Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin ß2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.

Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase.

Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.

High Elmo1 expression aggravates and low Elmo1 expression prevents diabetic nephropathy.

Human genome-wide association studies have demonstrated that polymorphisms in the engulfment and cell motility protein 1 gene (ELMO1) are strongly associated with susceptibility to diabetic nephropathy. However, proof of causation is lacking. To test whether modest changes in its expression alter the severity of the renal phenotype in diabetic mice, we have generated mice that are type 1 diabetic because they have the Ins2(Akita) gene, and also have genetically graded expression of Elmo1 in all tissues ranging in five steps from ∼30% to ∼200% normal. We here show that the Elmo1 hypermorphs have albuminuria, glomerulosclerosis, and changes in the ultrastructure of the glomerular basement membrane that increase in severity in parallel with the expression of Elmo 1. Progressive changes in renal mRNA expression of transforming growth factor ß1 (TGFß1), endothelin-1, and NAD(P)H oxidase 4 also occur in parallel with Elmo1, as do the plasma levels of cystatin C, lipid peroxides, and TGFß1, and erythrocyte levels of reduced glutathione. In contrast, Akita type 1 diabetic mice with below-normal Elmo1 expression have reduced expression of these various factors and less severe diabetic complications. Remarkably, the reduced Elmo1 expression in the 30% hypomorphs almost abolishes the pathological features of diabetic nephropathy, although it does not affect the hyperglycemia caused by the Akita mutation. Thus, ELMO1 plays an important role in the development of type 1 diabetic nephropathy, and its inhibition could be a promising option for slowing or preventing progression of the condition to end-stage renal disease.

Nicotinamide benefits both mothers and pups in two contrasting mouse models of preeclampsia.

Preeclampsia (PE) complicates ∼5% of human pregnancies and is one of the leading causes of pregnancy-related maternal deaths. The only definitive treatment, induced delivery, invariably results in prematurity, and in severe early-onset cases may lead to fetal death. Many currently available antihypertensive drugs are teratogenic and therefore precluded from use. Nonteratogenic antihypertensives help control maternal blood pressure in PE, but results in preventing preterm delivery and correcting fetal growth restriction (FGR) that also occurs in PE have been disappointing. Here we show that dietary nicotinamide, a nonteratogenic amide of vitamin B3, improves the maternal condition, prolongs pregnancies, and prevents FGR in two contrasting mouse models of PE. The first is caused by endotheliosis due to excess levels in the mothers of a soluble form of the receptor for vascular endothelial growth factor (VEGF), which binds to and inactivates VEGF. The second is caused by genetic absence of Ankiryn-repeat-and-SOCS-box-containing-protein 4, a factor that contributes to the differentiation of trophoblast stem cells into the giant trophoblast cells necessary for embryo implantation in mice; its absence leads to impaired placental development. In both models, fetal production of ATP is impaired and FGR is observed. We show here that nicotinamide decreases blood pressure and endotheliosis in the mothers, probably by inhibiting ADP ribosyl cyclase (ADPRC), and prevents FGR, probably by normalizing fetal ATP synthesis via the nucleotide salvage pathway. Because nicotinamide benefits both dams and pups, it merits evaluation for preventing or treating PE in humans.

A conversation with Oliver Smithies.

Professor Oliver Smithies is the Weatherspoon Eminent Distinguished Professor of Pathology and Laboratory Medicine at the University of North Carolina, Chapel Hill. Along with Mario Capecchi and Martin Evans, Oliver was awarded the Nobel Prize in Medicine in Physiology or Medicine in 2007 for his contributions to the development of gene targeting using homologous recombination in embryonic stem cells. This technique has had an immense impact on biomedical research over the past two decades. Professor Smithies has had a long and distinguished career as a researcher and mentor. Here, he provides an entertaining and enlightening discussion of his life in science.

Low TGFß1 expression prevents and high expression exacerbates diabetic nephropathy in mice.

