Kidney International, Vol. 63 (2003), pp. 607–616

VASCULAR BIOLOGY – HEMODYNAMICS – HYPERTENSION

Low protein diet mediated renoprotection in remnant kidneys:Renal autoregulatory versus hypertrophic mechanisms

KAREN A. GRIFFIN, MARIA PICKEN, ANITA GIOBBIE-HURDER, and ANIL K. BIDANI

Departments of Internal Medicine, Pathology, and Cooperative Studies Program Coordinating Center, Loyola UniversityMedical Center; and Edward Hines, Jr., Hospital, Maywood and Hines, Illinois

Low protein diet mediated renoprotection in remnant kidneys: by uninephrectomy combined with infarction of 2/3 ofRenal autoregulatory versus hypertrophic mechanisms. the other kidney, is characterized by adaptive increases

Background. The mechanism of low protein diet conferred in size and function of the remnant nephrons, which isrenoprotection in the ablation model remains controversial.followed by the development of proteinuria and progres-Blockade of glomerular hypertrophy, reduced preglomerular va-

sodilation, and preserved autoregulation have all been postulated. sive sclerosis of the initially normal remnant glomeruliThe potential differential impact of calcium channel blockers [1–6]. Substitution of a low protein for the standard pro-on these mechanisms and glomerulosclerosis was examined. tein diet has been demonstrated to substantially preventMethods. Rats with 5/6 renal ablation received either a 25%

the progressive glomerulosclerosis in this model [1–6].standard protein diet, an 8% low protein diet and a low proteindiet with either verapamil or amlodipine. Renal autoregulatory However, the mechanisms responsible for this renopro-and morphometric studies were performed at 3 weeks before tection remain controversial. Some investigators havethe development of significant injury, and the assessment of ascribed a primary protective importance to the inhibi-glomerulosclerosis after 7 weeks of continuous blood pressure

tory effects of a low protein diet on the compensatoryradiotelemetry in additional rats.Results. The preserved renal autoregulation in low protein hypertrophic responses to renal mass reduction and the

rats was abolished by both calcium channel blockers, with the associated increased expression of growth factors [7–10].impairment being either comparable to (low protein � vera- Others have attributed the protection to the concurrentpamil) or greater than the standard protein rats (low protein �

amelioration of the “maladaptive” hemodynamic changesamlodipine). Neither calcium channel blocker blocked the in-hibitory effects of low protein diet on renal blood flow, kidney and hyperfiltration [1, 3–6]. We have postulated a pri-weight, and glomerular volume. Results (mean � SE) for glo- mary hypertensive pathogenesis of glomerular injury inmerular volume (�m�3 � 10�6): low protein (N � 11), 1.6 � the 5/6 ablation model and suggested that a low protein0.1; low protein � verapamil (N � 10), 1.7 � 0.1; low protein �

diet provides protection in this model by preventing theamlodipine (N � 12), 1.7 � 0.2; versus standard protein (N �10), 2.2 � 0.1; P � 0.05. Only amlodipine, but not verapamil, impairment of renal autoregulation and the consequentreduced average systolic blood pressure (143 � 2 mm Hg versus enhanced glomerular transmission of systemic hyperten-low protein rats, 168 � 5 mm Hg, and standard rats, 170 � 6 sion observed in the 5/6 ablated rats fed a standard pro-mm Hg; P � 0.01). Nevertheless, the glomeruloprotection seen

tein diet [11]. We have also demonstrated that calciumin low protein (N � 15) as compared to standard protein (N � 14)rats (9% � 3% versus 28% � 6% glomerulosclerosis; P � 0.01) channel blockers further impair the already impaired renalwas abolished in both low protein � verapamil (N � 14, 32% � autoregulation in standard protein diet–fed rats and7%) and low protein � amlodipine rats (N � 16, 27% � 7%). thereby cause further enhancement of glomerular bloodConclusions. Preservation of renal autoregulation and not

pressure transmission and injury at any given blood pres-inhibition of hypertrophy is the critical component in low pro-tein diet-conferred glomeruloprotection. sure elevation in this model [12–14]. The present studies

were undertaken to examine if the adverse effects ofcalcium channel blockers on renal autoregulation would

The rat remnant kidney model has been widely used dissociate the effects of a low protein diet on glomerularto investigate the progressive nature of chronic human hypertrophy and renal autoregulation and thus allow anrenal disease [1–6]. The hypertensive model, produced assessment of their relative importance in the low protein

diet conferred renoprotection in this model.Key words: glomerulosclerosis, remnant kidney, autoregulation.

