Objectives: In this study, we aimed to ascertain the efficacy and determine the dose effects of a new analog of vitamin D, 2α-methyl-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2AMD), in decreasing fibrosis and improving renal function in a rat model of kidney disease.

Materials and Methods: Using the cyclosporine model of chronic kidney disease, we tested 4 dose regimens (2.5, 5, 10, and 20 ng/kg) of 2AMD by subcutaneous administration. The 2AMD analog was comparedwith another analog, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD), given at 5 ng/kg.

Results: After 28 days of cyclosporine administration with 5 ng/kg 2AMD or 2MD, blood urea nitrogen levels were decreased by 20% and 30%, with no increase in serum calcium. This dose significantly decreased collagen levels by 50%, as determined by relative measurements of birefringence elicited under polarized light following picrosirius red staining of kidney tissues. The 20 ng/kg dose of 2AMD was hypercalcemic, with consequent deleterious effects on measured parameters; however, all doses of2AMD tested decreased collagen as determined by picrosirius staining. In Western blot analysis of extracts from rat kidneys treated with cyclosporine and 5 ng/kg 2AMD, the fibrotic markers, fibronectin and vimentin, were decreased compared with animals treated only with cyclosporine.

Conclusions: We found that both vitamin D analogs are potent inhibitors of kidney fibrosis with potential renoprotective activity.

Key words : Collagen, Fibrosis, Kidney, Transplant

Introduction

Despite progress in the treatment of kidney disease, there is still a gap in therapies that prevent the need for dialysis or transplant.¹ Two salient histologic features that kidney disease and eventual graft failure have in common are tubular atrophy and fibrosis. Fibrosis is characterized by excess connective tissue deposition, primarily collagens, and inflammation in some cases.²

As shown in several observational studies, there is a survival benefit with the active metabolite of vitamin D, 1,25(OH)2D3 (calcitriol), for the treatment of patients with chronic kidney disease who are undergoing hemodialysis.^3-5 Vitamin D3, when converted in the kidney to calcitriol, acts as a potent steroid hormone on various organs, such as the intestine, bone, kidney, and parathyroid, primarily to promote homeostasis of blood calcium levels.^6,7 As a result of adverse events related to the deleterious effects of hypercalcemia, calcitriol analogs, such as paricalcitol, have been developed to circumvent this problem.^8,9 A number of clinical trials have demonstrated the efficacy of paricalcitol in decreasing proteinuria^10-12 and excessive parathyroid hormone (PTH) due to secondary hyperparathyroidism (SHPT) associated with chronic kidney disease^13-15 or following kidney transplant.^16,17 Despite the promising outcomes, there are still problems with hypercalcemia, albeit at lower rates.¹⁵ Novel selective analogs of 1,25(OH)2D3, such as 2-methylene-19-nor-(20S)-1α,25-dihydroxivitamin D3 (2MD), are known to be hyperactive vitamin D receptor agonists.^18,19 The 2MD analog has been shown to decrease PTH at doses that do not increase serum calcium or phosphorus levels in a uremic rat model of SHPT and in a cohort of postmenopausal women.¹⁹ At longer follow-up of 8 weeks, the study of uremic rats demonstrated that 2MD was about 4 times more effective in reducing PTH than paricalcitol. In addition, 2MD was effective in significantly reducing PTH in a cohort of 24 patients with SHPT on hemodialysis due to end-stage renal disease.²⁰ This was more recently confirmed in a double-blind, placebo-controlled trial that included dialysis patients who were previously administered a calcimimetic.²¹

In this study, we report the effects of 2α-methyl-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2AMD), an analog structurally similar to 2MD, on its ability to protect rat kidneys from cyclosporine (CsA)-induced nephrotoxicity. We compared its effects versus those of 2MD in a rat CsA model. The 2AMD analog was previously shown to inhibit the development of type 1 diabetes in the insulin 2-/- nonobese diabetes murine model,²² in which it was shown to preserve islet cell architecture, reduce T-cell invasion, and prevent diabetes. This report describes our findings regarding 2AMD and 2MD for renoprotection in the rodent CsA model, a model that approximates chronic kidney disease and is relevant for transplant studies. Cyclosporine is an immunosuppressant given to reduce organ rejection but that eventually promotes renal vasoconstriction, ending in epithelial cell apoptosis and interstitial fibrosis.^23,24

Neither 2AMD nor 2MD has been tested pre-viously for renoprotective or antifibrotic effects in models of kidney disease. We found that at 5 ng/kg both 2AMD and 2MD decreased kidney fibrosis and ameliorated nephrotoxic effects of CsA without raising serum calcium levels. In addition, we report the effects of higher 2AMD doses to be avoided due to toxicity in the CsA rat model.

