Histomorphometric effects of an antiretroviral treatment and obesity on the pancreas, liver, kidney and perivascular adipose tissue in a rat model
Date
2017-12-12
Authors
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Journal ISSN
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Stellenbosch : Stellenbosch University
Abstract
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Thesis (MSc)--Stellenbosch University, 2017
ENGLISH ABSTRACT : Human immunodeficiency virus (HIV)-infected patients receiving antiretroviral treatment (ART) may develop metabolic syndrome and lipodystrophy, leading to obesity rather than wasting. Obesity is associated with metabolic homeostasis disruptions leading to pancreatic toxicity, diabetes, renal vasodilation and compression as well as liver steatosis. In addition, patients on long-term combination antiretroviral treatment (cART) can develop pancreatitis, nephrotoxicity, hepatic steatosis, and diabetes. This study aims to investigate the effects of cART combined with a high-calorie diet (HCD) on the histomorphometry of the pancreas, kidney, liver, and aortic perivascular adipose tissue (PVAT) leptin expression in the rat. Male Wistar rats (N=40) were divided into four groups (n=10); C/ART- (lean control), C/ART+ (ART control), HCD/ART- (HCD control) and HCD/ART+ (HCD and ART). The aorta with surrounding PVAT, pancreas, liver and kidney were harvested and processed to wax. Pancreas sections underwent a double immunohistochemistry (IHC) labelling for insulin and glucagon and reactive cell areas were measured. Aortic PVAT was labelled with anti-leptin and the staining intensity classified accordingly. Kidney sections underwent haematoxylin and eosin (H&E) stain measuring the areas of the renal corpuscles, glomeruli, renal spaces as well as the renal arcuate arteries lumen diameter and artery wall thickness. Pancreatic, kidney and liver sections underwent H&E staining which were used for pathology assessment. The HCD/ART+ had a significant body mass increase compared to the C/ART-, suggesting ART combined with a HCD causes body mass increase. Unilocular and differentiating adipocytes in the C/ART+ had intense leptin staining, suggesting ART may lead to increased leptin expression in aortic PVAT. Unilocular and differentiating adipocytes in the HCD/ART- and HCD/ART+ had weak to no leptin intensity, suggesting possible leptin deficiency with a HCD. The C/ART+ showed a trend in smaller total pancreatic islet area compared to the C/ART-, suggesting that islets underwent possible hypotrophy with ART. Based on the average values, alpha-cells were increased in the HCD/ART-, whereas beta-cells decreased in the ART and HCD. This suggests that a HCD potentially increases glucagon secreting area, whereas ART and a HCD leads to β-cell mass loss. The HCD/ART- and HCD/ART+ showed a trend in increased number of islets per section compared to the C/ART-, suggesting that HCD leads to new forming islets. Based on the average values, the renal corpuscle, glomeruli and renal space sizes were increased in the HCD/ART- compared to the other groups, although not significantly. A trend in the HCD/ART+ showed increased arcuate artery lumen diameter and tunica media thickness, whereas the C/ART+ had increased tunica adventitia compared to the C/ART-. This suggests that ART combined with a HCD causes hypertrophic remodelling in the tunica media and hypotrophic remodelling in the tunica adventitia. All groups had mild vacuolisation and swelling of the proximal convoluted tubules in the kidney and granular appearance, vacuolisation, and fatty change in the liver. The six week cART and HCD used in the present study caused no significant morphometric or pathological changes within the pancreas, kidney, and liver, apart from significant leptin intensity staining with ART.
