Increased Hyaluronic Acid in the Inner Renal Medulla of Obese Dogs
Abstract Dogs placed on a high-fat diet develop obesity and hypertension associated with marked sodium retention that is due to increased tubular reabsorption. Previous studies showed that renal interstitial hydrostatic pressure is elevated in obese dogs compared with lean dogs, and histological studies revealed increases in medullary interstitial cells and expansion of the medullary but not the cortical extracellular matrix. This matrix stained intensively with Alcian Blue at pH 2.6, colloidal iron, and periodic acid–Schiff, suggesting increased glycosaminoglycans. The goal of this study was to quantitate medullary glycosaminoglycan content in obese (n=8) compared with lean (n=8) dogs. Measurement of total glycosaminoglycan content, estimated from uronic acid content, and of hyaluronate, the most abundant glycosaminoglycan in canine renal medulla, with an enzyme-linked immunosorbent assay indicated that there were no significant differences in total glycosaminoglycan or hyaluronate contents in the outer medulla of obese dogs compared with those in lean dogs. In contrast, in the inner medulla of obese dogs there was a 140% increase in hyaluronate compared with the content in lean dogs (4.3±0.5 versus 1.8±0.2 mg hyaluronate per gram wet tissue, respectively; P<.05); however, total glycosaminoglycan content was not significantly different (6.9±0.7 versus 6.2±0.5 mg uronic acid per gram wet tissue) in obese and lean dogs. These results suggest a change in the relative proportion of the glycosaminoglycan species in the inner medulla of obese dogs, with a selective increase in hyaluronate. These changes in extracellular matrix could contribute to the altered intrarenal physical forces, tubular compression, and increased tubular reabsorption observed in obese dogs.
- extracellular matrix
- hypertension, chronic
- hypertension, sodium-dependent
- water-electrolyte balance
Since the early 1920s, in several epidemiological studies a positive correlation has been found between body weight and arterial blood pressure.1 2 3 4 5 Weight gain elevates blood pressure in humans as well as in experimental animals, and weight loss produces a corresponding decrease in blood pressure.6 7 8 9 10 11 12 Weight gain also appears to be a major cause of increased blood pressure in many patients with essential hypertension.3 Although numerous mechanisms have been postulated to explain obesity-induced hypertension, most of our knowledge is based on correlations between body weight and various factors thought to cause hypertension.
Studies by Rocchini and colleagues6 7 and studies in our laboratory8 9 have demonstrated that dogs made obese by consumption of a high-fat diet develop many of the characteristics of obese hypertensive humans. For example, obese dogs develop insulin resistance and hyperinsulinemia, hypertension, and altered renal function, including marked sodium and water retention, which appear to be due primarily to increased tubular reabsorption.8 9 Nevertheless, the mechanisms responsible for the increased sodium reabsorption and hypertension in obesity have not been elucidated.
We recently found that in obese dogs there are striking changes in renal medullary histology that may contribute to altered renal function.13 14 The inner medulla, and to a lesser extent the outer medulla, of obese dogs showed marked increases in vacuolated interstitial cells and increased extracellular matrix material deposition between tubules. The cytoplasm of the medullary interstitial cells and the expanded acellular interstitium stained intensively with periodic acid–Schiff, suggesting the presence of glycoproteins. The extracellular material also stained with Alcian Blue, supporting the presence of glycosaminoglycans. This additional extracellular matrix material appeared to be causing distortion or compression of the renal tubules and vasa recta. Indeed, we have functional evidence for compression of the kidney; renal interstitial fluid hydrostatic pressure was markedly elevated in kidneys from obese dogs, averaging 19 mm Hg compared with only 9 mm Hg in lean dogs.14 Because the kidney is surrounded by a tight capsule, cell proliferation or matrix deposition between tubules could raise renal interstitial fluid hydrostatic pressure and solid tissue pressures, causing tubular compression. Tubular and vascular compression, especially in the very distensible thin loop of Henle and vasa recta, could reduce loop of Henle flow rate and medullary blood flow, thus increasing tubular reabsorption.
