Proteomic Analysis Reveals Alterations in the Renal Kallikrein Pathway during Hypoxia-Induced Hypertension

Abstract

Obstructive sleep apnea syndrome (OSAS), a disorder characterized by episodic hypoxia (EH) during sleep, is associated with systemic hypertension. We used proteomic analysis to examine differences in rat kidney protein expression during EH, and their potential relationship to EH-induced hypertension. Young male Sprague-Dawley rats were exposed to either EH or sustained hypoxia (SH) for 14 (EH14/SH14) and 30 (EH30/SH30) days. Mean arterial blood pressure was significantly increased only in EH30 (p < 0.0002). Kidney proteins were resolved by two-dimensional-PAGE and were identified by MALDI-MS. Renal expression of kallistatin, a potent vasodilator, was down-regulated in all animals. Expression of ␣-1-antitrypsin, an inhibitor of kallikrein activation, was up-regulated in EH but down-regulated in SH. Western blotting showed significant elevation of B 2 -bradykinin receptor expression in all normotensive animals but remained unchanged in hypertensive animals. Proteins relevant to vascular hypertrophy, such as smooth muscle myosin and protein-disulfide isomerase were up-regulated in EH30 but were down-regulated in SH30. These data indicate that EH induces changes in renal protein expression consistent with impairment of vasodilation mediated by the kallikrein-kallistatin pathway and vascular hypertrophy. In contrast, SH-induced changes suggest the kallikrein-and bradykinin-mediated compensatory mechanisms for prevention of hypertension and vascular remodeling. To test the hypothesis suggested by the proteomic data, we measured the effect of EH on blood pressure in transgenic hKLK1 rats that overexpress human kallikrein. Transgenic hKLK1 animals were protected from EH-induced hypertension. We conclude that EH-induced hypertension may result, at least in part, from altered regulation of the renal kallikrein system.Obstructive sleep apnea syndrome (OSAS), 1 a disorder characterized by episodic hypoxia (EH), is a major public health problem (1-3). OSAS affects 4 -5% of the general adult population in United States and 1-2% of children (4 -6). One of the major consequences of untreated OSAS is systemic hypertension (7, 8), with a prevalence ranging from 15 to 56% (8 -10). Indeed, OSAS has been demonstrated to be an independent risk factor for systemic hypertension, and the relative risk is elevated even in young children (8, 11). Moreover, OSAS has also been associated with both proteinuria and end-stage renal disease (12).The pathophysiology of systemic hypertension in OSAS is complex. Increased sympathetic nervous system activity induced by the intermittent hypoxia that characteristically accompanies OSAS appears to mediate hypertension during OSAS via a mechanism that requires changes in the vasoreactivity of resistance vascular beds such as the kidney (13-19). Indeed, sustained hypoxia (SH) does not lead to hypertension as do pharmacologically-mediated decreases of angiotensin II levels and blockade of angiotensin receptors (20,21). These data suggest that changes in renal vasoconstrictors play a major role in the dev...

Proteomic Identification and Immunolocalization of Increased Renal Calbindin-D28k Expression in OVE26 Diabetic Mice

“…One of the altered proteins with the greatest magnitude of change was calbindin-D28k, whose expression level increased 6.7-fold in diabetic kidney, as determined by 2-D proteomic analysis. Although MALDI-TOF MS followed by peptide mass fingerprinting is not a definitive protein identification method, we have frequently demonstrated that proteins identified by this method, with careful analysis using a vigorous scoring system to determine a significant hit (match), are consistent with those identified by other methods, especially 1-D and 2-D Western blot analyses [15,16,[19][20][21]. In the present study, the MALDI-MS-based proteomic data were again confirmed by Western blot analysis.…”

“…Protein extraction from the whole kidney was performed as described previously [15,16]. Briefly, the kidney was rapidly frozen in liquid nitrogen, ground to powder, resuspended in a buffer containing 50 mM Tris, 0.3% SDS, and 200 mM DTT, and incubated at 100 °C for 5 min.…”

Hypoxia: The Force that Drives Chronic Kidney Disease

“…292 In addition, intermittent hypoxia induces the sympathetic nervous system to increase vascular resistance, downregulates expression of the kallikrein-kallistatin vasodilator Shelley Transforming growth factor β inhibition 446 Pro-fibrotic metalloproteinase inhibition 447 Anti-fibrotic metalloproteinase activation 447 Destabilization of renal Remote ischemic pre-conditioning [384][385][386][387][388][389][390] hypoxia-inducible factor Prolyl hydroxylase inhibition 244,448 von Hippel-Lindau protein inhibition 449 pathway, and activates the renal renin-angiotensin-aldosterone system to cause vasoconstriction. [293][294][295][296][297][298][299] Together these processes conspire to produce renal fibrosis and sustained hypertension and their associated means of reducing renal oxygenation ( figure 1). 82,[300][301][302][303][304] Oxygenation is further compromised by nocturnal hypoxia altering parasympathetic control of heart rate and inducing left ventricular hypertrophy.…”

“…49,[379][380][381] The targeting of vasoconstriction pathways controlled through vasopressin, endothelin, and voltage dependent calcium channels are in their infancy, as is the use of vasodilators such as arginine and kallikrein. 155,250,268,298,379,382,383 Beyond systemic hypooxygenation and vasoconstriction, the hypoxia-dependent processes of microvascular rarefaction, oxidative stress, leukocyte recruitment, and fibrosis all contribute to the development of CKD. A host of potential therapeutic targets exists within these areas (Table 1).…”

γ-Amino Butyric Acid Type B Receptors Stimulate Neutrophil Chemotaxis during Ischemia-Reperfusion

“…Neutrophil lysates were subjected to isotype control or anti-Akt immunoprecipitation. Immunoprecipitated proteins were initially resolved by high-resolution 2D-PAGE as previously described by our group (22,23). MALDI analysis coupled with BLASTP analysis of Akt immunoprecipitates identified a C-terminal fragment of GABA B R2, as an Akt binding protein.…”