AFRIKAANSE OPSOMMING : Pasiënte wat met die menslike immuniteitsgebreksvirus (MIV) besmet is en antiretrovirale behandeling (ARB) ontvang, kan metaboliese sindroom en lipodistrofie ontwikkel, wat tot obesiteit eerder as uittering lei. Obesiteit word geassosieer met metaboliese homeostase-versteurings, wat lei tot pankreas-toksisiteit, diabetes, renale vasodilatasie en kompressie asook lewersteatose. Hierbenewens kan pasiënte wat langtermyn-kombinasie- antiretrovirale behandeling (kARB) ontvang pankreatitis, nier-toksisiteit, hepatiese steatose en diabetes ontwikkel. Hierdie studie was daarop gemik om die histomorfometriese effekte van kARB gekombineer met ʼn hoëkalorie-dieet (HKD) op die pankreas, niere, lewer en leptienuitdrukking van die aorta-perivaskulêre adiposisweefsel (PVAW) in ʼn rotmodel te ondersoek. Manlike Wistar-rotte (N=40) is in vier groepe verdeel (n=10); K/ARB- (vetvrye beheergroep), K/ARB+ (ARB-beheergroep), HKD/ARB- (HKD-beheergroep) en HKD/ARB+ (HKD en ARB). Die aorta met omliggende PVAW, pankreas, lewer en niere is geoes en histologies voorberei. Pankreasdele het ʼn dubbele immunohistochemie- (IHC-)merking vir insulien en glukagon ondergaan en reaktiewe seldele is gemeet. Aorta-PVAW is gemerk met antileptien en die kleuringintensiteit is dienooreenkomstig geklassifiseer. Nierdele is gekleur met hematoksilien- en eosien (H&E-) en die renale liggaampies, glomeruli, renale ruimtes asook die renale arkuate arterielumendeursnee en arteriewanddikte is gemeet. Pankreas-, nier- en lewerdele word met H&E gekleur, wat vir patologie-assessering gebruik is. Die HKD/ARB+ het ʼn aanmerklike liggaamsmassatoename getoon in vergelyking met die K/ARB-, wat aan die hand doen dat ARB gekombineer met ʼn HKD liggaamsmassatoename veroorsaak. Unilokulêre en differensiërende adiposiete in die K/ARB+ het intense leptienkleuring getoon, wat aan dui dat ARB tot verhoogde leptienuitdrukking in aorta-PVAW kan lei. Unilokulêre en differensiërende adiposiete in die HKD/ARB- en HKD/ARB+ het swak tot geen leptienintensiteit getoon, wat op moontlike leptientekort met HKD kan dui. Die K/ARB+ het ʼn neiging tot kleiner totale pankreas-eilandoppervlakte in vergelyking met die K/ARB- getoon, wat aan dui dat eilande moontlike hipotrofie met ARB ondergaan het. Alfa-selle het in die HKD/ARB- toegeneem, terwyl beta-selle in die ARB en HKD afgeneem het. Dit dui daarop dat ʼn HKD moontlik verhoogde alfa-sel area veroorsaak, terwyl ARB en ʼn HKD tot β-sel-massaverlies lei. Die HKD/ARB- en HKD/ARB+ het ʼn neiging getoon tot verhoogde aantal eilande per deel in vergelyking met die K/ARB-, wat aan die hand doen dat HKD tot die vorming van nuwe eilande lei. Renale liggaampies, glomeruli en renale ruimtegroottes is verhoog in die HKD/ARB- in vergelyking met die ander groepe, dog nie beduidend nie. ʼn Neiging in die HKD/ARB+ het verhoogde arkuate arterielumendeursnee en tunica mediadikte getoon, terwyl die K/ARB+ verhoogde tunica adventitia in vergelyking met die K/ARB- getoon het. Dit doen aan die hand dat ARB gekombineer met ʼn HKD hipertrofiese hermodellering in die tunica media en hipotrofiese hermodellering in die tunica adventitia veroorsaak. Alle groepe het matige vakuolisering en swelling van die proksimale kronkelbuise in die nier en granulêre voorkoms, vakuolisering en vetverandering in die lewer getoon. Die sesweek-kARB en HKD wat in die huidige studie gebruik is, het geen aanmerklike morfometriese of patologiese veranderinge in die pankreas, nier en lewer veroorsaak nie, buiten beduidende leptienintensiteitkleuring met ARB.
