effects of Klotho on endothelial function, smooth muscle cell differentiation, and the kidney, we hypothesized that a relationship might exist between hyalinosis as an aging-associated vascular lesion and Klotho deficiency as a state of accelerated aging.
Materials and Methods
All animal experiments were conducted in accordance with the NIH’s Guide for the Care and Use of Laboratory Animals.
Klotho−/− mice were housed at the central animal facility of the University Medical Center Groningen (Groningen, the Netherlands). Klotho−/− mice were housed in individually ventilated cages with abundant nesting material and wild-type (WT) or Klotho+/− buddies to prevent hypothermia. Klotho−/− mice were provided with wetted or soaked food and drinking water (with long drinking nipples to ensure easy accessibility) ad libitum. They were monitored daily. At the age of 7 weeks, mice were sacrificed under deep isoflurane anesthesia by cardiac puncture. Kidneys were collected and snap frozen in liquid nitrogen or fixed in formalin (after harvesting of tissues) and embedded in paraffin. Klotho−/− (n = 3), Klotho+/− (n = 4), and WT (n = 3) mice were analyzed for the presence of arteriolar hyalinosis. Kl/kl mice were housed at the Institute for Physiology at the University of Tübingen (Tübingen, Germany) and were not treated (n = 13) or treated with spironolactone (80 mg/L; n = 10) in drinking water for 8 weeks. Kidney tissue was used from 6- to 8-week–old β-actin–Cre/Klothoflox/flox mice, bred as previously described.
Different strains of mice were used to assess whether the hyalinosis phenotype is present or can be induced independent from the genetic background.
Paraffin sections (4 μm thick) were cut, deparaffinized in xylene, and rehydrated in a graded ethanol series. Periodic acid-Schiff (PAS) staining was performed by incubating sections in 1% periodic acid for 10 minutes and in Schiff's reagent for 15 minutes (or for 5 minutes as a mild counterstaining after immunohistochemistry with diaminobenzidine), followed by hematoxylin counterstaining. Von Kossa staining was performed by incubating sections in 1% silver nitrate for 1 hour under sunlight exposure, followed by incubation in 3% sodium thiosulfate for 5 minutes and counterstaining with nuclear fast red. Masson trichrome staining was performed by incubating sections for 5 minutes in celestine blue, 5 minutes in hematoxylin, 5 minutes in a 1:2 mixture of 1% acid fuchsin and 1% xylidine ponceau, 5 minutes in 1% phosphomolybdic acid, and 1 minute in 1% aniline blue. Verhoeff staining was performed by incubating sections for 15 minutes in Verhoeff staining solution (5:2:2 mixture of 5% alcoholic hematoxylin, 10% ferric chloride, and 2% potassium iodide/1% iodine), followed by destaining 2% ferric chloride and staining with Van Gieson solution. Oil red O staining was performed on cryosections, which were fixed for 10 minutes in 8% formalin and dipped twice in 60% 2-propanol, followed by incubation for 10 minutes in oil red O solution, destaining in 60% 2-propanol, and counterstaining in hematoxylin.
Paraffin sections (4 μm thick) were cut, deparaffinized in xylene, and rehydrated in a graded ethanol series. Antigen retrieval was performed by heating sections at 500 W for 15 minutes in 10 mmol/L citric acid (pH 6) for α-smooth muscle actin (α-SMA), CD31, and collagen I, in 170 mmol/L Tris/1 mmol/L EDTA (pH 9) for renin and S100A4, and in 1 mmol/L EDTA (pH 8) for collagen III. After blocking endogenous peroxidase [0.3% H2O2/phosphate-buffered saline (PBS)] for 30 minutes, sections were incubated with primary antibodies [α-SMA: dilution 1:300; mouse monoclonal 1A4; Dako, Glostrup, Denmark; CD31: dilution 1:50; SZ31; Dianova, Hamburg, Germany; collagen I: dilution 1:100; 1310-01; Southern Biotech, Birmingham, AL; collagen III: dilution 1:75; 1330-01; Southern Biotech; renin: dilution 1:2500; polyclonal antibody kindly provided by Dr. Tadashi Inagami (Vanderbilt University School of Medicine, Nashville, TN); and S100A4: dilution 1:2000; A5114; Dako] for 1 hour in 1% bovine serum albumin/PBS. If necessary, avidin and biotin blocking solutions were applied (Vector Laboratories Inc., Burlingame, CA). Sections were incubated with the appropriate