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The Role of the Renin-Angiotensin System in Cholesterol and Puromycin Mediated Renal Injury

      ABSTRACT

      Background

      Puromycin aminonucleoside (PAN) nephropathy is a widely studied model of glomerular sclerosis (GS) in the rat, and cholesterol feeding exacerbates the injury induced by PAN. The importance of the interaction of angiotensin II (Ang II) with the AT2 receptor is unclear. We investigated the role of the renin-angiotensin system, particularly with regard to AT1 and AT2 receptor dynamics, in PAN and cholesterol-mediated GS.

      Methods

      Sprague-Dawley rats were given a 4% cholesterol diet (group II), subcutaneous PAN (group III), or a 4% cholesterol diet and PAN (group IV) and compared with a control group given PAN vehicle (group I). After 16 weeks, kidneys were harvested and tissue Ang II concentration, angiotensin-converting enzyme (ACE) activity, and ACE, AT1, and AT2 mRNA levels were determined.

      Results

      Compared with control rats, proteinuria was significantly higher in groups II to IV. Kidney ACE activity and ACE mRNA levels in groups III and IV were 2- and 3-fold higher than in groups I and II, respectively. Kidney Ang II concentration also was increased in the experimental groups. Whereas kidney AT1 mRNA was significantly lower in groups III and IV, kidney AT2 mRNA was significantly increased in groups II to IV.

      Conclusion

      In these experimental models of GS, there is significant activation of the tissue-based renin-angiotensin system. Puromycin with and without cholesterol decreased the AT1 receptor mRNA and increased the AT2 receptor mRNA. Up-regulation of AT2 receptors may be important in ameliorating the proliferative effects of Ang II, which presumably occur through the AT1 receptor.

      KEY INDEXING TERMS

      Puromycin aminonucleoside (PAN) nephropathy is a widely studied model of glomerular disease and has been considered analogous to the human disease of focal segmental glomerulosclerosis (GS).
      • Glasser R.J.
      • Velosa J.A.
      • Michael A.F.
      Experimental model of focal sclerosis. I Relationship to protein excretion in aminonucleoside nephrosis.
      • Chandra M.
      • Susin M.
      • Teichberg S.
      • et al.
      Experimental focal segmental glomerulosclerosis: correlation with protein excretion, glomerular filtration rate, and renal plasma flow.
      • Grond J.
      • Weening J.J.
      • Elemma J.D.
      Glomerular sclerosis in nephrotic rats: comparison of the long-term effects of adriamycin and aminonucleoside.
      A number of factors are pivotal in the progression of the disease, including altered renal hemodynamics, intrarenal generation of angiotensin II (Ang II), and/or lipid deposition, among other factors.
      • Trachtman H.
      • Schwob N.
      • Maesaka J.
      • et al.
      Dietary vitamin E supplementation ameliorates renal injury in chronic puromycin aminonucleoside nephropathy.
      • Olson J.L.
      • Hostetter T.H.
      • Rennke H.G.
      • et al.
      Altered glomerular permselectivity and progressive sclerosis following extreme ablation of renal mass.
      Ang II activity is increased in PAN nephropathy, and it may play a permissive, if not direct, role in the pathologic response.
      • Kashgarian M.
      Mesangium and glomerular disease.
      Consistent with this premise, cultured adult rat glomerular mesangial cells hypertrophy in the presence of Ang II,
      • Brenner B.M.
      • Meyer T.W.
      • Hostetter T.H.
      Dietary protein intake and the progressive nature of kidney disease: the role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation, and intrinsic renal disease.
      and Ang II induces cellular proliferation in human fetal mesangial cells.
      • Hostetter T.H.
      • Olson J.L.
      • Rennke H.G.
      • et al.
      Hyperfiltration in remnant nephrons: a potentially adverse response to renal ablation.
      Ang II also promotes macromolecule deposition in mesangial cells
      • Kiprov D.D.
      • Colvin R.B.
      • McCluskey R.T.
      Focal and segmental glomerulosclerosis and proteinuria associated with unilateral renal agenesis.
      and in vivo glomerular matrix protein synthesis in remnant kidney models of GS.
      • Couser W.G.
      • Stilmant M.M.
      Mesangial lesions and focal glomerular sclerosis in the aging rat.
      Rats developing GS after multiple subcutaneous injections of PAN also display widespread lipid deposition consisting primarily of cholesterol and cholesteryl esters.
      • Grond J.
      • Weening J.J.
      • Elemma J.D.
      Glomerular sclerosis in nephrotic rats: comparison of the long-term effects of adriamycin and aminonucleoside.
      The PAN model of GS is mimicked by cholesterol feeding in a number of animal species.
      • Kreisberg J.I.
      • Karnovsky M.J.
      Focal glomerular sclerosis in the fawn-hooded rat.
      • Fogo A.
      • Yoshida Y.
      • Glick A.D.
      • et al.
      Serial micropuncture analysis of glomerular function in two rat models of glomerular sclerosis.
      For example, guinea pigs fed normal diets demonstrate histologically normal glomeruli, tubules, and vessels.
      • Kreisberg J.I.
      • Karnovsky M.J.
      Focal glomerular sclerosis in the fawn-hooded rat.
      When provided a cholesterol supplemented diet for 30 days, these animals demonstrate an increase in mesangial matrix with mesangial hypercellularity, a finding that becomes more pronounced by day 70 of cholesterol feeding. Furthermore, Diamond and Karnovsky
      • Diamond J.R.
      • Karnovsky M.J.
      Exacerbation of chronic aminonucleoside nephrosis by dietary cholesterol supplementation.
      showed that a diet of 4% cholesterol with 1% cholic acid causes GS and exacerbates PAN-mediated sclerosis in Sprague-Dawley rats. Cholesterol-fed nephrotic animals have more profound morphologic changes, including a greater prevalence of mesangial foam cells within glomeruli and a higher percentage of GS lesions.
      • Fogo A.
      • Yoshida Y.
      • Glick A.D.
      • et al.
      Serial micropuncture analysis of glomerular function in two rat models of glomerular sclerosis.
      Although a high-cholesterol diet can stimulate increased renal matrix protein synthesis, which progresses to GS in the rat kidney,
      • Yoshida Y.
      • Fogo A.
      • Ichikawa I.
      Glomerular hemodynamic changes vs. hypertrophy in experimental glomerular sclerosis.
      the hypothesis that Ang II participates in cholesterol-mediated GS has not been directly tested.
      The growth-promoting effect of Ang II is mediated via the AT1 receptor, whereas the action of Ang II on the AT2 receptor mediates growth retardation.
      • Diamond J.R.
      • Anderson S.
      Irreversible tubulointerstitial damage associated with chronic aminonucleoside nephrosis. Amelioration by angiotensin I converting enzyme inhibition.
      Because matrix expansion is a predominant phenomenon in GS, the purpose of the present study was to investigate the role of the renin-angiotensin system (RAS), particularly in relationship to AT1 and AT2 receptor dynamics, in PAN- and cholesterol-mediated GS.