These preliminary findings suggest an important link between altered renal medullary histology and abnormal sodium handling by the kidney in obesity-induced hypertension, but the stimulus that leads to increased matrix material and increased cellularity in the renal medulla is still unclear. As the first step toward identifying the biochemical changes responsible for altered renal medullary histology associated with obesity, the present study was designed to determine whether total glycosaminoglycan content, as estimated by uronic acid content, was elevated in the renal medulla of obese dogs compared with that in lean dogs. Because hyaluronic acid is the most abundant glycosaminoglycan in canine renal medulla,15 we also measured total hyaluronate content in the renal medulla of obese and lean dogs.
The experimental protocols were reviewed and approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee and were carried out according to the guidelines for the care and use of laboratory animals of the National Institutes of Health and the guidelines of the Animal Welfare Act. We used conditioned mongrel dogs that were housed in individual metabolic cages and instrumented for continuous monitoring of systemic hemodynamics and renal function. Total sodium intake was fixed in both groups at approximately 75 mmol/L sodium per day by giving the dogs two cans of a low-sodium food (H/D, Hill’s Pet Nutrition, Inc) that provided 7 mmol/L sodium and 65 mmol/L potassium per day, plus an intravenous infusion of sterile isotonic saline that provided the rest of the sodium. Some dogs were made obese by the addition of 1 to 2 lb cooked beef fat to their regular daily diet for at least 5 weeks. Control lean dogs were fed the same standard kennel ration but were not given the beef fat supplement.
Tissue Sample Preparation
Kidneys from eight lean and eight obese dogs were perfused in situ with 300 mL of 0.1 mol/L phosphate-buffered saline (PBS). Tissue samples from the outer and inner medullas were immediately dissected on ice, placed in plastic cryovials, frozen in liquid nitrogen, and stored at −80°C until use. Nonextracted outer and inner medulla samples were analyzed for total uronic and hyaluronic acid content. In addition, we used a standard procedure to solubilize proteoglycans.16 17 Tissue samples from the outer and inner medullas were separated into three fractions: (1) a soluble fraction containing macromolecules soluble in 85 mmol/L PBS, (2) a dissociative fraction containing macromolecules disaggregated by 4 mol/L guanidine hydrochloride, and (3) an insoluble fraction. Tissue samples (250 mg) were finely minced at 4°C and mixed with 5 mL of a solution containing (mmol/L) NaCl 85, KCl 1.33, Na2HPO4 10, KH2PO4 1.7, sodium azide 2.5, and protease inhibitors at final concentrations of EDTA 20, benzamidine 50, ε-amino-n-caproic acid 100, N-ethylmaleimide 0.5, and phenylmethylsulfonyl fluoride (PMSF) 1, pH 7.0. Tissue samples were gently shaken for 24 hours at 4°C and then centrifuged for 20 minutes at 2500 rpm. The supernatant was the soluble fraction. The pellet was then resuspended in 5 mL of a solution containing 4 mol/L guanidine hydrochloride and a mixture of detergents and protease inhibitors at a final concentration of 4% (wt/vol) CHAPS, 1% (vol/vol) Triton X-100, and (mmol/L) N-ethylmaleimide 1, benzamidine 5, ε-amino-n-caproic acid 200, EDTA 1, PMSF 0.1, and sodium azide 2.5. The samples were gently mixed for 24 hours at 4°C and then centrifuged for 20 minutes at 2500 rpm. The supernatant was the dissociative fraction and the pellet was the insoluble fraction.
Nonextracted and insoluble tissue samples were mechanically homogenized for 4 minutes at 4°C in 20 mmol/L sodium phosphate buffer, pH 6.8. One-milliliter aliquots of the homogenates, including the soluble and dissociative supernatants, were digested with 15 μL papain (Sigma) for 3 to 5 hours at 60°C and centrifuged for 3 minutes at 7000g, and the supernatant was used for glycosaminoglycan precipitation with 5 mg cetyltrimethylammonium bromide (CTAB; Sigma). The precipitate was collected by centrifugation and then agitated vigorously in saturated ethanolic potassium thiocyanate solution to dissociate the CTAB-glycosaminoglycan complex. The precipitate was then washed twice with 95% ethanol and dissolved in 1 mL distilled water for uronic acid measurement.17 Papain-digested samples were used for hyaluronic acid measurement after papain was denatured by being heated at 100°C for 5 minutes.
