Kidney Injury by Unilateral Ureteral Obstruction in Mice Lacks Sex Differences.

INTRODUCTION: Renal fibrosis is a critical event in the development and progression of chronic kidney disease (CKD), and it is considered the final common pathway for all types of CKD. The prevalence of CKD is higher in females; however, males have a greater prevalence of end-stage renal disease. In addition, low birth weight and low nephron number are associated with increased risk for CKD. This study examined the development and severity of unilateral ureter obstruction (UUO)-induced renal fibrosis in male and female wild-type (ROP +/+) and mutant (ROP Os/+) mice, a mouse model of low nephron number. METHODS: Male and female ROP +/+ and ROP Os/+ mice were subjected to UUO, and kidney tissue was collected at the end of the 10-day experimental period. Kidney histological analysis and mRNA expression determined renal fibrosis, tubular injury, collagen deposition, extracellular matrix proteins, and immune cell infiltration. RESULTS: Male and female UUO mice demonstrated marked renal injury, kidney fibrosis, and renal extracellular matrix production. Renal fibrosis and α-smooth muscle actin were increased to a similar degree in ROP +/+ and ROP Os/+ mice with UUO of either sex. There were also no sex differences in renal tubular cast formation or renal infiltration of macrophage in ROP +/+ and ROP Os/+ UUO mice. Interestingly, renal fibrosis and α-smooth muscle actin were 1.5-3-fold greater in UUO-ROP +/+ compared to UUO-ROP Os/+ mice. Renal inflammation phenotypes following UUO were also 30-45% greater in ROP +/+ compared to ROP Os/+ mice. Likewise, expression of extracellular matrix and renal fibrotic genes was greater in UUO-ROP +/+ mice compared to UUO-ROP Os/+ mice. In contrast to these findings, ROP Os/+ mice with UUO demonstrated glomerular hypertrophy with 50% greater glomerular tuft area compared to ROP +/+ with UUO. Glomerular hypertrophy was not sex-dependent in any of the genotypes of ROP mice. These findings provide evidence that low nephron number contributes to UUO-induced glomerular hypertrophy in ROP Os/+ mice but does not enhance renal fibrosis, inflammation, and renal tubular injury. CONCLUSION: Taken together, we demonstrate that low nephron number contributes to enhanced glomerular hypertrophy but not kidney fibrosis and tubular injury. We also demonstrate that none of the changes caused by UUO was affected by sex in any of the ROP mice genotypes.

Long-term kidney function and survival in recipients of allografts from living kidney donors with hypertension: a national cohort study.

Allografts from living kidney donors with hypertension may carry subclinical kidney disease from the donor to the recipient and, thus, lead to adverse recipient outcomes. We examined eGFR trajectories and all-cause allograft failure in recipients from donors with versus without hypertension, using mixed-linear and Cox regression models stratified by donor age. We studied a US cohort from 1/1/2005 to 6/30/2017; 49 990 recipients of allografts from younger (<50 years old) donors including 597 with donor hypertension and 21 130 recipients of allografts from older (≥50 years old) donors including 1441 with donor hypertension. Donor hypertension was defined as documented predonation use of antihypertensive therapy. Among recipients from younger donors with versus without hypertension, the annual eGFR decline was -1.03 versus -0.53 ml/min/m2 (P = 0.002); 13-year allograft survival was 49.7% vs. 59.0% (adjusted allograft failure hazard ratio [aHR] 1.23; 95% CI 1.05-1.43; P = 0.009). Among recipients from older donors with versus without hypertension, the annual eGFR decline was -0.67 versus -0.66 ml/min/m2 (P = 0.9); 13-year allograft survival was 48.6% versus 52.6% (aHR 1.05; 95% CI 0.94-1.17; P = 0.4). In secondary analyses, our inferences remained similar for risk of death-censored allograft failure and mortality. Hypertension in younger, but not older, living kidney donors is associated with worse recipient outcomes.

Upregulated proteoglycan-related signaling pathways in fluid flow shear stress-treated podocytes.

