Increased salt intake does not worsen the progression of renal cystic disease in high water-loaded PCK rats.

The anti-diuretic hormone arginine vasopressin is thought to be a detrimental factor in polycystic kidney disease (PKD). We previously reported that high water intake (HWI) reduced urine osmolality and urinary arginine vasopressin, improved renal function, and reduced the kidney/body weight ratio in PCK rats, an orthologous model of human PKD. In PKD patients, however, it is reported that HWI increases total kidney volume, urine volume, and urine sodium excretion, which could be a consequence of high salt intake. In the current study, we loaded PCK rats with high salt concurrently with HWI to determine whether this human-imitated condition exacerbates disease progression. PCK rats were assigned into 4 groups: control group (CONT: distilled water), HWI group (HWI: 5% glucose in water), HWI with 0.2% NaCl group (HWI+0.2%NaCl), and HWI with 0.45% NaCl group (HWI+0.45%NaCl). Total water intake during the experimental period was increased by 1.86-, 2.02-, and 2.42-fold in HWI, HWI+0.2%NaCl, and HWI+0.45%NaCl, and sodium intake was increased by 2.55- and 5.83-fold in HWI+0.2%NaCl and HWI+0.45%NaCl, respectively, compared with CONT. Systolic blood pressure was higher in HWI+0.2%NaCl and HWI+0.45%NaCl than in both CONT and HWI. Serum urea nitrogen, kidney/body weight ratio, cAMP, cystic area, and fibrosis index were significantly lower in HWI compared with CONT, and these ameliorative effects were not abrogated in either HWI+0.2%NaCl or HWI+0.45%NaCl. The amount of sodium excreted into the urine was increased by 2.50- and 8.38-fold in HWI+0.2%NaCl and HWI+0.45%NaCl, respectively, compared with HWI. Serum sodium levels were not different between the groups. These findings indicate that the beneficial effect of HWI against the progression of cystic kidney disease was not affected even by high salt-overload in this rodent model of PKD.

Urinary angiotensinogen level is associated with potassium homeostasis and clinical outcome in patients with polycystic kidney disease: a prospective cohort study.

BACKGROUND: Guidelines for general hypertension treatment do not recommend the combined use of renin-angiotensin-aldosterone system (RAAS) inhibitors due to the risk of hyperkalemia. However, a recent clinical trial showed that polycystic kidney disease (PKD) patients had infrequent episodes of hyperkalemia despite receiving combined RAAS inhibitors. Because intrarenal RAAS is a main component for renal potassium handling, we further investigated the association between intrarenal RAAS activity and serum potassium level in patients with chronic kidney disease, particularly in PKD patients, and examined whether intrarenal RAAS activity has a prognostic role in patients with PKD. METHODS: A total of 1788 subjects from the KoreaN cohort study for Outcome in patients With Chronic Kidney Disease (KNOW-CKD) were enrolled in this study. Intrarenal RAAS activity was assessed by the measurement of urinary angiotensinogen (AGT). The primary outcome was the composite of all-cause mortality and renal function decline. RESULTS: Patients with PKD had a significantly lower serum potassium level in chronic kidney disease stages 1 to 3b than non-PKD patients. In logistic regression analysis, after adjusting for multiple confounders, PKD patients had a significantly lower risk of hyperkalemia than non-PKD patients. In multivariable linear regression analysis, the urinary AGT/creatinine (Cr) ratio was negatively correlated with the serum potassium level (ß = - 0.058, P = 0.017) and positively correlated with the transtubular potassium gradient (TTKG, ß = 0.087, P = 0.001). In propensity score matching analysis, after matching factors associated with serum potassium and TTKG, PKD patients had a significantly higher TTKG (P = 0.021) despite a lower serum potassium level (P = 0.004). Additionally, the urinary AGT/Cr ratio was significantly higher in PKD patients than in non-PKD patients (P = 0.011). In 293 patients with PKD, high urinary AGT/Cr ratio was associated with increased risk of the composite outcome (hazard ratio 1.29; 95% confidence interval, 1.07-1.55; P = 0.007). CONCLUSIONS: High activity of intrarenal RAAS is associated with increased urinary potassium excretion and low serum potassium level in patients with PKD. In addition, intrarenal RAAS activity can be a prognostic marker for mortality and renal function decline in these patients.

Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease.

OBJECTIVE: PKD is a genetic disease that is characterized by abnormally proliferative epithelial cells in the kidney and liver. Urinary exosomes have been previously examined as a source of unique proteins that may be used to diagnose and monitor the progression of PKD. Previous studies by our group have shown that AGS3, which is a receptor-independent regulator G-proteins, was markedly upregulated in RTECs during kidney injury including PKD. In this study, our goal was to determine whether AGS3 could be measured in exosomes using animals and humans with PKD. RESULTS: In our study, urinary exosomes were isolated from PCK rats and the control Sprague-Dawley (SD) rats. AGS3 expression was significantly increased (P < 0.05) in PKD versus SD rats at 16 weeks of age. This increase was detectable in a time-dependent manner from 8 weeks of age and peaked at ~ 16-20 weeks (length of study). Similarly, in exosomes from human urine samples with PKD, AGS3 expression was significantly increased (P < 0.05) compared to healthy human controls where AGS3 was largely undetectable. In conclusion, the detection of AGS3 in urinary exosomes may be a novel biomarker for PKD, and provide new insight into the biology of tubular epithelial cell function during cystic disease progression.

Urinary proteomics using capillary electrophoresis coupled to mass spectrometry for diagnosis and prognosis in kidney diseases.

PURPOSE OF REVIEW: Urine is the most useful of body fluids for biomarker research. Therefore, we have focused on urinary proteomics, using capillary electrophoresis coupled to mass spectrometry, to investigate kidney diseases in recent years. RECENT FINDINGS: Several urinary proteomics studies for the detection of various kidney diseases have indicated the potential of this approach aimed at diagnostic and prognostic assessment. Urinary protein biomarkers such as collagen fragments, serum albumin, α-1-antitrypsin, and uromodulin can help to explain the processes involved during disease progression. SUMMARY: Urinary proteomics has been used in several studies in order to identify and validate biomarkers associated with different kidney diseases. These biomarkers, with improved sensitivity and specificity when compared with the current gold standards, provide a significant alternative for diagnosis and prognosis, as well as improving clinical decision-making.

Development of a Targeted Urine Proteome Assay for kidney diseases.

PURPOSE: Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases. EXPERIMENTAL DESIGN: Potential biomarkers were identified by using a multiproteomics workflow to compare urine proteomes of kidney transplant patients with immediate and delayed graft function. Differentially expressed proteins were identified, and corresponding stable isotope labeled internal peptide standards were synthesized for scheduled MRM. RESULTS: The Targeted Urine Proteome Assay (TUPA) was then developed by identifying those peptides for which there were at least two transitions for which interference in a urine matrix across 156 MRM runs was <30%. This resulted in an assay that monitors 224 peptides from 167 quantifiable proteins. CONCLUSIONS AND CLINICAL RELEVANCE: TUPA opens the way for using a robust mass spectrometric technology, MRM, for quantifying and validating biomarkers from among 167 urinary proteins. This approach, while developed using differentially expressed urinary proteins from patients with delayed versus immediate graft function after kidney transplant, can be expanded to include differentially expressed urinary proteins in multiple kidney diseases. Thus, TUPA could provide a single assay to help diagnose, prognose, and manage many kidney diseases.

Urinary extracellular vesicles for biomarker source to monitor polycystic kidney disease.

Extracellular vesicles (EVs) are bilayered lipid vesicles, 50-1000 nm in diameter and secreted by most types of cells. They contain many proteins, mRNAs, miRNAs, and lipids that reflect the pathophysiological state of the cells they originate from, and are therefore considered to be a rich source of potential biomarkers. In this issue (Pocsfalvi, G. et al., Proteomics Clin. Appl. 2015, 9, 552-567), Pocsfalvi et al. conducted pioneering investigations to determine whether changes in the protein content of EVs occur during progression of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disorder that predominantly affects the kidneys. Most significantly, iTRAQ-based quantitative proteomics showed that cytoskeleton-regulating and Ca(2+) -binding proteins are differentially expressed in urinary EVs of ADPKD patients. Impressively, these proteins are involved in biological processes that are closely related to the pathogenic state of tubular epithelial cells in ADPKD, demonstrating the possibility to monitor the status of patients using urinary EVs.

Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease.

PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology.

Prescriptions for dietary sodium in patients with chronic kidney disease: how will this shake out?