Total Glycosaminoglycan Analysis
Total uronic acid was measured by the method of Bitter and Muir.18 This method allows estimation of d-glucuronic acid found in chondroitin sulfate and hyaluronic acid, and also of the C-5 epimer l-iduronic acid found in dermatan sulfate and heparin. d-Glucuronic acid was used as a standard. The coefficient of variability for the standard curve was 2.4%.
We used an enzyme-linked immunosorbent assay (ELISA), originally described by Goldberg,19 for hyaluronate. Standards and samples were analyzed in quadruplicate. Grade I hyaluronic acid (ICN Biochemicals) was used as a standard. In a preincubation step carried out in non–hyaluronate-coated plates, 15 μL bovine nasal cartilage proteoglycan solution (0.04 mg/mL; ICN Biochemicals) was added to each well containing 150 μL standard or sample; 30 μL of a mixture containing 1 mol/L PBS, 5% bovine serum albumin (BSA; Sigma), 0.05% Tween, and 0.05% sodium azide was then added. Total volume was adjusted to 300 μL by the addition of 5% BSA in 0.1 mol/L PBS. After 18 hours of incubation at 4°C, 200 μL of sample was transferred to hyaluronate-coated wells (grade III hyaluronic acid; ICN Biochemicals) and incubated for 24 hours at 4°C. After the plates were washed with 0.05% Tween-PBS buffer, 200 μL monoclonal antibody to keratan sulfate (1:2000; ICN Biochemicals) was added to each well. The plates were then incubated for 1 hour at 37°C and washed again, and 200 μL peroxidase-conjugated anti-mouse IgG antibody (1:500; Bio-Rad) was then added. After 1 hour of incubation at 37°C, the plates were washed and incubated for 5 minutes with 200 μL freshly prepared substrate containing 0.2 mmol/L tetramethylbenzidine dihydrochloride (Pierce), 6.5% dimethyl sulfoxide (Sigma), and 0.03% hydrogen peroxide (Sigma) in 0.2 mmol/L sodium acetate, pH 5.0. Color development was stopped after 5 minutes by addition of 20 μL 25% sulfuric acid solution. The plates were read at 450 nm with a 96-well microtiter plate reader (Tecan US Inc). Data were fitted to a logistic curve. The coefficient of variability for the standard curve was between 5% and 15%, and for the samples it was less than 10%. The sensitivity of the assay was 10 ng/mL.
Results are expressed as mean±SEM. Statistically significant differences (P<.05) between tissue samples from kidneys of lean and obese dogs were determined by Student’s t test for unpaired data.
Uronic Acid Content
Results for glycosaminoglycan content are shown in Fig 1⇓. Glycosaminoglycans from either the outer or inner medulla of lean and obese dogs were poorly extracted in the soluble fraction, accounting for only 8% of the total glycosaminoglycan content. Similarly, only 5% of the total glycosaminoglycans were disaggregated, indicating that guanidine hydrochloride, a chaotropic agent, was unable to dissociate glycosaminoglycans from canine renal medulla. The majority of the glycosaminoglycans were left in the insoluble fraction, because they were released only after mechanical homogenization and digestion with papain.
Uronic acid content in the insoluble outer medulla fraction was not significantly different in lean and obese dogs (0.86±0.21 versus 1.01±0.17 mg/g wet tissue, respectively). Similar results were found for total uronic acid from nonextracted outer medulla samples (2.85±0.35 versus 2.71±0.28 mg/g wet tissue). In contrast, uronic acid content in the insoluble inner medulla fractions of obese dogs was elevated by 67% compared with that in lean dogs (4.73±0.63 versus 2.84±0.31 mg/g wet tissue, P<.05). However, the total uronic acid content of nonextracted inner medulla was not significantly different in lean and obese dogs (5.67±0.45 versus 6.87±0.70 mg/g wet tissue). In addition, the inner medulla of both lean and obese dogs had approximately twice as much total uronic acid as the outer medulla.
