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As opposed to diseases such as cancer, autoimmune disease, and diabetes, identifying drugs to treat CKD has proven significantly more challenging. Over the past 2 decades, new potential therapeutic targets have been identified as genetically altered proteins involved in rare monogenetic kidney diseases. Other possible target genes have been implicated through common genetic polymorphisms associated with CKD in the general population. Significant challenges remain before translating these genetic insights into clinical therapies for CKD. This paper will discuss how genetic variants may be leveraged to develop drugs and will especially focus on those genes associated with CKD to exemplify the value and challenges in including genetic information in the drug development pipeline.

Innovation in kidney diseases is not commensurate with the effect of these diseases on human health and mortality or innovation in other key therapeutic areas. A primary cause of the dearth in innovation is that kidney diseases disproportionately affect a demographic that is largely disenfranchised, lacking sufficient advocacy, public attention, and funding. A secondary and likely consequent cause is that the existing infrastructure supporting nephrology research pales in comparison with those for other internal medicine specialties, especially cardiology and oncology. Citing such inequities, however, is not enough. Changing the status quo will require a coordinated effort to identify and redress the existing deficits. Specifically, these deficits relate to the need to further develop and improve the following: understanding of the disease mechanisms and pathophysiology, patient engagement and activism, clinical trial infrastructure, and investigational clinical trial designs as well as coordinated efforts among critical stakeholders. This paper identifies potential solutions to these barriers, some of which are already underway through the Kidney Health Initiative. The Kidney Health Initiative is unique and will serve as a current and future platform from which to overcome these barriers to innovation in nephrology.

Objective— The present studies aimed at elucidating the role of prostaglandin E 2 receptor subtype 3 (E-prostanoid [EP] 3) in regulating blood pressure. Methods and Results— Mice bearing a genetic disruption of the EP3 gene (EP 3 −/− ) exhibited reduced baseline mean arterial pressure monitored by both tail-cuff and carotid arterial catheterization. The pressor responses induced by EP3 agonists M&B28767 and sulprostone were markedly attenuated in EP3 −/− mice, whereas the reduction of blood pressure induced by prostaglandin E 2 was comparable in both genotypes. Vasopressor effect of acute or chronic infusion of angiotensin II (Ang II) was attenuated in EP3 −/− mice. Ang II-induced vasoconstriction in mesenteric arteries decreased in EP3 −/− group. In mesenteric arteries from wild-type mice, Ang II-induced vasoconstriction was inhibited by EP3 selective antagonist DG-041 or L798106. The expression of Arhgef-1 is attenuated in EP3 deficient mesenteric arteries. EP3 antagonist DG-041 diminished Ang II-induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1 in isolated mesenteric arteries. Furthermore, in vascular smooth muscle cells, Ang II–induced intracellular Ca 2+ increase was potentiated by EP3 agonist sulprostone but inhibited by DG-041. Conclusion— Activation of the EP3 receptor raises baseline blood pressure and contributes to Ang II–dependent hypertension at least partially via enhancing Ca 2+ sensitivity and intracellular calcium concentration in vascular smooth muscle cells. Selective targeting of the EP3 receptor may represent a potential therapeutic target for the treatment of hypertension.

Inhibition of p38 mitogen-activated protein kinase and cyclooxygenase-2 reduces albuminuria in models of chronic kidney disease marked by podocyte injury. Previously, we identified a feedback loop in podocytes whereby an in vitro surrogate for glomerular capillary pressure (i.e., mechanical stretch) along with prostaglandin E(2) stimulation of its EP4 receptor induced cyclooxygenase-2 in a p38-dependent manner. Here we asked whether stimulation of EP4 receptors would exacerbate glomerulopathies associated with enhanced glomerular capillary pressure. We generated mice with either podocyte-specific overexpression or depletion of the EP4 receptor (EP4(pod+) and EP4(pod-/-), respectively). Glomerular prostaglandin E(2)-stimulated cAMP levels were eightfold greater for EP4(pod+) mice compared with nontransgenic (non-TG) mice. In contrast, EP4 mRNA levels were >50% lower, and prostaglandin E(2)-induced cAMP synthesis was absent in podocytes isolated from EP4(pod-/-) mice. Non-TG and EP4(pod+) mice underwent 5/6 nephrectomy and exhibited similar increases in systolic BP (+25 mmHg) by 4 weeks compared with sham-operated controls. Two weeks after nephrectomy, the albumin-creatinine ratio of EP4(pod+) mice (3438 μg/mg) was significantly higher than that of non-TG mice (773 μg/mg; P < 0.0001). Consistent with more severe renal injury, the survival rate for nephrectomized EP4(pod+) mice was significantly lower than that for non-TG mice (14 versus 67%). In contrast, 6 weeks after nephrectomy, the albumin-creatinine ratio of EP4(pod-/-) mice (753 μg/mg) was significantly lower than that of non-TG mice (2516 μg/mg; P < 0.05). These findings suggest that prostaglandin E(2), acting via EP4 receptors contributes to podocyte injury and compromises the glomerular filtration barrier.