Patients with chronic kidney disease (CKD) are at risk of exhibiting expanded extracellular volume, and low-sodium diets are often prescribed to limit clinical complications from this condition. Fan et al. performed a post hoc study from the database of the Modification of Diet in Renal Disease Study. Their article, as well as other recent observations, suggests that a low-sodium diet may not be as beneficial as previously thought in all CKD patients.

Urinary sodium excretion and kidney failure in nondiabetic chronic kidney disease.

Current guidelines recommend under 2 g/day sodium intake in chronic kidney disease, but there are a few studies relating sodium intake to long-term outcomes. Here we evaluated the association of mean baseline 24-h urinary sodium excretion with kidney failure and a composite outcome of kidney failure or all-cause mortality using Cox regression in 840 participants enrolled in the Modification of Diet in Renal Disease Study. Mean 24-h urinary sodium excretion was 3.46 g/day. Kidney failure developed in 617 participants, and the composite outcome was reached in 723. In the primary analyses, there was no association between 24-h urine sodium and kidney failure (HR 0.99 (95% CI 0.91-1.08)) nor on the composite outcome (HR 1.01 (95% CI 0.93-1.09)), each per 1 g/day higher urine sodium. In exploratory analyses, there was a significant interaction of baseline proteinuria and sodium excretion with kidney failure. Using a two-slope model, when urine sodium was under 3 g/day, higher urine sodium was associated with increased risk of kidney failure in those with baseline proteinuria under 1 g/day and with lower risk of kidney failure in those with baseline proteinuria of ⩾ 1 g/day. There was no association between urine sodium and kidney failure when urine sodium was ⩾ 3 g/day. Results were consistent using first baseline and time-dependent urinary sodium excretion. Thus, we noted no association of urine sodium with kidney failure. Results of the exploratory analyses need to be verified in additional studies and the mechanism explored.

Development of a non-targeted metabolomics method to investigate urine in a rat model of polycystic kidney disease.

AIM: The purpose of this research was to use metabolomics to investigate the cystic phenotype in the Lewis polycystic kidney rat. METHODS: Spot urine samples were collected from four male Lewis control and five male Lewis polycystic kidney rats aged 5 weeks, before kidney function was significantly impaired. Metabolites were extracted from urine and analysed using gas chromatography-mass spectrometry. Principal component analysis was used to determine key metabolites contributing to the variance observed between sample groups. RESULTS: With the development of a metabolomics method to analyse Lewis and Lewis polycystic kidney rat urine, 2-ketoglutaric acid, allantoin, uric acid and hippuric acid were identified as potential biomarkers of cystic disease in the rat model. CONCLUSION: The findings of this study demonstrate the potential of metabolomics to further investigate kidney disease.

A metabolomics approach using juvenile cystic mice to identify urinary biomarkers and altered pathways in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and affects 1 in 1,000 individuals. Ultrasound is most often used to diagnose ADPKD; such a modality is only useful late in the disease after macroscopic cysts are present. There is accumulating evidence suggesting that there are common cellular and molecular mechanisms responsible for cystogenesis in human and murine PKD regardless of the genes mutated, and, in the case of complex metabolomic analysis, the use of a mouse model has distinct advantages for proof of principle over a human study. Therefore, in this study we utilized a urinary metabolomics-based investigation using gas chromatography-time of flight mass spectrometry to demonstrate that the cystic mouse can be discriminated from its wild-type counterpart by urine analysis alone. At day 26 of life, before there is serological evidence of kidney dysfunction, affected mice are distinguishable by urine metabolomic analysis; this finding persists through 45 days until 64 days, at which time body weight differences confound the results. Using functional score analysis and the KEGG pathway database, we identify several biologically relevant metabolic pathways which are altered very early in this disease, the most highly represented being the purine and galactose metabolism pathways. In addition, we identify several specific candidate biomarkers, including allantoic acid and adenosine, which are augmented in the urine of young cystic mice. These markers and pathway components, once extended to human disease, may prove useful as a noninvasive means of diagnosing cystic kidney diseases and to suggest novel therapeutic approaches. Thus, urine metabolomics has great diagnostic potential for cystic renal disorders and deserves further study.

Urinary Peptide patterns in native kidneys and kidney allografts.