secondary and tertiary antibodies for 30 minutes [goat anti-mouse–biotin, rabbit anti-rat–horseradish peroxidase (HRP), goat anti-rabbit–HRP, and rabbit anti-goat–HRP; Dako]; and the chromogenic reaction was performed using diaminobenzidine in 0.03% H2O2/PBS, followed by hematoxylin counterstaining. IgM and iC3b/c/d stainings were performed on frozen sections (4 μm thick), which were dried and fixed in acetone for 10 minutes at room temperature, followed by endogenous peroxidase blocking in 0.075% H2O2/PBS for 30 minutes and incubation with primary antibodies for 1 hour (iC3b/c/d: dilution 1:20; HM1065; Hycult Biotech, Uden, the Netherlands; IgM-HRP: dilution 1:100; 1020-05; Southern Biotech). Then, the appropriate secondary and tertiary antibodies were applied for 30 minutes (goat anti-mouse–HRP, rabbit anti-goat–HRP, and goat anti-rabbit–HRP; Dako); and the chromogenic reaction was performed with diaminobenzidine or 3-amino-9-ethylcarbazole (AEC). Nuclei were counterstained with hematoxylin. All slides were scanned using a Hamamatsu Nanozoomer 2.0HT (Hamamatsu Photonics, Hamamatsu, Japan), and scans were analyzed using Aperio ImageScope software version 184.108.40.20613 (Leica Microsystems B.V., Amsterdam, the Netherlands).
Paraffin sections (4 μm thick) were cut, deparaffinized in xylene, and rehydrated in a graded ethanol series. Antigen retrieval was performed in 10 mmol/L citric acid (pH 6), followed by a blocking step with 1% bovine serum albumin and 5% donkey serum in PBS for 80 minutes at room temperature. Sections were incubated with polyclonal rabbit anti-mouse phosphorylated Smad2/3 (dilution 1:150; sc-11769; Santa Cruz Biotechnology, Dallas, TX) and polyclonal goat anti-mouse CD31 (dilution 1:200; AF3628; R&D Systems, Minneapolis, MN) for 60 minutes at room temperature, followed by donkey anti-rabbit–Alexa Fluor 555 (dilution 1:500; ab150074; Abcam, Cambridge, UK) and donkey anti-goat–Alexa Fluor 647 (dilution 1:500; A21447; Invitrogen, Carlsbad, CA) for 70 minutes at room temperature, followed by DAPI counterstaining and imaging on an EVOS fluorescence imaging system (Thermo Fisher Scientific, Waltham, MA).
Normally distributed data are presented as means ± SD or median (interquartile range). Differences between groups were tested with analysis of variance, followed by Bonferroni post-hoc correction; t-test; a Kruskal-Wallis test, followed by Dunn post-hoc correction; or U-test, after prior Kolmogorov-Smirnov testing for normality. Spearman's ρ was used for correlation analysis. P < 0.05 was considered statistically significant. All data analysis was performed using SPSS version 23 (IBM, Armonk, NY) and GraphPad Prism version 5 (GraphPad Software, San Diego, CA).
Klotho Deficiency Induces Arteriolar Hyalinosis
In 7-week–old Klotho−/− mouse kidneys, vascular lesions that were strongly PAS positive (Figure 1, A, C, and E ) and were typically eosinophilic and glassy were detected on hematoxylin-eosin staining (Figure 1F), indicative of hyalinosis. These lesions were consistently observed in all Klotho−/− mice and were not observed in Klotho+/− or wild-type littermates (Figure 1, A–D). The Von Kossa staining was negative in these arterioles, indicating that these lesions did not represent calcifications (Supplemental Figure S1, A–C). Oil red O staining was also negative, indicating that these lesions did not contain a fatty component (Supplemental Figure S1, D and E).
Hyalinosis Is Mostly Found in Terminal Interlobular Arteries and Proximal Afferent Arterioles
The relatively large segmental arteries generally contained affected regions (Figure 2A), and luminal measurements of all α-SMA–positive arteries indicate that this pattern remained patchy, with some parts of interlobar and arcuate arteries being affected. Most affected arteries were interlobular arteries and proximal afferent arterioles (Figure 2B), with most having a lumen with a diameter of 6 to 42 μm (Figure 2D). However, the vast majority of arterioles <15 μm (including terminal afferent arterioles and efferent arterioles) were generally unaffected (Figure 2D) and renin/PAS double staining did not reveal colocalization of renin expression and hyalinosis in terminal afferent arterioles (Figure 2C).