Superoxide excess plays a central role in tissue damage that results from diabetes, but the mechanisms of superoxide overproduction in diabetic nephropathy (DN) are incompletely understood. In the present study, we investigated the enzyme superoxide dismutase (SOD), a major defender against superoxide, in the kidneys during the development of murine DN. We assessed SOD activity and the expression of SOD isoforms in the kidneys of two diabetic mouse models (C57BL/6-Akita and KK/Ta-Akita) that exhibit comparable levels of hyperglycemia but different susceptibility to DN. We observed down-regulation of cytosolic CuZn-SOD (SOD1) and extracellular CuZn-SOD (SOD3), but not mitochondrial Mn-SOD (SOD2), in the kidney of KK/Ta-Akita mice which exhibit progressive DN. In contrast, we did not detect a change in renal SOD expression in DN-resistant C57BL/6-Akita mice. Consistent with these findings, there was a significant reduction in total SOD activity in the kidney of KK/Ta-Akita mice compared with C57BL/6-Akita mice. Finally, treatment of KK/Ta-Akita mice with a SOD mimetic, tempol, ameliorated the nephropathic changes in KK/Ta-Akita mice without altering the level of hyperglycemia. Collectively, these results indicate that down-regulation of renal SOD1 and SOD3 may play a key role in the pathogenesis of DN.

Ischemia- or toxin-induced acute kidney injury is generally thought to affect the cells of the proximal tubule, but it has been difficult to define the involvement of other tubular segments because of the widespread damage caused by ischemia/reperfusion or toxin-induced injury in experimental models. For evaluation of whether thick ascending limb (TAL)-specific epithelial injury results in acute kidney injury, a novel transgenic mouse model that expresses the herpes simplex virus 1 thymidine kinase gene under the direction of the TAL-specific Tamm-Horsfall protein promoter was generated. After administration of gancyclovir, these mice demonstrated apoptosis only in TAL cells, with little evidence of neutrophil infiltration. Compared with control mice, blood urea nitrogen and creatinine levels were at least five-fold higher in the transgenic mice, which also developed oliguria and impaired urinary concentrating ability. These findings suggest that acute injury targeted only to the TAL is sufficient to cause severe acute kidney injury in mice with features similar to those observed in humans.

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone and is a new clinically available class of agents for improving of insulin resistance in both animals and humans with type 2 diabetes. These studies aimed to determine whether long-term treatment with a long-acting GLP-1 analog, exendin-4, delayed the progression of diabetes. Male db/db mice and db/m mice at 8 wk of age were treated with exendin-4 for 8 wk, whereas the control db/db mice received only vehicle. Urinary albumin excretion was significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in db/db mice that were treated with 0.5 nmol/kg exendin-4 and control db/db mice (P < 0.005). Intraperitoneal glucose tolerance test was improved in db/db mice that were treated with 1 nmol/kg exendin-4 compared with other groups (P < 0.05). Despite this, fasting blood glucose, glycated hemoglobin, and creatinine concentrations were not significantly different among db/db mice. Renal histology studies further demonstrated that glomerular hypertrophy, mesangial matrix expansion, TGF-beta1 expression, and type IV collagen accumulation and associated glomerular lipid accumulation were significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4. Furthermore, there were fewer infiltrating inflammatory cells and apoptotic cells in the glomeruli of db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in the other groups accompanied by an increase in the renal immunoreactivity of peroxisome proliferator-activated receptor alpha and GLP-1 receptor-positive cells and a decrease in 24-h urinary 8-hydroxy-deoxyguanosine levels (P < 0.01, respectively) along with decreases in lipid content. Taken together, exendin-4 treatment seems to ameliorate diabetic nephropathy together with improvement of the metabolic anomalies. These results suggest that exendin-4 could provide a therapeutic role in diabetic nephropathy that results from type 2 diabetes.