Nephropathy develops in many but not all patients with long-standing type 1 diabetes. Substantial efforts to identify genotypic differences explaining this differential susceptibility have been made, with limited success. Here, we show that the expression of the transforming growth factor ß1 gene (Tgfb1) affects the development of diabetic nephropathy in mice. To do this we genetically varied Tgfb1 expression in five steps, 10%, 60%, 100%, 150%, and 300% of normal, in mice with type 1 diabetes caused by the Akita mutation in the insulin gene (Ins2(Akita)). Although plasma glucose levels were not affected by Tgfb1 genotype, many features of diabetic nephropathy (mesangial expansion, elevated plasma creatinine and urea, decreased creatinine clearance and albuminuria) were progressively ameliorated as Tgfb1 expression decreased and were progressively exacerbated when expression was increased. The diabetic 10% hypomorphs had comparable creatinine clearance and albumin excretion to wild-type mice and no harmful changes in renal morphology. The diabetic 300% hypermorphs had ∼1/3 the creatinine clearance of wild-type mice, >20× their albumin excretion, ∼3× thicker glomerular basement membranes and severe podocyte effacement, matching human diabetic nephropathy. Switching Tgfb1 expression from low to high in the tubules of the hypomorphs increased their albumin excretion more than 10-fold but creatinine clearance remained high. Switching Tgfb1 expression from low to high in the podocytes markedly decreased creatinine clearance, but minimally increased albumin excretion. Decreasing expression of Tgfb1 could be a promising option for preventing loss of renal function in diabetes.

Endothelin-1 critically influences cardiac function via superoxide-MMP9 cascade.

We have generated low-expressing and high-expressing endothelin-1 genes (L and H) and have bred mice with four levels of expression: L/L, ∼20%; L/+, ∼65%; +/+ (wild type), 100%; and H/+, ∼350%. The hypomorphic L allele can be spatiotemporally switched to the hypermorphic H allele by Cre-loxP recombination. Young adult L/L and L/+ mice have dilated cardiomyopathy, hypertension, and increased plasma volumes, together with increased ventricular superoxide levels, increased matrix metalloproteinase 9 (Mmp9) expression, and reduced ventricular stiffness. H/+ mice have decreased plasma volumes and significantly heavy stiff hearts. Global or cardiomyocyte-specific switching expression from L to H normalized the abnormalities already present in young adult L/L mice. An epithelial sodium channel antagonist normalized plasma volume and blood pressure, but only partially corrected the cardiomyopathy. A superoxide dismutase mimetic made superoxide levels subnormal, reduced Mmp9 overexpression, and substantially improved cardiac function. Genetic absence of Mmp9 also improved cardiac function, but increased superoxide remained. We conclude that endothelin-1 is critical for maintaining normal contractile function, for controlling superoxide and Mmp9 levels, and for ensuring that the myocardium has sufficient collagen to prevent overstretching. Even a modest (∼35%) decrease in endothelin-1 gene (Edn1) expression is sufficient to cause cardiac dysfunction.

Transforming growth factor-ß1 and diabetic nephropathy.

Transforming growth factor-ß1 (TGF-ß1) is established to be involved in the pathogenesis of diabetic nephropathy. The diabetic milieu enhances oxidative stress and induces the expression of TGF-ß1. TGF-ß1 promotes cell hypertrophy and extracellular matrix accumulation in the mesangium, which decreases glomerular filtration rate and leads to chronic renal failure. Recently, TGF-ß1 has been demonstrated to regulate urinary albumin excretion by both increasing glomerular permeability and decreasing reabsorption in the proximal tubules. TGF-ß1 also increases urinary excretion of water, electrolytes and glucose by suppressing tubular reabsorption in both normal and diabetic conditions. Although TGF-ß1 exerts hypertrophic and fibrogenic effects in diabetic nephropathy, whether suppression of the function of TGF-ß1 can be an option to prevent or treat the complication is still controversial. This is partly because adrenal production of mineralocorticoids could be augmented by the suppression of TGF-ß1. However, differentiating the molecular mechanisms for glomerulosclerosis from those for the suppression of the effects of mineralocorticoids by TGF-ß1 may assist in developing novel therapeutic strategies for diabetic nephropathy. In this review, we discuss recent findings on the role of TGF-ß1 in diabetic nephropathy.

