Comparative Analysis of Hypertensive Tubulopathy in Animal Models of Hypertension and Its Relevance to Human Pathology.

Assessment of hypertensive tubulopathy for more than fifty animal models of hypertension in experimental pathology employs criteria that do not correspond to lesional descriptors for tubular lesions in clinical pathology. We provide a critical appraisal of experimental hypertension with the same approach used to estimate hypertensive renal tubulopathy in humans. Four models with different pathogenesis of hypertension were analyzed-chronic angiotensin (Ang) II-infused and renin-overexpressing (TTRhRen) mice, spontaneously hypertensive (SHR), and Goldblatt two-kidney one-clip (2K1C) rats. Mouse models, SHR, and the nonclipped kidney in 2K1C rats had no regular signs of hypertensive tubulopathy. Histopathology in animals was mild and limited to variations in the volume density of tubular lumen and epithelium, interstitial space, and interstitial collagen. Affected kidneys in animals demonstrated lesion values that are significantly different compared with healthy controls but correspond to mild damage if compared with hypertensive humans. The most substantial human-like hypertensive tubulopathy was detected in the clipped kidney of 2K1C rats. For the first time, our study demonstrated the regular presence of chronic progressive nephropathy (CPN) in relatively young mice and rats with induced hypertension. Because CPN may confound the assessment of rodent models of hypertension, proliferative markers should be used to verify nonhypertensive tubulopathy.

Prostaglandin E2 receptor EP1 (PGE2/EP1) deletion promotes glomerular podocyte and endothelial cell injury in hypertensive TTRhRen mice.

Prostaglandin E2 receptor EP1 (PGE2/EP1) promotes diabetic renal injury, and EP1 receptor deletion improves hyperfiltration, albuminuria, and fibrosis. The role of EP1 receptors in hypertensive kidney disease (HKD) remains controversial. We examined the contribution of EP1 receptors to HKD. EP1 null (EP1-/-) mice were bred with hypertensive TTRhRen mice (Htn) to evaluate kidney function and injury at 24 weeks. EP1 deletion had no effect on elevation of systolic blood pressure in Htn mice (HtnEP1-/-) but resulted in pronounced albuminuria and reduced FITC-inulin clearance, compared with Htn or wild-type (WT) mice. Ultrastructural injury to podocytes and glomerular endothelium was prominent in HtnEP1-/- mice; including widened subendothelial space, subendothelial lucent zones and focal lifting of endothelium from basement membrane, with focal subendothelial cell debris. Cortex COX2 mRNA was increased by EP1 deletion. Glomerular EP3 mRNA was reduced by EP1 deletion, and EP4 by Htn and EP1 deletion. In WT mice, PGE2 increased chloride reabsorption via EP1 in isolated perfused thick ascending limb (TAL), but PGE2 or EP1 deletion did not affect vasopressin-mediated chloride reabsorption. In WT and Htn mouse inner medullary collecting duct (IMCD), PGE2 inhibited vasopressin-water transport, but not in EP1-/- or HtnEP1-/- mice. Overall, EP1 mediated TAL and IMCD transport in response to PGE2 is unaltered in Htn, and EP1 is protective in HKD.

A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors: GPR40 and GPR84.

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases.

PBI-4050 via GPR40 activation improves adenine-induced kidney injury in mice.