      Methods

      Animals

      Male Sprague-Dawley rats (90–100 g) were purchased from Harlan (Indianapolis, IN) and housed in the Medical College of Virginia animal facility. The animals were maintained at 25°C with a 12-hour/12-hour light/dark cycle. They were provided free access to water and standard rodent laboratory chow or a 4% cholesterol and 1% cholic acid diet (Harlan Teklad). All procedures were approved by the animal research committee (IAUCC) of Virginia Commonwealth University.

      Induction of GS by PAN

      GS was induced by the method of Grond et al.
      • Grond J.
      • Weening J.J.
      • Elemma J.D.
      Glomerular sclerosis in nephrotic rats: comparison of the long-term effects of adriamycin and aminonucleoside.
      In brief, rats were injected subcutaneously with PAN (2 mg/100 g) for a period of 16 weeks. PAN was given weekly for the first 3 weeks and then on alternate weeks for the remaining 13 weeks. The high cholesterol-fed animals received a diet that contained 4% cholesterol and 1% cholic acid (Harlan Teklad). The following 4 groups of rats were studied: group I (n=7), normal diet; group II (n=7), cholesterol diet; group III (n=7), normal diet+PAN, group IV (n=5), cholesterol diet+PAN. Rats were weighed weekly and awake blood pressure was measured monthly by the tail cuff method with the use of a photoelectric sensor and pulse amplifier (IITC Life Science, Woodlands Hill, CA) connected to a 2-channel recorder.
      • Homma T.
      • Akai Y.
      • Burns K.D.
      • et al.
      Activation of S6 kinase activity by repeated cycles of stretching and relaxation in cultured rat glomerular mesangial cells. Evidence for involvement of protein kinase C.
      Animals were placed in individual metabolic cages monthly to determine urinary protein excretion. Rats were provided free access to water but not food while they were in the metabolic cages. At the end of 16 weeks, animals were killed and trunk blood was collected for the measurement of serum angiotensin-converting enzyme (ACE) and plasma Ang II. Kidneys were removed and immediately processed for subsequent RNA extraction and the later determination of Ang II and ACE activity.

      Determination of Cholesterol

      Cholesterol was measured spectrophotometrically using Sigma Diagnostic Kit (Sigma, St. Louis, MO). Manufacturer’s instructions were followed to determine serum cholesterol of the rats.

      Extraction of ACE from Kidney

      Membrane-bound kidney ACE was extracted using 0.5% Nonidet P-40.
      • Ray P.E.
      • Bruggeman L.A.
      • Horikoshi S.
      • et al.
      Angiotensin II stimulates human mesangial cell proliferation and fibronectin biosynthesis by binding to AT1receptors.
      Tissues were homogenized with a Polytron homogenizer for 10 seconds in a buffer solution containing 0.1 mol/L Tris-HCl, pH 7.8, 30 mmol/L KCl, 5 mmol/L MgCl2, 0.25 mol/L sucrose, and 0.5% Nonidet P-40. Homogenates were centrifuged at 20,000 g for 20 minutes at 4°C. Supernatants were stored at −70°C until ACE activity or total protein measurements were performed.