Primary aldosteronism and impaired natriuresis in mice underexpressing TGFß1.

To uncover the potential cardiovascular effects of human polymorphisms influencing transforming growth factor ß1 (TGFß1) expression, we generated mice with Tgfb1 mRNA expression graded in five steps from 10% to 300% normal. Adrenal expression of the genes for mineralocorticoid-producing enzymes ranged from 50% normal in the hypermorphs at age 12 wk to 400% normal in the hypomorphs accompanied with proportionate changes in plasma aldosterone levels, whereas plasma volumes ranged from 50% to 150% normal accompanied by marked compensatory changes in plasma angiotensin II and renin levels. The aldosterone/renin ratio ranged from 0.3 times normal in the 300% hypermorphs to six times in the 10% hypomorphs, which have elevated blood pressure. Urinary output of water and electrolytes are markedly decreased in the 10% hypomorphs without significant change in the glomerular filtration rate. Renal activities for the Na(+), K(+)-ATPase, and epithelial sodium channel are markedly increased in the 10% hypomorphs. The hypertension in the 10% hypomorphs is corrected by spironolactone or amiloride at doses that do not change blood pressure in wild-type mice. Thus, changes in Tgfb1 expression cause marked progressive changes in multiple systems that regulate blood pressure and fluid homeostasis, with the major effects being mediated by changes in adrenocortical function.

Transforming growth factor beta1 and aldosterone.

PURPOSE OF REVIEW: It is well established that blocking the renin-angiotensin-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of the RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here, we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. RECENT FINDINGS: TGFbeta1 suppresses the adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps, from 10 to 300% of normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10% hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. SUMMARY: These results identify TGFbeta signalling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism, as well as assist in the development of novel therapeutic strategies for hypertension.

Stable oligomeric clusters of gold nanoparticles: preparation, size distribution, derivatization, and physical and biological properties.

Reducing dilute aqueous HAuCl4 with NaSCN under alkaline conditions produces 2-3 nm diameter yellow nanoparticles without the addition of extraneous capping agents. We here describe two very simple methods for producing highly stable oligomeric grape-like clusters (oligoclusters) of these small nanoparticles. The oligoclusters have well-controlled diameters ranging from ∼5 to ∼30 nm, depending mainly on the number of subunits in the cluster. Our first ["delay-time"] method controls the size of the oligoclusters by varying from seconds to hours the delay time between making the HAuCl4 alkaline and adding the reducing agent, NaSCN. Our second ["add-on"] method controls size by using yellow nanoparticles as seeds onto which varying amounts of gold derived from "hydroxylated gold", Na(+)[Au(OH4-x)Clx](-), are added-on catalytically in the presence of NaSCN. Possible reaction mechanisms and a simple kinetic model fitting the data are discussed. The crude oligocluster preparations have narrow size distributions, and for most purposes do not require fractionation. The oligoclusters do not aggregate after ∼300-fold centrifugal-filter concentration, and at this high concentration are easily derivatized with a variety of thiol-containing reagents. This allows rare or expensive derivatizing reagents to be used economically. Unlike conventional glutathione-capped nanoparticles of comparable gold content, large oligoclusters derivatized with glutathione do not aggregate at high concentrations in phosphate-buffered saline (PBS) or in the circulation when injected into mice. Mice receiving them intravenously show no visible signs of distress. Their sizes can be made small enough to allow their excretion in the urine or large enough to prevent them from crossing capillary basement membranes. They are directly visible in electron micrographs without enhancement, and can model the biological fate of protein-like macromolecules with controlled sizes and charges. The ease of derivatizing the oligoclusters makes them potentially useful for presenting pharmacological agents to different tissues while controlling escape of the reagents from the circulation.