PBI-4050 (3-pentylbenzenacetic acid sodium salt), a novel first-in-class orally active compound that has completed clinical Phases Ib and II in subjects with chronic kidney disease (CKD) and metabolic syndrome respectively, exerts antifibrotic effects in several organs via a novel mechanism of action, partly through activation of the G protein receptor 40 (GPR40) receptor. Here we evaluate the effects of PBI-4050 in both WT and Gpr40-/- mice on adenine-induced tubulointerstitial injury, anemia and activation of the unfolded protein response (UPR) pathway. Adenine-induced CKD was achieved in 8-week-old C57BL/6 mice fed a diet supplemented with 0.25% adenine. After 1 week, PBI-4050 or vehicle was administered daily by oral-gavage for 3 weeks. Gpr40-/- mice were also subjected to adenine-feeding, with or without PBI-4050 treatment. PBI-4050 improved renal function and urine concentrating ability. Anemia was present in adenine-fed mice, while PBI-4050 blunted these effects and led to significantly higher plasma erythropoietin (EPO) levels. Adenine-induced renal fibrosis, endoplasmic reticulum (ER) stress and apoptosis were significantly decreased by PBI-4050. In parallel, Gpr40-/- mice were more susceptible to adenine-induced fibrosis, renal function impairment, anemia and ER stress compared with WT mice. Importantly, PBI-4050 treatment in Gpr40-/- mice failed to reduce renal injury in this model. Taken together, PBI-4050 prevented adenine-induced renal injury while these beneficial effects were lost upon Gpr40 deletion. These data reinforce PBI-4050's use as a renoprotective therapy and identify GPR40 as a crucial mediator of its beneficial effects.

PGE2 EP1 receptor inhibits vasopressin-dependent water reabsorption and sodium transport in mouse collecting duct.

PGE2 regulates glomerular hemodynamics, renin secretion, and tubular transport. This study examined the contribution of PGE2 EP1 receptors to sodium and water homeostasis. Male EP1-/- mice were bred with hypertensive TTRhRen mice (Htn) to evaluate blood pressure and kidney function at 8 weeks of age in four groups: wildtype (WT), EP1-/-, Htn, HtnEP1-/-. Blood pressure and water balance were unaffected by EP1 deletion. COX1 and mPGE2 synthase were increased and COX2 was decreased in mice lacking EP1, with increases in EP3 and reductions in EP2 and EP4 mRNA throughout the nephron. Microdissected proximal tubule sglt1, NHE3, and AQP1 were increased in HtnEP1-/-, but sglt2 was increased in EP1-/- mice. Thick ascending limb NKCC2 was reduced in the cortex but increased in the medulla. Inner medullary collecting duct (IMCD) AQP1 and ENaC were increased, but AVP V2 receptors and urea transporter-1 were reduced in all mice compared to WT. In WT and Htn mice, PGE2 inhibited AVP-water transport and increased calcium in the IMCD, and inhibited sodium transport in cortical collecting ducts, but not in EP1-/- or HtnEP1-/- mice. Amiloride (ENaC) and hydrochlorothiazide (pendrin inhibitor) equally attenuated the effect of PGE2 on sodium transport. Taken together, the data suggest that EP1 regulates renal aquaporins and sodium transporters, attenuates AVP-water transport and inhibits sodium transport in the mouse collecting duct, which is mediated by both ENaC and pendrin-dependent pathways.

Hyperfiltration in ubiquitin C-terminal hydrolase L1-deleted mice.

Neuronal ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that maintains intracellular ubiquitin pools and promotes axonal transport. Uchl1 deletion in mice leads to progressive axonal degeneration, affecting the dorsal root ganglion that harbors axons emanating to the kidney. Innervation is a crucial regulator of renal hemodynamics, though the contribution of neuronal UCHL1 to this is unclear. Immunofluorescence revealed significant neuronal UCHL1 expression in mouse kidney, including periglomerular axons. Glomerular filtration rate trended higher in 6-week-old Uchl1-/- mice, and by 12 weeks of age, these displayed significant glomerular hyperfiltration, coincident with the onset of neurodegeneration. Angiotensin converting enzyme inhibition had no effect on glomerular filtration rate of Uchl1-/- mice indicating that the renin-angiotensin system does not contribute to the observed hyperfiltration. DCE-MRI revealed increased cortical renal blood flow in Uchl1-/- mice, suggesting that hyperfiltration results from afferent arteriole dilation. Nonetheless, hyperglycemia, cyclooxygenase-2, and nitric oxide synthases were ruled out as sources of hyperfiltration in Uchl1-/- mice as glomerular filtration rate remained unchanged following insulin treatment, and cyclooxygenase-2 and nitric oxide synthase inhibition. Finally, renal nerve dysfunction in Uchl1-/- mice is suggested given increased renal nerve arborization, decreased urinary norepinephrine, and impaired vascular reactivity. Uchl1-deleted mice demonstrate glomerular hyperfiltration associated with renal neuronal dysfunction, suggesting that neuronal UCHL1 plays a crucial role in regulating renal hemodynamics.