The ultrafiltrate flow over the major processes and cell body generates fluid flow shear stress (FFSS) on podocytes. Hyperfiltration-associated increase in FFSS can lead to podocyte injury and detachment. Previously, we showed that FFSS-induced upregulation of the cyclooxygenase 2 (COX2)-PGE2-prostaglandin E receptor 2 (EP2) axis in podocytes activates Akt-glycogen synthase kinase-3ß-ß-catenin and MAPK/ERK signaling in response to FFSS. Integrative MultiOmics Pathway Resolution (IMPRes) is a new bioinformatic tool that enables simultaneous time-series analysis of more than two groups to identify pathways and molecular connections. In the present study, we used previously characterized COX2 [prostaglandin-endoperoxide synthase 2 (Ptgs2)], EP2 (Ptger2), and ß1-catenin (Ctnnb1) as "seed genes" from an array data set of four groups analyzed over a time course. The 3 seed genes shared 7 pathways and 50 genes of 14 pathways and 89 genes identified by IMPRes. A composite of signaling pathways highlighted the temporal molecular connections during mechanotransduction signaling in FFSS-treated podocytes. We investigated the "proteoglycans in cancer" and "galactose metabolism" pathways predicted by IMPRes. A custom-designed PCR array validated 60.7% of the genes predicted by IMPRes analysis, including genes for the above-named pathways. Further validation using Western blot analysis showed increased expression of phosho-Erbb2, phospho-mammalian target of rapamycin (mTOR), CD44, and hexokinase II (Hk2); decreased total Erbb2, galactose mutarotase (Galm), and ß-1,4-galactosyltransferase 1 (B4galt1); and unchanged total mTOR and AKT3. These findings corroborate our previously reported results. This study demonstrates the potential of the IMPRes method to identify novel pathways. Identifying the "proteoglycans in cancer" and "galactose metabolism" pathways has generated a lead to study the significance of FFSS-induced glycocalyx remodeling and possible detachment of podocytes from the glomerular matrix.

Urinary prostaglandin E2 is a biomarker of early adaptive hyperfiltration in solitary functioning kidney.

INTRODUCTION: Hyperfiltration is a major contributor to progression of chronic kidney disease (CKD) in diabetes, obesity and in individuals with solitary functioning kidney (SFK). We have proposed hyperfiltration-induced injury as a continuum of overlapping glomerular changes caused by increased biomechanical forces namely, fluid flow shear stress (FFSS) and tensile stress. We have shown that FFSS is elevated in animals with SFK and, it upregulates prostaglandin E2 (PGE2), cyclooxygenase-2 and PGE2 receptor EP2 in cultured podocytes and in uninephrectomized mice. We conceptualized urinary PGE2 as a biomarker of early effects of hyperfiltration-induced injury preceding microalbuminuria in individuals with SFK. We studied children with SFK to validate our hypothesis. METHODS: Urine samples from children with SFK and controls were analyzed for PGE2, albumin (glomerular injury biomarker) and epidermal growth factor (EGF, tubular injury biomarker). Age, gender, and Z-scores for height, weight, BMI, and blood pressure were obtained. RESULTS: Children with SFK were comparable to controls except for lower BMI Z-scores. The median values were elevated in SFK compared to control for urine PGE2 [9.1 (n = 57) vs. 5.7 (n = 72), p = 0.009] ng/mgCr and albumin [7.6 (n = 40) vs. 7.0 (n = 41), p = 0.085] µg/mgCr, but not for EGF [20098 (n = 44) vs. 18637 (n = 44), p = 0.746] pg/mgCr. Significant increase in urinary PGE2 (p = 0.024) and albumin (p = 0.019) but not EGF (p = 0.412) was observed using additional regression modeling. These three urinary analytes were independent of each other. CONCLUSION: Increased urinary PGE2 from elevated SNGFR and consequently increased FFSS during early stage of CKD precedes overt microalbuminuria and is a biomarker for early hyperfiltration-induced injury in individuals with SFK.

Vibrodissociation method for isolation of defined nephron segments from human and rodent kidneys.

