Most animal studies have used the 5/6 renal mass reduction model, characterized by early hyperfusion and hyperfiltration in the remnant with developing proteinuria and focal and segmental glomerulosclerosis (FSGS) (20). Fitzgibbon et al. (12) reported that the Wistar-Furth (WF) rat strain shows remarkable resistance to induced CRD by renal mass reduction. We subsequently confirmed this finding and reported that maintained renal NO production is seen in the WF compared with the vulnerable Sprague-Dawley (SD) and appears to be critical in protection against CRD, because low-dose NOS inhibition following ablation/infarction (A/I) in the WF causes severe kidney damage (11).

Another animal model that results in FSGS and CRD is induced by administration of puromycin aminonucleoside (PAN). An initial dose of PAN causes a nephrotic range proteinuria followed by a “recovery” phase, leading to a slowly evolving CRD that resembles human FSGS (2). Repeated injections of PAN result in an accelerated model of CRD (13).

The present study examined the renal NO system in PAN-induced CRD and investigated whether the WF would again exhibit protection against progression of kidney damage leading to CRD together with preserved renal NOS.

NOS activity was measured from the conversion of l-[³H]-arginine to l-[³H]-citrulline in the kidney cortex and medulla as described by us earlier (44). Briefly, tissues were homogenized in iced homogenization buffer, ultracentrifuged, and both supernatant (soluble) and membrane fractions were assayed; the soluble fraction contains predominantly neuronal (n) and inducible (i)NOS, whereas the membrane fraction contains mostly endothelial (e)NOS. Endogenous arginine was removed from the supernatant using Dowex while the pellet was reconstituted in 5 ml of homogenization buffer, ultracentrifuged, and resuspended in 350 μl Samples were run in triplicate at baseline and duplicate in the presence of nonselective NOS inhibitors N^G-monomethyl-l-arginine (5 mM), N^G-nitro-l-arginine methyl ester (10 mM), and the calcium chelator trifluperazine (2 mM). Data are expressed as picomoles of l-[³H]-arginine converted to l-[³H]-citrulline per minute per milligram of protein (pmol citrulline · min⁻¹ · mg protein⁻¹) minus any activity not inhibited by the NOS inhibitor cocktail and adjusted for background. Background was determined on heat-inactivated samples and represented <5% of the 100% standard, representing free tritium or noncationic tritiated species not bound by the Dowex resin. The 100% standard was prepared without Dowex, and this value represented the total number of counts available for conversion.

SD rats given intravenous PAN were severely proteinuric at week 1, and supplemental doses at weeks 3, 4, and 5 caused further increases in protein excretion (Fig. 1). Although total urinary protein excretion (U_pV) declined after week 8, U_pV was always higher in SD-PAN rats vs. the controls (SD sham), which showed only a mild age-dependent increase in protein excretion over the 15-wk observation period. The WF also showed a significant acute increase in total protein excretion with PAN, although the absolute level was blunted compared with the SD-PAN. The fold-increase was lower in WF at week 1 (17 ± 2) than in SD (26 ± 5, P = 0.05), which tended to persist although the strain difference in proteinuria diminished in the last few weeks. In WF sham, there was no tendency for U_pV to change with age (5 ± 1 and 4 ± 1 mg · 100 g BW⁻¹ · 24 h⁻¹, presurgery and week 15, respectively) as also seen with the SD (7 ± 1 and 9 ± 2 mg · 100 g BW⁻¹ · 24 h⁻¹) with a strain difference present at all times (P < 0.05).

SD-PAN had markedly increased BUN and plasma creatinine concentrations at 15 wk following the initial dose of PAN compared with sham rats, indicating decreased renal function, as shown by a creatinine clearance (C_cr) of <10% of sham values (Table 2). No increase in BUN or plasma creatinine (P_Cr) due to PAN treatment was seen in the WF at 15 wk and C_cr was also unchanged relative to WF sham rats. Also shown in Table 2, a strain difference exists in BP with WF having consistently lower BP than SD and PAN having no effect in either strain at 15 wk following the initial dose of PAN.

