**10. Effects of HE-86 administration on mRNA levels of TNF–α, Ang II and AT II R in renal tissue**

The effects of HE-86 on the gene expression of Ang II (Figure 3), AT1R (Figure 4) and TNF–(Figure 5) in the renal cortex were examined. We investigated the potential

Molecular Mechanisms of Nephro-Protective

**0**

compared to the normal sham-control

**0.5**

**1**

**1.5**

**2**

**2.5**

Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure 185

Sham group Control group Treatment group

\*

A B C

Fig. 4. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal

AT1RmRNA expression(B). and Semiquantitative analysis of the therapeutic effect of HE-86 on AT1RmRNA localization in the glomerulus and tubulointerstitium using the FR-2000 Image Analyze System. A: Degree of glomerular and tubulointerstitial AT1RmRNA expression in sham group. B: Numbers of AT1RmRNA expression in glomerular and tubulointerstitial without treatment C:Numbers of glomerular and tubulointerstitial cells with nuclear localization of AT1RmRNA in nephrectomized rats treated with HE-86 was decreased significantly. Each bar represents data (mean ± SEM) #, P < 0.05 and ##, P < 0.01, when compared to empty vector-treated controls; \*, P < 0.05 and \*\*, P < 0.01, when

#

mechanisms whereby HE-86 suppressed rat tubular interstitial fibrosis and glomerular cirrhosis. TNF-, being key proinflammatory cytokines in anti-GBM glomerulonephritis, and a group of chemotactic and adhesion molecules including ICAM-1, MCP-1, was examined. In vehicle-treated chronic renal failure rats, there was a substantial increase in renal mRNA expression of TNF-, Treatment with HE-86 significantly reduced upregulation of TNF- inflammatory genes examined (P<0.05). Furthermore, HE-86 was capable of attenuating renal cortical mRNAs for Ang II and AT1R as compared with the control uraemic rats when they were administered after the establishment of nephrectomized. However, the renal mRNA levels of Ang II and AT1R were markedly increased in control uraemic rats as compared with normal rats. The variation in the mRNA levels of TNF–, Ang II and AT1R in both HE-86-treated and control uraemic rats are related to variation in the extent of CRF.

Fig. 3. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal Ang II mRNA expression(A). and Semiquantitative analysis of the therapeutic effect of HE-86 on Ang II mRNA localization in the glomerulus and tubulointerstitium using the FR-2000 Image Analyze System. A: Degree of glomerular and tubulointerstitial Ang II mRNA expression in sham group. B: Numbers of Ang II mRNA expression in glomerular and tubulointerstitial without treatment C: Numbers of glomerular and tubulointerstitial cells with nuclear localization of Ang II mRNA in twelve rats treated with HE-86 was decreased significantly. Each bar represents data (mean ± SEM) #, P < 0.05 and ##, P < 0.01, when compared to empty vector-treated controls; \*, P < 0.05 and \*\*, P < 0.01, when compared to the normal sham-control.

mechanisms whereby HE-86 suppressed rat tubular interstitial fibrosis and glomerular

and a group of chemotactic and adhesion molecules including ICAM-1, MCP-1, was examined. In vehicle-treated chronic renal failure rats, there was a substantial increase in

capable of attenuating renal cortical mRNAs for Ang II and AT1R as compared with the control uraemic rats when they were administered after the establishment of nephrectomized. However, the renal mRNA levels of Ang II and AT1R were markedly increased in control uraemic rats as compared with normal rats. The variation in the