Diabetic nephropathy (DN) is a late diabetic complication that comprises progressively increasing albuminuria, declining GFR, and increased cardiovascular risk. Only a minority of patients with diabetes (25 to 40%) develop nephropathy, and there is evidence that heritable genetic factors predispose these "at-risk" individuals to DN. Comparing variability among inbred mouse strains with respect to a specific phenotype can model interhuman variability, and each strain represents a genetically homogeneous system with a defined risk for nephropathy. C57BL/6 mice, which are relatively resistant to DN, were mutagenized using N-ethyl-N-nitrosourea and screened for mutants that developed excess albuminuria on a sensitizing type 1 diabetic background contributed by the dominant Akita mutation in insulin-2 gene (Ins2(Akita)). Two of 375 diabetic G1 founders were found to exhibit albumin excretion rates persistently 10-fold greater than albumin excretion rates in nonmutagenized Ins2(Akita) controls. This albuminuria trait was heritable and transmitted to approximately 50% of Ins2(Akita) G2 and G3 progeny, consistent with a simple, dominantly inherited trait, but was never observed in nondiabetic offspring. During the course of 1 yr, albuminuric Ins2(Akita) G2 and G3 progeny developed reduced inulin clearance with elevated blood urea nitrogen and plasma creatinine. Glomerular histology revealed mesangial expansion, and glomerular basement membrane thickening as determined by electron microscopy was enhanced in diabetic mutant kidneys. Hereditary albuminuric N-ethyl-N-nitrosourea-induced mutants were redesignated as Nphrp1 (nephropathy1) and Nphrp2 (nephropathy2) mice for two generated lines. These novel mutants provide new, robust mouse models of DN and should help to elucidate the underlying genetic basis of predisposition to DN.

Functionally significant polymorphisms in endothelial nitric oxide synthase (eNOS) and reduced vascular eNOS activity have been associated with increased human diabetic nephropathy (DN), but the pathogenic role of eNOS deficiency in the development of DN has not yet been confirmed. This study characterizes the severity of DN in eNOS(-/-) mice that were backcrossed to C57BLKS/J db/db mice. Although the severity of hyperglycemia was similar to C57BLKS/J db/db mice, by 26 wk, eNOS(-/-) C57BLKS/J db/db mice exhibited dramatic albuminuria, arteriolar hyalinosis, increased glomerular basement membrane thickness, mesangial expansion, mesangiolysis, and focal segmental and early nodular glomerulosclerosis. Even more remarkable, eNOS(-/-) C57BLKS db/db exhibited decreases in GFR to levels

Accumulating evidence indicates cyclooxygenase (COX) 1 and COX2 differentially regulate cardiovascular and renal function. We have demonstrated previously in mice that COX2 inhibition enhances angiotensin II-induced hypertension, and COX1 inhibition attenuates the pressor effect of angiotensin II. To further elucidate the mechanism underlying the functional difference of COX1 versus COX2 inhibition, the present studies examined the prostaglandin (PG) profiles derived in COX1- or COX2-inhibited mouse kidney and aorta using gas chromatographic/mass spectrometric assays. PGE 2 is the most abundant prostanoid in both renal cortex and medulla in normal C57BL/6J mice, followed by PGI 2 , PGF 2α and thromboxane A 2 . In contrast PGI 2 was most abundant in aorta followed by thromboxane A 2 , PGE 2 , and PGF 2α . PGD 2 was undetectable in control kidney or aorta. At baseline, inhibition of COX1 decreased total prostaglandins in renal cortex, medulla, and aorta, whereas COX2 inhibition decreased total prostaglandins only in renal medulla. Angiotensin II infusion significantly increased COX2-dependent/COX1-independent PGE 2 and PGI 2 in renal cortex and medulla. Angiotensin II also significantly increased renal PGF 2α in cortex, but not in medulla, through both COX1- and COX2-dependent mechanisms. These studies demonstrate that although COX1 primarily contributes to basal prostanoid production in the kidney and aorta, angiotensin II increases renal vasodilator prostanoids predominately via COX2 activity. These effects may contribute to the specific effect of COX2 inhibitors to increase blood pressure.