Received for publication June 19, 2002 METHODSand in accepted form August 16, 2002Accepted for publication September 12, 2002 Two sets of investigations were performed in male

Sprague-Dawley rats (initial body weight 200 to 300 g) 2003 by the International Society of Nephrology

607

Griffin et al: Low protein diet protection in remnant kidney608

that were fed isocaloric diets with either a 25% standard studies, the kidneys were perfusion-fixed, harvested,weighed, and processed for morphometric studies, andprotein or an 8% low protein diet (Harland Teklad; Madi-

son, WI, USA), with or without the additional adminis- the rats were euthanized.tration of the calcium channel blockers, verapamil and

Blood pressure radiotelemetry studies for �7 weeksamlodipine, to the low protein diet–fed rats. The ratswere synchronized to a 12:12 hour light (6:00 to 18:00 For these studies, rats underwent 5/6 ablation as de-

scribed above for renal autoregulatory studies but werehour) and dark (18:00 to 6:00 hour) cycle and receivedfood and water ad libitum throughout the study. Studies additionally prepared for telemetric monitoring of blood

pressure (Data Science International, St. Paul, MN, USA)were performed at �3 weeks after renal ablation to as-sess the effects on renal hemodynamics, autoregulation, at the time of the renal ablation surgery, as previously

described [12–14]. Each rat had a blood pressure sensorand structural hypertrophy responses before the devel-opment of significant renal injury and at 7 weeks to assess (model TA11PA-C40) inserted into the aorta below the

level of the renal arteries, and the radio frequency trans-the severity of glomerulosclerosis and the relationshipbetween continuous radiotelemetrically measured blood mitter was fixed to the peritoneum. The rats were housed

individually in plastic cages that were placed on top ofpressure and glomerulosclerosis in the different experi-mental groups. the receiver. The signals from the pressure sensor were

converted, temperature compensated, and sent via theAutoregulatory and morphometric studies at 3 weeks radio frequency transmitter to the telemetry receiver.

The receiver was connected to a BCM-100 consolidationRats were anesthetized with sodium pentobarbital (45mg/kg intraperitoneally) and subjected to approximately matrix, which transmitted the information to the Data-

quest IV acquisition system. Systolic blood pressure in5/6 renal ablation (right nephrectomy and ligation of allbut one posterior extrarenal branch of the left renal each animal was continuously recorded at 10-minute in-

tervals with each blood pressure reading representingartery), as previously described [12–14]. Tail vein bloodsamples were obtained at 3 days for measurement of the average of �60 readings during a 10-second interval

[12–14]. As for autoregulatory studies, after 7 days, theserum creatinine as an index of the degree of renal massreduction [12–14]. After 7 days, the low protein diet–fed standard protein rats were left untreated and the low

protein diet–fed rats were randomly assigned to an un-rats were randomly assigned to an untreated group orreceived either verapamil (500 mg/L) or amlodipine (200 treated group or received verapamil or amlodipine in

their drinking water. After 7 weeks, tail vein serum creat-mg/L) in drinking water. The standard protein diet ratswere left untreated. At �3 weeks after 5/6 ablation, 24- inine and 24-hour urine collections for protein excretion

were obtained. At the conclusion of these studies, thehour urine protein excretion was measured and the ratswere anesthetized with intravenous sodium pentobarbi- kidneys were harvested for morphologic studies after

perfusion fixation before sacrifice, as previously de-tal (40 mg/kg) and surgically prepared for renal autoregu-latory studies, as previously described [12–14]. In brief, scribed [11–14].a tracheostomy was performed using polyethylene (PE-

Morphologic methods200) tubing, and a carotid artery was cannulated withPE-50 tubing and connected to a Windograf recorder The kidneys were fixed in situ by perfusion for 5 min-

utes at the measured blood pressure with 1.25% glutaral-(model 40-8474; Gould, Inc., Glen Burnie, MD, USA)for continuous recording of mean arterial pressure. A dehyde in 0.1 mol/L cacodylate buffer. Transverse sec-

tions through the papilla were cut at a thickness of �3femoral vein was cannulated with PE-50 tubing and 150mmol/L sodium chloride bolus equal to 1% of the body to 4 �m and stained with hematoxylin and eosin and

periodic acid-Schiff. Sections were evaluated systemati-weight was administered, followed by a continuous main-tenance infusion of 150 mmol/L sodium chloride at 0.055 cally in each kidney for glomerular injury (segmental

sclerosis and/or necrosis) in a blinded fashion by stan-mL/min for replacement of surgical and ongoing fluidlosses. A 1.0 mm R series flow probe (Transonic Systems, dard morphologic methods. At least 100 glomeruli in

each animal were evaluated, and the severity of injuryInc., Ithaca, NY, USA) was placed around the left renalartery for measurement of renal blood flow (RBF) by a was expressed as the percentage of glomeruli exhibiting

such lesions (% GS).flow meter, as described previously [12–14]. After themeasurement of RBF at the ambient mean arterial pres-

Morphometric methodssures, RBF autoregulatory studies were performed usingaortic miniclamps positioned above and below the left Glomerular volume was measured by area perimeter

analysis (Bioquant System IV software; R&M Biomet-renal artery to raise or lower renal perfusion pressure(RPP), as previously described. The RBF was allowed to rics, Inc., Nashville, TN, USA). The glomular cross-sec-

tional area (AG) of 75 consecutive glomerular profilesstabilize for 1 to 2 minutes at each pressure before RBFmeasurements were made. At the conclusion of these without evidence of injury contained in one kidney sec-

Griffin et al: Low protein diet protection in remnant kidney 609

tion for each animal was measured using a digitizing pad,as described previously [12, 13]. The mean glomerularvolume (VG) was then calculated from the respectivemean AG as VG � �/ (AG3/2), where � � 1.398 is thesize distribution coefficient and � 1.1 is the shapecoefficient for glomeruli idealized as spheres [15].