A modest decrease in endothelial NOS in mice comparable to that associated with human NOS3 variants exacerbates diabetic nephropathy.

Polymorphisms in the human endothelial nitric oxide synthase (eNOS) gene (NOS3) have been associated with advanced nephropathy in diabetic patients and with decreased expression in tissue culture. However, direct proof that modest genetic decreases in eNOS expression worsen diabetic nephropathy is lacking. To investigate this effect, we took advantage of the hybrid vigor and genetic uniformity of the F1 progeny (eNOS(+/+), eNOS(+/-), or eNOS(-/-) with or without diabetes) of a cross between heterozygous 129S6/SvEvTac eNOS(+/-) inbred females and heterozygous C57BL/6J eNOS(+/-) inbred males carrying the dominant Akita diabetogenic mutation Ins2(C96Y/+). Whereas all C57BL/6J inbred eNOS(-/-) and eNOS(+/-) diabetic mice died before 5 mo, almost half of the F1 hybrid eNOS(-/-) and eNOS(+/-) diabetic mice lived until killed at 7 mo. Heterozygous eNOS(+/-) diabetic mice expressed ∼35% eNOS mRNA in the kidney and ∼25% glomerular eNOS protein relative to their eNOS(+/+) diabetic littermates. These decreases in eNOS elevated blood pressure (BP) but not blood glucose. Urinary albumin excretion, mesangial expansion, glomerulosclerosis, mesangiolysis, and glomerular filtration rate increased in the order: eNOS(+/+) Akita < eNOS(+/-) Akita < eNOS(-/-) Akita, independently of BP. Glomerular basement membrane thickening depended on increased BP. Renal expression of tissue factor and other inflammatory factors increased with the nephropathy; Nos2 also increased. Surprisingly, however, decreased eNOS expression ameliorated the increases in oxidative stress and tubulointerstitial fibrosis caused by diabetes. Our data demonstrate that a modest decrease in eNOS, comparable to that associated with human NOS3 variants, is sufficient to enhance diabetic nephropathy independently of its effects on BP.

Mitochondrial DNA polymerase editing mutation, PolgD257A, reduces the diabetic phenotype of Akita male mice by suppressing appetite.

Diabetes and the development of its complications have been associated with mitochondrial DNA (mtDNA) dysfunction, but causal relationships remain undetermined. With the objective of testing whether increased mtDNA mutations exacerbate the diabetic phenotype, we have compared mice heterozygous for the Akita diabetogenic mutation (Akita) with mice homozygous for the D257A mutation in mitochondrial DNA polymerase gamma (Polg) or with mice having both mutations (Polg-Akita). The Polg-D257A protein is defective in proofreading and increases mtDNA mutations. At 3 mo of age, the Polg-Akita and Akita male mice were equally hyperglycemic. Unexpectedly, as the Polg-Akita males aged to 9 mo, their diabetic symptoms decreased. Thus, their hyperglycemia, hyperphagia and urine output declined significantly. The decrease in their food intake was accompanied by increased plasma leptin and decreased plasma ghrelin, while hypothalamic expression of the orexic gene, neuropeptide Y, was lower and expression of the anorexic gene, proopiomelanocortin, was higher. Testis function progressively worsened with age in the double mutants, and plasma testosterone levels in 9-mo-old Polg-Akita males were significantly reduced compared with Akita males. The hyperglycemia and hyperphagia returned in aged Polg-Akita males after testosterone administration. Hyperglycemia-associated distal tubular damage in the kidney also returned, and Polg-D257A-associated proximal tubular damage was enhanced. The mild diabetes of female Akita mice was not affected by the Polg-D257A mutation. We conclude that reduced diabetic symptoms of aging Polg-Akita males results from appetite suppression triggered by decreased testosterone associated with damage to the Leydig cells of the testis.

Lack of both bradykinin B1 and B2 receptors enhances nephropathy, neuropathy, and bone mineral loss in Akita diabetic mice.