      Determination of ACE in Serum and Kidney

      ACE activity was measured by a previously validated radioenzymatic assay (ALPCO, Windham, NH). In brief, serum or tissue was extracted and subsequently incubated with the substrate [3H]hippurylglycylglycine. After incubation for 60 minutes at 37°C, the reaction was stopped by the addition of 1 N HCl. The product, [3H]hippuric acid, was separated from unreacted substrate by extraction with scintillation cocktail, and measured in a beta counter. The sensitivities of the assay for serum and kidney were 122 pmol/mL/min and 0.25 nmol/min/mg of protein, respectively. The inter- and intra-assay coefficients of variation for ACE were 4.6 and 5.5%, respectively (n=10).

      Determination of Ang II in Plasma and Kidney

      At the time of death, trunk blood was obtained from the aorta and placed in prechilled tubes containing 10 mmol/L Na2EDTA and 100 μmol/L bestatin. The plasma was separated by centrifugation at 4°C and stored at −70°C. Kidneys were homogenized in prechilled methanol within 30 sec in a glass tissue grinder. The homogenate was centrifuged at 4°C for 30 minutes at 1200 rpm and the supernatant was dried in a vortex evaporator (HBI, Lenexa, KS). The dried residue was reconstituted with 50 mmol/L phosphate buffer, pH 7.4. Plasma and kidneys were processed for radioimmunoassay according to the published procedure of ALPCO. In brief, the samples were extracted on a phenylsilylsilica column with methanol and the eluant was dried and processed for radioimmunoassay using a specific antibody to Ang II (ALPCO). The sensitivities for the assay were 0.8 fmol/mL and 20 fmol/g for plasma and kidney protein, respectively. The inter- and intra-assay coefficients of variation for Ang II were 8.6 and 10.5% for plasma and kidney tissue, respectively (n=10).

      Preparation of Total RNA

      A coronal section of kidney was homogenized in 7.5 mL of guanidine isothiocyanate solution (4 mol/L guanidine isothiocyanate, 10 mmol/L Tris-HCl, pH 7.4, and 7% β-mercaptoethanol), and N-lauroyl sarcosine was added to a final concentration of 2%. This homogenate was passed through a 23-gauge needle and layered with 5.7 mol/L cesium chloride containing 10 mol/L EDTA, pH 7.4. The total RNA pellet obtained after centrifugation at 100,000 g for 16 hours was washed twice with 100% ethanol, dissolved in diethyl pyrocarbonate-treated water, precipitated with 2 volumes of 100% ethanol in the presence of 0.1 volume of 3 mol/L sodium acetate, pH 5.0, and stored at −20°C for overnight precipitation. Total RNA was pelleted by centrifugation at 12,000 g for 30 minutes, redissolved in diethyl pyrocarbonate-treated water, and quantified by measuring the absorbance at 260 nm.

      Northern Blot Analysis

      The vector containing the cDNA for ACE and the AT1 receptor were kindly provided by Dr. K. Bernstein (Emory University, Atlanta, GA). Total RNA (10 μg) was electrophoresed on a 1% agarose gel in the presence of formaldehyde. The RNA was stained with ethidium bromide, and the integrity of the isolated RNA was verified by the presence of 28S and 18S rRNA bands. The RNA was transferred to a GeneScreen membrane and hybridized with the 32P-labeled full-length cDNA probe for ACE and AT1 and a cyclophilin cDNA insert. The blots were washed under high stringencies [0.2 × standard saline citrate (SSC)+0.1% sodium dodecyl sulfate (SDS) at 65°C]. Positive hybridization was detected by exposure to Kodak XAR-2 films (Eastman Kodak, Rochester, NY) for 18 hours at −70°C. Radioactivity associated with each band was quantified using a personal densitometer from Amersham Biosciences (Piscataway, NJ). Values for kidney AT1 and ACE mRNA were normalized to the housekeeping gene cyclophilin.

      Quantitative Reverse Transciption-Polymerase Chain Reaction (RT-PCR)

      In Vitro Synthesis of AT2 RNA

      Plasmid containing full-length cDNA for human AT2 receptor was obtained from Dr. V. Dzau (Harvard Medical School, Boston, MA). Two primers were designed to generate an in vitro transcription template. The up-stream primer, AT2SenseLong, was 5′-GCAAAAACATTACCAGCGGTCTTC-3′ and the down-stream primer, AT2SP6, was 5′-ATTAGGTGACACTATAGAAGGAGAACTGCTTTTCCGGCA-AGAC-3′ (the underlined bases correspond to the SP6 promoter sequence). The PCR reaction contained 100 ng of plasmid DNA and 20 pmol of each primer. The following thermocycling conditions were used: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes, with a final extension of 10 minutes. The PCR product for the in vitro transcription template was purified using a PCR purification kit (QIAGEN, Valencia, CA), quantified, and stored at 4°C in small aliquots. The template (150–200 ng) was used for in vitro transcription using a MAXIscript kit (Ambion, Austin, TX) according to the manufacturer’s instructions. SP6 RNA polymerase was used to generate the control sense RNA with sequence identical to that of AT2 mRNA. This control sense RNA was quantified and stored in small aliquots at −20°C.