Sham group Control group Treatment group

\*

A B C

Fig. 3. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal Ang II mRNA expression(A). and Semiquantitative analysis of the therapeutic effect of HE-86 on Ang II mRNA localization in the glomerulus and tubulointerstitium using the FR-2000 Image Analyze System. A: Degree of glomerular and tubulointerstitial Ang II mRNA expression in sham group. B: Numbers of Ang II mRNA expression in glomerular and tubulointerstitial without treatment C: Numbers of glomerular and tubulointerstitial cells with nuclear localization of Ang II mRNA in twelve rats treated with HE-86 was decreased significantly. Each bar represents data (mean ± SEM) #, P < 0.05 and ##, P < 0.01, when compared to empty vector-treated controls; \*, P < 0.05 and \*\*, P < 0.01, when compared to

, being key proinflammatory cytokines in anti-GBM glomerulonephritis,

inflammatory genes examined (P<0.05). Furthermore, HE-86 was

, Ang II and AT1R in both HE-86-treated and control uraemic rats

, Treatment with HE-86 significantly reduced

#

cirrhosis. TNF-

upregulation of TNF-

mRNA levels of TNF–

**0**

the normal sham-control.

**0.1**

**0.2**

**0.3**

**0.4**

**0.5**

**0.6**

**0.7**

**0.8**

**0.9**

renal mRNA expression of TNF-

are related to variation in the extent of CRF.

Fig. 4. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal AT1RmRNA expression(B). and Semiquantitative analysis of the therapeutic effect of HE-86 on AT1RmRNA localization in the glomerulus and tubulointerstitium using the FR-2000 Image Analyze System. A: Degree of glomerular and tubulointerstitial AT1RmRNA expression in sham group. B: Numbers of AT1RmRNA expression in glomerular and tubulointerstitial without treatment C:Numbers of glomerular and tubulointerstitial cells with nuclear localization of AT1RmRNA in nephrectomized rats treated with HE-86 was decreased significantly. Each bar represents data (mean ± SEM) #, P < 0.05 and ##, P < 0.01, when compared to empty vector-treated controls; \*, P < 0.05 and \*\*, P < 0.01, when compared to the normal sham-control

Molecular Mechanisms of Nephro-Protective

Ang II and AT-1 receptors [40-41].

hypertension [34-36].

Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure 187

can prevent or slow the progression of kidney damage in patients with diabetes and

As expected, 5/6 renal ablation promoted growth retardation, systemic arterial hypertension, impaired renal function, and severe albuminuria. These functional changes were accompanied by severe glomerulosclerosis, as well as expansion and intense macrophage infiltration of the interstitial area. Mounting evidence indicates that these renal structural abnormalities, which are characteristic of the Nx and other models of progressive nephropathies, are a consequence of the concerted action of mechanical stress, caused by glomerular hypertension and hypertrophy [37-38], and inflammatory phenomena, comprising cell infiltration and/or proliferation and extracellular matrix accumulation [38-39]. Moreover, a causal relationship appears to exist between these phenomena, because the distension of the glomerular walls due to intracapillary hypertension may trigger the local release of cytokines, growth factors, and, particularly,

The beneficial effect of RAS suppressors was initially attributed to amelioration of the glomerular hemodynamic dysfunction associated with progressive nephropathies. However, recent observations suggest that the nonhemodynamic effects of RAS suppressors may be equally important, given the strong proinflammatory and profibrotic effects of Ang II [42]. A substantial fraction of this proinflammatory ANG II may originate in the renal parenchyma, rather than in renal vessels or in the systemic circulation [43]. Increased intrarenal production of ANG II was described in various models of renal fibrosis [44-46]. A preliminary report has suggested that, in the 5/6 renal ablation (Nx) model, ANG II is

Increasing evidence shows that angiotensin II (Ang II) plays a critical role in cardiovascular disease and is a key mediator in the process of vascular fibrosis, characterized by reduced lumen diameter and arterial wall thickening attributable to excessive deposition of extracellular matrix (ECM). Vascular fibrosis is a major complication of hypertension and diabetic mellitus. It has been shown that upregulated tissue rennin-angiotensin system is involved in development of vascular lesions in both human and experimental vascular diseases [48-49]. This observation is confirmed by the finding that infusion of Ang II is able to induce vascular fibrosis in rats [50]. The functional importance of Ang II in vascular fibrosis is further supported by the evidence that blockade of Ang II inhibits vascular

