Combination therapy of angiotensin-converting enzyme (ACE) inhibition and AT 1 receptor blockade has been shown to provide greater renoprotection than ACE inhibitor alone in human diabetic nephropathy, suggesting that ACEindependent pathways for ANG II formation are of major significance in disease progression. Studies were performed to determine the magnitude of intrarenal ACE-independent formation of ANG II in type II diabetes. Although renal cortical ACE protein activity [2.1 Ϯ 0.8 vs. 9.2 Ϯ 2.1 arbitrary fluorescence units (AFU) ⅐ mg Ϫ1 ⅐ min Ϫ1 ] and intensity of immunohistochemical staining were significantly reduced and ACE2 protein activity (16.7 Ϯ 3.2 vs. 7.2 Ϯ 2.4 AFU ⅐ mg Ϫ1 ⅐ min Ϫ1 ) and intensity elevated, kidney ANG I (113 Ϯ 24 vs. 110 Ϯ 45 fmol/g) and ANG II (1,017 Ϯ 165 vs. 788 Ϯ 99 fmol/g) levels were not different between diabetic and control mice. Afferent arteriole vasoconstriction due to conversion of ANG I to ANG II was similar in magnitude in kidneys of diabetic (Ϫ28 Ϯ 3% at 1 M) and control (Ϫ23 Ϯ 3% at 1 M) mice; a response completely inhibited by AT 1 receptor blockade. In control kidneys, afferent arteriole vasoconstriction produced by ANG I was significantly attenuated by ACE inhibition, but not by serine protease inhibition. In contrast, afferent arteriole vasoconstriction produced by intrarenal conversion of ANG I to ANG II was significantly attenuated by serine protease inhibition, but not by ACE inhibition in diabetic kidneys. In conclusion, there is a switch from ACE-dependent to serine proteasedependent ANG II formation in the type II diabetic kidney. Pharmacological targeting of these serine protease-dependent pathways may provide further protection from diabetic renal vascular disease. afferent arteriole; juxtamedullary nephron; db/db mouse; angiotensinconverting enzyme; serine protease; angiotensinogen; angiotensinconverting enzyme 2 DIABETIC NEPHROPATHY IS A microvascular complication of type II diabetes mellitus which causes progressive chronic kidney disease, often leading to end-stage renal disease. Pharmacological agents that inhibit the actions of ACE and AT 1 receptors delay the onset and slow the progression of diabetic nephropathy in humans, indicating the importance of the renin-angiotensin system (RAS) in diabetic renal disease. However, ACE inhibitors and AT 1 receptor blockers do not arrest disease progression to end-stage renal failure. Additionally, the demonstration that combined ACE inhibitor plus AT 1 receptor blocker lowers blood pressure (2, 25) and provides greater protection in diabetic nephropathy (13, 27) than ACE inhibitor alone suggests that suppression of the RAS is incomplete. It has been suggested that dual blockade of RAS with inhibition of ACE and AT 1 receptor blockade results in an additional reduction in proteinuria in patients with chronic kidney disease (5). Thus ACE inhibitor monotherapy may allow for the continued generation of ANG II via ACE-independent pathways.Recently, there has been growing interest in the role of ACE-independent AN...

The db/db mouse is a genetic model of type 2 diabetes that exhibits progressive renal disease. Obesity, hyperglycemia, and albuminuria (822 Ϯ 365 vs. 28 Ϯ 8 g/day) are evident in 18-wk-old db/db compared with db/m (lean littermate control) mice. Our goal was to determine the blood pressure (BP) phenotype of the db/db mouse. Mean arterial BP measured in conscious mice by radiotelemetry was not different between db/db (n ϭ 9) and db/m (n ϭ 12) mice, averaging 113 Ϯ 3 and 112 Ϯ 2 mmHg, respectively. The circadian BP profile of db/db mice was shifted to the left and exhibited a significant reduction in amplitude compared with db/m mice. Heart rate (487 Ϯ 9 vs. 542 Ϯ 7 beats/min; P Ͻ 0.05) and locomotor activity were significantly reduced in db/db compared with db/m mice. We tested the hypothesis that intact afferent arteriole (AA) responsiveness to increases in renal artery pressure (RAP) and angiotensin (ANG) II sensitivity contributes to normal BP in this diabetic model. AA diameters of in vitro blood-perfused juxtamedullary nephrons of db/db mice (15.7 Ϯ 0.5 m; n ϭ 38) were significantly larger than those of db/m mice (12.5 Ϯ 0.4 m; n ϭ 37). AA responses to increases in RAP and ANG II were not different between kidneys of db/db and db/m mice. Significant AA vasoconstriction to 1 nM ANG II was observed in kidneys of db/db mice (Ϫ11 Ϯ 4%), while 10 nM ANG II decreased AA diameter in both groups [db/db, Ϫ20 Ϯ 4%, (n ϭ 12); db/m, Ϫ26 Ϯ 4% (n ϭ 12)]. In summary, AA responses to increases in renal perfusion pressure and ANG II remain intact in db/db mice. Diabetic renal disease occurs in db/db mice independently of elevated BP. radiotelemetry; juxtamedullary nephron; angiotensin II; hyperglycemia TYPE 2 DIABETES MELLITUS AFFECTS 24 million Americans and 220 million people worldwide (1, 4) and is the sixth leading cause of death in the US. Obesity has been identified as the principal risk factor associated with the rising prevalence of type 2 diabetes (7). According to the American Diabetes Association, ϳ73% of adults with diabetes are hypertensive; therefore, over one-quarter of patients are normotensive. Diabetic nephropathy is the most common cause of end-stage renal disease in the Western world. Diabetic nephropathy is characterized by progressive albuminuria, declining glomerular filtration rate (GFR), and increased risk for cardiovascular disease.The db/db mouse was first identified at the Jackson Laboratory in 1966 (16). The diabetes mutation (db) is inherited as an autosomal recessive trait and resembles the metabolic disturbances in diabetes mellitus in humans. Thirty years later, it was discovered that the db mutation is a G-to-T point mutation in the gene encoding the leptin receptor. This mutation causes abnormal splicing and defective signaling of the adipocytederived hormone leptin (6, 19). The lack of leptin receptor signaling in the hypothalamus leads to persistent hyperphagia and obesity. The db/db mouse exhibits hyperleptinemia and hyperinsulinemia and develops hyperglycemia in association with insulin...