PGE2, Kidney Disease, and Cardiovascular Risk: Beyond Hypertension and Diabetes.

An important measure of cardiovascular health is obtained by evaluating the global cardiovascular risk, which comprises a number of factors, including hypertension and type 2 diabetes, the leading causes of illness and death in the world, as well as the metabolic syndrome. Altered immunity, inflammation, and oxidative stress underlie many of the changes associated with cardiovascular disease, diabetes, and the metabolic syndrome, and recent efforts have begun to elucidate the contribution of PGE2 in these events. This review summarizes the role of PGE2 in kidney disease outcomes that accelerate cardiovascular disease, highlights the role of cyclooxygenase-2/microsomal PGE synthase 1/PGE2 signaling in hypertension and diabetes, and outlines the contribution of PGE2 to other aspects of the metabolic syndrome, particularly abdominal adiposity, dyslipidemia, and atherogenesis. A clearer understanding of the role of PGE2 could lead to new avenues to improve therapeutic options and disease management strategies.

PGE2 receptor EP3 inhibits water reabsorption and contributes to polyuria and kidney injury in a streptozotocin-induced mouse model of diabetes.

AIMS/HYPOTHESIS: The first clinical manifestation of diabetes is polyuria. The prostaglandin E2 (PGE2) receptor EP3 antagonises arginine vasopressin (AVP)-mediated water reabsorption and its expression is increased in the diabetic kidney. The purpose of this work was to study the contribution of EP3 to diabetic polyuria and renal injury. METHODS: Male Ep 3 (-/-) (also known as Ptger3 (-/-)) mice were treated with streptozotocin (STZ) to generate a mouse model of diabetes and renal function was evaluated after 12 weeks. Isolated collecting ducts (CDs) were microperfused to study the contribution of EP3 to AVP-mediated fluid reabsorption. RESULTS: Ep 3 (-/-)-STZ mice exhibited attenuated polyuria and increased urine osmolality compared with wild-type STZ (WT-STZ) mice, suggesting enhanced water reabsorption. Compared with WT-STZ mice, Ep 3 (-/-)-STZ mice also had increased protein expression of aquaporin-1, aquaporin-2, and urea transporter A1, and reduced urinary AVP excretion, but increased medullary V2 receptors. In vitro microperfusion studies indicated that Ep 3 (-/-) and WT-STZ CDs responded to AVP stimulation similarly to those of wild-type mice, with a 60% increase in fluid reabsorption. In WT non-injected and WT-STZ mice, EP3 activation with sulprostone (PGE2 analogue) abrogated AVP-mediated water reabsorption; this effect was absent in mice lacking EP3. A major finding of this work is that Ep 3 (-/-)-STZ mice showed blunted renal cyclooxygenase-2 protein expression, reduced renal hypertrophy, reduced hyperfiltration and reduced albuminuria, as well as diminished tubular dilation and nuclear cysts. CONCLUSIONS/INTERPRETATION: Taken together, the data suggest that EP3 contributes to diabetic polyuria by inhibiting expression of aquaporins and that it promotes renal injury during diabetes. EP3 may prove to be a promising target for more selective management of diabetic kidney disease.

Prostaglandin E2 increases proximal tubule fluid reabsorption, and modulates cultured proximal tubule cell responses via EP1 and EP4 receptors.