Both the hemodynamic and proinflammatory effects of Ang II are mediated by AT-1 receptors (AT1R) [54], extensively expressed in renal tissue. In the normal rat kidney, AT1R are predominantly expressed in tubular cells and vessels [55]. Recent data obtained with the Nx model have suggested that AT1R expression is shifted from the glomerular to the tubulointerstitial compartment 4 wk after ablation [56]. However, the renal distribution of

Beyond its hemodynamic effects, Ang II is recognized as a cytokine with an active role in cardiovascular remodeling. It is well known that Ang II signals through its Ang II receptor 1 (AT1) receptor to exert most of its biological functions [57]. After binding to the AT1 receptor, Ang II activates multiple downstream intracellular signaling pathways, including tyrosine kinase, mitogen-activated protein kinase (MAPK), p38, and Janus family kinase

expressed in renal interstitial cells, paralleling the severity of renal injury [47].

fibrosis in diabetic and subtotal nephrectomy rats and NO-deficient mice [51-53].

AT1R in this model and its temporal evolution have not been established.

Fig. 5. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal TNF-mRNA expression(C). and Semiquantitative analysis of the therapeutic effect of HE-86 on TNF-mRNA within the glomerulus and tubulointerstitium using the FR-2000 Image Analyze System. A: Degree of glomerular and tubulointerstitial TNF-mRNA expression in sham group. B: Numbers of TNF-mRNA expression in glomerular and tubulointerstitial without treatment C: Numbers of glomerular and tubulointerstitial cells with nuclear localization of TNF-mRNA in nephrectomized rats treated with HE-86 was decreased significantly. Each bar represents data (mean ± SEM) #, P < 0.05 and ##, P < 0.01, when compared to empty vector-treated controls; \*, P < 0.05 and \*\*, P < 0.01, when compared to the normal sham-control.

#### **11. Discussion**

Renal fibrosis is a final common pathway to end-stage renal disease. Recent studies have shown that hypertensive nephropathy is a major leading cause of end-stage renal disease and the renin-angiotensin system plays a pivotal role in the development of progressive renal injury [34-35]. Clinical trials have shown that blocking the effects of angiotensin II (Ang II) with angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers

Sham group Control group Treatment group

\*

#

mRNA

mRNA expression in glomerular and

mRNA in nephrectomized rats treated with HE-86 was

A B C

mRNA expression(C). and Semiquantitative analysis of the therapeutic effect of HE-

mRNA within the glomerulus and tubulointerstitium using the FR-2000

tubulointerstitial without treatment C: Numbers of glomerular and tubulointerstitial cells

decreased significantly. Each bar represents data (mean ± SEM) #, P < 0.05 and ##, P < 0.01, when compared to empty vector-treated controls; \*, P < 0.05 and \*\*, P < 0.01, when

Renal fibrosis is a final common pathway to end-stage renal disease. Recent studies have shown that hypertensive nephropathy is a major leading cause of end-stage renal disease and the renin-angiotensin system plays a pivotal role in the development of progressive renal injury [34-35]. Clinical trials have shown that blocking the effects of angiotensin II (Ang II) with angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers

Fig. 5. Real-time PCR reveals the inhibitory effect of HE-86 liquid extract on renal

Image Analyze System. A: Degree of glomerular and tubulointerstitial TNF-

**0**

with nuclear localization of TNF-

compared to the normal sham-control.

expression in sham group. B: Numbers of TNF-

TNF-

86 on TNF-

**11. Discussion** 

**0.5**

**1**

**1.5**

**2**

**2.5**

**3**

**3.5**

**4**

**4.5**

can prevent or slow the progression of kidney damage in patients with diabetes and hypertension [34-36].

