Department of Nephrology and Department of Histology and Pathology, Universidad Austral de Chile, Valdivia, Chile

Correspondence: Leopoldo G. Ardiles, M.D., Department of Nephrology, Faculty of Medicine, Universidad Austral de Chile, PO Box 567, Valdivia, Chile. E-mail: lardiles@uach.cl

Abstract

Renal kallikrein-kinin system damage and salt sensitivity: Insights from experimental models. The importance of tubulointerstitial injury in the pathophysiology of human essential hypertension, and particularly salt sensitivity, is increasingly recognized. Since the renal kallikrein-kinin system (KKS) is located in the tubulointerstitial region of the kidney it is reasonable to expect that injury to this area, whatever the cause, may impair KKS production and compromise its role in blood pressure regulation.

In this review we discuss evidence of injury in the renal kallikrein-producing structures in three different experimental models characterized by prominent tubulointerstitial lesions: subtotal nephrectomy; inhibition of nitric oxide synthase; and overload proteinuria. These three experimental models have in common the development of important tubulointerstitial damage and salt-sensitive hypertension expressed after the initial injury has ceased. In these three models, reduced KKS activity may contribute to the establishment of a pathophysiologic state characterized by unopposed hyperactivity of the renin-angiotensin system, resulting in salt retention.

The structural integrity of the tubulointerstitial areas of the kidney plays an important role in the physiology of sodium balance. Recent investigations suggest that tubulointerstitial injury may be a critical feature in the development of essential hypertension and, particularly, in the development and maintenance of salt sensitivity¹.

Salt sensitivity is a term used to describe the hypertensive state that is significantly modified by variations in salt balance. Blood pressure is increased with a high-salt diet and is reduced by a reduction in salt intake, associated or not with furosemide administration^2,3. The pressure natriuresis curve is shifted to the right since a higher blood pressure is required to excrete an equivalent amount of sodium. Salt sensitivity may be a significant predictor of organ damage^3,4,5 and is such a common feature of essential hypertension, particularly in elderly individuals, that current recommendations for the treatment of hypertension includes restriction of dietary salt⁶.

The role of tubulointerstitial injury in the development of salt-sensitive hypertension is supported by the findings of focal microvascular damage in the renal interstitium of hypertensive patients^7,8. Identical intra-renal lesions may be induced with the administration of angiotensin II by subcutaneous minipumps in rats. This experimental manipulation also results in the subsequent development of hypertension if the rats are given a high-salt diet, while the animals remain normotensive if salt intake is not increased⁹. Similar findings were demonstrated in hypertension induced by the infusion of phenylephrine, where blood pressure was correlated with the degree of tubulointerstitial injury¹⁰.

Subsequent observations in other experimental models are consistent with the postulate that tubulointerstitial damage and loss of peritubular capillary area are common features in other types of salt-sensitive hypertension¹¹. In fact, the association of salt-sensitive hypertension, tubulointerstitial immune cell infiltration, and peritubular capillary loss has been found in several experimental conditions¹²

In general, the mechanisms proposed to explain salt sensitivity in these models center on, first, the reduction in peritubular capillary area which reduces the area available for the pressure-natriuresis mechanism to operate efficiently, and second, the participation of humoral factors that favor sodium retention resulting from the infiltration of immunocompetent cells in tubulointerstitial areas¹. Local up-regulation of the renin-angiotensin system, over-expression of angiotensin-converting-enzyme (ACE) at sites of interstitial injury, and expression of angiotensin II by infiltrating T cells, macrophages, and resident tubular cells, have been documented^13,14. Over-expression of endothelin-1, a potent vasoconstrictor, has also been demonstrated.

However, less attention has been devoted to the role of the vasodilator hormones in the phenomenon of salt sensitivity. Previous studies of the hypokalemia model showed a reduction in renal nitric oxide production, urinary prostaglandin excretion, and in the release of renal medullipin¹⁵. Human studies have reported reduction of renal prostaglandins¹⁶ and nitric oxide production¹⁷.