Renal prostaglandin (PG) E2 regulates salt and water transport, and affects disease processes via EP1-4 receptors, but its role in the proximal tubule (PT) is unknown. Our study investigates the effects of PGE2 on mouse PT fluid reabsorption, and its role in growth, sodium transporter expression, fibrosis, and oxidative stress in a mouse PT cell line (MCT). To determine which PGE2 EP receptors are expressed in MCT, qPCR for EP1-4 was performed on cells stimulated for 24 h with PGE2 or transforming growth factor beta (TGFß), a known mediator of PT injury in kidney disease. EP1 and EP4 were detected in MCT, but EP2 and EP3 are not expressed. EP1 was increased by PGE2 and TGFß, but EP4 was unchanged. To confirm the involvement of EP1 and EP4, sulprostone (SLP, EP1/3 agonist), ONO8711 (EP1 antagonist), and EP1 and EP4 siRNA were used. We first show that PGE2, SLP, and TGFß reduced H(3)-thymidine and H(3)-leucine incorporation. The effects on cell-cycle regulators were examined by western blot. PGE2 increased p27 via EP1 and EP4, but TGFß increased p21; PGE2-induced p27 was attenuated by TGFß. PGE2 and SLP reduced cyclinE, while TGFß increased cyclinD1, an effect attenuated by PGE2 administration. Na-K-ATPase α1 (NaK) was increased by PGE2 via EP1 and EP4. TGFß had no effect on NaK. Additionally, PGE2 and TGFß increased fibronectin levels, reaching 12-fold upon co-stimulation. EP1 siRNA abrogated PGE2-fibronectin. PGE2 also increased ROS generation, and ONO-8711 blocked PGE2-ROS. Finally, PGE2 significantly increased fluid reabsorption by 31 and 46% in isolated perfused mouse PT from C57BL/6 and FVB mice, respectively, and this was attenuated in FVB-EP1 null mice. Altogether PGE2 acting on EP1 and EP4 receptors may prove to be important mediators of PT injury, and salt and water transport.

Chronic kidney disease: targeting prostaglandin E2 receptors.

Chronic kidney disease is a leading cause of morbidity and mortality in the world. A better understanding of disease mechanisms has been gained in recent years, but the current management strategies are ineffective at preventing disease progression. A widespread focus of research is placed on elucidating the specific processes implicated to find more effective therapeutic options. PGE2, acting on its four EP receptors, regulates many renal disease processes; thus EP receptors could prove to be important targets for kidney disease intervention strategies. This review summarizes the major pathogenic mechanisms contributing to initiation and progression of chronic kidney disease, emphasizing the role of hyperglycemia, hypertension, inflammation, and oxidative stress. We have long recognized the multifaceted role of PGs in both the initiation and progression of chronic kidney disease, yet studies are only now seriously contemplating specific EP receptors as targets for therapy. Given the plethora of renal complications attributed to PG involvement in the kidney, this review highlights these pathogenic events and emphasizes the PGE2 receptor targets as options available to complement current therapeutic strategies.

PTGER1 deletion attenuates renal injury in diabetic mouse models.

