call for papers renal hemodynamics - semantic scholar · 2017-06-17 · 19 2 mmhg in fhh rats when...

CALL FOR PAPERS Renal Hemodynamics

Temporal characterization of the development of renal injury in FHH ratsand FHH.1BN congenic strains

Jan Michael Williams,1 Marilyn Burke,1 Jozef Lazar,2 Howard J. Jacob,2 and Richard J. Roman1

1Departments of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi; and 2Humanand Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 7 May 2010; accepted in final form 29 October 2010

Williams JM, Burke M, Lazar J, Jacob HJ, Roman RJ.Temporal characterization of the development of renal injury inFHH rats and FHH.1BN congenic strains. Am J Physiol RenalPhysiol 300: F330 –F338, 2011. First published November 3, 2010;doi:10.1152/ajprenal.00261.2010.—The present study examined theeffect of transfer of portions of chromosome 1 that includes (FHH.1BN

AR� strain) or excludes (control FHH.1BN AR� strain) a 4.3-Mbregion from the Brown Norway (BN) rat that restores the autoregu-lation (AR) of renal blood flow (RBF) on the development of hyper-tension and renal injury in congenic strains of Fawn Hooded Hyper-tensive (FHH) rats. FHH and control AR� rats exhibited poor auto-regulation of RBF, and glomerular capillary pressure (Pgc) rose by19 � 2 mmHg in FHH rats when renal perfusion pressure (RPP) wasincreased from 100 to 150 mmHg. In contrast, RBF was well auto-regulated in the AR� strain, and Pgc only increased by 3 � 1 mmHgwhen RPP was increased over this range. Baseline mean arterialpressure (MAP) at 12 wk of age was similar in all strains and averaged122 mmHg. MAP increased significantly in FHH rats and wassignificantly higher by 12 mmHg in 21-wk-old FHH rats than in theFHH.1BN congenic strains. Protein excretion rose from 5 � 1 to397 � 29 mg/day in 6- vs. 21-wk-old FHH rats. In contrast, proteinexcretion only increased to 139 � 21 mg/day in the control AR�

strain, and it did not increase significantly in the AR� strain. Glo-merular permeability to albumin was similar in all strains at 6 wk ofage. It increased significantly in 9-wk-old FHH and control AR� rats,but not in the AR� strain. The levels of matrix metalloproteinase(MMP)-2 and transforming growth factor (TGF)-�2 protein weresignificantly higher in the renal cortex of 9-wk-old FHH rats com-pared with the levels seen in the AR� strain. These data indicate thattransfer of a 4.3-Mb region of BN chromosome 1 into the FHHgenetic background improves autoregulation of RBF, normalizes Pgc,and slows the progression of renal disease.

Fawn Hooded Hypertensive rats; renal hemodynamics; renal bloodflow; renal injury; transforming growth factor-�2; matrix metallopro-teinase-2

THE FAWN HOODED HYPERTENSIVE (FHH) rat is a renin-dependent(15, 34) model of renal disease that develops progressiveproteinuria, focal glomerulosclerosis, and end-stage renal dis-ease (12, 15–16, 25, 28–31, 34). We have reported that FHHrats exhibit impaired autoregulation of RBF that is associatedwith a defect in the myogenic response of preglomerular renalarterioles (31). More recently, we found that transfer of a12.9-Mb region of chromosome 1 from the Brown Norway(BN) rat onto the FHH genetic background improves autoreg-

ulation of renal blood flow (RBF) and attenuates the develop-ment of proteinuria in a FHH.1BN congenic strain (12). How-ever, the mechanism by which restoration of RBF autoregula-tion alters the development proteinuria and renal disease in thisFHH.1BN congenic strain is unclear. Moreover, it remains to bedetermined whether changes in arterial pressure precede thedevelopment of renal injury in FHH rats. In the present study,we examined the hypothesis that impaired autoregulation ofRBF promotes renal injury by elevating glomerular capillarypressure (Pgc), leading to upregulation of the expression oftransforming growth factor (TGF)-� and matrix metallopro-teinase (MMP)-2. The rise in TGF-� and MMP-2 levels thendamages the glomerular permeability barrier and promotesepithelial to mesenchymal transition (EMT) and stimulatesrenal interstitial fibrosis. To test this hypothesis, the timecourse of changes in arterial pressure and proteinuria, perme-ability to albumin (Palb), renal TGF-�, and MMP-2 levels wascompared in FHH rats and in two genetically similar FHH.1BN

congenic strains that differ by the inclusion (FHH.1BN AR�

strain) or exclusion (control FHH.1BN AR� strain) of a 4.3-Mbregion of BN chromosome 1 that restored autoregulation ofRBF.

METHODS

General. Experiments were performed on 124, 6- to 21-wk-oldmale FHH and FHH.1BN congenic rats that were obtained from inbredcolonies maintained at the Medical College of Wisconsin. All of therats were housed in the Animal Care Facility at the Medical Collegeof Wisconsin, which is approved by the American Association for theAccreditation of Laboratory Animal Care. The rats had free access tofood and water throughout the study. All protocols received approvalby the Animal Care Committee of the Medical College of Wisconsin.The breeding colonies were maintained on a rodent diet purchasedfrom LabDiet (PMI Nutrition International, Brentwood, MO) contain-ing 0.28% NaCl. After weaning, the pups were switched to a purifiedAIN-76 rodent diet containing 0.4% NaCl (Dyets, Bethlehem, PA).