Angiotensin (ANG) type 1A (AT(1A)) receptor-null (AT(1A)(-/-)) mice exhibit reduced afferent arteriolar (AA) constrictor responses to ANG II compared with wild-type (WT) mice, whereas efferent arteriolar (EA) responses are absent (Harrison-Bernard LM, Cook AK, Oliverio MI, and Coffman TM. Am J Physiol Renal Physiol 284: F538-F545, 2003). In the present study, the renal arteriolar constrictor responses to norepinephrine (NE) and/or ANG II were determined in blood-perfused juxtamedullary nephrons from kidneys of AT(1A)(-/-), AT(1B) receptor-null (AT(1B)(-/-)), and WT mice. Baseline AA diameter in AT(1A)(-/-) mice was not different from that in WT mice (13.1 +/- 0.9 and 12.6 +/- 0.9 microm, n = 7 and 8, respectively); however, EA diameters were significantly larger (17.3 +/- 1.4 vs. 11.7 +/- 0.4 microm, n = 10 and 8) in AT(1A)(-/-) than in WT mice. Constriction of AA (-40 +/- 8 and -51 +/- 6% at 1 microM NE) and EA (-29 +/- 6 and -38 +/- 3% at 1 microM NE) in response to 0.1-1 microM NE was similar in AT(1A)(-/-) and WT mice. Baseline diameters of AA (13.5 +/- 0.7 and 14.2 +/- 0.9 microm, n = 9 and 10) and EA (15.4 +/- 1.0 and 15.0 +/- 0.7 microm, n = 11 and 9) and ANG II (0.1-10 nM) constrictor responses of AA (-25 +/- 4 and -31 +/- 5% at 10 nM) and EA (-32 +/- 6 and -35 +/- 7% at 10 nM) were similar in AT(1B)(-/-) and WT mice, respectively. ANG II-induced constrictions were eliminated by AT(1) receptor blockade with 4 microM candesartan. Taken together, our data demonstrate that AA and EA responses to NE are unaltered in the absence of AT(1A) receptors, and ANG II responses remain intact in the absence of AT(1B) receptors. Therefore, we conclude that AT(1A) and AT(1B) receptors are functionally expressed on the AA, whereas the EA exclusively expresses the AT(1A) receptor.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. Significance: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Diabetic nephropathy is a major cause of end-stage renal disease worldwide. The current studies were performed to determine the later stages of the progression of renal disease in type II diabetic mice (BKS; db/db). Methodology was developed for determining glomerular filtration rate (GFR) in conscious, chronically instrumented mice using continuous intravenous infusion of FITC-labeled inulin to achieve a steady-state plasma inulin concentration. Obese diabetic mice exhibited increased GFR compared with control mice. GFR averaged 0.313 Ϯ 0.018 and 0.278 Ϯ 0.007 ml/min in 18-wk-old obese diabetic (n ϭ 11) and control (n ϭ 13) mice, respectively (P Ͻ 0.05). In 28-wk-old obese diabetic (n ϭ 10) and control (n ϭ 15) mice, GFR averaged 0.348 Ϯ 0.030 and 0.279 Ϯ 0.009 ml/min, respectively (P Ͻ 0.05). GFR expressed per gram BW was significantly reduced in 18-and 28-wk-old obese diabetic compared with control mice (5.9 Ϯ 0.3 vs. 9.0 Ϯ 0.3; 6.6 Ϯ 0.6 vs. 7.8 Ϯ 0.3 l·min Ϫ1 ·g body wt Ϫ1 ), respectively (P Ͻ 0.05). However, older nonobese type II diabetic mice had significantly reduced GFR (0.179 Ϯ 0.023 ml/min; n ϭ 6) and elevated urinary albumin excretion (811 Ϯ 127 g/day) compared with obese diabetic and control mice (514 Ϯ 54, 171 Ϯ 18 g/day), which are consistent with the advanced stages of renal disease. These studies suggest that hyperfiltration contributes to the progression of renal disease in type II diabetic mice. renal inulin clearance; plasma volume; intravenous infusion; db/db mouse; Evans blue dye; indicator dilution technique TYPE II DIABETES MELLITUS is the most common endocrine disease affecting 250 million people worldwide (1, 4). Obesity has been identified as the principal risk factor associated with the rising prevalence of type II diabetes (12), which is predicted to reach 9% of the US population by 2025 (2). Diabetic nephropathy, a major cause of end-stage renal disease, is characterized by progressive albuminuria, declining glomerular filtration rate (GFR), and increased risk for cardiovascular disease.The obese leptin receptor-deficient type II diabetic db/db mouse exhibits metabolic disturbances of diabetes mellitus similar to the characteristics of humans, thus making it a valuable model of type II diabetic disease (5,7,16,28). The lack of leptin receptor signaling leads to persistent hyperphagia and obesity. The db/db mouse exhibits hyperleptinemia, hyperinsulinemia, and develops hyperglycemia in association with insulin resistance. Most importantly, this model exhibits a robust albuminuria, renal and glomerular hypertrophy, thick-