Analyses, calculations, and statistics

Urinary protein was measured by the quantitative sul-fosalicylic acid method, with human serum albumin serv-ing as the standard and serum creatinine was measuredusing a creatinine analyzer (Beckman Instruments, Inc.,Fullerton, CA, USA) [11–14]. Autoregulatory index (AI)was calculated by the method of Semple and de Wardeneras follows: AI � [(RBF2 � RBF1)/RBF1]/[RPP2 � RPP1)/RPP1] [16]. An AI of 0 indicates perfect autoregulation,whereas an AI of 1 indicates that the vessels act as passiveconduits for blood flow. Statistical analysis was performedusing analysis of variance, followed by Student-Newman-Keuls test or by Kruskal-Wallis nonparametric analysisof variance followed by Dunn’s multiple comparisontests, as appropriate [17]. Linear regression analysis wasused to calculate the slopes and intercepts of the relation-ship between blood pressure and glomerulosclerosis ineach group. Analysis of covariance was used to comparethe slopes and intercepts between the groups [17]. A Pvalue of less than 0.05 was considered statistically sig-nificant. All results are expressed as mean � SEM.

RESULTS

Autoregulatory and morphometric studies at 3 weeks

Table 1 provides the baseline body weight and serumcreatinine, serum creatinine at 3 days, and the final renalfunctional parameters at 3 weeks for the animals under-going autoregulatory studies. No significant differenceswere present between the groups for the initial body weightand serum creatinine or for serum creatinine at 3 days.However, at 3 weeks, body weights and proteinuria weresignificantly lower in all three low protein diet–fed groupsas compared to standard protein diet–fed rats. AmbientAP under anesthesia was significantly lower in the am-lodipine, but not the verapamil-treated low protein diet–fed rats as compared to both low protein and standardprotein untreated groups, which were not significantlydifferent from each other. By contrast, RBF was signifi-cantly lower in all three groups of low protein diet–fedrats as compared to the standard protein diet–fed rats.

The results of the RBF autoregulation studies are shownin Figure 1. Due to difficulty in achieving the necessarystep increases in renal perfusion pressures in 1/11 lowprotein, 1/10 low protein � verapamil and 4/12 lowprotein � amlodipine rats, data are only presented forthe remaining rats in each group in whom autoregulatory

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Griffin et al: Low protein diet protection in remnant kidney610

Fig. 2. Kidney weights and glomerular volume at 3 weeks after ablationin rats. Rats had received either a standard (�; N �10) or low protein

Fig. 1. Renal blood flow autoregulatory studies at 3 weeks after 5/6 (�; N � 11) diet. After 7 days, the low protein diet–fed rats were leftrenal ablation in rats. Rats had received either 25% standard protein untreated or had received either verapamil (low protein � verapamil)(SP) (�; N � 10) or an 8% low protein (LP) (�; N � 10) diet. After ( ; N � 10) or amlodipine (low protein � amlodipine) ( ; N � 12)day 7, the LP diet–fed rats were left untreated or had additionally in their drinking water. Kidney weight and glomerular volume werereceived either verapamil (V) (LP � V) (�; N � 9) or amlodipine (A) significantly lower in all three low protein diet–fed groups as compared(LP � A) (*; N � 8) in their drinking water. Graded changes in renal to the standard protein diet–fed rats, *P � 0.05 maximum.perfusion pressure were produced by aortic miniclamps and resultedin significant changes in RBF with every change in renal perfusionpressure in SP, LP � V, and LP � A, but not in LP rats (P � 0.05maximum). The bottom of the figure shows the calculated autoregula-tory indices in the changes in renal blood flow for perfusion pressure and glomeruli volumes in the two calcium channel blocker–changes in the range between 150 and 100 mm Hg in the different

treated low protein diet–fed rats were not significantlygroups. The autoregulation indices for the two-step changes in RPPwere not significantly different from each other; therefore, a single different from the untreated low protein rats or fromautoregulation index for the renal blood flow change between 150 and each other but were significantly lower than in the stan-100 mm Hg is presented. The autoregulation index was significantly

dard protein rats.lower in LP and significantly higher in the LP � A compared to theLP � V or SP groups (P � 0.05 maximum).