Protocol 1. Comparison of autoregulation of RBF in FHH rats andFHH.1BN congenic strains. These experiments were performed on 9-to 12-wk-old FHH rats and FHH.1BN congenic strains. The controlAR� congenic strain is genetically identical to the AR� strain exceptthat they lack a 4.3-Mb AR� region of chromosome 1 from the BNrat. The rats were anesthetized with ketamine (30 mg/kg im; PhoenixPharmaceutical, St. Joseph, MO) and inactin (50 mg/kg ip; Sigma, St.Louis, MO), and catheters were placed in the femoral artery for themeasurement of mean arterial pressure (MAP) and femoral vein for anintravenous infusion of 6% BSA in a 0.9% NaCl solution at a rate of100 �l/min. After surgery, MAP was increased to 150 mmHg bytying of the celiac and mesenteric arteries. Next, RBF was mea-sured using a ultrasound flow probe (Transonic System, Ithaca,

Address for reprint requests and other correspondence: R. J. Roman, Dept.of Pharmacology and Toxicology, Univ. of Mississippi Medical Center, 2500North State St., Jackson, MS 39216 (e-mail: [email protected]).

Am J Physiol Renal Physiol 300: F330–F338, 2011.First published November 3, 2010; doi:10.1152/ajprenal.00261.2010.

0363-6127/11 Copyright © 2011 the American Physiological Society http://www.ajprenal.orgF330

by 10.220.32.247 on June 17, 2017http://ajprenal.physiology.org/

Dow

nloaded from

http://ajprenal.physiology.org/

NY) on the renal artery as renal perfusion pressure (RPP) wasvaried from 150 to 80 mmHg in steps of 10 mmHg by tighteninga clamp on the abdominal aorta above the left renal artery aspreviously described (12). RBF autoregulatory indexes over therange of pressures from 100 to 140 mmHg were calculated by themethod of Semple and De Wardener (21).

Protocol 2. Comparison of RBF, glomerular filtration rate, andPgc in FHH and FHH.1BN AR� congenic rats. These experimentswere performed on 9- to 12-wk-old FHH and FHH.1BN AR� congenicrats. The rats were surgically prepared for the measurement of RBF,glomerular filtration rate (GFR), and Pgc as described above andinfused with 2% BSA in 0.9% NaCl solution at a rate of 100 �l/min.A catheter was inserted in the left ureter for the collection of urine,and FITC-labeled inulin (2 mg/ml; Sigma) was added to the intrave-nous infusion solution for the measurement of GFR. After surgery,RPP was lowered to 100 mmHg by tightening the aortic clamp. Aftera 30-min equilibration period, urine and plasma samples were col-lected during a 20-min control period. RPP was then increased to�150 mmHg by releasing the aortic clamp and tying off the celiac andmesenteric arteries, and, after a 10-min equilibration period, urine andplasma samples were collected during a 20-min experimental period.At the end of each experiment, the left kidney was removed andweighed, and the concentration of inulin in urine and plasma sampleswas determined using a plate reader fluorometer.

Additional experiments were performed to estimate autoregulationof Pgc by micropuncture. The left kidney was placed in a stainlesssteel holder and surrounded with 2.5% agar (Sigma). The surface ofthe kidney was constantly bathed with a saline solution. Pgc wasestimated indirectly in the absence of tubuloglomerular feedback bymeasuring plasma oncotic pressure and stop flow pressures in three tofive early proximal tubules after blocking the tubular lumen withhigh-vacuum grease as previously described (31).

Protocol 3. Comparison of the time course of the development ofhypertension, proteinuria, and Palb in FHH and FHH.1BN congenicrats. These experiments were performed on 11-wk-old FHH rats andthe FHH.1BN congenic strains. Telemetry transmitters (modelTA11PA-C40; Data Sciences International, St. Paul, MN) were im-planted in the femoral artery, and the transmitters were placed underthe skin. After a 1-wk recovery period, MAP was recorded between1:00 and 4:00 P.M. when the rats were 12, 15, 18, and 21 wk of age.At each time point, the rats were placed in metabolic cages for 24 hfor the determination of protein excretion. The concentration ofprotein in the urine samples was measured using the Bradford method

using BSA as the standard (Bio-Rad Laboratories, Hercules, CA). Atthe end of the protocol, rats were anesthetized, and a catheter wasplaced in the aorta below the kidneys. The kidneys were flushed with10 ml of saline, collected, weighed, and fixed in a 10% bufferedformalin solution. Paraffin sections (3 �m) were cut and stained withMason’s trichrome to determine the degree of glomerular injury. Thedegree of injury was assessed on 30 glomeruli/section as the percent-age of the glomerular capillary area filled with matrix material on a0–4 scale with 0 representing no injury, 2 indicating loss of 50% ofglomerular capillary area, and 4 representing the complete obstructionof glomerular capillaries (18). Additional analysis was performed todetermine the degree of renal interstitial fibrosis. Images were cap-tured using a Nikon Eclipse 55i microscope equipped with a NikonDS-Fi1 color camera (Nikon, Melville, NY) and analyzed for thepercentage of the image stained blue using the NIS-Elements D 3.0software. Ten to twenty representative fields were analyzed perkidney. The assessment of glomerular injury and renal interstitialfibrosis was done blinded and was performed by the same observer.