ENGLISH ABSTRACT : Human immunodeficiency virus (HIV)-infected patients receiving antiretroviral treatment (ART) may develop metabolic syndrome and lipodystrophy, leading to obesity rather than wasting. Obesity is associated with metabolic homeostasis disruptions leading to pancreatic toxicity, diabetes, renal vasodilation and compression as well as liver steatosis. In addition, patients on long-term combination antiretroviral treatment (cART) can develop pancreatitis, nephrotoxicity, hepatic steatosis, and diabetes. This study aims to investigate the effects of cART combined with a high-calorie diet (HCD) on the histomorphometry of the pancreas, kidney, liver, and aortic perivascular adipose tissue (PVAT) leptin expression in the rat. Male Wistar rats (N=40) were divided into four groups (n=10); C/ART- (lean control), C/ART+ (ART control), HCD/ART- (HCD control) and HCD/ART+ (HCD and ART). The aorta with surrounding PVAT, pancreas, liver and kidney were harvested and processed to wax. Pancreas sections underwent a double immunohistochemistry (IHC) labelling for insulin and glucagon and reactive cell areas were measured. Aortic PVAT was labelled with anti-leptin and the staining intensity classified accordingly. Kidney sections underwent haematoxylin and eosin (H&E) stain measuring the areas of the renal corpuscles, glomeruli, renal spaces as well as the renal arcuate arteries lumen diameter and artery wall thickness. Pancreatic, kidney and liver sections underwent H&E staining which were used for pathology assessment. The HCD/ART+ had a significant body mass increase compared to the C/ART-, suggesting ART combined with a HCD causes body mass increase. Unilocular and differentiating adipocytes in the C/ART+ had intense leptin staining, suggesting ART may lead to increased leptin expression in aortic PVAT. Unilocular and differentiating adipocytes in the HCD/ART- and HCD/ART+ had weak to no leptin intensity, suggesting possible leptin deficiency with a HCD. The C/ART+ showed a trend in smaller total pancreatic islet area compared to the C/ART-, suggesting that islets underwent possible hypotrophy with ART. Based on the average values, alpha-cells were increased in the HCD/ART-, whereas beta-cells decreased in the ART and HCD. This suggests that a HCD potentially increases glucagon secreting area, whereas ART and a HCD leads to β-cell mass loss. The HCD/ART- and HCD/ART+ showed a trend in increased number of islets per section compared to the C/ART-, suggesting that HCD leads to new forming islets. Based on the average values, the renal corpuscle, glomeruli and renal space sizes were increased in the HCD/ART- compared to the other groups, although not significantly. A trend in the HCD/ART+ showed increased arcuate artery lumen diameter and tunica media thickness, whereas the C/ART+ had increased tunica adventitia compared to the C/ART-. This suggests that ART combined with a HCD causes hypertrophic remodelling in the tunica media and hypotrophic remodelling in the tunica adventitia. All groups had mild vacuolisation and swelling of the proximal convoluted tubules in the kidney and granular appearance, vacuolisation, and fatty change in the liver. The six week cART and HCD used in the present study caused no significant morphometric or pathological changes within the pancreas, kidney, and liver, apart from significant leptin intensity staining with ART.