Results for hyaluronate content are shown in Fig 2⇓. Hyaluronate from either the outer or inner medulla of lean and obese dogs was poorly extracted in the soluble fraction, accounting for only 7% of the total hyaluronate. Similarly, only 5% of the total hyaluronate was disaggregated by the chaotropic agent. Most of the hyaluronate was left in the insoluble fraction. Hyaluronate in the insoluble outer medulla fraction was not significantly different in lean and obese dogs (0.53±0.16 versus 0.48±0.11 mg/g wet tissue, respectively). Similar results were found for total hyaluronate from nonextracted outer medulla of lean and obese dogs (0.74±0.09 versus 0.77±0.08 mg/g wet tissue). In contrast, hyaluronate content in the insoluble inner medulla fraction of obese dogs was elevated by 140% compared with that in lean dogs (3.22±0.62 versus 1.24±0.07 mg/g wet tissue, P<.05). Total hyaluronate from nonextracted inner medulla was also elevated in obese dogs by 160% compared with that in lean dogs (4.27±0.52 versus 1.85±0.21 mg/g wet tissue, P<.05). In addition, the inner medulla of lean dogs had twice as much hyaluronate as the outer medulla, and the inner medulla of obese dogs had almost five times more hyaluronate than the outer medulla.
The most important findings of the present study are that (1) total glycosaminoglycan content is elevated in the insoluble inner medulla fraction of obese dogs compared with that in lean dogs and (2) hyaluronate appears to be an important glycosaminoglycan involved in the altered extracellular matrix found in the inner medulla of obese dogs.
Previous theoretical and experimental studies have suggested that in all forms of hypertension, there is an abnormality of renal sodium handling characterized by a shift of pressure natriuresis.20 21 Our previous studies8 9 have shown that obesity in dogs leads to hypertension characterized by increased tubular reabsorption and a shift of pressure natriuresis. Increased tubular reabsorption appears to occur at a site beyond the proximal tubule, because fractional lithium reabsorption, a marker for proximal tubular sodium reabsorption, was not different in lean compared with obese dogs. The cause of this shift of pressure natriuresis in obesity is not clear, but it could be the result of abnormal intrarenal changes as well as neurohumoral mechanisms that influence kidney function.
One mechanism by which obesity could lead to increased tubular reabsorption is through physical changes in the kidney. Preliminary evidence from our laboratory showed that obesity is associated with remarkable intrarenal histological changes that may lead to tubular compression, primarily in the renal medulla.13 14 For instance, the renal medullary histological changes included marked increases in vacuolated interstitial cells with abundant rough endoplasmic reticulum and marked increases in extracellular matrix deposition between renal tubules. This matrix material appeared to be distorting or compressing adjacent tubules and vasa recta. Because the kidney is surrounded by a tight capsule with a low compliance, cell proliferation or extracellular matrix deposition between tubules in the renal medulla could raise renal interstitial hydrostatic pressure as well as solid tissue pressure. The increase in total tissue pressure would tend to cause tubular and vascular compression. Indeed, functional evidence of compression of the kidney has been obtained from measurements of renal interstitial hydrostatic pressure made by use of implanted polyethylene matrix capsules.22 In one study, renal interstitial hydrostatic pressure averaged 19.0±2.4 mm Hg in kidneys from obese dogs, compared with 9.6±2.2 mm Hg in kidneys from lean dogs.13 Because tubular pressure in the thin loop of Henle is normally about 8 to 12 mm Hg, this large interstitial fluid pressure in obese dogs could tend to cause tubular compression, especially in the thin loop of Henle, which is very distensible.23 Compression of the thin loops of Henle and vasa recta in the renal medulla would tend to slow down loop of Henle flow rate and medullary blood flow, thus explaining in part the increased tubular reabsorption observed in obese dogs. Increased arterial pressure would help to maintain patency of the tubules and allow relatively normal urine output despite a tendency toward compression of the tubules. Although this hypothesis must be considered speculative until direct measurements of tubular pressures are made, it is almost certain that loop of Henle hydrostatic pressure must be increased to at least 19 mm Hg in obese dogs to keep the loops of Henle patent and allow urine to flow through the tubules.