Measurement of glomerular Palb. These experiments determinedwhether Palb differs in 12- and 21-wk-old FHH rats vs. the corre-sponding values seen in the FHH.1BN congenic strains. The rats wereanesthetized with pentobarbital sodium (60 mg/kg), and the kidneyswere collected. Glomeruli were isolated using the sieving method ina 5% albumin solution. Palb was determined by measuring the changein glomerular volume (�V) after exchange of the bath with freshmedium containing 1% albumin as previously described (6, 20, 24,35). Palb was calculated as 1 � (�Vexperimental/�Vcontrol), where�Vcontrol was taken as the mean change in volume measured in allglomeruli isolated from 6-wk-old control Sprague-Dawley rats, whichare assumed to have no preexisting renal damage. A minimum of 10glomeruli were studied from each rat, and these experiments wereperformed using a minimum of four rats per strain.

Protocol 4. Filtration of FITC-labeled albumin in the kidneys ofFHH and FHH.1BN congenic rats. Experiments were performed inFHH and FHH.1BN congenic strains to confirm our Palb measurementsindicating that transfer of the AR� region attenuates filtration ofprotein by glomerular capillaries in vivo. A catheter was placed in thefemoral vein for the infusion of FITC-labeled albumin (20 mg/kg;Sigma) at a rate of 100 �l/min for 15 min. At the end of this period,kidneys were collected and fixed in a 10% buffered formalin solution.Paraffin sections (3 �m) were prepared and counterstained withAlcian blue. Images were obtained using a fluorescence microscopeequipped with a high-sensitivity camera (Q-Imaging digital camera;

Fig. 1. Genetic map showing the introgressedregions in control FHH.1BN AR� and FHH.1BN

AR� congenic strains. Left: location of geneticmarkers used to genotype the animals and thelocation of the previously identified renal failure1 (Rf-1) and renal failure 2 (Rf-2) quantitativetrait loci (QTLs) on chromosome 1 in FawnHooded Hypertensive (FHH) rats. Open bars,FHH genome; filled bars, Brown Norway (BN)genomes. Right, the candidate genes in the4.3-Mb region of interest.

F331RENAL INJURY IN FHH RATS

AJP-Renal Physiol • VOL 300 • FEBRUARY 2011 • www.ajprenal.org


Dow

nloaded from


W. Nuhsbaum, McHenry, IL), and the overlaid images were createdusing Imaging Pro Plus software.

Protocol 5. Comparison of excretion of protein, Palb, and renalTGF-�2 and MMP-2 levels in 6- to 9-wk-old FHH and FHH.1BN

congenic rats. These experiments were performed in younger animalsto determine when differences in protein excretion and Palb firstappear in FHH and FHH.1BN congenic rats. The rats were placed inmetabolic cages for 24 h at 6 and 9 wk of age for the determinationof protein excretion. When the rats were 9 wk of age, they wereanesthetized with isoflurane, and the kidneys were harvested. Glomer-uli were isolated from one kidney to measure Palb, and the otherkidney was collected for biochemical analysis. The renal cortex wasseparated and homogenized in 1 ml of RIPA buffer (Sigma) formeasurement of the expression of TGF-� and MMP-2 protein levels.The homogenates were centrifuged at 5,000 g for 5 min and 11,000 gfor 15 min, and the supernatant was collected and frozen in liquid N2

and stored at �80°C. Western blot analysis for TGF-�2 was per-formed using a 1:500 dilution of TGF-�2 primary antibody (sc90;Santa Cruz Biotechnology, Santa Cruz, CA) and a 1:4,000 dilution ofa horseradish peroxidase-coupled secondary antibody (sc2004; SantaCruz Biotechnology) for 1 h. This antibody recognizes the active formof TGF-�2 (�12 kDa) and the mature form associated with a latent

Fig. 2. Relationship between renal perfusion pressure (RPP) and renal bloodflow (RBF) in volume-expanded FHH and FHH.1BN congenic strains (A) andRBF autoregulation indexes (B). Nos. in parentheses indicate the no. of ratsstudied/group. *Significant difference (P � 0.05) from the corresponding RBFvalue at RPP of 100 mmHg within the same strain. †Significant difference(P � 0.05) from the corresponding value in FHH rats.

Fig. 3. Comparison of the effect of elevations of RPP on RBF (A), glomerularfiltration rate (GFR; B), and glomerular capillary pressure (Pgc; C) in FHH andFHH.1BN AR� congenic strains in which autoregulation of RBF is restored.Nos. in parentheses indicate the no. of rats studied/group. *Significant differ-ence (P � 0.05) from the corresponding RBF value at RPP of 100 mmHgwithin the same strain. †Significant difference (P � 0.05) from the correspond-ing value in FHH rats.

F332 RENAL INJURY IN FHH RATS



Dow

nloaded from


protein (TGF-�2/LAP, �75 kDa). Equal loading of homogenateprotein was confirmed by stripping and reprobing the membranesusing a 1:8,000 dilution of a �-actin primary antibody (Sigma) and1:8,000 dilution of horseradish peroxidase-coupled secondary anti-body (Santa Cruz Biotechnology) for 1 h. Levels of MMP-2 in therenal cortical homogenates were measured by using a MMP-2 ELISA(R&D Systems, Minneapolis, MN).

Statistical analysis. Data are presented as means � SE. Signifi-cance of differences in mean values was determined using a one- ortwo-way repeated-measures ANOVA and the Holm-Sidak test forpreplanned comparisons. A P � 0.05 was considered to be significant.