PURPOSE OF REVIEW Advances in mouse genetics have made this species particularly useful as a model for human disease. This review will summarize recent advances regarding the pathogenesis of diabetic nephropathy discovered in mice. RECENT FINDINGS Diabetic nephropathy has been characterized in novel genetic models of murine diabetes including the Akita, Ove26, and ICER-Igamma mice. Mutagenesis resources targeting every gene of the genome and the importance of inbred genetic background are discussed. SUMMARY Through the use of these resources mouse models should provide new insight into the pathogenesis of diabetic nephropathy, and complement human studies and validate the identity of candidate genes contributing to diabetic nephropathy.

Nestin, an intermediate filament protein, is widely used as stem cell marker. Nestin has been shown to interact with other cytoskeleton proteins, suggesting a role in regulating cellular cytoskeletal structure. These studies examined renal nestin localization and developmental expression in mice. In developing kidney, anti-nestin antibody revealed strong immunoreactivity in vascular cleft of the S-shaped body and vascular tuft of capillary loop-stage glomerulus. The nestin-positive structures also were labeled by endothelial cell markers FLK1 and CD31 in immature glomeruli. Nestin was not detected in epithelial cells of immature glomeruli. In contrast, in mature glomerular, nestin immunoreactivity was observed only outside laminin-positive glomerular basement membrane, and co-localized with nephrin, consistent with podocyte nestin expression. In adult kidney, podocytes were the only cells that exhibited persistent nestin expression. Nestin was not detected in ureteric bud and its derivatives throughout renal development. Cell lineage studies, using a nestin promoter-driven Cre mouse and a ROSA26 reporter mouse, showed a strong beta-galactosidase activity in intermediate mesoderm in an embryonic day 10 embryo and all of the structures except those that were derived from ureteric bud in embryonic kidney through adult kidney. These studies show that nestin is expressed in progenitors of glomerular endothelial cells and renal progenitors that are derived from metanephric mesenchyme. In the adult kidney, nestin expression is restricted to differentiated podocytes, suggesting that nestin could play an important role in maintaining the structural integrity of the podocytes.

Mice provide an experimental model of unparalleled flexibility for studying mammalian diseases. Inbred strains of mice exhibit substantial differences in their susceptibility to the renal complications of diabetes. Much remains to be established regarding the course of diabetic nephropathy (DN) in mice as well as defining those strains and/or mutants that are most susceptible to renal injury from diabetes. Through the use of the unique genetic reagents available in mice (including knockouts and transgenics), the validation of a mouse model reproducing human DN should significantly facilitate the understanding of the underlying genetic mechanisms that contribute to the development of DN. Establishment of an authentic mouse model of DN will undoubtedly facilitate testing of translational diagnostic and therapeutic interventions in mice before testing in humans.

Cyclooxygenases (COX) are the target of non-steroidal anti-inflammatory drugs (NSAIDs) which exert their therapeutic effect by blocking COX's capacity to metabolize arachidonate to a series of biologically active fatty acids, designated prostaglandins. NSAID use is associated with two major tonicities: gastrointestinal bleeding and renal dysfunction. In the setting of significant physiologic stress, renal function becomes dependent upon prostaglandins and NSAID use may be associated with acute deterioration of renal function, including development of sodium retention, edema, hypertension, hyperkalemia, and or papillary necrosis. Two isoforms, COX1 and COX2, have been identified. They are products of distinct genes and their expression is under different regulatory control. Both COX1 and COX2 are highly expressed in the kidney and both are inhibited by conventional NSAIDs. Accumulating data using recently developed selective COX2 inhibitors suggest that while these agents spare the gastrointestinal tract they have similar renal effects as non-selective NSAIDs. Therefore, caution should be taken when prescribing selective COX2 inhibitor to patients, especially to patients with predisposed physiologic stress.