We hypothesized that the EP1 receptor promotes renal damage in diabetic nephropathy. We rendered EP1 (PTGER1, official symbol) knockout mice (EP1(-/-)) diabetic using the streptozotocin and OVE26 models. Albuminuria, mesangial matrix expansion, and glomerular hypertrophy were each blunted in EP1(-/-) streptozotocin and OVE26 cohorts compared with wild-type counterparts. Although diabetes-associated podocyte depletion was unaffected by EP1 deletion, EP1 antagonism with ONO-8711 in cultured podocytes decreased angiotensin II-mediated superoxide generation, suggesting that EP1-associated injury of remaining podocytes in vivo could contribute to filtration barrier dysfunction. Accordingly, EP1 deletion in OVE26 mice prevented nephrin mRNA expression down-regulation and ameliorated glomerular basement membrane thickening and foot process effacement. Moreover, EP1 deletion reduced diabetes-induced expression of fibrotic markers fibronectin and α-actin, whereas EP1 antagonism decreased fibronectin in cultured proximal tubule cells. Similarly, proximal tubule megalin expression was reduced by diabetes but was preserved in EP1(-/-) mice. Finally, the diabetes-associated increase in angiotensin II-mediated constriction of isolated mesenteric arteries was blunted in OVE26EP1(-/-) mice, demonstrating a role for EP1 receptors in the diabetic vasculature. These data suggest that EP1 activation contributes to diabetic nephropathy progression at several locations, including podocytes, proximal tubule, and the vasculature. The EP1 receptor facilitates the actions of angiotensin II, thereby suggesting that targeting of both the renin-angiotensin system and the EP1 receptor could be beneficial in diabetic nephropathy.

NADPH oxidases, reactive oxygen species, and the kidney: friend and foe.

Reactive oxygen species (ROS) play an important role in normal cellular physiology. They regulate different biologic processes such as cell defense, hormone synthesis and signaling, activation of G protein-coupled receptors, and ion channels and kinases/phosphatases. ROS are also important regulators of transcription factors and gene expression. On the other hand, in pathologic conditions, a surplus of ROS in tissue results in oxidative stress with various injurious consequences such as inflammation and fibrosis. NADPH oxidases are one of the many sources of ROS in biologic systems, and there are seven isoforms (Nox1-5, Duox1, Duox2). Nox4 is the predominant form in the kidney, although Nox2 is also expressed. Nox4 has been implicated in the basal production of ROS in the kidney and in pathologic conditions such as diabetic nephropathy and CKD; upregulation of Nox4 may be important in renal oxidative stress and kidney injury. Although there is growing evidence indicating the involvement of NADPH oxidase in renal pathology, there is a paucity of information on the role of NADPH oxidase in the regulation of normal renal function. Here we provide an update on the role of NADPH oxidases and ROS in renal physiology and pathology.

Celecoxib modifies glomerular basement membrane, mesangium and podocytes in OVE26 mice, but ibuprofen is more detrimental.

The role of COXs/PGs (cyclo-oxygenases/prostaglandins) in diabetic kidneys remains unclear. NSAIDs (non-steroidal anti-inflammatory drugs) that inhibit COXs/PGs are known for their renal toxicity, and COX-2 inhibitors worsen cardiovascular outcomes in susceptible individuals. Given the renal controversies concerning COX-2 inhibitors, we compared the effect of chronic NSAIDs (non-selective, ibuprofen; COX-2-selective, celecoxib) on diabetic kidneys in OVE26 mice from 8 weeks of age. Systolic BPs (blood pressures) were increased by NSAIDs in diabetic mice at 20 weeks, but were unchanged at 32 weeks. Although NSAIDs further increased diabetic kidney/body weight ratios, they did not affect albuminuria. Mesangial matrix was increased 2-fold by celecoxib but not ibuprofen. Electron microscopy revealed that NSAIDs reduced GBM (glomerular basement membrane) thickness and slit pore diameters. Although diabetics had increased glomerular diameters and reduced foot process densities, these were unaltered by NSAIDs. Celecoxib does not exacerbate the diabetic state, but PG inhibition may contribute to disease progression by modifying the GBM, mesangial area and podocyte structure in OVE26 mice. Despite these findings, celecoxib remains safer than a similar dose of ibuprofen. The present study substantiates the need to more closely consider selective COX-2 inhibitors such as celecoxib as alternatives to non-selective NSAIDs for therapeutic management in a setting of chronic kidney disease.

Renoprotective effects of a novel Nox1/4 inhibitor in a mouse model of Type 2 diabetes.