An insertion polymorphism of the angiotensin-I converting enzyme gene (ACE) is common in humans and the higher expressing allele is associated with an increased risk of diabetic complications. The ACE polymorphism does not significantly affect blood pressure or angiotensin II levels, suggesting that the kallikrein-kinin system partly mediates the effects of the polymorphism. We have therefore explored the influence of lack of both bradykinin receptors (B1R and B2R) on diabetic nephropathy, neuropathy, and osteopathy in male mice heterozygous for the Akita diabetogenic mutation in the insulin 2 gene (Ins2). We find that all of the detrimental phenotypes observed in Akita diabetes are enhanced by lack of both B1R and B2R, including urinary albumin excretion, glomerulosclerosis, glomerular basement membrane thickening, mitochondrial DNA deletions, reduction of nerve conduction velocities and of heat sensation, and bone mineral loss. Absence of the bradykinin receptors also enhances the diabetes-associated increases in plasma thiobarbituric acid-reactive substances, mitochondrial DNA deletions, and renal expression of fibrogenic genes, including transforming growth factor beta1, connective tissue growth factor, and endothelin-1. Thus, lack of B1R and B2R exacerbates diabetic complications. The enhanced renal injury in diabetic mice caused by lack of B1R and B2R may be mediated by a combination of increases in oxidative stress, mitochondrial DNA damage and over expression of fibrogenic genes.

Heterogeneous myocyte enhancer factor-2 (Mef2) activation in myocytes predicts focal scarring in hypertrophic cardiomyopathy.

Unknown molecular responses to sarcomere protein gene mutations account for pathologic remodeling in hypertrophic cardiomyopathy (HCM), producing myocyte growth and increased cardiac fibrosis. To determine if hypertrophic signals activated myocyte enhancer factor-2 (Mef2), we studied mice carrying the HCM mutation, myosin heavy-chain Arg403Gln, (MHC(403/+)) and an Mef2-dependent ß-galactosidase reporter transgene. In young, prehypertrophic MHC(403/+) mice the reporter was not activated. In hypertrophic hearts, activation of the Mef2-dependent reporter was remarkably heterogeneous and was observed consistently in myocytes that bordered fibrotic foci with necrotic cells, MHC(403/+) myocytes with Mef2-dependent reporter activation reexpressed the fetal myosin isoform (ßMHC), a molecular marker of hypertrophy, although MHC(403/+) myocytes with or without ßMHC expression were comparably enlarged over WT myocytes. To consider Mef2 roles in severe HCM, we studied homozygous MHC(403/403) mice, which have accelerated remodeling, widespread myocyte necrosis, and neonatal lethality. Levels of phosphorylated class II histone deacetylases that activate Mef2 were substantially increased in MHC(403/403) hearts, but Mef2-dependent reporter activation was patchy. Sequential analyses showed myocytes increased Mef2-dependent reporter activity before death. Our data dissociate myocyte hypertrophy, a consistent response in HCM, from heterogeneous Mef2 activation and reexpression of a fetal gene program. The temporal and spatial relationship of Mef2-dependent gene activation with myocyte necrosis and fibrosis in MHC(403/+) and MHC(403/403) hearts defines Mef2 activation as a molecular signature of stressed HCM myocytes that are poised to die.

eNOS deficiency acts through endothelin to aggravate sFlt-1-induced pre-eclampsia-like phenotype.