Our current knowledge of the properties of renal ion channels responsible for electrolytes and cell energy homeostasis mainly relies on rodent studies. However, it has not been established yet to what extent their characteristics can be generalized to those of humans. The present study was designed to develop a standardized protocol for the isolation of well-preserved glomeruli and renal tubules from rodent and human kidneys and to assess the functional suitability of the obtained materials for physiological studies. Separation of nephron segments from human and rodent kidneys was achieved using a novel vibrodissociation technique. The integrity of isolated renal tubules and glomeruli was probed via electrophysiological analysis and fluorescence microscopy, and the purity of the collected fractions was confirmed using quantitative RT-PCR with gene markers for specific cell types. The developed approach allows rapid isolation of well-preserved renal tubules and glomeruli from human and rodent kidneys amenable for electrophysiological, Ca2+ imaging, and omics studies. Analysis of the basic electrophysiological parameters of major K+ and Na+ channels expressed in human cortical collecting ducts revealed that they exhibited similar biophysical properties as previously reported in rodent studies. Using vibrodissociation for nephron segment isolation has several advantages over existing techniques: it is less labor intensive, requires little to no enzymatic treatment, and produces large quantities of well-preserved experimental material in pure fractions. Applying this method for the separation of nephron segments from human and rodent kidneys may be a powerful tool for the indepth assessment of kidney function in health and disease.

Increased ENaC activity during kidney preservation in Wisconsin solution.

BACKGROUND: The invention of an effective kidney preservation solution capable of prolonging harvested kidney viability is the core of kidney transplantation procedure. Researchers have been working on upgrading the preservation solution quality aiming at prolonging storage time while maintaining utmost organ viability and functionality. For many years, the University of Wisconsin (UW) solution has been considered the gold standard solution for kidney preservation. However, the lifespan of kidney preservation in the UW solution is still limited. Its impact on the epithelial Na+ channel (ENaC) activity and its mediated processes is unknown and the primary goal of this study. METHODS: Kidneys harvested from 8 weeks old Sprague Dawley rats were divided into 4 groups depending upon the period of preservation in UW solution. Additional analysis was performed using dogs' kidneys. ENaC activity was measured using patch clamp technique; protein expression and mRNA transcription were tested through Western blot and RT-qPCR, respectively. A colorimetric LDH level estimation was performed at different time points during UW solution preservation. RESULTS: Kidney preservation in Wisconsin solution caused reduction of the kidney size and weight and elevation of LDH level. ENaC activity increased in both rat and dog kidneys preserved in the UW solution as assessed by patch clamp analysis. On the contrary, ENaC channel mRNA levels remained unchanged. CONCLUSIONS: ENaC activity is significantly elevated in the kidneys during preservation in UW solution, which might affect the immediate post-implantation allograft function and trajectory post-transplant.

Mechanotransduction signaling in podocytes from fluid flow shear stress.

Recently, we and others have found that hyperfiltration-associated increase in biomechanical forces, namely, tensile stress and fluid flow shear stress (FFSS), can directly and distinctly alter podocyte structure and function. The ultrafiltrate flow over the major processes and cell body generates FFSS to podocytes. Our previous work suggests that the cyclooxygenase-2 (COX-2)-PGE2-PGE2 receptor 2 (EP2) axis plays an important role in mechanoperception of FFSS in podocytes. To address mechanotransduction of the perceived stimulus through EP2, cultured podocytes were exposed to FFSS (2 dyn/cm2) for 2 h. Total RNA from cells at the end of FFSS treatment, 2-h post-FFSS, and 24-h post-FFSS was used for whole exon array analysis. Differentially regulated genes ( P < 0.01) were analyzed using bioinformatics tools Enrichr and Ingenuity Pathway Analysis to predict pathways/molecules. Candidate pathways were validated using Western blot analysis and then further confirmed to be resulting from a direct effect of PGE2 on podocytes. Results show that FFSS-induced mechanotransduction as well as exogenous PGE2 activate the Akt-GSK3ß-ß-catenin (Ser552) and MAPK/ERK but not the cAMP-PKA signal transduction cascades. These pathways are reportedly associated with FFSS-induced and EP2-mediated signaling in other epithelial cells as well. The current regimen for treating hyperfiltration-mediated injury largely depends on targeting the renin-angiotensin-aldosterone system. The present study identifies specific transduction mechanisms and provides novel information on the direct effect of FFSS on podocytes. These results suggest that targeting EP2-mediated signaling pathways holds therapeutic significance for delaying progression of chronic kidney disease secondary to hyperfiltration.