Blood pressure radiotelemetry studies for 7 weeks

Table 2 shows that the initial body weight, serum creat-inine, and 24-hour urine protein excretion for the fourtory capacity was preserved in the untreated low proteingroups were not significantly different from each other.rats. By contrast, the standard protein rats demonstratedSimilarly, the serum creatinine at 3 days was not signifi-significant impairment in RBF autoregulation, that is, therecantly different between the groups, indicating compara-were significant changes in RBF with each change inble renal mass reduction in all groups. The final bodyrenal perfusion pressure. The concurrent administrationweight was significantly lower in the verapamil-treatedof both calcium channel blockers to low protein diet–fedrats as compared to the low protein and standard proteinrats resulted in significant impairment in renal autoregu-groups. The amlodipine-treated rats had a final bodylation. In the case of verapamil, the degree of impairmentweight that was significantly lower than the standardwas similar to that seen in untreated standard proteinprotein but not the untreated low protein rats.diet–fed rats as indicated by the lack of significant differ-

Figure 3 shows the course of weekly averages of radio-ences in the calculated autoregulatory indices betweentelemetrically monitored systolic blood pressure afterthem. In contrast, RBF autoregulation was even more�5/6 renal ablation in the four groups of rats over 7severely impaired by amlodipine, such that the amlodi-weeks. For the first week before the initiation of amlodi-pine-treated low protein diet–fed rats exhibited an auto-pine or verapamil therapy, the blood pressure was similarregulatory index of �1.0, indicating a complete loss ofin all four groups. There were no significant differencesautoregulatory vascular responses in this group.in systolic blood pressure between the standard proteinFigure 2 presents the results of the assessment of theand low protein diet–fed rats during the entire 7 weekseffects of dietary protein content and calcium channelof observation. The initiation of treatment with bothblockers on the renal hypertrophy response parameters.amlodipine and verapamil significantly reduced systemicAt 3 weeks after renal ablation, the kidney weight (gramblood pressure, but the antihypertensive effects tendedper kilogram body weight) and glomeruli volume wereto wane after the first 2 to 3 weeks of therapy, particularlysignificantly lower in the low protein rats as comparedin the verapamil-treated low protein rats whose averageto the standard protein rats. Neither calcium channel

blocker had an effect on these parameters. Kidney weights systolic blood pressure during the final 3 weeks did not

Griffin et al: Low protein diet protection in remnant kidney 611

Fig. 3. Course of systolic blood pressure averages over 7 weeks after5/6 renal ablation in rats. Rats had received a standard protein (�; N �14) or a low protein (�; N � 15) diet. After 7 days, the low proteindiet–fed rats were left untreated or had additionally received eitherverapamil (low protein � verapamil) (�; N � 14) or amlodipine (lowprotein � amlodipine) (�; N � 16) in their drinking water. Bloodpressure was radiotelemetrically recorded continuously at 10-minuteintervals starting at 6 p.m. on the day of renal ablation surgery tosacrifice �7 weeks later; *P � 0.05 maximum versus untreated lowprotein or standard protein diet–fed rats; P � 0.05 maximum versusall other groups; �P � 0.05 maximum versus untreated low proteindiet–fed rats.

Fig. 4. Average systolic blood pressure during the final 6 weeks after5/6 renal ablation and % glomerulosclerosis at 7 weeks in rats. Rats hadreceived either a standard protein (�; N � 10) or a low protein (�; N �15) diet. After day 7, the low protein diet–fed rats were left untreatedor had additionally received either verapamil (low protein � verapamil)( ; N � 10) or amlodipine (low protein � amlodipine) ( ; N � 12)in their drinking water; *P � 0.05 maximum versus all other groups.

differ significantly from that of the untreated standardprotein and low protein rats. This is further illustratedby a comparison of the overall averaged systolic bloodpressure during the final 6 weeks (the mean of all �6000systolic blood pressure readings after day 7) as an indexof average ambient blood pressure load (Fig. 4). This

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Griffin et al: Low protein diet protection in remnant kidney612

Fig. 5. Correlations of percentage of glomeruli with sclerosis at �7 weeks in individual rats with 5/6 ablation in the four groups with their averagesystolic blood pressure during the final 6 weeks. (A) After the first 7 days, when the rats received either a standard protein (�; N � 14) or lowprotein (�; N � 15) diet, the low protein diet–fed rats were left untreated or had received either verapamil (low protein � verapamil) (�; N �14) or amlodipine (low protein � amlodipine) (�; N � 16) in their drinking water. (B) Linear regression analysis of the relationship betweenaverage blood pressure during the final 6 weeks and the % glomerulosclerosis in each of the four groups. Standard protein (SP) rats (�; N � 14),r � 0.70, P � 0.005, slope 0.77 � 0.23, � intercept, 133 mm Hg; low protein (LP) rats (�; N � 15), r � 0.71, P � 0.005, slope 0.39 � 0.11, �intercept, 150 mm Hg; low protein � verapamil (LP � V) (�; N � 14), r � 0.57, P � 0.05, slope 0.89 � 0.37, � intercept, 123 mm Hg; and lowprotein � amlodipine (LP � A) (�; N � 16), r � 0.58, P � 0.02, slope 2.1 � 0.78, � intercept, 130 mm Hg. Both, the � intercept (P � 0.01) andthe slope (P � 0.02) were significantly different for the untreated low protein group with preserved autoregulation when compared to the combineddata of the three groups with impaired autoregulation, but statistical significance for the individual comparisons between low protein and othergroups was only achieved for the difference in the slope between low protein and low protein � amlodipine groups (P � 0.02).