Diabetic nephropathy is the major cause of ESRD in the United States.1 Unfortunately, only angiotensin-converting enzyme inhibitors or angiotensin type 1 receptor blockers are registered for slowing the progression of diabetic nephropathy—but they do not halt progression.2–5 Given that diabetic nephropathy costs the health care system close to $30 billion per year,6 one would hope to see a flurry of active clinical trials aimed at registering new therapies to slow progression. Instead, the sad truth is that only 31 interventional phase 2 or 3 trials are currently listed on ClinicalTrials.gov and most only study various angiotensin-converting enzyme inhibitor/angiotensin type 1 receptor blocker and renin inhibitor combinations. In striking contrast, there are 757 breast cancer and 989 lung cancer interventional survival trials and 175 interventional major adverse cardiac event outcomes trials in the same database.7 Numerous hurdles exist to closing this gap for diabetic nephropathy, including the identification of dynamic biomarkers that reflect not just the presence of disease but also the rate of loss of kidney function.8,9 Discovery of these biomarkers and novel therapies will require a deeper understanding of the pathophysiology of diabetic nephropathy. Because of limited access to diseased human kidney tissues, validated and clinically relevant animal models of diabetic nephropathy would significantly facilitate these …

Abstract —Four E-prostanoid (EP) receptors, designated EP 1 , EP 2 , EP 3 , and EP 4 , mediate the cellular effects of prostaglandin E 2 (PGE 2 ). The present studies pharmacologically characterize the vasopressor and vasodepressor EP receptors in wild-type mice (EP 2 +/+ mice) and mice with targeted disruption of the EP 2 receptor (EP 2 −/− mice). Mean arterial pressure (MAP) was measured via a carotid artery catheter in anesthetized male mice. Intravenous infusion of PGE 2 decreased MAP in EP 2 +/+ mice but increased MAP in EP 2 −/− mice. Infusion of EP 3 -selective agonists, including MB28767, SC46275, and sulprostone, increased MAP in both EP 2 +/+ and EP 2 −/− mice. Pretreatment with SC46275 desensitized mice to the subsequent pressor effect of sulprostone, but the vasodepressor effect of PGE 2 in EP 2 +/+ mice remained intact. Although PGE 2 alone increased MAP in EP 2 −/− mice, prior desensitization of the pressor effect with SC46275 allowed a residual vasodepressor effect of PGE 2 to be seen in the EP 2 −/− mice. An EP 4 -selective agonist (prostaglandin E 1 -OH) functioned also as a vasodepressor in both EP 2 −/− and EP 2 +/+ mice. High levels of EP 3 receptor mRNA were detected in mouse aortas and rabbit preglomerular arterioles by nuclease protection, with lower expressions of EP 1 , EP 2 , and EP 4 mRNA. The findings suggest that combined vasodepressor effects of EP 2 and EP 4 receptors normally dominate, accounting for the depressor effects of PGE 2 . In contrast, in EP 2 −/− mice, EP 4 receptor activity alone is insufficient to overcome the EP 3 vasopressor effect. These findings suggest that a balance between pressor and depressor PGE 2 receptors determines its net effect on arterial pressure and that these receptors may be important therapeutic targets.

In the years after the introduction of life-saving insulin therapy for the treatment of diabetes, diabetic nephropathy emerged as a complication of chronic diabetes.[1][1],[2][2] Diabetic nephropathy has since metamorphosed from a clinical rarity to the single major cause of kidney failure in the

Cyclooxygenase (COX)-inhibiting NSAIDs are widely prescribed to treat arthritis, exerting analgesic and antiinflammatory effects via their capacity to block endogenous prostaglandin synthesis.1,2 Unfortunately, their widespread use has been associated with an increased risk of developing hypertension,3,4 and clinical experience with COX-2 selective inhibitors suggests they similarly predispose hypertension.5 The physiological mechanisms through which COX-2 inhibitors increase blood pressure are gradually being elucidated. Experimental evidence from animal models suggests an important vasodilator role for endogenous COX-2–derived prostaglandins, reducing the systemic pressor effects of angiotensin II.6 These findings underscore the importance of COX-2 activity in regulating the tone of peripheral resistance vessels. COX-2, but not COX-1, inhibition also reduces renal medullary blood flow and urine salt excretion,6 which is in agreement with clinical …