Although small increases in renal interstitial hydrostatic pressure tend to decrease tubular reabsorption,24 25 large increases in interstitial pressure may increase it. Burnett and Knox24 reported that in volume-expanded dogs, elevations in interstitial hydrostatic pressure to 20 to 40 mm Hg were associated with marked increases in sodium reabsorption. Micropuncture studies have localized this response to the loop of Henle; increased renal venous pressure in volume-expanded rats was associated with increased reabsorption in the loop of Henle and marked elevation of distal tubular pressure and prolongation of loop transit time.26
It is possible that elevated renal interstitial hydrostatic pressure results from external compression of the renal pelvis, including the renal vein and ureter, by increased abdominal fat deposition. In this case, the increased cellularity and extracellular matrix deposition in the renal medulla could be a compensatory response to increased intratubular and intravascular pressures within the kidney to offset the increased renal interstitial hydrostatic pressure. Further studies are needed to test this hypothesis.
The exact cause of increased renal interstitial hydrostatic pressure in obesity and its contribution to hypertension and alterations in tubular function remain to be established. However, increased matrix deposition may play an important role in the functional changes observed in the kidneys of obese dogs. As the first step toward identifying the biochemical changes responsible for altered renal medullary histology associated with obesity, we measured uronic acid as an estimate of total glycosaminoglycan content in outer and inner medulla samples. Our uronic acid data showed no major changes in total glycosaminoglycan content in the nonextracted outer medulla or the insoluble outer medulla fractions of obese dogs compared with the content in lean dogs. There was no significant difference between lean and obese dogs in total glycosaminoglycan content in nonextracted inner medulla, although a tendency toward higher values was observed in obese dogs. In contrast, the insoluble inner medulla fraction of obese dogs showed a 67% increase in uronic acid content compared with that in lean dogs. These results suggest a change in the relative proportion of the glycosaminoglycan species in the inner medulla of obese dogs compared with that in lean dogs.
Previous studies by Castor and Greene15 have shown that hyaluronate is the major glycosaminoglycan found in canine renal medulla. Therefore, we measured hyaluronate content in the renal medulla of obese and lean dogs using a specific assay. Our hyaluronate data confirmed our results from measurements of uronic acid content. There were no major changes in the amount of hyaluronate in the outer medulla of obese dogs compared with that in lean dogs. Total hyaluronate in the insoluble inner medulla fraction of obese dogs was signficantly elevated by 140% compared with that in lean dogs. In addition, there was a 160% increase in the amount of total hyaluronate in nonextracted inner medulla of obese dogs compared with that in lean dogs. Therefore, hyaluronate appears to be a major macromolecule involved in the altered extracellular matrix deposition observed in the inner medulla of obese dogs. However, there may be other glycosaminoglycans or other proteins that contribute to the altered matrix deposition. Further analysis of the extracellular matrix components of the medulla of obese dogs is needed.
In summary, histological and biochemical changes in the renal medulla of obese dogs may play an important role in the etiology of obesity-induced hypertension. We have previously shown that renal interstitial hydrostatic pressure is markedly elevated in obese dogs compared with lean dogs.8 13 In addition, the results of the present study demonstrated that there is a change in the relative proportion of the glycosaminoglycan species in the renal medulla of obese dogs compared with that in lean dogs. Hyaluronate appears to be one of the major macromolecules involved in the altered extracellular matrix deposition observed primarily in the inner medulla. This change in the extracellular matrix could contribute to compression of the inner medulla and lead to increased tubular reabsorption in obesity.
This work was supported by National Institutes of Health grants HL-51971, HL-39399, and HL-23502.
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