BACKGROUND: The use of biopsies to determine kidney health after kidney transplantation is an invasive procedure with some risk to the patient. Consequently, a noninvasive test for transplanted kidney health would provide a significant advantage over current clinical practice. METHODS: Urines from kidney donors before nephrectomy, pretransplant patients with native kidney disease, and posttransplant kidney recipients were examined for protein biomarkers to diagnose or prognose kidney disease. Proteins were extracted by C4 reverse phase affinity and analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Urine from individuals with healthy kidneys showed few components other than two ubiquitous saposin B glycoisoforms. Patients with kidney disease lacked saposin B and showed new components in two patterns: the most common contained beta-2 microglobulin (B2M, m/z=11,732) plus one or more peaks at m/z=10,350, 9480, 4337, and 4180. Pattern 2 lacked beta-2 microglobulin but contained several degradation products of alpha-1 antitrypsin. Other pathologic components included urinary protein 1 (m/z=15,835), transthyretin (m/z=13,880), and a component at m/z=13,350. CONCLUSIONS: Patients with acute rejection showed profiles that ranged from those of kidney donors to those of advanced kidney disease. The range of patterns may be useful for analysis of transplant patients without complications and persons with developing kidney disease before or after transplant.

The effect of angiotensin-converting-enzyme inhibitors on progression of advanced polycystic kidney disease.

BACKGROUND: It is not known whether angiotensin-converting-enzyme (ACE) inhibitors slow the progression of polycystic kidney disease (PKD). We performed a patient-level meta-analysis to compare the effect of antihypertensive regimens, including ACE inhibitors, to those without ACE inhibitors (controls) on kidney disease progression in patients with PKD. METHODS: We analyzed a database of 11 randomized controlled trials including 1860 patients with nondiabetic kidney disease. We compared randomized groups for the decline in urine protein excretion and kidney disease progression (combined outcome of doubling of baseline serum creatinine or onset of kidney failure). We also performed multivariable linear regression and Cox proportional hazards analyses. Based on previous findings, we searched for interactions between the treatment effect (effect of ACE inhibitors vs. controls) and baseline urine protein excretion in both models. RESULTS: Eight studies included a total of 142 subjects with PKD: 68 (48%) were randomized to ACE inhibitors and 74 (52%) were randomized to the control. Baseline mean (SD) urine protein excretion was 0.92 (1.40) g/day: 1.08 (1.50) g/day in the ACE inhibitor and 0.76 (1.28) g/day in the control group. During a mean follow-up of 2.3 years, mean (SD) urine protein excretion declined by 0.33 (1.11) g/day in the ACE inhibitor group and increased by 0.19 (0.88) g/day in the control group (P < 0.001). Kidney disease progression occurred in 50 patients: 20 patients (29%) in the ACE inhibitor group and 30 patients (41%) in the control group (P= 0.17). ACE inhibitors had a greater effect on lowering urine protein excretion and slowing kidney disease progression in patients with higher levels of baseline urine protein excretion (interaction P < 0.001 and P= 0.03, respectively). CONCLUSION: As in other causes of non-diabetic kidney disease, antihypertensive regimens with ACE inhibitors are more effective in lowering urine protein excretion in patients with advanced PKD compared to regimens without ACE inhibitors, and this benefit is greater in patients with higher levels of baseline urine protein excretion. The effect of ACE inhibitors to slow kidney disease progression in PKD is inconclusive.

Gross hematuria rapidly deteriorated renal function in a patient with polycystic kidney disease and Klippel-Trenaunay-Weber syndrome.

A case of polycystic kidney disease (PKD) associated with Klippel-Trenaunay-Weber syndrome is described. A 58-year-old man with chronic renal failure experienced urinary retention following gross hematuria. Intermittent drainage was necessary for significant urination for five days. Thereafter his urinary retention was relieved, but renal failure progressively developed and hemodialysis was started. Right hydronephrisis and hydroureter disappeared one month later. In spite of relief of obstruction, of which the cause was likely blood clots, renal function was not restored. Obstructive nephropathy was most likely explicable for notable deterioration in renal function. Our case might have susceptibilities to PKD development in terms of angiogenesis.

An endocytosis defect as a possible cause of proteinuria in polycystic kidney disease.