Materials and Methods

Cyclosporine rat model
All work was conducted under protocol M5421 and was reviewed and approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. Animals were maintained in humidity- and temperature-controlled rooms under 12:12-h light/dark cycles. All rats were provided with food and water ad libitum as per standard procedure. Animal treatment groups are outlined in Table 1.

Male Sprague-Dawley rats (Harlan/Envigo, Madison, WI, USA) weighing approximately 250 g were started on a low-sodium diet (TD94268, 0.1% NaCl; Teklad, Madison, WI, USA) 1 week before start of CsA treatment. Rats were randomly assigned to groups of 6 each. Cyclosporine (Sandimmune, Novartis, East Hanover, NJ, USA) in Cremophor EL (Sigma, St. Louis, MO, USA) and 33% ethanol was administered subcutaneously at 20 mg/kg/day under 2% isoflurane for 28 days. Vitamin D analogs were dissolved in polyethylene glycol, 0.5% ethanol and administered subcutaneously at 2.5, 5, 10, and 20 ng/kg for 2AMD and at 5 ng/kg for 2MD for 28 days under 2% isoflurane. Vehicle-only animals received polyethylene glycol and Cremophor-33% ethanol; vehicle plus CsA animals received CsA plus polyethylene glycol. Animals received the last dose approximately 24 hours before blood collection and organ retrieval. Animals were placed under anesthesia, blood (aorta) was collected, and kidneys were removed, with subsequent death by pneumothorax puncture.

Kidneys were sectioned longitudinally, with one-half placed into 10% formalin followed by embedding in paraffin and sectioned for further histochemistry. The remaining kidney half was snap frozen in liquid nitrogen. Blood was allowed to clot for about 1 hour, which was followed by spinning and removal of serum. Serum and frozen kidney tissues were stored at -80°C.

Serum tests
Serum was analyzed for blood urea nitrogen (BUN), creatinine, and calcium levels. Blood urea nitrogen and creatinine levels were measured using IDEXX cartridges on a VetTest 8008 bioanalyzer (IDEXX Laboratories, West Sacramento, CA, USA). Calcium levels were measured in 0.1% lanthanum chloride by atomic absorption spectroscopy (Pinnacle 500, Perkin-Elmer, Waltham, MA, USA).

Histochemistry
Kidneys fixed in formalin were embedded in paraffin, sectioned, and stained with hematoxylin and eosin and picrosirius red, which stains mostly collagens I and III.25 Sections were imaged using the ×20 objective on an upright bright-field Nikon Eclipse 600 microscope with polarizing capabilities to assess the birefringence of the picrosirius red-stained collagen fibers. Quantitation of birefringence was carried out using ImageJ software26 from 6 images from each rat section obtained at random within the central cortical area and excluding large blood vessels and capsules.

Tissue extraction and Western blotting
Kidney tissues were homogenized in RIPA buffer containing 1% deoxycholate at 0.1 g/mL of buffer and spun at 4°C. The resulting pellets were resuspended in buffer containing 4 M urea, 4% sodium dodecyl sulfate, and 1 mM dithiothreitol. Resuspended pellets were vortexed and heated to 95°C for 5 minutes. This latter pool constituted the tissue fraction containing extracellular matrix (ECM) proteins.^27,28 We obtained the protein concentration in the ECM fraction using the DC protein assay kit (Bio-Rad, Hercules, CA, USA), with albumin standards diluted in the corresponding buffer. Extracellular matrix fractions (1% deoxycholate-insoluble) or 1% deoxycholate-soluble fractions (lysates) were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 10 to 20 μg/well, transferred to nitrocellulose, and incubated with antibodies to fibronectin (Abcam, Cambridge, MA, USA) or vimentin (eBioscience/Thermo, Waltham, MA, USA) followed by horseradish peroxidase-conjugated antirabbit or mouse immunoglobulin G (LifeTech/Thermo, Waltham, MA, USA). Relative quantitation of specific blotted protein was performed by assessing relative band intensities using ImageJ and normalizing to bands obtained with anti-histone 3 rabbit antibody (Cell Signal Technology, Danvers, MA, USA) or anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-horseradish peroxidase (Genscript, Piscataway, NJ, USA), used as loading controls for ECM fractions or lysate fractions, respectively.

Statistics
Graphpad Prism (La Jolla, CA, USA) software was used to determine significant differences among treatment groups, with results analyzed with one-way analysis of variance and Bonferroni test or t test, as indicated in the text. P ≤ .05 was considered significant.