PAS/CD31 double staining indicated that the endothelial lining of affected arterioles is generally still intact (Figure 3A). Verhoeff staining indicated that the elastic lamina is intact in unaffected arterioles but cannot be discerned in affected areas (Figure 3B). PAS/α-SMA double staining indicated that arteries and arterioles affected by hyalinosis lose their smooth muscle cell expression of α-SMA (Figure 3C).
Hyaline Depositions Contain Plasma Proteins
Using immunohistochemistry, hyalinous lesions in Klotho−/− kidneys were found to be positive for IgM (Figure 4, A–C) and for activated C3 proteins iC3b/c/d (Figure 4, D–F), indicating that the integrity of the endothelial barrier function is locally compromised and that leaked plasma proteins accumulate in the subendothelial space. Vascular positivity for IgM and iC3b/c/d was not found in WT kidneys (Figure 4, A and D).
Dedifferentiation of Smooth Muscle Cells to a Synthetic Phenotype
In Klotho−/− mouse arteries, lesional smooth muscle cells were found to have lost expression of α-SMA, a contractile apparatus protein that is also a marker for differentiated SMCs (Figures 2A and 5H). In turn, lesional SMCs gained expression of S100A4 (Figure 5J), which is considered a marker of a synthetic phenotype in SMCs. Indeed, in Klotho−/− kidneys, collagen I, collagen III, and Masson trichrome positivity were also detected, most prominently in hyalinized parts of segmental arteries (Figure 5, B, D, and F), indicating increased deposition of extracellular matrix proteins. WT renal arteries of similar size did not have medial/subendothelial collagen expression (Figure 5, A, C, and E), loss of α-SMA expression (Figure 5G), or SMC expression of S100A4 (Figure 5I).
Hyalinous Lesions Are Associated with Increased TGF-β1 Signaling
The key pathway known to be involved in the development of arteriolar hyalinosis is transforming growth factor (TGF)-β1 signaling, of which Klotho is a known inhibitor. We, therefore, hypothesized that loss of Klotho leads to deranged TGF-β1 signaling and promotes the development of arteriolar hyalinosis. Phosphorylated Smad2/3 expression was highly increased ubiquitously in Klotho−/− mouse kidney, with strong nuclear expression in glomerular, tubular, and vascular cells alike (Figure 6, C–F), compared with low, constitutive expression levels in WT mice (Figure 6, A–C). Particularly high phosphorylated Smad2/3 expression, however, was found in the vascular cells associated with hyalinous lesions (Figure 6F), suggesting that not only is TGF-β1 signaling constitutively activated in Klotho−/− kidney, activation of TGF-β1 signaling could play a role in the development of arteriolar hyalinosis in Klotho deficiency. Conjugate control stainings for phosphorylated Smad2/3 and CD31 were negative (Figure 6, G–I).
Vascular Lesions in Different Klotho Knockout Mouse Strains
Because not only the development of hyalinosis but also the lack of vascular calcification in Klotho−/− mice is an unconventional finding, it was assessed whether hyalinosis is a trait also found in other strains of Klotho knockout mice. Therefore, PAS and Von Kossa staining was performed on kidney sections from kl/kl mice (which have a disrupted promoter) and β-actin–Cre/Klothoflox/flox (or β-actin–KL−/−) mice (which have a deletion of exon 2 of the Klotho gene in cells that express β-actin, which is ubiquitous), to compare with Klotho−/− mice (which have a deletion of exon 2 in all cells). The kl/kl mouse kidneys almost exclusively displayed vascular calcification (Figure 7, A and D), whereas β-actin–KL−/− mice exhibited a mix of vascular calcification and some hyalinosis (Figure 7, B and E) and Klotho−/− mice only had prominent hyalinosis (Figure 7, C and F). Quantitative assessment is depicted in Figure 7, G–I. These findings indicate that at least β-actin–KL−/− mice can also develop arteriolar hyalinosis, although their phenotype is variable, possibly because of slight variations in recombination.
Spironolactone Inhibits Vascular Calcification and Allows for Its Replacement by Hyalinosis
Finally, to further examine whether the development of arteriolar hyalinosis is a consequence of Klotho deficiency in general, it was studied whether inhibition of vascular calcification in kl/kl would also potentiate the development of arteriolar hyalinosis. The kl/kl mice were treated with 80 mg/L spironolactone for 8 weeks, which was previously found to inhibit the development of vascular calcification.