WF sham had no injury (Figs. 2 and ​and3)3) except for occasional small foci of interstitial lymphocytes. SD sham rats had little injury, showing no more than 5% glomerulosclerosis with occasional areas of interstitial inflammation and tubular casts. Glomerular injury was present in two-thirds of WF treated with puromycin; however, the injury was mild in three (segmental sclerosis affecting fewer than 10% of glomeruli with little tubulointerstitial change) and moderate in only one. The difference in segmental sclerosis in WF-PAN was not different compared with WF sham (Fig. 2), although analysis of the ranks of overall injury showed a statistical significance (P < 0.01; Fig. 3). Administration of PAN to SD caused considerable injury with segmental sclerosis present in more than one-third of glomeruli in all animals. An additional 25–30% of glomeruli demonstrated global sclerosis. Extensive tubular injury was characterized by dilated tubules containing large casts and often surrounded by inflammatory infiltrates comprised largely of lymphocytes. This group showed much more injury than either the SD sham or WF-PAN rats by the analysis of both extent of glomerulosclerosis (Fig. 2) and the rank of overall injury (Fig. 3). Of note, creatinine clearance was reduced ~90%, whereas glomerulosclerosis affected ~65% glomeruli in the SD-PAN, suggesting concomitant renal vasoconstriction in our model at the time of harvest. Also, extensive tubulointerstitial injury/inflammation was observed in the SD-PAN, which could further contribute to reduced clearance.

Both the WF and SD given PAN showed a similar transient, marked decline in total NO production (determined from 24-h urinary NOx excretion, U_NOXV) at week 1 (Fig. 4). As renal injury developed, SD-PAN showed a persistent decline in total NO production with U_NOXV that was significantly lower than all other groups by week 6 and thereafter. In WF given PAN, U_NOXV rebounded to baseline values after week 1 (and despite 3 maintenance doses of PAN) so that at week 15 WF-PAN had the same total NO production as in the baseline state and as the 15-wk WF sham rats. In the baseline state, before PAN, WF had significantly more total NO production compared with SD and this difference was maintained at 15 wk. Despite the marked increase in plasma NOx in the SD-PAN (Table 2), when the loss of renal function is corrected by factoring plasma NOx (P_NOx) by P_Cr, a significant reduction in the P_NOx/P_cr ratio was seen in SD-PAN, which together with the decreased U_NOxV strongly suggests reduced overall NO production (Table 2). In WF, there was no change in plasma NO_x (P_NOx) or P_NOx/P_cr at 15 wk after PAN administration.

As shown in Fig. 5, sham WF have elevated nNOS protein abundance compared with control SD. PAN caused a significant decrease in cortical nNOS abundance in the WF and a similar trend (of ~70%) in the SD but because of the higher baseline values, WF-PAN had similar levels to SD sham rats. Medullary nNOS was greatly reduced in the SD-PAN but unchanged in the WF-PAN (Fig. 5). Cortical eNOS showed no change due to treatment or strain, whereas medullary eNOS was reduced in the SD-PAN but unchanged in the WF-PAN (Fig. 6).

Although abundance of nNOS in the cortex was higher in the sham WF, in vitro NOS activity of the soluble fraction of the cortex (location of the nNOS) was greater in the SD sham rats (Fig. 7) as also reported by us previously (11). After PAN treatment, NOS activity was maintained in the WF (despite a decline in abundance) but significantly reduced in the SD (as predicted by the fall in abundance). Both medullary eNOS and nNOS abundance were reduced in the SD, and parallel changes were seen in the soluble and membrane fraction NOS activity (Fig. 7). Only a mild decrease was observed in the soluble fraction of the medulla in the WF-PAN, although abundance of the constitutive NOS isoforms was maintained. Of note, NOS activity was higher in the SD vs. WF sham in all fractions measured but reversed with PAN as SD-PAN declined to levels significantly below those in WF-PAN.