As expected, 5/6 renal ablation promoted growth retardation, systemic arterial hypertension, impaired renal function, and severe albuminuria. These functional changes were accompanied by severe glomerulosclerosis, as well as expansion and intense macrophage infiltration of the interstitial area. Mounting evidence indicates that these renal structural abnormalities, which are characteristic of the Nx and other models of progressive nephropathies, are a consequence of the concerted action of mechanical stress, caused by glomerular hypertension and hypertrophy [37-38], and inflammatory phenomena, comprising cell infiltration and/or proliferation and extracellular matrix accumulation [38-39]. Moreover, a causal relationship appears to exist between these phenomena, because the distension of the glomerular walls due to intracapillary hypertension may trigger the local release of cytokines, growth factors, and, particularly, Ang II and AT-1 receptors [40-41].

The beneficial effect of RAS suppressors was initially attributed to amelioration of the glomerular hemodynamic dysfunction associated with progressive nephropathies. However, recent observations suggest that the nonhemodynamic effects of RAS suppressors may be equally important, given the strong proinflammatory and profibrotic effects of Ang II [42]. A substantial fraction of this proinflammatory ANG II may originate in the renal parenchyma, rather than in renal vessels or in the systemic circulation [43]. Increased intrarenal production of ANG II was described in various models of renal fibrosis [44-46]. A preliminary report has suggested that, in the 5/6 renal ablation (Nx) model, ANG II is expressed in renal interstitial cells, paralleling the severity of renal injury [47].

Increasing evidence shows that angiotensin II (Ang II) plays a critical role in cardiovascular disease and is a key mediator in the process of vascular fibrosis, characterized by reduced lumen diameter and arterial wall thickening attributable to excessive deposition of extracellular matrix (ECM). Vascular fibrosis is a major complication of hypertension and diabetic mellitus. It has been shown that upregulated tissue rennin-angiotensin system is involved in development of vascular lesions in both human and experimental vascular diseases [48-49]. This observation is confirmed by the finding that infusion of Ang II is able to induce vascular fibrosis in rats [50]. The functional importance of Ang II in vascular fibrosis is further supported by the evidence that blockade of Ang II inhibits vascular fibrosis in diabetic and subtotal nephrectomy rats and NO-deficient mice [51-53].

Both the hemodynamic and proinflammatory effects of Ang II are mediated by AT-1 receptors (AT1R) [54], extensively expressed in renal tissue. In the normal rat kidney, AT1R are predominantly expressed in tubular cells and vessels [55]. Recent data obtained with the Nx model have suggested that AT1R expression is shifted from the glomerular to the tubulointerstitial compartment 4 wk after ablation [56]. However, the renal distribution of AT1R in this model and its temporal evolution have not been established.

Beyond its hemodynamic effects, Ang II is recognized as a cytokine with an active role in cardiovascular remodeling. It is well known that Ang II signals through its Ang II receptor 1 (AT1) receptor to exert most of its biological functions [57]. After binding to the AT1 receptor, Ang II activates multiple downstream intracellular signaling pathways, including tyrosine kinase, mitogen-activated protein kinase (MAPK), p38, and Janus family kinase

Molecular Mechanisms of Nephro-Protective

as urine TGF-ß and renal tissue TNF-α, NF-

II, AT1R and its derivatives as TNF-

prevent further renal deterioration.

**12. References** 

with traditional Chinese medicine prescription.