AFRIKAANSE OPSOMMING : Pasiënte wat met die menslike immuniteitsgebreksvirus (MIV) besmet is en antiretrovirale behandeling (ARB) ontvang, kan metaboliese sindroom en lipodistrofie ontwikkel, wat tot obesiteit eerder as uittering lei. Obesiteit word geassosieer met metaboliese homeostase-versteurings, wat lei tot pankreas-toksisiteit, diabetes, renale vasodilatasie en kompressie asook lewersteatose. Hierbenewens kan pasiënte wat langtermyn-kombinasie- antiretrovirale behandeling (kARB) ontvang pankreatitis, nier-toksisiteit, hepatiese steatose en diabetes ontwikkel. Hierdie studie was daarop gemik om die histomorfometriese effekte van kARB gekombineer met ʼn hoëkalorie-dieet (HKD) op die pankreas, niere, lewer en leptienuitdrukking van die aorta-perivaskulêre adiposisweefsel (PVAW) in ʼn rotmodel te ondersoek. Manlike Wistar-rotte (N=40) is in vier groepe verdeel (n=10); K/ARB- (vetvrye beheergroep), K/ARB+ (ARB-beheergroep), HKD/ARB- (HKD-beheergroep) en HKD/ARB+ (HKD en ARB). Die aorta met omliggende PVAW, pankreas, lewer en niere is geoes en histologies voorberei. Pankreasdele het ʼn dubbele immunohistochemie- (IHC-)merking vir insulien en glukagon ondergaan en reaktiewe seldele is gemeet. Aorta-PVAW is gemerk met antileptien en die kleuringintensiteit is dienooreenkomstig geklassifiseer. Nierdele is gekleur met hematoksilien- en eosien (H&E-) en die renale liggaampies, glomeruli, renale ruimtes asook die renale arkuate arterielumendeursnee en arteriewanddikte is gemeet. Pankreas-, nier- en lewerdele word met H&E gekleur, wat vir patologie-assessering gebruik is. Die HKD/ARB+ het ʼn aanmerklike liggaamsmassatoename getoon in vergelyking met die K/ARB-, wat aan die hand doen dat ARB gekombineer met ʼn HKD liggaamsmassatoename veroorsaak. Unilokulêre en differensiërende adiposiete in die K/ARB+ het intense leptienkleuring getoon, wat aan dui dat ARB tot verhoogde leptienuitdrukking in aorta-PVAW kan lei. Unilokulêre en differensiërende adiposiete in die HKD/ARB- en HKD/ARB+ het swak tot geen leptienintensiteit getoon, wat op moontlike leptientekort met HKD kan dui. Die K/ARB+ het ʼn neiging tot kleiner totale pankreas-eilandoppervlakte in vergelyking met die K/ARB- getoon, wat aan dui dat eilande moontlike hipotrofie met ARB ondergaan het. Alfa-selle het in die HKD/ARB- toegeneem, terwyl beta-selle in die ARB en HKD afgeneem het. Dit dui daarop dat ʼn HKD moontlik verhoogde alfa-sel area veroorsaak, terwyl ARB en ʼn HKD tot β-sel-massaverlies lei. Die HKD/ARB- en HKD/ARB+ het ʼn neiging getoon tot verhoogde aantal eilande per deel in vergelyking met die K/ARB-, wat aan die hand doen dat HKD tot die vorming van nuwe eilande lei. Renale liggaampies, glomeruli en renale ruimtegroottes is verhoog in die HKD/ARB- in vergelyking met die ander groepe, dog nie beduidend nie. ʼn Neiging in die HKD/ARB+ het verhoogde arkuate arterielumendeursnee en tunica mediadikte getoon, terwyl die K/ARB+ verhoogde tunica adventitia in vergelyking met die K/ARB- getoon het. Dit doen aan die hand dat ARB gekombineer met ʼn HKD hipertrofiese hermodellering in die tunica media en hipotrofiese hermodellering in die tunica adventitia veroorsaak. Alle groepe het matige vakuolisering en swelling van die proksimale kronkelbuise in die nier en granulêre voorkoms, vakuolisering en vetverandering in die lewer getoon. Die sesweek-kARB en HKD wat in die huidige studie gebruik is, het geen aanmerklike morfometriese of patologiese veranderinge in die pankreas, nier en lewer veroorsaak nie, buiten beduidende leptienintensiteitkleuring met ARB.
Keywords
Immunohistochemistry, UCTD, Rats -- Antiretroviral treatment, Rats as laboratory animals, Obesity -- Animal models, Organs (Anatomy) -- Side effects