tein rats and, although somewhat lower in the verapamil- and between groups. Glomerulosclerosis decreased withdecreasing blood pressure within each group, althoughtreated low protein rats, the difference was not statisti-

cally significant. By contrast, the amlodipine-treated low the relationship between blood pressure and glomerulo-sclerosis and the strength of the correlation differed be-protein rats had a significantly lower overall average sys-

tolic blood pressure as compared to the other three groups. tween the groups. The stronger correlations were ob-served in the untreated low protein and standard proteinFigure 4 also shows the quantitated histologic glomerular

injury expressed as the percentage of glomeruli exhibiting rats, while the correlations were somewhat weaker inthe calcium channel blocker–treated low protein rats,lesions of segmental sclerosis and/or necrosis. Significant

glomeruloprotection was observed in the untreated low indicating a greater heterogeneity of the relationshipbetween blood pressure and glomeruloscerosis. As com-protein group as compared to the standard protein rats.

However, this glomeruloprotection in the low protein– pared to the other groups, the � intercept was shifted tothe right in the low protein rats and the slope of thetreated rats was abolished in those that had concurrently

received either calcium channel blocker. Glomeruloscle- relationship between blood pressure and glomerulosclero-sis (increase in % glomerulosclerosis/mm Hg increaserosis in these groups at 7 weeks was not significantly differ-

ent than the untreated standard protein rats. A similar in systolic blood pressure) was also flatter. However,because of the scatter in the data, individual comparisonsclear separation was not observed for proteinuria results

(Table 2). While the low protein and standard protein between the low protein and other groups did not reachstatistical significance except for the differences in therats exhibited significant differences in proteinuria, par-

alleling the differences in glomerulosclerosis in the two slope between low protein and low protein � amlodipinerats (P � 0.02). But, if the low protein group (preservedgroups, the values for low protein rats treated with either

verapamil or amlodipine were intermediate between the autoregulation) is compared to combined data of theother groups with impaired autoregulation, both the �low protein and standard protein rats and not signifi-

cantly different from either group. intercept (P � 0.01) and the slope (P � 0.02) weresignificantly different, indicating a lower threshold forFigure 5A shows the correlation of % glomeruli with

the averaged systolic blood pressure in individual ani- glomerular injury and greater glomerulosclerosis at anygiven blood pressure elevation in rats with impaired auto-mals of all groups, while Figure 5B presents the results

of linear regression analysis of this relationship within regulation.

Griffin et al: Low protein diet protection in remnant kidney 613

DISCUSSION differences in blood pressure transmission or the totalglomerular capillary pressure load. In addition, such mea-The provision of low protein diets (�12% proteinsurements are potentially compromised by anesthesia-content) has long been known to ameliorate the progres-induced activation of neurohumoral systems with theirsive glomerular injury and nephron loss that is character-independent impact on segmental vascular resistancesistically observed after severe ( 50%) renal ablation in[21, 25]. Nevertheless, the observed elevated glomerularrats fed a standard protein diet ( 20% protein content)capillary pressure values in standard protein and normal[1–6]. Despite extensive investigations, the mechanismsvalues in low protein diet–fed rats after renal ablationthrough which such renoprotection is conferred remainare consistent with significant differences in glomerularcontroversial. This is at least in part due to the fact thatblood pressure transmission [1, 4–6]. In this context, ita low protein diet has concurrent potentially beneficialis of note that high and low protein diets even in normaleffects on several pathways postulated to be of pathoge-rats produce directionally similar effects on renal auto-netic importance in the development of progressive glo-regulatory mechanisms, although the precise molecularmerulosclerosis in this hypertensive model. These includemediators remain unknown [26, 27].(1) structural glomerular hypertrophy and the associated