      Quantitation of AT2 mRNA in Total Kidney RNA by RT-PCR

      Human and rat AT2 cDNA sequences were compared and 2 primers were designed to amplify the 482 base-pair PCR product. The sequence of the downstream primer, AT2AntisenseShort, 5′-ACCACTGAGCATATTTCTCAGG-3′, was identical in both species. However, degeneracy at position 14 and 17 was introduced in the upstream primer, AT2Redun, 5′-CAGATAAGCATTTA/GGAA/TGCAATTCC-3′, so it could be used in RT-PCR, starting from the sense RNA generated above and total rat kidney RNA. The sense RNA was diluted serially and used for the first strand cDNA synthesis using the Preamplification kit from Invitrogen (Carlsbad, CA). The antisense primer used for first-strand cDNA synthesis was identical to AT2SP6 without the SP6 promoter sequence. One tenth of the first strand reaction was amplified using 20 pmol of AT2AntisenseShort and AT2Redun in a 100-mL reaction. The thermocycling conditions were as follows: denaturation at 94°C for 30 seconds, annealing at 55°C for 1 minute, and extension at 72°C for 2 minutes, with a final extension of 10 min in the 25th cycle. One tenth of the PCR reaction was separated on a 1.2% agarose gel, transferred to a GeneScreen membrane, and probed with 32P-labeled full-length AT2 cDNA. After 2 washes with 2× SSC containing 0.1% SDS at room temperature, and 1 stringent wash with 0.2× SSC containing 0.1% SDS, the blot was exposed to Kodak film for 18 hours. The autoradiogram was scanned using AlphaImager (Alpha Innotech Corporation, San Leandro, CA) and a standard curve was generated. Using this standard curve, levels of AT2 RNA were determined in total RNA from rat kidney. Total RNA (10 μg) was used for the first-strand cDNA synthesis and processed as described above. Two concentrations of control sense RNA in the linear range of the standard curve were processed along with all samples.

      Histology

      Five animals from each group were studied for histological changes in the kidney. The left kidney was cut longitudinally and immersion-fixed in 100% ethanol for light microscopy. The basic scoring system described by Diamond and Karnovsky
      • Diamond J.R.
      • Karnovsky M.J.
      Exacerbation of chronic aminonucleoside nephrosis by dietary cholesterol supplementation.
      was employed, in which the fraction of total glomeruli showing segmental sclerosis and/or hyalinosis, mesangial “foam” cells, and segmental mesangial proliferation was determined. Between 50 and 100 glomeruli were scored per animal by an observer blinded to the origin of the tissue. Sections were cut at 2-μm thickness and stained with periodic acid-Schiff.

      Statistics

      The results were expressed as mean ± SD. Analysis of variance was used to compare the differences between the groups, and the statistical significance was evaluated after Bonferroni correction.

      Results

      Proteinuria in Rats

      Proteinuria is a common finding in both experimental and human GS. As illustrated in Figure 1, group II (cholesterol), group III (puromycin), and group IV (cholesterol+puromycin) had significantly greater degrees of proteinuria (P < 0.05) than group I (control). The between-group increase in proteinuria ranked as follows: group IV > group III > group II and were all significantly (P < 0.05) increased compared with each other group and controls.
      Figure thumbnail gr1
      Figure 1Effect of puromycin administration and cholesterol feeding on proteinuria in rats. Protein excretion (mg/24 hours) at 16 weeks is expressed as mean ± SD. Compared with I (control), II (cholesterol), III (puromycin), and IV (cholesterol+puromycin) had significantly higher proteinuria (P < 0.05). The increase in proteinuria between the groups was statistically significant: IV > III > II > I (P < 0.05).

      Blood Pressure

      During the first 8 weeks after PAN, the mean arterial blood pressure (MAP) remained unchanged among groups. However, at 12 weeks, the MAP in the group IV animals was significantly higher than that of group I or II (P < 0.05) (Table 1). By 16 weeks, MAP groups III and IV was significantly higher than that in groups I and II. The group IV animals (cholesterol+puromycin) had a significantly higher MAP than group III animals.
      Table 1Effect of Cholesterol, Puromycin, and Combination of Cholesterol and Puromycin on Blood Pressure, Serum ACE Activity, and Plasma Ang II Concentration
      Group
      IIIIIIIV
      MAP (mm Hg)
      12 weeks125 ± 8127 ± 9.7133 ± 8.8142 ± 6.8
      P < 0.05 compared with group I.
      16 weeks124 ± 7128 ± 11140 ± 8
      P < 0.05 compared with group I.
      151 ± 5
      P < 0.05 compared with group I.
      Group IV significantly greater than groups II and III (P < 0.05).
      Serum ACE (nmol/mL/min)59.6 ± 12.753.8 ± 12.548.3 ± 10.952.15 ± 6.1
      Plasma Ang II (fmol/mL)59.4 ± 7.862.3 ± 1858.7 ± 1159.4 ± 15.8
      Cholesterol (mg/dL)110.6 ± 9942.2 ± 198.1
      P < 0.05 compared with group I.
      828 ± 137
      P < 0.05 compared with group I.
      2150 ± 587
      P < 0.05 compared with group I.
      Group IV significantly greater than groups II and III (P < 0.05).
      Ang II, angiotensin II; MAP, mean arterial pressure; ACE, angiotensin-converting enzyme.
      Values are mean ± SD.
      * P < 0.05 compared with group I.
      Group IV significantly greater than groups II and III (P < 0.05).