Nox (NADPH oxidase)-derived ROS (reactive oxygen species) have been implicated in the development of diabetic nephropathy. Of the Nox isoforms in the kidney, Nox4 is important because of its renal abundance. In the present study, we tested the hypothesis that GKT136901, a Nox1/4 inhibitor, prevents the development of nephropathy in db/db (diabetic) mice. Six groups of male mice (8-week-old) were studied: (i) untreated control db/m, (ii) low-dose GKT136901-treated db/m (30 mg/kg of body weight per day), (iii) high-dose GKT136901-treated db/m (90 mg/kg of body weight per day), (iv) untreated db/db; (v) low dose GKT136901-treated db/db; and (vi) high-dose GKT136901-treated db/db. GKT136901, in chow, was administered for 16 weeks. db/db mice developed diabetes and nephropathy as evidenced by hyperglycaemia, albuminuria and renal injury (mesangial expansion, tubular dystrophy and glomerulosclerosis). GKT136901 treatment had no effect on plasma glucose or BP (blood pressure) in any of the groups. Plasma and urine TBARSs (thiobarbituric acid-reacting substances) levels, markers of systemic and renal oxidative stress, respectively, were increased in diabetic mice. Renal mRNA expression of Nox4, but not of Nox2, increased, Nox1 was barely detectable in db/db. Expression of the antioxidant enzyme SOD-1 (superoxide dismutase 1) decreased in db/db mice. Renal content of fibronectin, pro-collagen, TGFß (transforming growth factor ß) and VCAM-1 (vascular cell adhesion molecule 1) and phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) were augmented in db/db kidneys, with no change in p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). Treatment reduced albuminuria, TBARS and renal ERK1/2 phosphorylation and preserved renal structure in diabetic mice. Our findings suggest a renoprotective effect of the Nox1/4 inhibitor, possibly through reduced oxidative damage and decreased ERK1/2 activation. These phenomena occur independently of improved glucose control, suggesting GKT136901-sensitive targets are involved in complications of diabetes rather than in the disease process.

Sex-dependent effects of Canagliflozin on kidney protection in mice with combined hypertension-type 1 diabetes.

Canagliflozin (CANA) is a sodium-glucose cotransporter 2 (SGLT2) inhibitor with blood glucose lowering effects. CANA also promotes kidney protection in patients with cardiovascular diseases and type 2 diabetes (T2D), as well as in normoglycemic patients with hypertension or heart failure. Clinical studies, although conduct in both sexes, do not report sex-dependent differences in T2DM treated with CANA. However, the impact of CANA in type 1 diabetes, as well in sex-dependent outcomes in such cohort needs further understanding. To analyze the effects of CANA in mice with combined hypertension and type 1 diabetes, diabetes was induced by STZ injection (5 days, 50mg/kg/day) in both male and female 8 weeks old genetic hypertensive mice (Lin), whereas the control (Lin) received 0.1M sodium citrate injections. 8 weeks after STZ. Mice were fed either regular or CANA-infused diet for 4 weeks. 8 weeks after STZ, hyperglycemia was present in both male and female mice. CANA reversed BG increase mice fed regular diet. Male LinSTZ mice had elevated water intake, urine output, urinary albumin to creatinine ratio (ACR), kidney lesion score, and creatinine clearance compared to the Lin control group. Kidney injury was improved in male LinSTZ + CANA group in male mice. Water intake and urine output were not statistically significantly different in female LinSTZ compared to female LinSTZ+ CANA. Moreover, CANA did not improve kidney injury in female mice, showing no effect in creatinine clearance, lesion score and fibrosis when compared to LinSTZ fed regular diet. Here we show that Canagliflozin might exert different kidney protection effects in male compared to female mice with hypertension and type 1 diabetes. Sex-dimorphisms were previously found in the pathophysiology of diabetes induced by STZ. Therefore, we highlight the importance of in-depth investigation on sex-dependent effects of CANA, taking in consideration the unique characteristics of disease progression for each sex.

High fat diet is protective against kidney injury in hypertensive-diabetic mice, but leads to liver injury.