Nephrol. 1997;17:448–454

Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure 189

mounting evidence to implicate TNF-α in the pathogenesis of glomeruli of rodents with experimental nephritis, and is found in renal biopsies, sera and urine of patients with different types of glomerulonephritis [88-91]; In vitro and in vivo studies document that TNF-α is produced locally within inflamed glomeruli by mesangial and epithelial cells, as well as by infiltrating monocytes/macrophages [89,91]; Systemic administration of TNF-α results in glomerular damage in rabbits [92] and exacerbates the degree of glomerular injury in nephrotoxic nephritis in rats [93]; and blocking endogenous TNF-α in nephrotoxic nephritis in rats ameliorates acute glomerular inflammation [94], and down-regulates

Treatment of Nx rats with the HE-86 promoted a significant regression of hypertension, high level of creatinine and blood urea nitrogen, albuminuria, and inflammatory signs such

parameters of renal structural tissue injury were strongly attenuated, compared with pretreatment levels. The protection achieved with effective unit from clinical prescription treatment was much greater than that obtained with traditional prescription alone. On the basis of the present study, we cannot exclude the hypothesis that the success of HE-86 was due to a particularly effective hemodynamic action, although previous observations from this laboratory [96] indicated that NOF, a new nonsteroidal anti-inflammatory, had no significant effect on glomerular hemodynamics. Because treatment with NOF alone had no effect on blood pressure, it seems unlikely that the hemodynamic effect of NOS was directly intensified by its association with NOF. Therefore, the efficacy of extract HE-86 was likely due to the simultaneous blockade of the hemodynamic and proinflammatory actions of Ang

interplay between hypertension and inflammation. The present findings support other scholars' observations of the Nx model, which similarly indicated the superiority of the combination of a RAS suppressor with an anti-inflammatory agent [97-99]. It is noteworthy that HE-86 afforded partial regression of the nephropathy associated with Nx even though it was started 4 week after surgery, when renal injury was already established. This observation suggests that both continued stimulation of Ang II and AT1 receptors and production of inflammatory factors continue to play an important pathogenic role even during the late phases of the process, necessitating vigorous and persistent treatment to

Taken together with our previous data and the present results, it is likely that HE-86 induced reduction of renal rennin-angentensin system is mediated, at least partly, by reducing the overload of inflammatory factors activity on remnant kidney unit. In summary, HE-86effective composition coming from clinical validly treating patients with chronic renal failure especially for early and middle stage, partially reversed the nephropathy and renal inflammation associated with the Nx model, showing much more effective protection than

[1] Wolf G, Ziyadeh FN. Renal tubular hypertrophy induced by angiotensin II. Semin

, NF-

B, Ang II and AT1R expression, whereas the

B, TGF-ß and by abrogation of the complex

glomerular IL-1βmRNA and circulating TNF-α concentrations [95].

[58]. Activation of these pathways leads to numerous heterogeneous downstream events that play essential roles in the biological activities of Ang II, such as cell growth and migration, ECM production, and apoptosis [58].

Renal expression of AT1R in rats appeared mostly in tubular cells, and to a lesser extent, at the interstitial area, whereas weaker expression was seen in vessels and glomeruli. This pattern was completely disrupted after Nx, when dense AT1R expression could be demonstrated in interstitial cells, far exceeding in intensity the expression of AT1R in tubules. The exact meaning of this finding and the cell types involved are uncertain. Several inflammatory cells known to infiltrate the renal interstitium in the Nx model have the potential to express AT1R, such as lymphocytes [59] and macrophages [60]. In addition, AT1R may be expressed by myofibroblasts originating from tubular cell transdifferentiation [61]. This hypothesis is particularly attractive because it helps to explain the progressive shift in AT1R expression, from tubules to the interstitial area, observed in Nx rats, and also because tubular cells already express AT1R under normal conditions. The simultaneous presence at the interstitial area of large amounts of Ang II and of the AT1R may accelerate the progression of the nephropathy by a positive-feedback mechanism. Consistent with this view is the aggravation of the renal structural injury of Nx, which was paralleled by the intensity of the inflammatory infiltration and of the interstitial expression of Ang II.