      Serum Cholesterol

      Compared with the control (group I) the serum cholesterol level of group II was significantly higher (P < 0.05). The animals receiving puromycin (group III) had 7-fold higher mean cholesterol than group I (P < 0.05) but were not significantly different from group II. Cholesterol concentration of group IV was 20-fold higher than that of group I (P < 0.05) and was also significantly higher than that of groups II and III (P < 0.05;Table 1).

      Serum ACE and Plasma Ang II

      Serum ACE activity and plasma Ang II concentration were not significantly different between groups (Table 1). The plasma Ang II concentration and values for serum ACE activity are comparable with those previously reported for normal Sprague-Dawley rats.
      • Schunkert H.
      • Ingelfinger J.R.
      • Hirsch A.T.
      • et al.
      Feedback regulation of angiotensin converting enzyme activity and mRNA levels by angiotensin II.
      • Fox J.
      • Guan S.
      • Hymel A.A.
      • et al.
      Dietary Na and ACE inhibition effects on renal tissue angiotensin I and II and ACE activity in rats.

      Kidney ACE Activity and mRNA

      In contrast to serum, there were significant differences in kidney ACE activity among groups (Figure 2). Kidney ACE activity of groups III and IV was 2- and 3-fold higher than that of groups I and II, respectively (P < 0.05). Kidney ACE activity in the group IV animals was significantly higher than that observed in group III (P < 0.05). Kidney ACE activity was not significantly different between groups I and II. The change in kidney ACE mRNA was similar to that observed in ACE activity (Figure 3). The differences in ACE mRNA between groups I and II were not statistically significant, whereas groups III and IV had significantly higher mRNA (P < 0.05) than groups I and II. As with enzyme activity, kidney ACE mRNA in group IV was significantly higher (P < 0.05) than that observed in group III.
      Figure thumbnail gr2
      Figure 2Effect of puromycin administration and cholesterol feeding on kidney ACE activity in rats. Kidney ACE activity is expressed as mean ± SD nmol/min/mg of protein. Kidney ACE activity of groups III (puromycin) and IV (cholesterol+puromycin) was significantly higher (P < 0.05) than that of groups I (control) and II (cholesterol). Group IV had significantly higher kidney ACE activity than group III (P < 0.05).
      Figure thumbnail gr3
      Figure 3Changes in kidney ACE mRNA after puromycin administration and cholesterol feeding. The kidney ACE mRNA is expressed as mean ± SD. The ACE mRNA of groups III (puromycin) and IV (puromycin+cholesterol) were significantly higher (P < 0.05) than that of I and II (control and cholesterol). Kidney ACE mRNA of group IV was significantly higher (P < 0.05) than that of group III.

      Kidney Ang II

      The average concentration of kidney Ang II in the control group was similar to published values for Sprague-Dawley rat kidney.
      • Schunkert H.
      • Ingelfinger J.R.
      • Hirsch A.T.
      • et al.
      Feedback regulation of angiotensin converting enzyme activity and mRNA levels by angiotensin II.
      Kidney Ang II concentrations were significantly different (P < 0.05) compared with each other group as well compared with controls (Figure 4)
      Figure thumbnail gr4
      Figure 4Effect of puromycin administration and cholesterol feeding on kidney Ang II concentration. Kidney Ang II concentration is expressed as mean ± SD. The concentration of Ang II was highest in group IV (cholesterol+puromycin) followed by group III (puromycin), group II (cholesterol), and group I (control). The differences between the groups (IV > III > II > I) were statistically significant (P < 0.05).

      Kidney AT1 mRNA

      Changes in AT1 mRNA are shown in Figure 5. The AT1 mRNA of the group I and II animals were not significantly different. However, the AT1 mRNA of groups III and IV were significantly lower than that of group I and II (P < 0.05). The difference between groups III and IV was not statistically significant.
      Figure thumbnail gr5
      Figure 5Effect of puromycin and cholesterol feeding on kidney AT1 mRNA. Kidney AT1 mRNA is expressed as mean ± SD. Groups III (puromycin) and IV (cholesterol+puromycin) had significantly higher kidney AT1 mRNA levels than groups I (control; P < 0.05) and II (cholesterol; P < 0.05). The difference between groups III and IV was not statistically significant.