The kl/kl mice spontaneously displayed severe nephrocalcinosis (86% ± 8% calcified arteries) (Figure 8, A, B, and D ) and little hyalinosis (4% ± 2%) (Figure 8G). However, spironolactone treatment allowed for the replacement of calcified arteries (51% ± 27%) with hyalinosis (32% ± 23%) (Figure 8, C–G).
This study shows that Klotho deficiency induces arteriolar hyalinosis. The emergence of an aging-related vascular lesion as part of the phenotype of Klotho deficiency compounds the hypothesis that Klotho exerts antiaging effects. Given the prevalent view that arteriolar hyalinosis, particularly in afferent arterioles, leads to the loss of renal autoregulation and contributes to the aging-related loss of renal function,
the potential involvement of Klotho is of particular interest to the aging population. The aging-related decrease in Klotho expression may be causally involved in this process; and considering the protective effects Klotho has been shown to have experimentally on renal function,
increasing Klotho levels may prove to be a promising approach to the preservation of renal function during aging.
Klotho deficiency allows for plasma proteins to accumulate in the subendothelial space in hyaline lesions, attesting to the poor barrier function of the endothelium. Indeed, endothelial hyperpermeability
have been shown before to develop in the absence of Klotho. It is, therefore, clear that adequate Klotho levels are paramount to endothelial integrity. However, more interesting in this study is the observed plasticity of the smooth muscle cell phenotype in Klotho deficiency, considering the previously reported direct in vitro effects of Klotho on SMC differentiation.
In vivo genetic evidence for suppressing vascular and soft-tissue calcification through the reduction of serum phosphate levels, even in the presence of high serum calcium and 1,25-dihydroxyvitamin d levels.
in particular of the media, as occurs in chronic kidney disease or in aging. However, likely due to differences in genetic background, diet, and other environmental factors, Klotho-deficient mice displayed a certain phenotypic variability. Klotho−/− mice had not developed vascular calcification in the kidney at the age of 7 weeks. We hypothesize that the accumulated procalcific triggers were not potent enough to induce vascular calcification, which, in turn, allowed for the development of arteriolar hyalinosis instead. The Klotho−/− mice have a mixed genetic background, but one that is mostly C57BL/6, which is generally more resistant to the development of vascular calcification. To address this phenomenon, kl/kl mice (on a mixed background, which was mostly 129/Sv) and β-actin–KL−/− mice (also on a mixed background, predominantly C57BL/6
) were studied; kl/kl mice displayed severe vascular calcification at 7 to 8 weeks of age. The observation that β-actin–KL−/− mice spontaneously display some arteriolar hyalinosis and that kl/kl mice do so after treatment with spironolactone, which inhibited the development of calcification by blocking vascular SMC–derived aldosterone, inducing calcification via a Pit1-dependent mechanism,
allows us to speculate that the development of hyalinosis is a function of the degree to which vascular calcification develops, which can be influenced by the genetic background, as well as by dietary factors. The development of arteriolar hyalinosis in lieu of vascular calcification in spironolactone-treated mice lets us conclude that, in the absence of sufficiently strong procalcific stimulus, smooth muscle cells dedifferentiate to a more synthetic phenotype. We hypothesize that Klotho inhibits pathologic dedifferentiation of smooth muscle cells and the consequently assumed phenotype is the net result of the stimuli to which the SMCs are exposed. In the case of hyalinosis, the subendothelial presence of accumulated plasma proteins likely triggers the SMC response of dedifferentiation and synthesis of ECM proteins.
This concept is schematically depicted in Figure 9.