It is well accepted that NF-B is a key transcriptional factor to regulate a variety of inflammatory responses [75]. NF-B is composed of p50 and p65 subunits, among which p65 is a potent transcriptional activator, strongly promoting inflammatory reaction in kidney diseases [76]. NFB total protein expression, and inflammation, which may have resulted from blockade of the oxidative stress pathway [77-78]. This was accompanied by a substantial attenuation in renal fibrosis, which might have resulted from the modulating actions of vitamins on lipid peroxidation and profibrotic activity involved in renal tissue damage [79-82]. In this study, marked activation of NF-B was closely correlated with the renal inflammation. In our study, using liquid extract isolated from clinical effective Chinese prescription, we were able to show that overexpression activation of NF-B was substantially suppressed as compared with control group. These findings are consistent with the improving renal function and correcting high blood pressure.

Tumour necrosis fator-α(TNF-α)is a potent pro-inflammatory cytokine which is produced by many cell types including monocytes/macrophages, and renal mesangial and epithelial cells. It induces the expression of major histocompatibility complex (MHC) class I and II molecules, endothelial adhesion molecules and procoagulant activity of endothelium. TNFα stimulates the release of other pro-inflammatory cytokines, chemokines and growth factors, including interleukin-1β(IL-1β), monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-β(TFG-β) [83-84]. The biological effects of TNF-α are mediated by binding to specific receptors which are widely distributed. TNF-α binds to two types of receptor: TNF receptor type 1 and TNF receptor type 2, which have molecular weights of 55 kDa (p55) and 75 kDa (p75), respectively. Both receptors are necessary and act synergistically for cell proliferation and maturation, cytotoxicity and antiviral activity, but p55 is responsible for activation of NFкB and mediation of apoptosis [85].

TNF- may contribute to renal damage by inciting an inflammatory response within the kidney via induction of a variety of chemokines and adhesion molecules [86-87]. There is a

[58]. Activation of these pathways leads to numerous heterogeneous downstream events that play essential roles in the biological activities of Ang II, such as cell growth and

Renal expression of AT1R in rats appeared mostly in tubular cells, and to a lesser extent, at the interstitial area, whereas weaker expression was seen in vessels and glomeruli. This pattern was completely disrupted after Nx, when dense AT1R expression could be demonstrated in interstitial cells, far exceeding in intensity the expression of AT1R in tubules. The exact meaning of this finding and the cell types involved are uncertain. Several inflammatory cells known to infiltrate the renal interstitium in the Nx model have the potential to express AT1R, such as lymphocytes [59] and macrophages [60]. In addition, AT1R may be expressed by myofibroblasts originating from tubular cell transdifferentiation [61]. This hypothesis is particularly attractive because it helps to explain the progressive shift in AT1R expression, from tubules to the interstitial area, observed in Nx rats, and also because tubular cells already express AT1R under normal conditions. The simultaneous presence at the interstitial area of large amounts of Ang II and of the AT1R may accelerate the progression of the nephropathy by a positive-feedback mechanism. Consistent with this view is the aggravation of the renal structural injury of Nx, which was paralleled by the

intensity of the inflammatory infiltration and of the interstitial expression of Ang II.

p65 is a potent transcriptional activator, strongly promoting inflammatory reaction in

resulted from blockade of the oxidative stress pathway [77-78]. This was accompanied by a substantial attenuation in renal fibrosis, which might have resulted from the modulating actions of vitamins on lipid peroxidation and profibrotic activity involved in renal tissue

renal inflammation. In our study, using liquid extract isolated from clinical effective Chinese

substantially suppressed as compared with control group. These findings are consistent