      Kidney AT2 mRNA

      Because AT2 mRNA was undetectable by Northern blot, quantitative RT-PCR was used to measure AT2 mRNA. In group I, the AT2 mRNA was barely detectable; in fact, AT2 mRNA expression was absent in 3 of 4 animals. Hence, for comparison purposes, the values were considered zero, which explains the high SD observed in group I (Figure 6). The AT2 mRNAs in groups II to IV were significantly higher than that found in group I (P < 0.05). However, the differences between groups II, III, and IV did not reach statistical significance.
      Figure thumbnail gr6
      Figure 6Effect of puromycin and cholesterol feeding on kidney AT2 mRNA. Kidney AT2 mRNA is expressed as mean ± SD. Groups II (cholesterol), III (puromycin), and IV (cholesterol+puromycin) had significantly higher kidney AT1 mRNA than group I (control; P < 0.05). The differences between groups II, III, and IV were not statistically significant.

      Histology

      Histological results are summarized in Table 2. Control animals (group I) had healthy glomeruli (Fig 7A). Animals fed cholesterol (group II) developed segmental sclerosis (Fig 7B) and mesangial foam cells. These abnormalities were more pronounced in animals receiving PAN (group III) and even more so in those animals receiving PAN and a high-cholesterol diet (Group IV) (Figure 7C). Mesangial cell hypercellularity was present only in glomeruli of group III and IV animals and was infrequent, occurring in <4% of group III and IV glomeruli.
      Table 2Summary of Morphologic Abnormalities Observed in the Kidney after Treatment with Cholesterol (II), Puromycin (III), and Puromycin+Cholesterol (IV).
      Group
      IIIIIIV
      % Glomeruli with segmental sclerosis/hyalnosis10.5 ± 2.720.6 ± 5.6
      P < 0.05
      33.2 ± 3.3
      P < 0.05
      % Glomeruli with foam cells4.5 ± 0.513.4 ± 2.4
      P < 0.05
      25.4 ± 6.5
      P < 0.05
      The control group showed no foam cell formation or sclerosis/hylanosis.
      IV > III > II
      Values are mean ± SD.
      * P < 0.05
      Figure thumbnail gr7
      Figure 7Representative glomeruli from a normal rat (group I, A), an animal fed a high-cholesterol diet (group II, B), and a rat given PAN and fed a high-cholesterol diet (group IV, C). Arrowhead, segmental sclerosis; arrow, mesangial foam cells.