The correlations between blood pressure and glomer-increased expression of profibrotic growth factors suchulosclerosis in the present studies provide support foras angiotensin II, platelet-derived growth factor, and trans-both, a predominantly hypertensive pathogenesis of glo-forming growth factor-� (TGF-�) [7–10, 18]; (2) the func-merulosclerosis in this model and the protective impor-tional hemodynamic changes, including renal vasodilation,tance of intact renal autoregulatory mechanisms. Thethat mediate the characteristic glomerular hyperfiltrationsignificant leftward shift of the � intercept indicating ain remnant nephrons [1, 3–6]; and (3) the impairment oflower blood pressure threshold for glomerular injury inrenal autoregulation with the associated enhanced glomer-rats with impaired autoregulation and the steeper slopeular transmission of systemic pressures and hypertensiveof the relationship between blood pressure and glomeru-glomerular injury [11]. Since all of these intrinsic struc-losclerosis (increase in % glomerulosclerosis/mm Hg in-

tural and functional adaptations are simultaneously andcrease in systolic blood presssure) are consistent with such

substantially abrogated by the substitution of a low pro- interpretations [13, 25]. The adverse effects of calciumtein diet, the difficulty in separating these effects has re- channel blockers on renal autoregulation, even in low pro-sulted in continued controversy as to their relative inde- tein diet–fed rats, are not unexpected, given that the auto-pendent pathogenetic importance. regulatory responses initiated by blood pressure increases

The present results suggest that the beneficial effects and the resultant depolarization are critically dependentof a low protein diet on renal autoregulation may be central on calcium entry through voltage gated calcium channelsto its renoprotective ability in this hypertensive model. [28, 29]. The weaker correlation between blood pressureNormally, proportionate preglomerular autoregulatory va- and glomerulosclerosis in calcium channel blocker–treatedsoconstriction in response to increases in systemic blood low protein rats, may in part reflect the greater heteroge-pressure, episodic or sustained, prevent such blood pres- neity in autoregulatory impairment over time in these lowsure increases from being transmitted to the glomerular protein diet–fed rats. The results were particularly notablecapillaries, and renal damage (malignant nephrosclerosis) in the amlodipine-treated rats, who displayed the great-only ensues when blood pressure exceeds the autoregula- est autoregulatory impairment and the steepest relation-tory range [11, 19–21] By contrast, when autoregulatory ship between blood pressure and glomerulosclerosis.mechanisms are impaired (preglomerular vasoconstric- Thus, renoprotection was not achieved despite substan-tion is inadequate to the blood pressure increase), even tial blood pressure reductions. The greater deleteriousmodest increases in systemic blood pressure are likely effects of the dihydropyridine calcium channel blockerto be accompanied by increases in glomerular capillary on renal autoregulation are similar to that observed inpressure [11, 22]. Moreover, given the spontaneous, fre- standard protein diet–fed rats [12–14] and probably stemquent, and large blood pressure fluctuations that are from their binding site being very proximate to the volt-observed in these animals in the unanesthetized state age sensor segment (S4) of the �1 subunit of the calcium[23–25], renal autoregulatory efficiency is likely to be channel [30]. However, in contrast to the present resultsthe primary determinant of the degree to which such in low protein rats, even higher doses of verapamil failedfluctuations reach the glomerular capillaries. Such blood to produce an additional impairment of the already im-pressure lability also substantially limits the validity of paired renal autoregulation in the standard protein diet–interpretations based on conventional tail-cuff blood fed rats [13]. It is possible that the adverse effects ofpressure measurements to assess the renal blood pres- verapamil on renal autoregulation can only be elicitedsure burden [12–14, 23–25]. For the same reason, isolated in states of preserved autoregulation such as normal ratsglomerular capillary pressure measurements under anes- [28, 29] or low protein diet–fed 5/6 ablated rats. Until

the precise cellular and molecular mediators of renalthesia also may not provide an adequate index of the

Griffin et al: Low protein diet protection in remnant kidney614

autoregulatory impairment after renal mass reduction the issue of the pathogenetic contribution of glomerularare identified, the reasons for such differential effects of hypertrophy to glomerulosclerosis. It has been suggestedcalcium channel blockers are likely to remain obscure. that the glomerular hypertrophy response may be mal-However, it is of interest in this context that the adverse adaptive and possibly pathogenetically more importanteffects of calcium channel blockers on renal autoregula- than the hemodynamic alterations, including elevatedtion in the present study were observed without significant glomerular capillary pressure in the development of glo-vasodilation in the low protein diet–fed rats, suggesting merulosclerosis [7–10, 37, 38]. Such postulates are pri-that the “steady-state” vasodilatory and the autoregula- marily based on the commonly observed association be-tory effects of dietary protein may be mediated by differ- tween glomerular hypertrophy and glomerulosclerosisent cellular pathways. Such a dissociation is consistent in both renal mass reduction and diabetic states, and relywith the observations that all renal vasodilators do not on interpretations that have used glomerular capillarynecessarily impair renal autoregulation and, conversely, pressure measurements as a definitive index to assessinterventions that cause renal vasoconstriction and/or the relative contribution of hypertensive versus hyper-reduce vasodilation after renal mass reduction do not trophic mechanisms in the pathogenesis of glomerulo-improve renal autoregulation [19, 29–33]. sclerosis in these models [6–10, 36–41]. As noted earlier,