An important vasodilator system located in the renal tubulointerstitial space is the kallikrein-kinin system (KKS)¹⁸. This is a multienzymatic complex in which the major components are an enzyme (renal kallikrein), its substrates (renal high- and low-molecular weight kininogens), effector bioactive peptides (kinins, lys-bradykinin and bradykinin), the kinin-metabolizing enzymes (ACE and neutral endopeptidase), the kinin receptors (B1 and B2), and several activators/inhibitors of the system¹⁹. This system behaves as a paracrine renal complex in which the kinin peptides are formed and act within the kidney mainly through the activation of B2 receptors, which appear to be constitutively expressed not only in the kidney but also in many other cell types¹⁸. One of the key targets of kinin action is the collecting duct that expresses B2 receptors on both basolateral and luminal cell membranes²⁰. After a delay period that may be as short as three minutes, kinins cause a four- to six-fold increase in the formation of prostaglandin E2 (PGE₂) but only when added to the luminal surface of canine cortical collecting duct cells in culture²¹. The formation of PGE₂ in the collecting duct fluid could mediate the natriuretic action of kinins, as PGE₂ has been reported to inhibit Na⁺ reabsorption in isolated rabbit collecting ducts²². In contrast, kinins inhibit the arginine vasopressin-stimulated water permeability and Na⁺ reabsorption in isolated collecting ducts only when the peptide is applied on the serosal side of the tubule^23,24. This effect is likely the result of inhibition of an arginine vasopressin–dependent increase of cAMP, the major second messenger produced after vasopressin V₂ receptor activation²⁵. Furthermore, kinin B2 receptor blockade enhances Cl^- and water absorption in the medullary collecting ducts, a finding that supports a role of renal kinins in the regulation of NaCl and water excretion²⁶. B2 receptors have also been found in proximal straight tubule, the distal straight tubule (thick ascending limb of Henle), the connecting tubule, and afferent arterioles^20,27.

Kallikrein production is carried out in connecting tubule cells, in the renal cortex²⁸, whereas its substrate, kininogen, is synthesized downstream in collecting tubules²⁹. Anatomic vicinity of cells responsible for the synthesis of both components makes possible kinin formation and action at the luminal side of collecting tubule cells, as well as in the peritubular space affecting renal blood flow and electrolytes and water excretion. The site of kallikrein synthesis is in close anatomical proximity to the site of renin production since in the rat³⁰ and in humans³¹ both, the distal connecting tubule returns to the vicinity of the vascular pole after its outward trajectory to subcapsular areas.

Abundant evidence indicates that the KKS plays an important role in blood pressure regulation, salt sensitivity, water balance, electrolyte excretion, and growth-modulating activity^32,33,34. Indeed, essential hypertension has been characterized as a low urinary kallikrein excretion state³⁵, and the subset of salt-sensitive patients show lower levels of urinary kallikrein compared with those salt resistant³⁶. Furthermore, normotensive offspring of hypertensive parents and salt retainers have shown low levels of urinary kallikrein excretion^35,37,38,39. The role of the KKS is emphasized by the fact that when kallikrein is administered in pharmacologic doses to hypertensive patients, their blood pressure is reduced⁴⁰, and this effect is more pronounced in salt-sensitive than salt-resistant individuals⁴¹. Experimental work has given comparable results; a rat strain inbred for reduced urinary kallikrein excretion differs from normal-kallikrein Wistar rats not only in basal levels of blood pressure, but also in the modifications of blood pressure resulting from variations in sodium balance^42,43. The protective role of the KKS against increments in blood pressure is further emphasized by the finding of the early onset of salt-sensitive hypertension in the kinin B2 receptor null mice⁴⁴. On the other hand, promotion of renal KKS activity by inhibition of kinin catabolism, results in increased sodium excretion and prevention of the development of hypertension⁴⁵. Interesting pharmacologic studies⁴⁶ have shown that long-term infusion of a subdepressor dose (700 ng/day, intravenously) of purified rat urinary kallikrein attenuates renal injury in salt-induced hypertension in Dahl salt-sensitive rats in association with stimulation of the KKS-prostaglandin and nitric oxide axes. Moreover, adenovirus-mediated delivery of human tissue kallikrein (KLK1) results not only in high-efficiency expression and blood pressure reduction in the spontaneously hypertensive rat (SHR)^47,48,49, but also protects against renal injury and cardiac remodeling in the rat remnant kidney model of chronic renal failure⁵⁰.