      Discussion

      It takes 6 to 12 months of puromycin administration to generate progressive GS in rats
      • Glasser R.J.
      • Velosa J.A.
      • Michael A.F.
      Experimental model of focal sclerosis. I Relationship to protein excretion in aminonucleoside nephrosis.
      • Chandra M.
      • Susin M.
      • Teichberg S.
      • et al.
      Experimental focal segmental glomerulosclerosis: correlation with protein excretion, glomerular filtration rate, and renal plasma flow.
      ; thus, the findings of the present study can be taken to represent an early stage of GS. These results demonstrate that in the early stages of cholesterol- and PAN-mediated nephrosis, a significant down-regulation of AT1 and up-regulation of AT2 receptors occur, as reflected by lower kidney AT1 and higher kidney AT2 mRNA expression. Although serum ACE activity and serum Ang II concentration were not different among the 4 experimental groups, kidney ACE activity and ACE mRNA levels of group III (PAN) and IV (PAN+cholesterol) were significantly higher, suggesting a role for intrarenal RAS activity in the early developmental stages of GS. Intrarenal activation of the RAS, as well as independent effects of cholesterol and hypertension on progressive GS, may all be important pathophysiologic factors.
      Intrarenal Ang II might cause a differential growth-modulating effect dependent upon the ratio of Ang II receptor subtypes found on a given cell. The growth-promoting properties of Ang II are mediated by the AT1 receptor and are associated with increased growth factor expression.
      • Abboud H.E.
      Role of platelet-derived growth factor in renal injury.
      • Kagami S.
      • Border W.A.
      • Miller D.E.
      • et al.
      Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells.
      • Stoll M.
      • Meffert S.
      • Stroth U.
      • et al.
      Growth or antigrowth: angiotensin and the endothelium.
      Ang II blockade can be renoprotective by limiting both mesangial cell proliferation and matrix synthesis, which results from the direct action of Ang II on the AT1 receptor
      • Keane W.F.
      Lipids and progressive renal failure.
      and autocrine activation of growth factors.
      • Abboud H.E.
      Role of platelet-derived growth factor in renal injury.
      • Kagami S.
      • Border W.A.
      • Miller D.E.
      • et al.
      Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells.
      • Stoll M.
      • Meffert S.
      • Stroth U.
      • et al.
      Growth or antigrowth: angiotensin and the endothelium.
      Recently, AT1 receptor mRNA expression was shown to be significantly lower in glomeruli of patients with glomerulonephritis compared with control tumor nephrectomy biopsy samples.
      • Wagner J.
      • Gehlen F.
      • Ciechanowicz A.
      • et al.
      Angiotensin II receptor type 1 gene expression in human glomerulonephritis and diabetes mellitus.
      The high concentration of kidney Ang II was probably responsible for the down-regulation of kidney AT1 receptors found for groups III and IV. The AT1-receptor pattern was somewhat different in the animals fed cholesterol alone (group II). Group II animals showed no change in AT1 receptor mRNA expression, although the kidney concentration of Ang II was 24% higher than that found in control animals, whereas the Ang II concentration increased by 56 and 66% in groups III and IV, respectively. It can be speculated that the modest increase in tissue Ang II content in the group II animals was not sufficient to decrease AT1 receptor mRNA expression.
      Although the physiological role of the AT2 receptor is not clearly understood, it has been shown that although the chronic blockade of AT2 receptors in Ang II-induced hypertensive rats has no effect on blood pressure, it will potentiate Ang II-mediated hypertrophy and fibrosis.
      • Levy B.I.
      • Benessiano J.
      • Henrion D.
      • et al.
      Chronic blockade of AT2-subtype receptors prevents the effect of angiotensin II on the rat vascular structure.
      It has been proposed that, under pathophysiological conditions, AT2 receptors increase to curb excessive growth, regardless of whether growth is mediated via the AT1 receptor or other Ang II-independent growth factors.
      • Stoll M.
      • Meffert S.
      • Stroth U.
      • et al.
      Growth or antigrowth: angiotensin and the endothelium.
      For example, in coronary endothelial cells, the antiproliferative actions of Ang II, derived from AT2 receptor stimulation, offset the growth-promoting events mediated by AT1 receptor stimulation.
      • Stoll M.
      • Steckelings U.M.
      • Paul M.
      • et al.
      The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells.
      It has also been observed that increased proliferation of mesangial cells in rats prone to hypertensive stroke results from the lower expression of AT2 receptors.
      • Goto M.
      • Mukoyama M.
      • Suga S.
      • et al.
      Growth-dependent induction of angiotensin II type 2 receptor in rat mesangial cells.
      AT2 receptors have previously been observed to up-regulate in the presence of Ang II.
      • Dudley D.T.
      • Summerfelt R.M.
      Regulated expression of angiotensin II (AT2) binding sites in R3T3 cells.
      The increased tissue Ang II in group II is likely to have influenced for AT2 mRNA expression. This could then represent the increase of renal AT2 receptors and the observed changes in Ang II in this experimental model; the change in Ang II was of sufficient magnitude to induce AT2 message expression but not great enough to suppress AT1 mRNA expression. Alternatively, other AT2 receptor ligands, such as Ang I or Ang III, could have changed in our experimental model and increased the number of cell-surface AT2 receptors.
      • Csikos T.
      • Balmforth A.J.
      • Grojec M.
      • et al.
      Angiotensin AT2receptor degradation is prevented by ligand occupation.
      As in our observations, down-regulation of AT1 receptors and up-regulation of AT2 receptors have been observed in idiopathic dilated cardiomyopathy,
      • Tsutsumi Y.
      • Matsubara H.
      • Ohkubo N.
      • et al.
      Angiotensin II type 2 receptor is upregulated in human hearts with interstitial fibrosis, and cardiac fibroblasts are the major cell type for its expression.
      whereas up-regulation of AT2 receptors alone has been detected in rat cortical cells after exposure to Ang II
      • Shibata K.
      • Makino I.
      • Shibaguchi H.
      • et al.
      Up-regulation of angiotensin type 2 receptor mRNA by angiotensin II in rat cortical cells.
      and wound healing.
      • Viswanathan M.
      • Saavedra J.M.
      Expression of angiotensin II AT2receptors in the rat skin during experimental wound healing.
      PAN causes marked elevation of plasma cholesterol secondary to nephrotic syndrome,
      • Vaziri N.D.
      • Liang K.
      Up-regulation of acyl-coenzyme A: cholesterol acyltransferase (ACAT) in nephrotic syndrome.
      and it is unclear whether this hypercholesterolemia contributes to stimulation of the RAS. To delineate this, cholesterol-fed animals were also studied because this diet results in plasma cholesterol concentrations that are similar to those of PAN-treated animals. Increased dietary cholesterol is linked to the development of hypercholesterolemia, proteinuria, and mild GS in a number of animal species.