The lack of significant vasodilatory effects of calcium due to the lability of glomerular pressures parallelingchannel blockers in low protein diet–fed rats may also the lability of systemic pressures in states of impairedbe relevant with respect to the proteinuria results in the autoregulation, such interpretations may not be valid. Inpresent study. Previous studies have indicated that the any event, the precise intermediate mechanisms remaineffects of dietary protein on proteinuria are associated controversial and range from altered growth factor ex-with concurrent and directionally similar changes in RBF pression [7–10] to adverse effects on glomerular capillaryand are thought to be mediated by parallel changes in wall tension as predicted by the Laplace Law (tension �both ambient glomerular capillary pressure and glomeru- pressure � radius) [39–41]. However, in normotensivelar permselectivity [1, 4–6, 34, 35]. The lack of significant models of renal mass reduction that do not exhibit sub-increases in proteinuria at �3 weeks in calcium channel stantial glomerular hypertension, glomerular hypertro-blocker–treated low protein diet-–fed rats before the phy comparable to that in hypertensive models is seendevelopment of significant glomerulosclerosis may there- without concomitant glomerulosclerosis [39, 42, 43]. Con-fore be due to the lack of significant concurrent vasodila- versely, calcium channel blockers impair renal autoregu-tion. But, the lack of consistent increases in proteinuria lation and promote hypertensive glomerular injury with-even at 7 weeks, despite the development of glomerulo- out impacting glomerular hypertrophy in either low proteinsclerosis in the calcium channel blocker–treated low pro-

or standard protein fed rats. Collectively, these data indi-tein rats, suggest that low protein diets may have glomer-

cate that glomerular hypertrophy is neither necessaryulosclerosis-independent effects on proteinuria, eithernor sufficient for the development of glomerulosclerosiswith respect to the glomerular permselectivity or by stim-after renal mass reduction. Such data, however, do notulating postfiltration protein conservation/reabsorptionexclude a permissive role for glomerular hypertrophy inby the renal tubules [34, 35]. Such interpretations are alsomagnifying hypertensive glomerular injury through La-consistent with the observations that it is the uninjuredplace Law effects [39–43]. Such an interpretation can pro-glomeruli that are the source of most of the proteinuriavide an explanation for why better correlations are ob-in this model [36].served between glomerular size and glomerulosclerosisIn contrast to the quantitatively differential effectswithin the kidney of an individual rat where the glomer-of verapamil and amlodipine on renal autoregulation,uli are exposed to a similar pressure profile than betweenneither agent had an effect on glomerular hypertrophy,animals where the glomeruli of different rats are exposedsuggesting separate mediation of these two effects. Into different pressure profiles [38]. Similarly, these dataany event, a notable feature of the present result is thatare also not inconsistent with the postulated role of growththe deleterious effects of calcium channel blockers onfactors as molecular mediators of glomerulosclerosis.the development of glomerulosclerosis in the low proteinHowever, pathologic glomerular hypertension seems todiet–fed rats were observed, despite the continued inhibi-be necessary for the development of glomerulosclerosis,tion of glomerular hypertrophy during the time frameat least in renal mass reduction models [42, 44]. Thewhen glomerular injury starts to develop in this modelunderlying pathogenetic mechanisms and their modifi-(3 to 4 weeks). Our data do not permit any conclusionscations by low protein diets may well be different inas to the possible mediation of the effects of dietaryother normotensive models of renal damage [45]. Forprotein on glomerular hypertrophy beyond the fact thatinstance, it has been suggested that a low protein dietcalcium channel blockers do not alter these responsesprovides protection in experimental glomerulonephritiseither in low protein (present study) or standard protein

diet–fed rats [12, 13]. However, they are relevant to through the concurrent restriction in l-arginine intake

Griffin et al: Low protein diet protection in remnant kidney 615

2. Klahr S, Schreiner G, Ichikawa I: The progression of renal[45], but arginine supplementation has been shown todisease. N Engl J Med 318:1657–1666, 1988

be beneficial in the 5/6 ablation model [46]. 3. Olson JL, Heptinstall RH: Non-immunologic mechanisms ofglomerular injury. Lab Invest 59:564–578, 1988With respect to the clinical implications of these data,

4. Hostetter TH, Olson HJL, Rennke HG, et al: Hyperfiltration init is of relevance that unlike the consistent effectivenessremnant nephrons: A potentially adverse response to renal abla-

of low protein diets in retarding progressive glomerulo- tion. Am J Physiol 241:F85–F93, 19815. Brenner BM, Meyer TW, Hostetter TH: Dietary protein intakesclerosis and nephron loss in rodent models, the benefits

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6. Neuringer JR, Brenner BM: Hemodynamic theory of progressivefor such differences that have been discussed previouslyrenal disease: A 10-year update in brief review. Am J Kidney Dis