Further evidence for the responsibility of this vasodilator system in blood pressure control is found in hypertensive disorders of pregnancy, where low levels of urinary kallikrein have been also described⁵¹. In summary, accumulated evidence about the physiologic role of the KKS in the control of blood pressure and salt excretion modulation, as well as its location in the renal tubulointerstitium, makes it plausible that it may be involved in blood pressure modifications induced by changes in sodium balance.

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METHODS

In this communication we present evidence of KKS injury found in three experimental models of renal tubulointerstitial damage characterized by the subsequent development of salt-sensitive hypertension. We have focused our attention in the structural appearance and tissue expression of a pivotal component of the system, the enzyme kallikrein, evaluated with immunohistochemical techniques using sections of rat renal tissue previously fixed in 4% formalin in phosphate-buffered saline (PBS), and embedded in paraffin. Kallikrein detection was achieved using a polyclonal antibody directed to rat urinary kallikrein. As previously reported, the cross-reactivity of this antiserum assessed by dot-blot immunoassay was strong for rK1, moderate for rK2 and rK7, and minimal for rK9⁵². Bound rabbit immunoglobulins were detected using the labeled streptavidin-biotin system (LSAB+) peroxidase system. The extension and the intensity of the labeled surface area were evaluated by using a computerized imaging system previously described in detail^53,54. Briefly, the degree of staining was calculated by the ratio of suitable binary threshold image and the total field area, and integrating the intensity of the staining in the specific areas, thus avoiding variability induced by differences in the amount of total tissue examined. For each sample, a mean value was obtained by analysis of 20 different fields (at 20 or 40, depending of the amount of tissue evaluated) and excluding glomeruli and blood vessels.

The nitric oxide inhibition model

Nitric oxide (NO), endothelium-derived relaxing factor^55,56 has a crucial role in the regulation of vascular tone and renal blood flow. Chronic inhibition of NO synthesis, achieved by the oral administration of N-nitro-L-arginine methyl ester (L-NAME) for 4 to 6 weeks, induces a significant rise in blood pressure accompanied by severe vascular glomerular damage and proteinuria⁵⁷. Renal injury and severity of hypertension are directly related to the level of salt intake⁵⁸.

Most of the vasodilatory effects of kinins appear to be mediated by the release of NO from endothelial cells, since endothelial denudation of large arteries abrogates the vasorelaxant response to kinins⁵⁹. B2 receptors have been identified in the endothelium of large arteries and smooth muscle layers in arterioles⁶⁰. Within the kidney, the vasodilatory response to bradykinin (B2 agonist) is mediated by arachidonic acid metabolites and, to a lesser extent, by NO^61,62,63.

Tubulointerstitial damage is likely to reduce kallikrein-producing tubular areas. Such damage has been reported after long-term nitric oxide synthase inhibition⁶⁴, an experimental manipulation that results in interstitial inflammation, increased local activity of the renin-angiotensin system, and salt sensitive hypertension⁶⁵

We have examined the expression of renal kallikrein in male rats sacrificed after receiving L-NAME for 21 and 28 days at a dose of 50 mg/kg while hydrated with 1% salt in the drinking water [abstract; Ardiles et al, Book of the XX Chilean Congress of Nephrology, Hypertension and Transplantation, 2002, pp 28). These animals showed an important elevation of blood pressure after the first week, whereas control animals receiving only 1% salt in the drinking water remained normotensive. Kidneys of experimental animals showed important tubulointerstitial damage with patchy areas of cellular infiltration and destruction of tubular structures. Immunohistochemistry demonstrated kallikrein-immunoreactive connecting tubules in inflammatory areas and reduction in the immunoexpression of the enzyme after the third week Figure 1.

Figure 1.

(A) Morphologic appearance of tubulointerstitial damage in a representative case of L-NAME treated animal. (B) Quantitative analysis of the immunohistochemical kallikrein expression by computerized imaging system is shown. *P < 0.05 compared with controls. (C) Immunoreactive kallikrein expression in a control and (D) L-NAME treated animal.