      • Diamond J.R.
      Role of cholesterol in glomerular injury.
      Moreover, it has been shown that increasing dietary cholesterol significantly exacerbates puromycin-induced nephrosis
      • Keane W.F.
      Lipids and progressive renal failure.
      and increases the severity of glomerular disease observed with reduced renal mass and hypertension.
      • Diamond J.R.
      Role of cholesterol in glomerular injury.
      In the present investigation, a 4% cholesterol diet caused both proteinuria and an elevation in the Ang II content of the kidney. When this diet was given to puromycin-treated animals, both of these parameters increased well beyond the values observed after puromycin alone.
      In this study, we have observed a 7- to 8-fold increase in hypercholesterolemia in the animals of groups II and III and a 20-fold increase in serum cholesterol in the animals of group IV. The association between alimentary hypercholesterolemia and Ang II is only partially developed. In a recent study, Song et al
      • Song K.
      • Shiota N.
      • Takai S.
      • et al.
      Induction of angiotensin converting enzyme and angiotensin II receptors in the atherosclerotic aorta of high-cholesterol fed Cynomolgus monkeys.
      suggested that the production of Ang II might be increased in the aorta of cholesterol-fed monkeys. In addition, it has been observed that hypercholesterolemia increases glomerular macrophage number
      • Wintroub B.U.
      • Klickstein L.B.
      • Dzau V.J.
      • et al.
      Granulocyte-angiotensin system. Identification of angiotensinogen as the plasma protein substrate of leukocyte cathepsin G.
      and that cathepsin G, present in monocytes/macrophages, converts angiotensinogen to Ang II.
      • Hollenberg N.K.
      • Fisher N.D.
      • Price D.A.
      Pathways for angiotensin II generation in intact human tissue: evidence from comparative pharmacological interruption of the renin system.
      Because cholesterol feeding did not affect kidney ACE but increased kidney Ang II concentration, it is possible that the hypercholesterolemia seen in this study increased macrophage infiltration, which, in turn, permitted an increase in Ang II production via cathepsin G and/or another ACE-independent mechanism for Ang II production
      Finally, an additional component of this model is that of a progressive form of hypertension, which was most severe in the group IV (PAN+cholesterol-fed) animals but was also evident in the group III (PAN alone) animals. In the instance of the latter, MAP began rising by week 12 of the study and was significantly different from that of group I and II animals at week 16. Hypertension has proven to be an intermittent finding in PAN nephrosis. When present, its development is generally viewed as being without a clear cause. Previous work by Diamond and Karnovsky
      • Diamond J.R.
      • Karnovsky M.J.
      Exacerbation of chronic aminonucleoside nephrosis by dietary cholesterol supplementation.
      suggested that a high-cholesterol diet accentuated hypertension, a finding also observed in our study.
      Although prior observations have not been entirely consistent, there seems not to be significant activation of the circulating RAS in this model; thus, the hypertension found in this model is probably not renin-dependent. It might have been suspected that intrarenal RAS activation accelerates the process of GS and indirectly aggravates hypertension in part because prior studies have demonstrated an increase in tissue ACE in PAN nephrosis.
      • Arevalo A.E.
      • Ibarra-Rubio M.E.
      • Cruz C.
      • et al.
      Angiotensin-I converting enzyme activity in puromycin aminonucleoside-nephrotic syndrome.
      Thus, it is certainly plausible that tissue-based production of Ang II might occur independently, a finding we observed in experimental groups II to IV, although the ACE activity and mRNA were not different from control values in each of these groups. In kidney, although Ang II generation is heavily renin-dependent, at least 40% of the conversion of Ang I to Ang II occurs by pathways other than ACE, presumably by a chymase-dependent pathway, although other enzyme pathways exist. Moreover, the contribution of non-ACE pathways to Ang II production is substantially greater in disease states.
      • Wintroub B.U.
      • Klickstein L.B.
      • Dzau V.J.
      • et al.
      Granulocyte-angiotensin system. Identification of angiotensinogen as the plasma protein substrate of leukocyte cathepsin G.
      Because the Ang II concentration in the group II was 24% over control values and tissue ACE was not elevated in this group, it is likely that an ACE-independent mechanism for Ang II generation is triggered early and with as slight a stimulus as 4% cholesterol feeding. Even though there are similarities between PAN- and cholesterol-induced nephrosis, there are unique differences. At the end of the fourth month, PAN significantly increased MAP, kidney ACE activity, and kidney ACE mRNA levels; these changes were not observed in the cohort of animals receiving the cholesterol-enriched diet. However, cholesterol feeding augmented the effect of PAN on MAP and kidney ACE activity and mRNA levels, as demonstrated in group IV animals (cholesterol-fed and PAN-administered). The group IV animals had significantly higher proteinuria than the other groups, suggesting that a cholesterol-enriched diet can amplify PAN-induced nephrosis.
      In summary, in evaluating a model of puromycin-induced GS, we have identified a tissue-based activation of the renin-angiotensin axis within the kidney. This renal activation of the renin-angiotensin axis developed independently of systemic renin axis activation and was more profound in the setting of a high-cholesterol diet. Moreover, cholesterol feeding alone resulted in a similar, but less significant, activation of the renal renin-angiotensin axis. Puromycin alone, as well as cholesterol, decreased the AT1 receptor mRNA expression and increased the message for the AT2 receptor mRNA. During early stages of renal injury, in the absence of an ACE inhibitor or an AT1 receptor antagonist, the normal physiological response to thwart the progression of GS would be to decrease mesangial cell proliferation by decreasing Ang II formation, down-regulating AT1 receptors, and/or up-regulating AT2 receptors. Ang II regulation is more effective than AT2 receptor regulation because the production of Ang II is not limited to a single pathway. The degree of proteinuria and increase in blood pressure in these studies was more severe in the puromycin and puromycin/cholesterol treatment groups, suggesting an association between the degree of structural change and local renin-angiotensin axis activation. A more complete description of the role of local renin-angiotensin axis activation in the origin and/or progression of puromycin-induced GS awaits further studies with compounds that inhibit activity in this axis, such as ACE inhibitors and/or angiotensin receptor antagonists.

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