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promoters in the development of sclerosis. Semin Nephrol 9:329–consumption may only become quantitatively significant342, 1990

with respect to renal autoregulatory capacity after sub- 8. Ketteler M, Noble NA, Border WA: Transforming growth fac-tor-� and angiotensin II: The missing link from glomerular hyper-stantial loss of functional renal mass. Nevertheless, thefiltration to glomerulosclerosis? Annu Rev Physiol 57:279–295,present data do suggest that to the degree to which a low 1995

protein diet confers protection in chronic human renal 9. Tanaka R, Sugihara K, Tatematsu A, Fogo A: Internephronheterogeneity of growth factors and sclerosis: Modulation of plate-disease, such protection may be abrogated by calciumlet-derived growth factor by angiotensin II. Kidney Int 47:131–139,channel blockers, particularly those of the dihydropyri- 1995

dine class. However, in the clinical context, the adverse 10. Fogo AB: Glomerular hypertension, abnormal glomerular growth,and progression of renal diseases. Kidney Int 57(Suppl 75):S15–S21,effects of calcium channel blockers on renal autoregula-2000tion and blood pressure transmission are counteracted 11. Bidani AK, Schwartz MM, Lewis EJ: Renal autoregulation and

by the blood pressure reduction achieved by calcium vulnerability to hypertensive injury in remnant kidney. Am J Phys-iol 252:1003–1010, 1987channel blockers. The greater the achieved blood pres-

12. Griffin KA, Picken MM, Bidani AK: Deleterious effects of cal-sure reduction, the less would be the deleterious impact cium channel blockade on pressure transmission and glomerularof the adverse effects on renal autoregulation. This may injury in rat remnant kidneys. J Clin Invest 96:793–800, 1995

13. Griffin KA, Picken MM, Bakris GL, Bidani AK: Class differ-account for the rather variable results that have beenences in the effects of calcium blockers in the rat remnant kidneyobserved with calcium channel blockers, both in experi- model. Kidney Int 55:1849–1860, 1999

mental animal models [12, 14] and in clinical trials with 14. Griffin KA, Picken MM, Bakris GL, Bidani AK: Comparativeeffects of T- and L-type calcium channel blockade in the remnantrespect to both proteinuria and glomerular filtration ratekidney model. Hypertension 37:1268–1272, 2001.

loss [49]. Nevertheless, it is of note that several recent 15. Weibel ER: Stereological Methods: Practical Methods for Biologi-cal Morphometry, Vol 1, London, Academia, 1979, pp 51–57trials have shown that regimens including the dihydro-

16. Semple SJG, DeWardener HE: Effect of increased renal venouspyridine calcium channel blockers are not only less effec-pressure on circulatory “autoregulation” of isolated dog kidneys.

tive than renin-angiontension system blockade–based Circ Res 7:643–648, 195917. Woolson RF: Statistical methods for the analysis of biomedicalregimens [21], but also as compared to beta blockers–

data, Wiley series, in Probability and Mathematical Statistics, Phila-based regimens [21, 49, 50]. Collectively, these data sug-delphia, John Wiley & Sons, Inc., 1987, pp 314–344, 366–373

gest that the calcium channel blockers, particularly the 18. Rosenberg ME, Kren SM, Hostetter TH: Effects of dietary pro-tein on the renin-angiotensin system in subtotally nephrectomizeddihydropyridine calcium channel blockers should berats. Kidney Int 38:240–248, 1990avoided if possible in patients with chronic renal disease

19. Navar LG: Renal autoregulation: perspective from whole kidneyand if used, more emphasis be given to achieving lower and single nephron studies. Am J Physiol 234:F357–F370, 1978

20. Hayashi K, Epstein M, Loutzenhiser R: Pressure induced vaso-blood pressure goals.constriction of renal microvessels in normotensive rats: Studies inthe isolated perfused hydronephrotic kidney. Circ Res 65:1475–

ACKNOWLEDGMENTS 1484, 198921. Bidani AK, Griffin KA: Long-term renal consequences of hyper-The authors thank Wanda Plott, Shilpa Gude, and Jennifer Quinn

tension for normal and diseased kidneys. Curr Opin Nephrol Hyp-for technical assistance and Martha Prado for secretarial assistance.ertens 11:73–80, 2002This material is based upon work supported by the National Institutes 22. Pelayo JC, Westcott JY: Impaired autoregulation of glomerularof Health grant DK-40426 and Office of Research and Development capillary hydrostatic pressure in the rat remnant nephrons. J Clin

of the Department of Veterans Affairs. Invest 88:101–105, 199123. Bidani AK, Griffin KA, Picken M, Lansky DM: Continuous

Reprint requests to Karen A. Griffin, M.D., Hines VA Hospital, Mail telemetric BP monitoring and glomerular injury in the rat remnantSlot 151, Bldg. 1, Room C246, Hines, IL 60141, USA. kidney model. Am J Physiol 265:F391–F398, 1993E-mail address: Martha.Prado@med.va.gov 24. Griffin KA, Picken M, Bidani AK: Radiotelemetric BP monitor-

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