Results

Serum calcium levels were obtained from blood collected at organ retrieval, approximately 24 hours after the last vitamin D analog dose. As shown in Figure 1 (left), serum calcium levels increased with dose of 2AMD. The CsA rats treated with 2AMD at 10 and 20 ng/kg demonstrated increased serum calcium levels of about 20% and 30%, respectively, compared with animals treated with the vehicle alone or animals treated with CsA plus vehicle. At 5 ng/kg or lower, neither 2AMD nor 2MD raised serum calcium levels significantly.

As shown in Figure 1 (middle), the percent weight gain in grams per rat from day 0 to day 28 for vehicle-treated rats without CsA was approximately 28%, whereas rats that received vehicle plus CsA gained only 15%, indicating the deleterious impact of CsA in this model of nephrotoxicity.^23,24 The CsA rats given 10 or 20 ng/kg 2AMD did not gain weight or lost up to 8% of their weight, respectively, suggesting a worsening of CsA pathology with hypercalcemia. However, when 2AMD or 2MD was administered at 2.5 or 5 ng/kg, rat weight increased by 20%, indicating a trend in amelioration of the deleterious effects caused by CsA.

Compared with results in vehicle only-treated rats, BUN levels (Figure 1, right) were 2.8-fold higher when CsA was added. However, the addition of 2AMD at 5 and 10 ng/kg and 2MD at 5 ng/kg elicited a significant decrease of about 18%, 24%, and 33%, respectively, suggesting improvements in the pathologic effects of CsA. Serum creatinine levels were measured, but there was no significant increase with CsA compared with vehicle alone.

Representative images of hematoxylin and eosin-stained cortical kidney sections from the CsA-treated cohorts are shown in Figure 2. Sections from rats that received CsA plus vehicle exhibited the typical areas of tubular atrophy and increased fibrosis apparent as basophilia, likely due to mesenchymal cell proliferation and inflammatory cell infiltration typical for CsA-treated kidneys.^23,24 Mesenchymal cells are responsible for the production of excess collagens.² Vehicle-treated kidney sections were mostly acidophilic, with intact tubules and no apparent areas of interstitial cell growth. Kidney sections from CsA rats also treated with 2AMD or 2MD at 5 ng/kg showed fewer basophilic areas and an increase in acidophilic staining, indicative of preserved tubular cytoplasm. Kidney sections from CsA rats treated with 2.5 or 10 ng/kg 2AMD showed areas of tubular atrophy and interstitial cellularity.

Effects on fibrosis were measured by quantifying microscopic images of birefringence elicited from picrosirius red staining of collagen I and III fibers, reflected with use of polarized light.25 Quantitation of images is depicted in Figure 3. The central cortical regions of kidneys from CsA-treated rats showed 2.5-fold higher collagen content than the vehicle only-treated animals. Addition of 2MD at 5 ng/kg and 2AMD at all concentrations tested decreased collagen present in the cortical regions of kidneys of CsA rats by 50% to 60%.

Both fibronectin, another ECM protein, and vimentin, a fibroblast intermediate filament protein, are recognized markers of fibrotic tissue in the CsA model.^23,29,30 We aimed to ascertain the effects on these markers in CsA-treated rats given 5 ng/kg 2AMD. The Figure 4 immunoblots show ECM and lysate fractions reacted for fibronectin using histone 3 as loading control for the ECM fractions or for vimentin using GAPDH as loading control for lysates. The intensities of the corresponding bands for fibronectin and vimentin were measured using ImageJ and normalized to loading controls, as depicted in Figure 4C. We observed a significant increase in fibronectin with CsA, which was ameliorated by approximately 37% with 5 ng/kg 2AMD. Albeit more variable, the trending increase in vimentin with CsA was reduced by approximately 50% with 2AMD compared with CsA rats treated only with vehicle.

Discussion

We tested 2AMD for renoprotective and antifibrotic activities in a CsA animal model of kidney disease. The effects of 2AMD at 4 different doses were compared to 2MD at 5 ng/kg in the CsA model, a concentration known to be effective in other measures of renal function.^19,20 Induction of nephrotoxicity was evident in our CsA model, as shown by histologic changes (including increased tubular atrophy and interstitial cellularity) and by significant rises in BUN, increased collagen, fibronectin and vimentin content, and decreased body weight.