Chronic kidney disease (CKD) is a worldwide health burden with increases risk of end-stage renal function if left untreated. CKD induced in the context of metabolic syndrome (MS) increases risks of hypertension, hyperglycemia, excess body fat and dyslipidemia. To test if combining a high-fat diet (HFD) regimen onto the hypertensive/ diabetic phenotype would mimic features of MS induced-CKD in mice, hyperglycemia was induced in genetically hypertensive mice (Lin), followed by HFD regimen. For that, 8-week-old male were subjected to streptozotocin (STZ) intraperitoneal (i.p.) injections (50 mg/kg, 5 days consecutive). LinSTZ were fed a 60% kCal HFD for 8 weeks. Lin mice treated with STZ developed polydipsia, became hypertensive and hyperglycemic. HFD induced weight gain, protected against glomerular hypertrophy, scarring, and albuminuria at endpoint compared to regular diet fed LinSTZ. On the other hand, HFD induced steatosis, liver fibrosis, inflammation, and increase in AST/ALT ratio, characteristics of non-alcoholic liver disease. Taken together, our results show that LinSTZ mice fed a HFD did not lead to a more robust model of MS-induced CKD, protected against kidney injury, but inducing liver damage. More studies are necessary to understand the kidney protective mechanisms of HFD when superimposed with hypertension and type 1 diabetes.

Hypertonicity increases sodium transporters in cortical collecting duct cells independently of PGE2.

Cyclooxygenase-2 (COX-2) expression is increased by hypertonicity. Therefore we hypothesized that hypertonicity increased PGE(2) can modulate the sodium transporters (Na(+)/K(+)-ATPase: NKA, epithelial sodium channel: ENaC, and sodium hydrogen exchanger: NHE) in M1 cortical collecting duct (CCD) cells. We demonstrated by immunoblotting a 2-fold increase in NKA expression and activity following hypertonic treatment. α-ENaC was also increased, however sgk1, an ENaC activator, decreased in response to hypertonicity. Other CCD sodium transporters (ß-ENaC, NHE) were unchanged. Hypertonicity also increased PGE(2) but EP(4) receptor mRNA was unaltered. PGE(2) increased intracellular Na(+) and cAMP production in M1 cells, but PGE(2)-stimulated cAMP response was attenuated by hypertonicity. Overall, PGE(2) had no effect on sodium transporter levels. Since neither COX inhibition nor EP(4) siRNA altered the induction of NKA, we propose that sodium transporter regulation by hypertonicity is independent of PGE(2). Altogether, these data indicate that despite a concomitant increase in PGE(2) production and sodium transporter expression in hypertonicity, both pathways are acting independently of each other.

Hyperfiltration and effect of nitric oxide inhibition on renal and endothelial function in humans with uncomplicated type 1 diabetes mellitus.

Studies of experimental diabetes mellitus (DM) suggest that increased nitric oxide (NO) bioactivity contributes to renal hyperfiltration. However, the role of NO in mediating hyperfiltration has not been fully elucidated in humans. Our aim was to examine the effect of NO synthase inhibition on renal and peripheral vascular function in normotensive subjects with uncomplicated type 1 DM. Renal function and brachial artery flow-mediated vasodilatation (FMD) were measured before and after an intravenous infusion of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NMMA) in 21 healthy control and 37 type 1 DM patients. Measurements in DM participants were made under clamped euglycemic conditions. The effect of l-NMMA on circulating and urinary NO metabolites (NO(x)) and cGMP and on urinary prostanoids was also determined. Baseline characteristics were similar in the two groups. For analysis, the DM patients were divided into those with hyperfiltration (DM-H, n = 18) and normal glomerular filtration rate (GFR) levels (DM-N, n = 19). Baseline urine NO(x) and cGMP were highest in DM-H. l-NMMA led to a decline in GFR in DM-H (152 ± 16 to 140 ± 11 ml·min(-1)·1.73 m(-2)) but not DM-N or healthy control participants. The decline in effective renal plasma flow in response to l-NMMA (806 ± 112 to 539 ± 80 ml·min(-1)·1.73 m(-2)) in DM-H was also exaggerated compared with the other groups (repeated measures ANOVA, P < 0.05), along with declines in urinary NO(x) metabolites and cGMP. Baseline FMD was lowest in DM-H compared with the other groups and did not change in response to l-NMMA. l-NMMA reduced FMD and plasma markers of NO bioactivity in the healthy control and DM-N groups. In patients with uncomplicated type 1 DM, renal hyperfiltration is associated with increased NO bioactivity in the kidney and reduced NO bioactivity in the systemic circulation, suggesting a paradoxical state of high renal and low systemic vascular NO bioactivity.