Role of biomechanical forces in hyperfiltration-mediated glomerular injury in congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) including solitary kidney constitute the main cause of progressive chronic kidney disease (CKD) in children. Children born with CAKUT develop signs of CKD only during adolescence and do not respond to renin-angiotensin-aldosterone system blockers. Early cellular changes underlying CKD progression to end-stage renal disease by early adulthood are not well understood. The mechanism of maladaptive hyperfiltration that occurs from loss of functional nephrons, including solitary kidney, is not clear. We re-examine the phenomenon of hyperfiltration in the context of biomechanical forces with special reference to glomerular podocytes. Capillary stretch exerts tensile stress on podocytes through the glomerular basement membrane. The flow of ultrafiltrate over the cell surface directly causes fluid flow shear stress (FFSS) on podocytes. FFSS on the podocyte surface increases 1.5- to 2-fold in animal models of solitary kidney and its effect on podocytes is a subject of ongoing research. Podocytes (i) are mechanosensitive to tensile and shear forces, (ii) use prostaglandin E2, angiotensin-II or nitric oxide for mechanoperception and (iii) use specific signaling pathways for mechanotransduction. We discuss (i) the nature of and differences in cellular responses to biomechanical forces, (ii) methods to study biomechanical forces and (iii) effects of biomechanical forces on podocytes and glomeruli. Future studies on FFSS will likely identify novel targets for strategies for early intervention to complement and strengthen the current regimen for treating children with CAKUT.

Cyclooxygenase-2, prostaglandin E2, and prostanoid receptor EP2 in fluid flow shear stress-mediated injury in the solitary kidney.

Hyperfiltration subjects podocytes to increased tensile stress and fluid flow shear stress (FFSS). We showed a 1.5- to 2.0-fold increase in FFSS in uninephrectomized animals and altered podocyte actin cytoskeleton and increased synthesis of prostaglandin E2 (PGE2) following in vitro application of FFSS. We hypothesized that increased FFSS mediates cellular changes through specific receptors of PGE2. Presently, we studied the effect of FFSS on cultured podocytes and decapsulated isolated glomeruli in vitro, and on solitary kidney in uninephrectomized sv129 mice. In cultured podocytes, FFSS resulted in increased gene and protein expression of cyclooxygenase (COX)-2 but not COX-1, prostanoid receptor EP2 but not EP4, and increased synthesis and secretion of PGE2, which were effectively blocked by indomethacin. Next, we developed a special flow chamber for applying FFSS to isolated glomeruli to determine its effect on an intact glomerular filtration barrier by measuring change in albumin permeability (Palb) in vitro. FFSS caused an increase in Palb that was blocked by indomethacin (P < 0.001). Finally, we show that unilateral nephrectomy in sv129 mice resulted in glomerular hypertrophy (P = 0.006), increased glomerular expression of COX-2 (P < 0.001) and EP2 (P = 0.039), and increased urinary albumin excretion (P = 0.001). Activation of the COX-2-PGE2-EP2 axis appears to be a specific response to FFSS in podocytes and provides a mechanistic basis for alteration in podocyte structure and the glomerular filtration barrier, leading to albuminuria in hyperfiltration-mediated kidney injury. The COX-2-PGE2-EP2 axis is a potential target for developing specific interventions to ameliorate the effects of hyperfiltration-mediated kidney injury in the progression of chronic kidney disease.

Fluid flow shear stress upregulates prostanoid receptor EP2 but not EP4 in murine podocytes.

Podocytes in the glomerular filtration barrier regulate the passage of plasma proteins into urine. Capillary pressure and ultrafiltration impact the structure and function of podocytes. The mechanism of podocyte injury by fluid flow shear stress (FFSS) from hyperfiltration in chronic kidney disease (CKD) is not completely understood. Recently, we demonstrated increased synthesis of prostaglandin E2 in podocytes exposed to FFSS. Here, we determine the effect of FFSS on prostanoid receptors EP1-EP4 in cultured podocytes and in Os/+ mouse kidney, a model of hyperfiltration. Results of RT-PCR, qRT-PCR, immunoblotting and immunofluorescence studies indicate that cultured podocytes express EP1, EP2 and EP4 but not EP3. FFSS resulted in upregulated expression of only EP2 in podocytes. Kidney immunostaining showed significantly increased expression of EP2 in Os/+ mice compared with littermate controls. These novel results suggest that EP2 may be responsible for mediating podocyte injury from hyperfiltration-induced augmented FFSS in CKD.