RESULTS

Protocol 1. Comparison of autoregulation of RBF in FHHand FHH.1BN congenic strains. We previously reported thatautoregulation of RBF is impaired in FHH rats and that transferof a 12.9-Mb region of chromosome 1 from the BN rat ontoFHH genetic background in a congenic strain (AR� original;Fig. 1) restores autoregulation of RBF and reduces proteinuria(12). In the present study, we characterized renal function intwo additional FHH.1BN congenic strains that were derived

from the original congenic strain and are genetically identicalto each other, except for the inclusion (FHH.1BN AR� strain)or exclusion (control FHH.1BN AR� strain) of a 4.3-Mbpregion of interest located between the genetic makersD1Rat20G17B-6 and D1Rat888 of BN chromosome 1 (Fig. 1).These two strains also carry a common segment of BN chro-mosome 1 from markers D1Rat183 to D1Mit18 that are sharedwith the original congenic strain that encompasses the renalfailure 2 (Rf-2) quantitative trait loci (QTL) associated with aknockout of the Rab38 gene in the FHH rat that influences coatcolor, a bleeding disorder, and the reuptake of filtered protein(8, 11, 17, 19) but not autoregulation of RBF.

In the present study, autoregulation of RBF in response to anelevation in RPP from 100 to 140 mmHg was nearly absent inFHH rats after acute volume expansion to blunt tubuloglo-merular feedback responsiveness (Fig. 2A), and the autoregu-latory index averaged 0.8 (Fig. 2B). Similar results wereobtained in the control AR� strain (Fig. 2A). In contrast,autoregulation of RBF was restored in the AR� strain, andRBF remained relatively constant when RPP was increasedover this range. The RBF autoregulatory index was signifi-cantly lower in the AR� strain than the corresponding valuesseen in the FHH rats and the control AR� congenic strain.

Protocol 2. Comparison of RBF, GFR, and Pgc in FHH ratsand the AR� strain. The relationships between RBF, GFR, andPgc in response to an elevation in RPP from 100 to 150 mmHgare presented in Fig. 3. RBF increased significantly in FHHrats, whereas RBF was not significantly altered in the AR�

strain (Fig. 3A). GFR doubled in FHH rats when RPP wasincreased from 100 to 150 mmHg, whereas no significantchange was observed in the AR� strain (Fig. 3B). Pgc in-creased significantly by 19 � 2 mmHg in FHH rats in responseto elevations in RPP from 100 to 150 mmHg (Fig. 3C). Incontrast, Pgc only increased by 3 � 1 mmHg in the AR� strain.Fig. 4. Time course of the development of hypertension (A) and proteinuria (B)

in 12- to 21-wk-old FHH and FHH.1BN congenic rats. Nos. in parenthesesindicate the no. of rats studied/group. *Significant difference (P � 0.05) fromthe corresponding value at 12 wk of age within the same strain. †Significantdifference (P � 0.05) from the corresponding value in FHH rats.

Fig. 5. Comparison of glomerular permeability to albumin (Palb) measured inFHH and FHH.1BN congenic strains at 12 and 21 wk of age. Nos. inparentheses indicate the no. of glomeruli and rats studied/group. †Significantdifference (P � 0.05) from the corresponding value in FHH rats.




Dow

nloaded from


Protocol 3. Comparison of the time course of the develop-ment of hypertension and proteinuria and renal injury in FHHrats and FHH.1BN congenic strains. The results of theseexperiments are presented in Fig. 4. Baseline MAP was notsignificantly different in 12-wk-old FHH rats and the FHH.1BN

congenic strains (Fig. 4A). MAP increased significantly by�15 mmHg in the FHH rats over the 9-wk course of the studyand was significantly higher by 12 mmHg in 21-wk-old FHHrats than the corresponding values observed in the FHH.1BN

congenic strains.Baseline proteinuria was already twice as high in 12-wk-old

FHH rats as that measured in either of the FHH.1BN congenicstrains (Fig. 4B). The degree of proteinuria increased signifi-cantly to 397 � 29 mg/day in 21-wk-old FHH rats. In contrast,proteinuria increased significantly less to only 139 � 21mg/day in the control AR� strain, and it did not increasesignificantly over the 9-wk course of the study in the AR�

strain.A comparison of changes in Palb in FHH rats and the

FHH.1BN congenic strains is presented in Fig. 5. Palb wassignificantly lower in both the 12- and 21-wk-old AR� con-genic strain compared with the corresponding values seen inFHH rats and the control AR� congenic strain.

A comparison of the degree of renal injury in 21-wk-old FHHrats and the FHH.1BN congenic strains is presented in Fig. 6. The

kidneys from FHH rats and the control AR� strain exhibitedsevere renal injury with glomerulosclerosis, renal interstitialfibrosis, tubular necrosis, inflammation, and the formation ofprotein casts. The degree of renal injury was markedly reducedin the AR� strain (Fig. 6A). The glomerular injury score (Fig.6B) and the percentage of renal fibrosis (Fig. 6C) was signif-icantly higher in FHH rats than in both FHH.1BN congenicstrains, and the degree of glomerular injury and renal fibrosiswas significantly greater in the control AR� strain than in theAR� strain.