Testing a range of 2AMD dose regimens allowed us to identify an optimal therapeutic dose and allowed important assessments of the possible effects of a range of relevant doses. We observed a direct correlation between 2AMD dosage and levels of serum calcium, indicating the potential for tight control of serum calcium with 2AMD administration. There was no change in serum calcium levels with 2MD or 2AMD at doses ≤ 5 ng/kg. We observed a significant decrease in BUN with 2AMD at 5 and 10 ng/kg and 2MD at 5 ng/kg. However, serum calcium levels increased and weight decreased in animals treated with the 10 ng/kg 2AMD plus CsA compared with vehicle plus CsA. In rats that received 20 ng/kg 2AMD, serum calcium and BUN levels were significantly increased and weight was significantly decreased, suggesting generalized toxicity. Toxicity was also evident by histopathology of hematoxylin and eosin-stained kidney sections, in which mesenchymal cell proliferation, infiltrating leukocytes, and tubular atrophy were observed (not shown), as expected for the CsA model.²³ These histologic pathologies were observed to a lesser degree in kidneys from CsA rats treated with 2AMD at 10 ng/kg. We observed improvement of tubular atrophy and reduced interstitial growth in sections from CsA rats treated with 2AMD or 2MD at 5 ng/kg.

The worsened nephrotoxic effects obtained with 10 or 20 ng/kg 2AMD may be due to the recognized ability of CsA (and tacrolimus but not rapamycin) to increase serum calcitriol levels.^31,32 This would suggest that, in the absence of CsA or tacrolimus, 2AMD or 2MD may have a wider therapeutic window. In previous studies,^19,20 2MD was shown to suppress PTH in uremic rats, postmenopausal women, and hemodialysis patients with SHPT. The latter finding was from a dosing trial in which patients received oral 2MD 3 times weekly for 4 weeks and the effective dose range was 330 to 770 ng/dose (or 131-330 ng/day), which is equivalent to 2 to 4.7 ng/kg, assuming patient weight of 70 kg. This is thus consistent with our findings that 5 ng/kg is a safe and effective dose for 2AMD or 2MD in the CsA and other models associated with kidney disease.

It is well recognized that vitamin D, in addition to its central role in the regulation of calcium and the musculoskeletal system, exerts a wide range of pleiotropic protective effects in multiple systems, including immunity, autoimmunity, cardiovascular disease, and cancer.6,33 Although there are many different causative pathologies, both kidney disease and eventual graft failure are associated with inflammation and fibrosis, the latter characterized by excess connective tissue deposition.2,34,35 Chronic injury leads to scarring with excessive myofibroblast proliferation and consequent production of ECM proteins, particularly collagens.

In this study, we noted strong antifibrotic effects by 2AMD and 2MD. All doses of 2AMD tested (2.5-20 ng/kg) and 2MD tested at 5 ng/kg decreased collagen by about 50%. The antifibrotic effect of 2AMD at 5 ng/kg was corroborated by Western blotting, which showed a decrease in the fibrotic markers fibronectin and vimentin in extracted kidneys. That the highest dose of 2AMD (20 ng/kg) promoted toxicity while also decreasing fibrosis may be due to higher dysregulation of ion transporter receptors in renal epithelial cells compared with lower susceptibility to calcium regulation in interstitial cells responsible for the production of collagen.³⁶

In a small pilot study using a unilateral ureteral obstruction model, a harsher kidney disease model than the CsA model with frank fibrosis at day 14, oral 2AMD significantly decreased collagen (by 60%) in obstructed kidneys, even at 2.5 ng/kg (not shown). Thus, the antifibrotic activities of 2AMD extend to a wide dose range in the CsA model, to chronic and acute models of kidney disease, and 2 venues of administration.

Thus, vitamin D and its non-calcemic analogs are potent antifibrotic agents. There is ample mechanistic understanding of the antifibrotic actions of vitamin D, including the following: (1) suppression of the renin-angiotensin and nuclear factor κB systems,8,37,38 (2) increased expression of hepatocyte growth factor,39 (3) direct repression of collagen I expression,40 and (4) interference with transforming growth factor-beta-promoted activation of fibrogenic genes by direct dislodging by activated vitamin D receptor complexes of SMAD3 binding to its DNA-binding elements.^41,42

We postulate that the clear decrease in fibrosis elicited by 2AMD and 2MD will lead to frank improvement in renal function with extended therapy. Based on our results and the literature described here, further investigations of vitamin D and its noncalcemic analogs as potential therapeutics in the small molecule tool box of antifibrotics43 are warranted, with future conclusive testing in randomized clinical trials for the prevention and treatment of fibrosis associated with graft failure in transplanted kidneys.

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