Salt-sensitive hypertension after reversal of unilateral ureteral obstruction.

The incidence of ureter obstruction is increasing and patients recovering from this kidney injury often progress to chronic kidney injury. There is evidence that a long-term consequence of recovery from ureter obstruction is an increased risk for salt-sensitive hypertension. A reversal unilateral ureteral obstruction (RUUO) model was used to study long-term kidney injury and salt-sensitive hypertension. In this model, we removed the ureteral obstruction at day 10 in mice. Mice were divided into four groups: (1) normal salt diet, (2) high salt diet, (3) RUUO normal salt diet, and (4) RUUO high salt diet. At day 10, the mice were fed a normal or high salt diet for 4 weeks. Blood pressure was measured, and urine and kidney tissue collected. There was a progressive increase in blood pressure in the RUUO high salt diet group. RUUO high salt group had decreased sodium excretion and glomerular injury. Renal epithelial cell injury was evident in RUUO normal and high salt mice as assessed by neutrophil gelatinase-associated lipocalin (NGAL). Kidney inflammation in the RUUO high salt group involved an increase in F4/80 positive macrophages; however, CD3+ positive T cells were not changed. Importantly, RUUO normal and high salt mice had decreased vascular density. RUUO was also associated with renal fibrosis that was further elevated in RUUO mice fed a high salt diet. Overall, these findings demonstrate long-term renal tubular injury, inflammation, decreased vascular density, and renal fibrosis following reversal of unilateral ureter obstruction that could contribute to impaired sodium excretion and salt-sensitive hypertension.

Identification of nephropathy candidate genes by comparing sclerosis-prone and sclerosis-resistant mouse strain kidney transcriptomes.

BACKGROUND: The genetic architecture responsible for chronic kidney disease (CKD) remains incompletely described. The Oligosyndactyly (Os) mouse models focal and segmental glomerulosclerosis (FSGS), which is associated with reduced nephron number caused by the Os mutation. The Os mutation leads to FSGS in multiple strains including the ROP-Os/+. However, on the C57Bl/6J background the mutation does not cause FSGS, although nephron number in these mice are equivalent to those in ROP-Os/+ mice. We exploited this phenotypic variation to identify genes that potentially contribute to glomerulosclerosis. METHODS: To identify such novel genes, which regulate susceptibility or resistance to renal disease progression, we generated and compared the renal transcriptomes using serial analysis of gene expression (SAGE) from the sclerosis-prone ROP-Os/+ and sclerosis resistant C57-Os/+ mouse kidneys. We confirmed the validity of the differential gene expression using multiple approaches. We also used an Ingenuity Pathway Analysis engine to assemble differentially regulated molecular networks. Cell culture techniques were employed to confirm functional relevance of selected genes. RESULTS: A comparative analysis of the kidney transcriptomes revealed multiple genes, with expression levels that were statistically different. These novel, candidate, renal disease susceptibility/resistance genes included neuropilin2 (Nrp2), glutathione-S-transferase theta (Gstt1) and itchy (Itch). Of 34 genes with the most robust statistical difference in expression levels between ROP-Os/+ and C57-Os/+ mice, 13 and 3 transcripts localized to glomerular and tubulointerstitial compartments, respectively, from micro-dissected human FSGS biopsies. Network analysis of all significantly differentially expressed genes identified 13 connectivity networks. The most highly scored network highlighted the roles for oxidative stress and mitochondrial dysfunction pathways. Functional analyses of these networks provided evidence for activation of transforming growth factor beta (TGFß) signaling in ROP-Os/+ kidneys despite similar expression of the TGFß ligand between the tested strains. CONCLUSIONS: These data demonstrate the complex dysregulation of normal cellular functions in this animal model of FSGS and suggest that therapies directed at multiple levels will be needed to effectively treat human kidney diseases.

Lack of xanthine dehydrogenase leads to a remarkable renal decline in a novel hypouricemic rat model.