Protocol 4. Filtration of FITC-labeled albumin in the kid-neys of FHH rats and the AR� strain. The results of theseexperiments are presented in Fig. 7. FITC-labeled albumin waspresent in glomerular capillaries of FHH rats and the AR�

strain. High levels of albumin were filtered and found in thelumen of many proximal tubules in the kidneys of FHH rats(Fig. 7, A-D). In contrast, high levels of albumin were notreadily apparent in the lumen of proximal tubules in the AR�

strain.Protocol 5. Comparison of protein excretion, Palb, and levels

of TGF-�1 and MMP-2 in 6- to 9-wk-old FHH rats andFHH.1BN congenic strains. The results of these experimentsare presented in Fig. 8. The excretion of protein was similar in6-wk-old FHH rats and the FHH.1BN congenic strains (Fig.8A). Protein excretion increased significantly in all three strains

Fig. 6. Comparison of the renal histopathology (A), glomerular injury scores (B), and percentage renal interstitial fibrosis (C) in FHH and FHH.1BN congenicrats at 21 wk of age. Nos. in parentheses indicate the no. of rats studied/group. †Significant difference (P � 0.05) from the corresponding value in FHH rats.#Significant difference (P � 0.05) from the corresponding value in the control AR� congenic strain.




Dow

nloaded from


over the course of the study, but it was twofold higher in9-wk-old FHH rats compared with the corresponding valuesseen in either of the FHH.1BN congenic strains. Consistent withthese findings, Palb was similar in 6-wk-old FHH rats and theFHH.1BN congenic strains (Fig. 8B). Palb increased signifi-cantly in both the FHH and the control AR� strain as these ratsaged from 6 to 9 wk old. In contrast, Palb did not increase in theAR� strain in which autoregulation is intact, and it remainedsignificantly lower than the corresponding values seen in eitherFHH rats or the control AR� strain.

A comparison of the expression of TGF-�2 and MMP-2protein in the renal cortex of 6- and 9-wk-old FHH and theAR� strain is presented in Fig. 9. The expression of TGF-�2protein was not different in the renal cortex of 6-wk-old FHHrats and the AR� strain (Fig. 9A). However, the expression ofTGF-�2 protein increased and was significantly elevated in9-wk-old FHH rats compared with values seen in the AR�

strain. Similarly, the levels of MMP-2 protein in the renalcortex were also significantly greater in 6- and 9-wk-old FHHrats than the levels seen in the AR� strain (Fig. 9B).

DISCUSSION

Previous studies have indicated that FHH rats develop focalglomerulosclerosis and progressive proteinuria that leads toend-stage renal disease (12, 15–16, 25, 28–31, 34). However,the genes and mechanisms involved remain to be determined.Recently, we reported that autoregulation of RBF was impairedin FHH rats and that transfer of a 12.9-Mb region of chromo-some 1 containing 83 genes from the BN rat onto the FHHgenetic background in a congenic strain restored autoregulationof RBF and attenuated the development of proteinuria andrenal injury (12). In the present study, we phenotyped a newFHH.1BN congenic strain that narrows the region of interest forautoregulation of RBF from 12.9 to 4.3 Mb. Transfer of the

region of chromosome 1 from markers D1Rat20G17B-6 toD1Rat888 from BN into the FHH genetic background restoredautoregulation of RBF in the AR� strain relative to the im-paired autoregulation of RBF seen in both FHH rats and thecontrol AR� strain, which is genetically identical to the AR�

strain except for the region of interest. This finding decreasesthe number of genes in the candidate region for autoregulationof RBF from 83 to 23 genes, of which only 11 have any knownfunction. There are only three candidate genes in this regionthat have been reported to affect cardiovascular function. Theyare soluble aminopeptidase 1 (Xpnpep1), adducin 3 (Add3),and dual-specificity phosphatase (Dusp5). However, furthersequencing, expression, and transgenic studies will be neededto determine which of these potential positional candidategenes contributes to the vascular dysfunction in FHH rats.

The purpose of the present study was to determine what role,if any, alterations in RBF autoregulation play in the develop-ment of proteinuria and renal disease in FHH rats by compar-ing the time course changes in MAP, protein excretion, andPalb in FHH rats and two genetically similar FHH.1BN congenicstrains that differ only by the inclusion (AR� strain) or exclu-sion (control AR� strain) of a 4.3-Mbp region of BN chromo-some 1 that restores autoregulation of RBF. We found that Pgcincreased in proportion to the rise in RBF in FHH rats as RPPincreased from 100 to 150 mmHg. In contrast, Pgc increasedby only 3 mmHg in the AR� strain. This finding is consistentwith the view that the control of preglomerular vascular resis-tance is impaired in FHH rats (31). GFR more than doubledwhen RPP was increased from 100 to 150 mmHg in FHH rats.This finding is consistent with a marked increase in net filtra-tion pressure, which was estimated to increase from 5 to 35mmHg when RPP was increased. In addition, it is likely a risein that glomerular capillary filtration coefficient also rises andcontributes to the elevation in GFR in FHH rats when RPP is

Fig. 7. Representation example showing thefiltration of FITC-labeled albumin in the kid-ney of FHH and FHH.1BN AR� congenicstrains between 15 and 21 wk of age. Mag-nification equals 100 in A and B and 400in C and D.




Dow

nloaded from


elevated due to an increase in the length of the glomerularcapillaries available for filtration before filtration pressureequilibrium is achieved.