Tumour necrosis fator-α(TNF-α)is a potent pro-inflammatory cytokine which is produced by many cell types including monocytes/macrophages, and renal mesangial and epithelial cells. It induces the expression of major histocompatibility complex (MHC) class I and II molecules, endothelial adhesion molecules and procoagulant activity of endothelium. TNFα stimulates the release of other pro-inflammatory cytokines, chemokines and growth factors, including interleukin-1β(IL-1β), monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-β(TFG-β) [83-84]. The biological effects of TNF-α are mediated by binding to specific receptors which are widely distributed. TNF-α binds to two types of receptor: TNF receptor type 1 and TNF receptor type 2, which have molecular weights of 55 kDa (p55) and 75 kDa (p75), respectively. Both receptors are necessary and act synergistically for cell proliferation and maturation, cytotoxicity and antiviral activity, but

 may contribute to renal damage by inciting an inflammatory response within the kidney via induction of a variety of chemokines and adhesion molecules [86-87]. There is a

prescription, we were able to show that overexpression activation of NF-

B is a key transcriptional factor to regulate a variety of

B total protein expression, and inflammation, which may have

B is composed of p50 and p65 subunits, among which

B was closely correlated with the

B was

damage [79-82]. In this study, marked activation of NF-

with the improving renal function and correcting high blood pressure.

p55 is responsible for activation of NFкB and mediation of apoptosis [85].

migration, ECM production, and apoptosis [58].

It is well accepted that NF-

kidney diseases [76]. NF

TNF-

inflammatory responses [75]. NF-

mounting evidence to implicate TNF-α in the pathogenesis of glomeruli of rodents with experimental nephritis, and is found in renal biopsies, sera and urine of patients with different types of glomerulonephritis [88-91]; In vitro and in vivo studies document that TNF-α is produced locally within inflamed glomeruli by mesangial and epithelial cells, as well as by infiltrating monocytes/macrophages [89,91]; Systemic administration of TNF-α results in glomerular damage in rabbits [92] and exacerbates the degree of glomerular injury in nephrotoxic nephritis in rats [93]; and blocking endogenous TNF-α in nephrotoxic nephritis in rats ameliorates acute glomerular inflammation [94], and down-regulates glomerular IL-1βmRNA and circulating TNF-α concentrations [95].

Treatment of Nx rats with the HE-86 promoted a significant regression of hypertension, high level of creatinine and blood urea nitrogen, albuminuria, and inflammatory signs such as urine TGF-ß and renal tissue TNF-α, NF-B, Ang II and AT1R expression, whereas the parameters of renal structural tissue injury were strongly attenuated, compared with pretreatment levels. The protection achieved with effective unit from clinical prescription treatment was much greater than that obtained with traditional prescription alone. On the basis of the present study, we cannot exclude the hypothesis that the success of HE-86 was due to a particularly effective hemodynamic action, although previous observations from this laboratory [96] indicated that NOF, a new nonsteroidal anti-inflammatory, had no significant effect on glomerular hemodynamics. Because treatment with NOF alone had no effect on blood pressure, it seems unlikely that the hemodynamic effect of NOS was directly intensified by its association with NOF. Therefore, the efficacy of extract HE-86 was likely due to the simultaneous blockade of the hemodynamic and proinflammatory actions of Ang II, AT1R and its derivatives as TNF-, NF-B, TGF-ß and by abrogation of the complex interplay between hypertension and inflammation. The present findings support other scholars' observations of the Nx model, which similarly indicated the superiority of the combination of a RAS suppressor with an anti-inflammatory agent [97-99]. It is noteworthy that HE-86 afforded partial regression of the nephropathy associated with Nx even though it was started 4 week after surgery, when renal injury was already established. This observation suggests that both continued stimulation of Ang II and AT1 receptors and production of inflammatory factors continue to play an important pathogenic role even during the late phases of the process, necessitating vigorous and persistent treatment to prevent further renal deterioration.

Taken together with our previous data and the present results, it is likely that HE-86 induced reduction of renal rennin-angentensin system is mediated, at least partly, by reducing the overload of inflammatory factors activity on remnant kidney unit. In summary, HE-86effective composition coming from clinical validly treating patients with chronic renal failure especially for early and middle stage, partially reversed the nephropathy and renal inflammation associated with the Nx model, showing much more effective protection than with traditional Chinese medicine prescription.