To expand on the topic of phenotypic variability in Klotho deficiency, it has long been known that different Klotho levels induce different vascular phenotypes. Klotho−/− mice display severe vascular calcification,
which probably does not develop in Klotho−/− mice because of the dominance of the calcification phenotype. Of note, arteriolar hyalinosis and arterial stiffening have in common the aberrant SMC behavior of excessive ECM deposition. Naturally, the short lifespan of full knockout mice also limits the window for the development of other pathologies. The same probably holds true for the development of hypertension from 15 to 16 weeks of age onward in Klotho+/− mice,
This study indicates that a similar example of phenotypic divergence is the uncovering of arteriolar hyalinosis if the development of calcification is mildly delayed and/or inhibited. The comparison of these phenotypes between different Klotho knockout genotypes under different conditions may shed light on the development of vascular disease in patients. The rapid and severe development of vascular calcification in kl/kl mice may make them a suitable model for vascular pathologies in chronic kidney disease patients, who are also more severely Klotho deficient than the general aging population. Heterozygous mice may constitute a suitable model for general aging or other patients with intermediate Klotho levels, displaying milder pathologies, like arterial stiffening, endothelial dysfunction, and hypertension. This study suggests that even in aging patients in whom vascular calcification in the kidney plays a limited role, Klotho deficiency may still contribute to the development of arteriolar hyalinosis and the subsequent decline in renal function.
The development of hyalinosis has not been widely studied in animal models, but calcineurin appears to be a key modulator. Calcineurin inhibitors, like tacrolimus and cyclosporine A, genetic deletion of calcineurin α,
leading to less activation of TGF-βRI, it appears that Klotho may act upstream of calcineurin inhibitor–induced hyalinosis. Indications of constitutively and ubiquitously active TGF-β1 signaling in the absence of Klotho and in particular of strong phosphorylated Smad2/3 expression in vascular cells associated with hyalinous lesions were also found in this study, suggesting a role for TGF-β1 signaling. Together, these lines of evidence lead to the hypothesis that Klotho may be able to mitigate the TGF-β1–induced adverse effects of calcineurin inhibitor use. Of note are also the down-regulation of Klotho in cyclosporine A nephropathy,
Investigation of Klotho as a therapeutic target in inhibiting development of chronic graft dysfunction in renal transplantation patients may, therefore, also be warranted. Furthermore, although interventional studies are certainly necessary for more solid conclusions, the evidence that Klotho and TGF-β1 signaling may act in a common pathway in the development of arteriolar hyalinosis may be a step in expanding our understanding of this aging-related vasculopathy and its implications in the aging process.
In summary, arteriolar hyalinosis appears to be an important feature of the Klotho deficiency phenotype, which is likely generally masked by the development of vascular calcification and is uncovered on inhibition of or resistance against vascular calcification. The finding that Klotho deficiency induces arteriolar hyalinosis raises new questions and hypotheses on the effects of Klotho on endothelial integrity and smooth muscle cell dedifferentiation, on the role of Klotho in aging and in aging-related renal function decline, and on the potential of Klotho in aging and in calcineurin inhibitor nephrotoxicity.
We thank Wierd Kooistra and Marian Reinders for technical support.
R.M., H.v.G., F.L., G.K., and J.-L.H. designed the study; R.M., A.T.U., L.M.W., J.V., G.H., M.B., and H.O. performed experiments; R.M. analyzed data and wrote the manuscript; J.V., H.O., F.L., L.Q.-M., H.v.G., and J.-L.H. edited the manuscript.
Composition of hyalinous lesions. A: Periodic acid-Schiff staining on Klotho−/− kidney, showing arteriolar hyalinosis. B: Von Kossa staining on the same arteriole, showing no calcification. C: Positive control for the Von Kossa staining (human placenta). D: Oil red O staining on Klotho−/− kidney, showing arteriolar hyalinosis, without a fatty component. E: Positive control for the oil red O staining (human carotid artery plaque). Arrows indicate hyalinous lesions. Scale bars: 50 μm (A, B, and D); 200 μm (C and E). Original magnification: ×400 (A, B, and D); ×100 (C and E).
Arteriolar sclerosis in hypertensive and non-hypertensive individuals.
In vivo genetic evidence for suppressing vascular and soft-tissue calcification through the reduction of serum phosphate levels, even in the presence of high serum calcium and 1,25-dihydroxyvitamin d levels.
Supported by a Dutch Kidney Foundation Consortium grant CP10.11 (NIGRAM [NIerGerichte Research: van Arterie naar Mens] principal investigators: Joost Hoenderop and René Bindels [ Radboud University Medical Center , Nijmegen, the Netherlands]; Piet ter Wee and Marc Vervloet [ Amsterdam University Medical Centers - Free Univeristy of Amsterdam (VUmc) , Amsterdam, the Netherlands]; and Gerjan Navis, Martin de Borst, and J.-L.H. [ University Medical Center Groningen ]); and the University Medical Center Groningen M.D./Ph.D. program (R.M.).