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The model of 5/6 renal ablation

This model is characterized by progressive tubulointerstitial damage with inflammation and fibrosis⁶⁶ and subsequent salt sensitivity⁶⁷, and it has been shown to be suitable for testing the potential participation of KKS in the pathogenesis of hypertension. Tubulointerstitial damage is a recognized characteristic of this model^68,69,70, and a reduction in urinary kallikrein levels has been demonstrated^71,72. Figure 2 shows the remnant kidney of 5/6 nephrectomized male Sprague-Dawley rats after 4 weeks of surgery demonstrating severe tubulointerstitial injury characterized by inflammatory cellular infiltration, dilation, and atrophy of tubular structures and a marked reduction in the tissue expression of kallikrein coincidental with an elevation in blood pressure. Interestingly, when these animals were treated with mycophenolate mofetil, attenuation of the tubulointerstitial lesions, an increase in kallikrein tissue expression and normalization of blood pressure were observed⁵⁴. Experiments in which kallikrein gene is delivered into rats with 5/6 reduction in renal mass show that this experimental maneuver attenuates hypertension and protects nephrectomized rats against renal injury and cardiac remodeling⁵⁰. Similar to kallikrein infusion experiments, kallikrein gene delivery significantly decreased proteinuria and increased levels of urinary kinins and cyclic guanosine monophosphate (GMP), while decreasing peripheral vascular resistance. Interestingly, kallikrein gene transfer also reduced glomerular sclerotic lesions, tubular damage, and interstitial inflammation in the kidney⁵⁰. In summary, in the 5/6 renal ablation model, as in other experimental models, tubulointerstitial damage in association with reduction in kallikrein expression may predispose for salt sensitivity and its functional recovery prevents the development of hypertension.

Figure 2.

(A) Morphologic appearance of tubulointerstitial damage in a representative case of 5/6 nephrectomized (Nx) animal. (B) Quantitative analysis of the immunohistochemical kallikrein expression by computarized imaging system *P < 0.05 compared with sham). Immunoreactive kallikrein expression in a (C) sham and (D) Nx animal.

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The overload proteinuria model

This model has been used to study factors triggered by non-immune interstitial inflammation that play a role in the progression of renal diseases^{73,74,75,76,77,78,79,80,81,82,83}. Proteinuria is induced by the daily intraperitoneal injection of 1 to 2 g of bovine serum albumin (BSA) to rats with or without previous nephrectomy. This challenge induces massive proteinuria, followed by substantial renal interstitial inflammation and tubular destruction. Previous studies with this model have not focused on blood pressure changes but recent investigations have shown that the administration of a high-salt diet, a week after the administration of intraperitoneal BSA has stopped, induces salt-sensitive hypertension in female Lewis rats⁸⁴. In these studies, the salt-sensitive hypertension is prevented if an immunosuppressive drug, mycophenolate mofetil, is administered during protein overload⁸⁴, suggesting that the reduction in tubulointerstitial immune infiltration and interstitial angiotensin II activity resulting from this therapy, play a critical role in the pathogenesis of salt retention. We have performed similar experiments using Sprague-Dawley that received 2 g of BSA intraperitoneally for 2 weeks, while control rats received equivalent volumes of vehicle. During the proteinuric stage, the animals developed hypertension after the third dose, which remained significantly higher than the control during the rest of the experimental period. The histologic examination of the kidneys Figure 3 disclosed severe tubulointerstitial damage, including focal inflammation, protein casts, and tubular atrophy with tubular dilation. Immunohistochemistry showed dilated kallikrein-containing tubules (connecting segments), composed by flattened cells considerably less stained than those in vehicle-treated animals [abstract; Ardiles et al, J Am Soc Nephrol 12:460A, 2001].

Figure 3.

(A) Morphologic appearence of tubulointerstitial damage in a representative case of bovine serum albumin (BSA) treated animal. (B) Quantitative analysis of the immunohistochemical kallikrein expression by computerized imaging system. *P < 0.05 compared with control. Immunoreactive kallikrein expression in (C) control and (D) BSA treated animals.

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Recent unpublished experiments performed in our laboratory have shown that stimulation of kallikrein production with high-potassium diet before overload proteinuria was induced, increasing renal kallikrein immunohistochemical expression and urinary activity, reducing blood pressure levels during the experiment. It appears likely that extensive damage in kallikrein-producing structures may disrupt the balance between vasocontrictors/salt-retaining and vasodilators/salt-excreting factors, thus setting the stage for salt retention and arterial hypertension.