Proteinuria and Palb were similar in all the strains at 6 wk ofage. However, Palb increased significantly in both the FHH ratsand the control AR� strain by the time the rats were 9 wk ofage. In contrast, Palb did not increase in the AR� strainthroughout the 15-wk study in which autoregulation of RBFand Pgc is intact. Damage to the glomerular permeabilitybarrier in the FHH rats was confirmed in vivo by showing thatthe filtration of FITC-labeled protein was greater in 12-wk-oldFHH rats than that seen in the kidneys of the AR� congenicstrain. This indicates that the increase in Palb and proteinuria islinked to the lack of RBF autoregulation, and the critical timewindow for the onset of renal injury in FHH rats is between 6and 9 wk of age, which clearly precedes the development ofhypertension. Both the FHH and control AR� rats developedproteinuria, glomerulosclerosis, and renal interstitial fibrosis.The degree of renal injury was significantly less in the 21-wk-old AR� strain relative to the levels seen in FHH rats or thegenetically similar control AR� strain that does not autoregu-late RBF.

Interestingly, protein excretion was significantly lower inthe control AR� strain compared with FHH rats. This is notdue to alterations in renal hemodynamics, since this strainfailed to autoregulate RBF, and Palb was elevated to thesame extent in 9-, 12-, and 21-wk-old FHH rats and thecontrol AR� strain. Moreover, the control AR� strain stillexhibited about the same degree of glomerulosclerosis asFHH rats although the degree of renal interstitial fibrosiswas not as severe. The reason as to why the protein excre-tion in the FHH rats may be higher than that seen in thecontrol AR� strain is that the control AR� strain carries aregion of BN chromosome 1 from D1Rat210 to D1Rat47,which contains the Rf-2 QTL. In this regard, Rangel-Filho etal. (19) reported that there is an A to G mutation at the startsite of transcription of the Rab38 gene in FHH rats thatknockout the expression of this protein. This gene is thoughtto regulate the reuptake of filtered protein in the proximaltubule. Thus protein excretion may be lower in the controlAR� strain relative to FHH rats due to increased reuptakeand processing of filtered protein by the proximal tubule.

The mechanism by which elevations in Pgc contribute toelevations in Palb and glomerular disease remains to be deter-mined. However, previous studies have indicated the renallevels of TGF-� and MMP are elevated in several models ofglomerular disease (1–3, 6–7, 9–10, 13, 22–23). Both TGF-�and MMP are associated with the development of EMT,glomerulosclerosis, and renal interstitial fibrosis (2, 4–5, 26,33, 36–37). Indeed, TGF-� promotes the synthesis of extra-cellular matrix proteins such as fibronectin and collagen infibroblasts, podocytes, and renal tubular and mesangial cells.Previous studies have also linked TGF-� to the disruption ofthe glomerular permeability barrier causing glomeruli to be-come leaky to protein (6, 24). In the current study, we foundthat the levels of TGF-� in the renal cortex were elevated in9-wk-old FHH rats compared with the levels seen in the AR�

strain. Interestingly, MMP-2 levels were also increased as earlyas 6 wk of age in FHH rats. MMP-2 is thought to promote therelease of TGF-� from the large latent associated proteinbound to extracellular matrix (4, 14, 27, 32). Recent studieshave also indicated that TGF-� and MMP-2 are involved in thedevelopment of EMT leading to the development of chronicrenal injury (2, 4–5, 26, 33, 36–37). Collectively, these datasuggest that the lack of autoregulation in FHH rats, whichincreases transmission of systemic pressure to the glomer-ulus, may trigger elevated production of MMP-2 that in-creases the release of the active form of TGF-� in theglomerulus. The upregulation of the expression of TGF-�then disrupts the glomerular filtration barrier allowing glo-meruli to leak protein into the tubular lumen stimulating theproduction of MMP-2 and TGF-� in the renal tubules. Overtime, this process could promote EMT and accelerate thedevelopment of glomerulosclerosis and renal interstitialfibrosis in FHH rats and the control AR� strain that exhibitimpaired autoregulation of RBF.

Perspectives

The results from the present study indicate that transfer of a4.3-Mb region of BN chromosome 1 containing just 13 genesrestores the autoregulation of RBF and delays the developmentof renal injury in FHH rats. It also indicates that the lack of

Fig. 8. Comparison of protein excretion and glomerular Palb in 6- and 9-wk-oldFHH and FHH.1BN congenic strains. Nos. in parentheses indicate the no. ofrats studied/group at each time period. *Significant difference (P � 0.05) fromthe corresponding value at 6 wk of age within a strain. †Significant difference(P � 0.05) from the corresponding value in FHH rats.




Dow

nloaded from


autoregulation of RBF in FHH rats may contribute to the earlyonset of renal disease by causing glomerular hyperfiltrationand increasing Pgc. The rise in Pgc is associated with theupregulation of MMP-2 levels, which increase the expres-sion of TGF-�. TGF-� increases glomerular permeabilityand eventually leads to glomerulosclerosis and renal inter-stitial fibrosis. The current data also suggest that this cas-cade of events happens before the development of hyper-tension in FHH rats.