Many different versions of the PAN model have been employed. A single intravenous dose of 50 mg/kg PAN caused a severe initial proteinuria resulting in a nephrotic syndrome, followed by a slowly developing CRD over many months (2, 8). Other workers have given multiple intraperitoneal injections to accelerate the development of the CRD (13, 17). We adapted the model to give an initial intravenous bolus and three intravenous maintenance doses that resulted in severe CRD in the SD by 15 wk. The exact biochemical events that cause PAN nephropathy are not known, although the early effects are primarily on the podocytes and likely involve oxidative damage (13). In the chronic phase of the disease, glomerular hypertension has been reported (2) but because FSGS develops in the absence of high glomerular blood pressure in a different PAN model (14), this is unlikely to be a primary cause. Of note, the course of PAN-induced CRD is different from the commonly used 5/6 renal mass reduction models, characterized by glomerular hypertension and hyperfiltration of remaining nephrons (13). Nevertheless, there are also similarities between the two models, with both early and late inflammatory cell infiltration, increased extracellular matrix (ECM) synthesis, and decreased ECM degradation as well as protection vs. progression of CRD with both angiotensin inhibition and low protein feeding (13). Another common finding is the decrease in both total NO production and in renal NO-generating capacity in rats with renal mass reduction (1, 3, 11, 31, 39) and the PAN CRD model, as shown in the present study.

Recent observations have supported the suggestion that NO deficiency is both a cause and consequence of CRD (4). Chronic, experimentally induced NOS inhibition causes renal injury, proteinuria, and glomerular hypertension (45). Clinical studies in CRD and ESRD patients show reduced total NO production (32-34), whereas animal models of CRD exhibit decreased renal NOS abundance and activity as well as reduced total NO production (1, 3, 11, 31, 39, 40). Furthermore, stimulation of endogenous NO synthesis with l-arginine supplementation is beneficial in several animal models of CRD while chronic NOS inhibition can enhance progression (3, 15, 22). Together, this evidence suggests a central role for NO deficiency in the progression of CRD.

Our previous studies in animal models of CRD showed quite variable responses in the renal eNOS with no change in chronic glomerulonephritis (40), a decrease in age-dependent injury (10), an increase in the obese diabetic Zucker rat (9), and an increase, decrease, and no change have been shown in the remnant kidney depending on the duration and model (1, 11, 16, 21, 36, 39). These differences between models suggest a disease-specific response of eNOS rather than a generalized change due to CRD. In the present study, eNOS levels were unchanged in PAN CRD with the exception of a reduction in eNOS abundance in the medulla of SD-PAN, which correlated with reduced NOS activity in the membrane fraction (primary location of eNOS) of the SD-PAN medulla.

Unlike eNOS, renal nNOS abundance and activity change consistently in several models of CRD. Downregulation of renal nNOS in CRD was first shown by Roczniak et al. (31) in the remnant kidney (polectomy) model. Reduced renal nNOS abundance and NOS activity have since been reported by us in rats at 4–11 wk post-5/6 A/I, accelerated 5/6 A/I (2–3 wk post-5/6 A/I + high-sodium and -protein intake), age-dependent injury, chronic glomerulonephritis (10, 11, 36, 40), and also in the diabetic obese Zucker rat (9). In the 5/6 A/I model of CRD studied at various levels of severity determined by time after A/I (2–11 wk), the level of nNOS protein in both the cortex and medulla correlated with the severity of glomerulosclerosis (36). In the PAN model, nNOS abundance and NOS activity in the soluble fraction were markedly reduced in both the cortex and medulla of the susceptible SD. Although it is difficult to dissociate cause and effect, it is possible that an initial renal nNOS deficiency contributes to further progression of CRD. Medullary NO derived from nNOS is involved in the regulation of sodium balance and inner medullary nNOS inhibition causes a salt-dependent hypertension (26). Furthermore, PAN nephrosis is associated with sodium retention, and the loss of inner medullary NO may contribute (28). However, because systemic hypertension was not present in our model of PAN-induced CRD, the significance of decreased medullary nNOS in SD-PAN is unclear. Cortical nNOS, mainly expressed in macula densa, acts as a brake on tubuloglomerular feedback (TGF) (42). However, although acute nNOS inhibition exacerbates TGF-induced renal vasoconstriction, nNOS knockout mice and rats given chronic nNOS inhibition show normal TGF responsiveness (29, 30, 38). Thus, although it is unclear how decreased renal nNOS influences the progression of PAN-induced CRD, our recent observations in the C57BL6 mouse, showing that selective nNOS inhibition greatly potentiates progression of CRD (27), support a causal relationship.