Excess soluble fms-like tyrosine kinase 1 (sFlt-1) of vascular endothelial growth factor receptor 1 secreted from the placenta causes pre-eclampsia-like features by antagonizing vascular endothelial growth factor signaling, which can lead to reduced endothelial nitric oxide synthase (eNOS) activity; the effect of this concomitant decrease in eNOS activity is unknown. We tested whether the decrease in nitric oxide occurring in female mice lacking eNOS aggravates the pre-eclampsia-like phenotype induced by increased sFlt-1. Untreated eNOS-deficient female mice had higher BP than wild-type mice. Adenovirus-mediated overexpression of sFlt-1 increased systolic BP by approximately 27 mmHg and led to severe loss of fenestration of glomerular capillary endothelial cells in both eNOS-deficient and wild-type mice. However, only the eNOS-deficient sFlt-1 mice exhibited severe foot process effacement. Compared with wild-type sFlt-1 mice, eNOS-deficient sFlt-1 mice also showed markedly higher urinary albumin excretion (467±74 versus 174±23 µg/d), lower creatinine clearance (126±29 versus 452±63 µl/min), and more severe endotheliosis. Expression of preproendothelin-1 (ET-1) and its ET(A) receptor in the kidney was higher in eNOS-deficient sFlt-1 mice than in wild-type sFlt-1 mice. Furthermore, the selective ET(A) receptor antagonist ambrisentan attenuated the increases in BP and urinary albumin excretion and ameliorated endotheliosis in both wild-type and eNOS-deficient sFlt-1 mice. Ambrisentan improved creatinine clearance and podocyte effacement in eNOS-deficient sFlt-1 mice. In conclusion, reduced maternal eNOS/nitric oxide exacerbates the sFlt1-related pre-eclampsia-like phenotype through activation of the endothelin system.

Null mutations at the p66 and bradykinin 2 receptor loci induce divergent phenotypes in the diabetic kidney.

Candidate genes have been identified that confer increased risk for diabetic glomerulosclerosis (DG). Mice heterozygous for the Akita (Ins2(+/C96Y)) diabetogenic mutation with a second mutation introduced at the bradykinin 2 receptor (B2R(-/-)) locus express a disease phenotype that approximates human DG. Src homology 2 domain transforming protein 1 (p66) controls mitochondrial metabolism and cellular responses to oxidative stress, aging, and apoptosis. We generated p66-null Akita mice to test whether inactivating mutations at the p66 locus will rescue kidneys of Akita mice from disease-causing mutations at the Ins2 and B2R loci. Here we show null mutations at the p66 and B2R loci interact with the Akita (Ins2(+/C96Y)) mutation, independently and in combination, inducing divergent phenotypes in the kidney. The B2R(-/-) mutation induces detrimental phenotypes, as judged by increased systemic and renal levels of oxidative stress, histology, and urine albumin excretion, whereas the p66-null mutation confers a powerful protection phenotype. To elucidate the mechanism(s) of the protection phenotype, we turned to our in vitro system. Experiments with cultured podocytes revealed previously unrecognized cross talk between p66 and the redox-sensitive transcription factor p53 that controls hyperglycemia-induced ROS metabolism, transcription of p53 target genes (angiotensinogen, angiotensin II type-1 receptor, and bax), angiotensin II generation, and apoptosis. RNA-interference targeting p66 inhibits all of the above. Finally, protein levels of p53 target genes were upregulated in kidneys of Akita mice but unchanged in p66-null Akita mice. Taken together, p66 is a potential molecular target for therapeutic intervention in DG.

The kallikrein-kinin system in diabetic nephropathy.

Diabetic nephropathy is the major cause of end-stage renal disease worldwide. Although the renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy, angiotensin I-converting enzyme inhibitors have a beneficial effect on diabetic nephropathy independently of their effects on blood pressure and plasma angiotensin II levels. This suggests that the kallikrein-kinin system (KKS) is also involved in the disease. To study the role of the KKS in diabetic nephropathy, mice lacking either the bradykinin B1 receptor (B1R) or the bradykinin B2 receptor (B2R) have been commonly used. However, because absence of either receptor causes enhanced expression of the other, it is difficult to determine the precise functions of each receptor. This difficulty has recently been overcome by comparing mice lacking both receptors with mice lacking each receptor. Deletion of both B1R and B2R reduces nitric oxide (NO) production and aggravates renal diabetic phenotypes, relevant to either lack of B1R or B2R, demonstrating that both B1R and B2R exert protective effects on diabetic nephropathy presumably via NO. Here, we review previous epidemiological and experimental studies, and discuss novel insights regarding the therapeutic implications of the importance of the KKS in averting diabetic nephropathy.