GRANTS

This work was supported, in part, by National Institutes of Health GrantsHL-36279, HL-29587, HL-2958727S1, and DK-79306 and a United NegroCollege Fund/Merck Postdoctoral Science Research Fellowship awarded to J.Williams.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

Fig. 9. Comparison of the expression of transforminggrowth factor (TGF)-�2 protein (A) and the levels ofmatrix metalloproteinase (MMP)-2 (B) in the renal cortexof 6- and 9-wk-old FHH and FHH.1BN AR� congenicstrains. Nos. in parentheses indicate the no. of rats studied/group. *Significant difference (P � 0.05) from the corre-sponding value in 6-wk-old rats within a strain. †Signifi-cant difference (P � 0.05) from the corresponding value inthe FHH rats.




Dow

nloaded from


REFERENCES

1. Bolbrinker J, Markovic S, Wehland M, Melenhorst WB, van Goor H,Kreutz R. Expression and response to angiotensin-converting enzymeinhibition of matrix metalloproteinases 2 and 9 in renal glomerular damagein young transgenic rats with renin-dependent hypertension. J PharmacolExp Ther 316: 8–16, 2006.

2. Bottinger EP, Bitzer M. TGF-beta signaling in renal disease. J Am SocNephrol 13: 2600–2610, 2002.

3. Camp TM, Smiley LM, Hayden MR, Tyagi SC. Mechanism of matrixaccumulation and glomerulosclerosis in spontaneously hypertensive rats. JHypertens 21: 1719–1727, 2003.

4. Cheng S, Lovett DH. Gelatinase A (MMP-2) is necessary and sufficientfor renal tubular cell epithelial-mesenchymal transformation. Am J Pathol162: 1937–1949, 2003.

5. Cheng S, Pollock AS, Mahimkar R, Olson JL, Lovett DH. Matrixmetalloproteinase 2 and basement membrane integrity: a unifying mech-anism for progressive renal injury. Faseb J 20: 1898–1900, 2006.

6. Dahly-Vernon AJ, Sharma M, McCarthy ET, Savin VJ, LedbetterSR, Roman RJ. Transforming growth factor-beta, 20-HETE interaction,and glomerular injury in Dahl salt-sensitive rats. Hypertension 45: 643–648, 2005.

7. Dahly AJ, Hoagland KM, Flasch AK, Jha S, Ledbetter SR, RomanRJ. Antihypertensive effects of chronic anti-TGF� antibody therapy inDahl S rats. Am J Physiol Regul Integr Comp Physiol 283: R757–R767,2002.

8. Datta YH, Wu FC, Dumas PC, Rangel-Filho A, Datta MW, Ning G,Cooley BC, Majewski RR, Provoost AP, Jacob HJ. Genetic mappingand characterization of the bleeding disorder in the fawn-hooded hyper-tensive rat. Thromb Haemost 89: 1031–1042, 2003.

9. Harendza S, Schneider A, Helmchen U, Stahl RA. Extracellular matrixdeposition and cell proliferation in a model of chronic glomerulonephritisin the rat. Nephrol Dial Transplant 14: 2873–2879, 1999.

10. Kuroda T, Yoshida Y, Kamiie J, Kovalenko P, Nameta M, FujinakaH, Yaoita E, Endo T, Ishizuka S, Nakabayashi K, Yamada A, Naga-sawa T, Yamamoto T. Expression of MMP-9 in mesangial cells and itschanges in anti-GBM glomerulonephritis in WKY rats. Clin Exp Nephrol8: 206–215, 2004.

11. Loftus SK, Larson DM, Baxter LL, Antonellis A, Chen Y, Wu X,Jiang Y, Bittner M, Hammer JA 3rd, Pavan WJ. Mutation of melano-some protein RAB38 in chocolate mice. Proc Natl Acad Sci USA 99:4471–4476, 2002.

12. Lopez B, Ryan RP, Moreno C, Sarkis A, Lazar J, Provoost AP, JacobHJ, Roman RJ. Identification of a QTL on chromosome 1 for impairedautoregulation of RBF in fawn-hooded hypertensive rats. Am J PhysiolRenal Physiol 290: F1213–F1221, 2006.

13. Luft FC. Transforming growth factor beta-angiotensin II interaction:implications for cardiac and renal disease. J Mol Med 77: 517–518, 1999.

14. Maeda S, Dean DD, Gomez R, Schwartz Z, Boyan BD. The first stageof transforming growth factor beta1 activation is release of the large latentcomplex from the extracellular matrix of growth plate chondrocytes bymatrix vesicle stromelysin-1 (MMP-3). Calcif Tissue Int 70: 54–65, 2002.

15. Mattson DL, Dwinell MR, Greene AS, Kwitek AE, Roman RJ, CowleyAW Jr, Jacob HJ. Chromosomal mapping of the genetic basis ofhypertension and renal disease in FHH rats. Am J Physiol Renal Physiol293: F1905–F1914, 2007.

16. Mattson DL, Kunert MP, Roman RJ, Jacob HJ, Cowley AW Jr.Substitution of chromosome 1 ameliorates L-NAME hypertension andrenal disease in the fawn-hooded hypertensive rat. Am J Physiol RenalPhysiol 288: F1015–F1022, 2005.

17. Oiso N, Riddle SR, Serikawa T, Kuramoto T, Spritz RA. The rat Ruby(R) locus is Rab38: identical mutations in Fawn-hooded and Tester-Moriyama rats derived from an ancestral Long Evans rat sub-strain.Mamm Genome 15: 307–314, 2004.