Uric acid (UA) is the final metabolite in purine catabolism in humans. Previous studies have shown that the dysregulation of UA homeostasis is detrimental to cardiovascular and kidney health. The Xdh gene encodes for the Xanthine Oxidoreductase enzyme group, responsible for producing UA. To explore how hypouricemia can lead to kidney damage, we created a rat model with the genetic ablation of the Xdh gene on the Dahl salt-sensitive rat background (SSXdh-/-). SSXdh-/- rats lacked UA and exhibited impairment in growth and survival. This model showed severe kidney injury with increased interstitial fibrosis, glomerular damage, crystal formation, and an inability to control electrolyte balance. Using a multi-omics approach, we highlighted that lack of Xdh leads to increased oxidative stress, renal cell proliferation, and inflammation. Our data reveal that the absence of Xdh leads to kidney damage and functional decline by the accumulation of purine metabolites in the kidney and increased oxidative stress.

Accelerated lysine metabolism conveys kidney protection in salt-sensitive hypertension.

Hypertension and kidney disease have been repeatedly associated with genomic variants and alterations of lysine metabolism. Here, we combined stable isotope labeling with untargeted metabolomics to investigate lysine's metabolic fate in vivo. Dietary 13C6 labeled lysine was tracked to lysine metabolites across various organs. Globally, lysine reacts rapidly with molecules of the central carbon metabolism, but incorporates slowly into proteins and acylcarnitines. Lysine metabolism is accelerated in a rat model of hypertension and kidney damage, chiefly through N-alpha-mediated degradation. Lysine administration diminished development of hypertension and kidney injury. Protective mechanisms include diuresis, further acceleration of lysine conjugate formation, and inhibition of tubular albumin uptake. Lysine also conjugates with malonyl-CoA to form a novel metabolite Nε-malonyl-lysine to deplete malonyl-CoA from fatty acid synthesis. Through conjugate formation and excretion as fructoselysine, saccharopine, and Nε-acetyllysine, lysine lead to depletion of central carbon metabolites from the organism and kidney. Consistently, lysine administration to patients at risk for hypertension and kidney disease inhibited tubular albumin uptake, increased lysine conjugate formation, and reduced tricarboxylic acid (TCA) cycle metabolites, compared to kidney-healthy volunteers. In conclusion, lysine isotope tracing mapped an accelerated metabolism in hypertension, and lysine administration could protect kidneys in hypertensive kidney disease.

p66Shc-mediated hydrogen peroxide production impairs nephrogenesis causing reduction of number of glomeruli.

AIMS: Adaptor protein p66Shc, encoded by Shc1 gene, contributes to the pathogenesis of oxidative stress-related diseases. p66Shc ability to promote oxidative stress-related diseases requires phosphorylation of serine 36 residue (Ser36) and depends on translocation of p66Shc to the mitochondria. We tested the hypothesis that abnormal p66Shc-mediated reactive oxygen species (ROS) production could be critically involved in nephrons development during nephrogenesis. MAIN METHODS: We have generated unique mutant rats (termed p66Shc-Del), which express endogenous p66Shc with a 9-amino acid deletion, and lack regulatory Ser36. H2O2 renal production was measured by enzymatic microelectrode biosensors. Nephron numbers in 3-5 weeks old p66Shc-Del rats were quantified using the acid maceration method. KEY FINDINGS: p66Shc-Del rats, as wild type salt sensitive rats, display increased mean arterial blood pressure following chronic exposure to a high salt diet. In contrast to wild type rats, p66Shc-Del rats display increased H2O2 renal production and are characterized by a reduction in renal function. The number of glomeruli is significantly reduced in adult p66Shc-Del rats. SIGNIFICANCE: Since low nephron number is an established risk factor for kidney disease and hypertension in humans and rodents, our data suggest that H2O2 renal production, caused by irregular signaling of p66Shc, could be critical in regulating nephrogenesis and that abnormal p66Shc signaling negatively impacts kidney development and renal function by increasing susceptibility to diabetic nephropathy and hypertension-induced nephropathy.

Role of opioid signaling in kidney damage during the development of salt-induced hypertension.