One major finding in the present study is the impressive resistance of the WF to the chronic phase of PAN-induced renal injury, despite an active acute response. Previous studies showed that the WF is resistant to renal mass reduction (11, 12) and that enhanced/preserved NO production (compared with the SD) conferred protection (11). As also seen in our previous study in which we compared the response of SD and WF to 5/6 A/I, here we observed that in the baseline state WF had elevated total NO production (from U_NOXV) vs. SD. After the insult (either 5/6 A/I or PAN), the strain difference in UNOXV was maintained and in both models the injured SDs exhibited reduced total NO production compared with all other groups. Total NO production also fell acutely with PAN in both strains, and while it is unclear what caused this acute fall in U_NOXV, this is unlikely to be related to the development of CRD because WF do not progress. Walker et al. (41) also report a decrease in U_NOXV at 7 days postintravenous injection in SD.

Strain differences, leading to variable chronic responses after acute kidney injury, have been reviewed by Fogo (13). She points out the marked variation in susceptibility to renal ablation-induced CRD in mice that may segregate with renin gene status (13). Indeed, progression of CRD in vulnerable rat strains, after both renal ablation and PAN, is markedly attenuated by chronic angiotensin inhibition (3, 13, 46). Of relevance to the present study, the piebald viral glaxo (PVG)-inbred rat strain is also resistant to PAN-induced chronic renal damage (19) as well as to aging and uninephrectomy-induced renal damage (18). The renin-angiotensin-aldosterone system (RAAS) in the WF is altered with documented resistance to mineralocorticoid-induced hypertension (37); however, there is no information on the RAAS in PVG rats. One known similarity between these two resistant stains is the high glomerular number relative to SD and Wistars (11, 18), supporting the hypothesis that nephron number is a critical determinant of rate of progression of CRD (5). The greater BP between the strains could certainly be a risk factor predisposing to greater injury in the SD; however, in our terminal study, there was no elevation in BP with PAN CRD in either strain, and thus the higher baseline BP is not the cause of the greatly accelerated injury in the SD-PAN. The hypertrophic response of the kidney following PAN was much greater in the SD than the WF, which could also contribute to the greater injury in the SD. However, the exaggerated renal hypertrophic response might also be a consequence of the marked loss in function in the SD, so causality is hard to determine. We also suggest that the preserved renal NO-generating capacity seen in the WF may play a critical role in reducing progression of CRD, as discussed above (there is no information on the PVG rat).

The regulation of renal nNOS activity in the SD-PAN was quite straightforward with parallel declines in nNOS protein abundance and NOS activity in the soluble fraction of both the cortex and medulla. This was exactly the same pattern seen in the SD after 5/6 A/I (11). The changes in the WF were more complex because although nNOS abundance was greater in control WF vs. SD kidney cortex, the SD sham had significantly higher NOS activity compared with the WF. Furthermore, whereas absolute nNOS abundance fell in the cortex of WF-PAN, the NOS activity was maintained, relative to shams. Again, this was the pattern that we saw in the WF after 5/6 A/I (11). The tissue NOS activity is measured in vitro with an excess of substrate and cofactor present and should therefore reflect abundance and inherent enzyme activity. As reviewed recently, many proteins interact with the nNOS and regulate activity, including heat shock protein 90, caveolin, the protein inhibitor of nNOS, and a number of proteins that interact through the PDZ domain. These may regulate activity by control of phosphorylation, dimerization, etc., and nNOS activity is also influenced by nitrosylation, oxidant status, substrate availability, etc. (23, 43). Therefore, it would be naive to expect NOS activity and abundance to always change in parallel. In the case of the WF, it appears that for a given abundance of renal nNOS, the protein possesses a marked “reserve” capacity for increased activity that is not seen in the SD.

We do not know what signals renal nNOS abundance to decline in states of compromised renal function, although the effect is clearly posttranscriptional in view of the tendency for upregulation of nNOS mRNA in SD rats given PAN. In fact, all three NOS mRNAs were upregulated in the SD-PAN, suggesting a secondary feedback response to low ambient NO (6, 7). This possibility is strengthened by the finding that there was no change in any NOS mRNAs in the WF kidneys, where overall NO generation was likely maintained due to the maintained NOS activity. Because renal nNOS protein abundance declines in multiple models of renal disease (9-11, 31, 36, 40) and given our finding that nNOS decreases with increasing renal dysfunction (36), the signal(s) may be related to renal parenchymal damage.