18. Raij L, Chiou XC, Owens R, Wrigley B. Therapeutic implications ofhypertension-induced glomerular injury. Comparison of enalapril and acombination of hydralazine, reserpine, and hydrochlorothiazide in anexperimental model. Am J Med 79: 37–41, 1985.

19. Rangel-Filho A, Sharma M, Datta YH, Moreno C, Roman RJ,Iwamoto Y, Provoost AP, Lazar J, Jacob HJ. RF-2 gene modulates

proteinuria and albuminuria independently of changes in glomerular per-meability in the fawn-hooded hypertensive rat. J Am Soc Nephrol 16:852–856, 2005.

20. Savin VJ, Sharma R, Lovell HB, Welling DJ. Measurement of albuminreflection coefficient with isolated rat glomeruli. J Am Soc Nephrol 3:1260–1269, 1992.

21. Semple SJ, De Wardener HE. Effect of increased renal venous pressureon circulatory autoregulation of isolated dog kidneys. Circ Res 7: 643–648, 1959.

22. Sharma K, McGowan TA. TGF-beta in diabetic kidney disease: role ofnovel signaling pathways. Cytokine Growth Factor Rev 11: 115–123,2000.

23. Sharma K, Ziyadeh FN. Renal hypertrophy is associated with upregula-tion of TGF-�1 gene expression in diabetic BB rat and NOD mouse. AmJ Physiol Renal Fluid Electrolyte Physiol 267: F1094–F1101, 1994.

24. Sharma R, Khanna A, Sharma M, Savin VJ. Transforming growthfactor-beta1 increases albumin permeability of isolated rat glomeruli viahydroxyl radicals. Kidney Int 58: 131–136, 2000.

25. Simons JL, Provoost AP, Anderson S, Troy JL, Rennke HG, Sand-strom DJ, Brenner BM. Pathogenesis of glomerular injury in the fawn-hooded rat: early glomerular capillary hypertension predicts glomerularsclerosis. J Am Soc Nephrol 3: 1775–1782, 1993.

26. Stahl PJ, Felsen D. Transforming growth factor-beta, basement mem-brane, and epithelial-mesenchymal transdifferentiation: implications forfibrosis in kidney disease. Am J Pathol 159: 1187–1192, 2001.

27. Sun SZ, Wang Y, Li Q, Tian YJ, Liu MH, Yu YH. Effects of benazeprilon renal function and kidney expression of matrix metalloproteinase-2 andtissue inhibitor of metalloproteinase-2 in diabetic rats. Chin Med J (Engl)119: 814–821, 2006.

28. van Dokkum RP, Jacob HJ, Provoost AP. Blood pressure and thesusceptibility to renal damage after unilateral nephrectomy and L-NAME-induced hypertension in rats. Nephrol Dial Transplant 15: 1337–1343,2000.

29. van Dokkum RP, Jacob HJ, Provoost AP. Difference in susceptibility ofdeveloping renal damage in normotensive fawn-hooded (FHL) and August Copenhagen Irish (ACI) rats after N(omega)-nitro-L-arginine methyl esterinduced hypertension. Am J Hypertens 10: 1109–1116, 1997.

30. Van Dokkum RP, Jacob HJ, Provoost AP. Genetic differences defineseverity of renal damage after L-NAME-induced hypertension in rats. JAm Soc Nephrol 9: 363–371, 1998.

31. van Dokkum RP, Sun CW, Provoost AP, Jacob HJ, Roman RJ.Altered renal hemodynamics and impaired myogenic responses in thefawn-hooded rat. Am J Physiol Regul Integr Comp Physiol 276: R855–R863, 1999.

32. Wang M, Zhao D, Spinetti G, Zhang J, Jiang LQ, Pintus G, Monti-cone R, Lakatta EG. Matrix metalloproteinase 2 activation of transform-ing growth factor-beta1 (TGF-beta1) and TGF-beta1-type II receptorsignaling within the aged arterial wall. Arterioscler Thromb Vasc Biol 26:1503–1509, 2006.

33. Wang SN, LaPage J, Hirschberg R. Role of glomerular ultrafiltration ofgrowth factors in progressive interstitial fibrosis in diabetic nephropathy.Kidney Int 57: 1002–1014, 2000.

34. Weichert W, Paliege A, Provoost AP, Bachmann S. Upregulation ofjuxtaglomerular NOS1 and COX-2 precedes glomerulosclerosis in fawn-hooded hypertensive rats. Am J Physiol Renal Physiol 280: F706–F714,2001.

35. Williams JM, Sharma M, Anjaiahh S, Falck JR, Roman RJ. Role ofendogenous CYP450 metabolites of arachidonic acid in maintaining theglomerular protein permeability barrier. Am J Physiol Renal Physiol 293:F501–F505, 2007.

36. Yard BA, Chorianopoulos E, Herr D, van der Woude FJ. Regulationof endothelin-1 and transforming growth factor-beta1 production in cul-tured proximal tubular cells by albumin and heparan sulphate glycosami-noglycans. Nephrol Dial Transplant 16: 1769–1775, 2001.

37. Zeisberg M, Bonner G, Maeshima Y, Colorado P, Muller GA, StrutzF, Kalluri R. Renal fibrosis: collagen composition and assembly regulatesepithelial-mesenchymal transdifferentiation. Am J Pathol 159: 1313–1321, 2001.




Dow

nloaded from


call for papers renal hemodynamics - semantic scholar · 2017-06-17 · 19 2 mmhg in fhh rats when...

Documents