Troglitazone induces extracellular matrix and cytoskeleton remodeling in mouse collecting duct cells.

Peroxisome proliferator-activated receptor (PPARγ) has been shown to have a protective role in the nephron through its ability to inhibit a transforming growth factor- (TGF-ß) mediated fibrotic response. In contrast, PPARγ was also shown to induce a mesenchymal transformation in epithelial intestinal cells. A fibrotic response in the collecting duct has only recently been established; however, the entire collecting duct has not been fully examined. Inner medullary collecting duct cells (IMCD-K2) and mouse cortical collecting duct cells (M1), representing the cortical and medullary collecting duct, were exposed to 5-10 µM troglitazone for 24 hours. Troglitazone resulted in an elongated morphology, 60% decreases in E-cadherin and ß-catenin, a 35% decrease in α-catenin, and a 1.5-fold increase in fibronectin. These effects were not reversed with PPARγ antagonists or affected with PPARγ overexpression. Our results indicate that troglitazone induced a mesenchymal-like transformation in M1 and IMCD-K2 epithelial cells independently of PPARγ.

Comparative analysis of hypertensive nephrosclerosis in animal models of hypertension and its relevance to human pathology. Glomerulopathy.

Current research on hypertension utilizes more than fifty animal models that rely mainly on stable increases in systolic blood pressure. In experimental hypertension, grading or scoring of glomerulopathy in the majority of studies is based on a wide range of opinion-based histological changes that do not necessarily comply with lesional descriptors for glomerular injury that are well-established in clinical pathology. Here, we provide a critical appraisal of experimental hypertensive glomerulopathy with the same approach used to assess hypertensive glomerulopathy in humans. Four hypertensive models with varying pathogenesis were analyzed-chronic angiotensin II infused mice, mice expressing active human renin in the liver (TTRhRen), spontaneously hypertensive rats (SHR), and Goldblatt two-kidney one-clip rats (2K1C). Analysis of glomerulopathy utilized the same criteria applied in humans-hyalinosis, focal segmental glomerulosclerosis (FSGS), ischemic, hypertrophic and solidified glomeruli, or global glomerulosclerosis (GGS). Data from animal models were compared to human reference values. Kidneys in TTRhRen mice, SHR and the nonclipped kidneys in 2K1C rats had no sign of hyalinosis, FSGS or GGS. Glomerulopathy in these groups was limited to variations in mesangial and capillary compartment volumes, with mild increases in collagen deposition. Histopathology in angiotensin II infused mice corresponded to mesangioproliferative glomerulonephritis, but not hypertensive glomerulosclerosis. The number of nephrons was significantly reduced in TTRhRen mice and SHR, but did not correlate with severity of glomerulopathy. The most substantial human-like glomerulosclerotic lesions, including FSGS, ischemic obsolescent glomeruli and GGS, were found in the clipped kidneys of 2K1C rats. The comparison of affected kidneys to healthy control in animals produces lesion values that are numerically impressive but correspond to mild damage if compared to humans. Animal studies should be standardized by employing the criteria and classifications established in human pathology to make experimental and human data fully comparable for comprehensive analysis and model improvements.