Opioid use is associated with predictors of poor cardiorenal outcomes. However, little is known about the direct impact of opioids on podocytes and renal function, especially in the context of hypertension and CKD. We hypothesize that stimulation of opioid receptors (ORs) contributes to dysregulation of intracellular calcium ([Ca2+]i) homeostasis in podocytes, thus aggravating the development of renal damage in hypertensive conditions. Herein, freshly isolated glomeruli from Dahl salt-sensitive (SS) rats and human kidneys, as well as immortalized human podocytes, were used to elucidate the contribution of specific ORs to calcium influx. Stimulation of κ-ORs, but not µ-ORs or δ-ORs, evoked a [Ca2+]i transient in podocytes, potentially through the activation of TRPC6 channels. κ-OR agonist BRL52537 was used to assess the long-term effect in SS rats fed a high-salt diet. Hypertensive rats chronically treated with BRL52537 exhibited [Ca2+]i overload in podocytes, nephrinuria, albuminuria, changes in electrolyte balance, and augmented blood pressure. These data demonstrate that the κ-OR/TRPC6 signaling directly influences podocyte calcium handling, provoking the development of kidney injury in the opioid-treated hypertensive cohort.

IgM nephropathy - Successful treatment with rituximab.

Immunoglobulin M nephropathy (IgMN) is a primary glomerulonephritis which is characterized by variable degrees of morphological features ranging from minimal glomerular involvement to segmental or global sclerosis. No specific treatment is known to date for this disease because of uncertainties in etiopathogenesis. The mainstay treatment for this disease has been corticosteroids, which has varying degrees of resistance ranging from 0% to 50%. We present the case of a 59-year-old Caucasian male who was referred to the outpatient nephrology clinic for the evaluation of proteinuria and was diagnosed with IgMN. We successfully treated the patient with rituximab with resolution of his proteinuria.

Improving sensitivity of amyloid detection by Congo red stain by using polarizing microscope and avoiding pitfalls.

Systemic amyloidosis is a devastating group of disorders for which there is no current cure. The treatment goal is to reduce the burden of amyloidogenic protein precursors. The treatment is only effective if applied early in the disease process before significant and irreversible end organ damage has taken place. Congo red is still the standard stain used in most histopathology laboratories to identify amyloid material in tissues. The identification of Congophilic amyloid material is challenging because of multiple interfering factors. Here we describe improved sensitivity of identifying Congophilic materials in histologic sections using a metallurgical polarized microscope specifically constructed for polarized microscopy. The microscope is equipped with strain-free optics, matching polarizers, dis-integrated compensators, and a circular mobile stage. Compared to a standard clinical microscope, this setup significantly improves sensitivity of identifying amyloid material in Congo red-stained slides. We also describe the deleterious effect of plastic coverslip which can interfere with the ability to examine the slides under polarized light. We present a series of 10 different patients who had cardiac, brain, and salivary gland biopsies that were either equivocal or deemed negative using a standard clinical microscope but were positive using the equipment described above. These samples were confirmed to be positive by other methods including electron microscopy. We conclude that use of the correct equipment is needed before ruling out amyloidosis in tissue sections.

Hyperfiltration-mediated Injury in the Remaining Kidney of a Transplant Donor.

Kidney donors face a small but definite risk of end-stage renal disease 15 to 30 years postdonation. The development of proteinuria, hypertension with gradual decrease in kidney function in the donor after surgical resection of 1 kidney, has been attributed to hyperfiltration. Genetic variations, physiological adaptations, and comorbidities exacerbate the hyperfiltration-induced loss of kidney function in the years after donation. A focus on glomerular hemodynamics and capillary pressure has led to the development of drugs that target the renin-angiotensin-aldosterone system (RAAS), but these agents yield mixed results in transplant recipients and donors. Recent work on glomerular biomechanical forces highlights the differential effects of tensile stress and fluid flow shear stress (FFSS) from hyperfiltration. Capillary wall stretch due to glomerular capillary pressure increases tensile stress on podocyte foot processes that cover the capillary. In parallel, increased flow of the ultrafiltrate due to single-nephron glomerular filtration rate elevates FFSS on the podocyte cell body. Although tensile stress invokes the RAAS, FFSS predominantly activates the cyclooxygenase 2-prostaglandin E2-EP2 receptor axis. Distinguishing these 2 mechanisms is critical, as current therapeutic approaches focus on the RAAS system. A better understanding of the biomechanical forces can lead to novel therapeutic agents to target FFSS through the cyclooxygenase 2-prostaglandin E2-EP2 receptor axis in hyperfiltration-mediated injury. We present an overview of several aspects of the risk to transplant donors and discuss the relevance of FFSS in podocyte injury, loss of glomerular barrier function leading to albuminuria and gradual loss of renal function, and potential therapeutic strategies to mitigate hyperfiltration-mediated injury to the remaining kidney.