Because proteinuria has been demonstrated in patients with autosomal-dominant polycystic kidney disease (ADPKD), we have investigated whether proteinuria also occurs in the (cy/+) rat, a widely used model for ADPKD. Increased urinary excretion of proteins, in particular of albumin, can be found in 16-wk-old (cy/+) rats, with a gel electrophoresis pattern compatible with a tubular origin of proteinuria. Using FITC-labeled dextran as an in vivo tracer for renal tubular endosomal function, we could show that portions of cyst-lining epithelia from proximal tubules have lost the ability to endocytose, which is necessary for the reabsorption of low-molecular-weight proteins. By immunohistochemistry, the expression of other proteins implicated in endocytosis, such as the chloride channel ClC-5 and the albumin receptor megalin, correlated well with the presence and absence of FITC-dextran in cysts. As an example of growth factor systems possibly being affected by this endocytosis defect, we could detect increased urinary levels of insulin-like growth factor-I protein in (cy/+) animals. These data indicate that proteinuria and albuminuria in the aforementioned rat model for ADPKD are due to a loss of the endocytic machinery in epithelia of proximal tubular cysts. This may also affect the concentration of different growth factors and hormones in cyst fluids and thus modulate cyst development.

Endotoxin and nanobacteria in polycystic kidney disease.

BACKGROUND: Microbes have been suspected as provocateurs of polycystic kidney disease (PKD), but attempts to isolate viable organisms have failed. Bacterial endotoxin is the most often reported microbial product found in PKD fluids. We assessed potential microbial origins of endotoxin in cyst fluids from 13 PKD patients and urines of PKD and control individuals. METHODS: Fluids were probed for endotoxin and nanobacteria, a new bacterium, by the differential Limulus Amebocyte Lysate assay (dLAL), genus-specific antilipopolysaccharide (LPS) antibodies, monoclonal antibodies to nanobacteria, and hyperimmune serum to Bartonella henselae (HS-Bh). Selected specimens were also assessed by transmission electron microscopy (TEM) and nanobacterial culture methods. RESULTS: LPS or its antigenic metabolites were found in more than 75% of cyst fluids tested. Nanobacteria were cultured from 11 of 13 PKD kidneys, visualized in 8 of 8 kidneys by TEM, and immunodetected in all 13 PKD kidneys. By immunodetection, nanobacterial antigens were found in urine from 7 of 7 PKD males, 1 of 7 PKD females, 3 of 10 normal males, and 1 of 10 normal females. "Nanobacterium sanguineum" was dLAL positive and cross-reactive with antichlamydial LPS and HS-Bh. Some cyst fluids were also positive for LPS antigens from Escherichia coli, Bacteroides fragilis and/or Chlamydia, and HS-Bh, as were liver cyst fluids from one patient. Tetracycline and citrate inhibited nanobacterial growth in vitro. CONCLUSION: Nanobacteria or its antigens were present in PKD kidney, liver, and urine. The identification of candidate microbial pathogens is the first step in ascertaining their contribution, if any, to human disease.

Gender-dependent effect of L-NAME on polycystic kidney disease in Han:SPRD rats.

To determine whether the renal nitric oxide (NO) system has a role in the pathogenesis of polycystic kidney disease (PKD) in Han:Sprague-Dawley (SPRD) rats, the NO synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), 70 mg/L, or L-arginine, 0.5 g/L, was administered to heterozygous diseased (cy/+) and homozygous normal animals. Urine nitrate and nitrite excretion was reduced by L-NAME treatment and, in the male cy/+ rats, increased by L-arginine administration. The administration of L-NAME significantly increased blood pressure in all groups, whereas L-arginine had no effect. L-NAME and L-arginine had a modest but significant overall effect on the severity of cystic disease in male rats, reflected by relative kidney weights and cyst volume densities. This effect was gender dependent because it was not observed in female animals. The administration of L-NAME resulted in a significant increase in plasma creatinine concentration of the cy/+ rats, which was more marked in male than female animals. These observations support the recently reported gender differences in the renal NO system and a small role for NO synthesis that can be inhibited by L-NAME in the pathogenesis of PKD in Han:SPRD rats. These observations do not exclude a more important role for the endogenous renal NO production in the pathogenesis of PKD in view of a recent report of a major NOS resistant to conventional inhibitors in the rat kidney.

The pathogenesis of dysplastic kidney in a urinary tract obstruction in the female fetal lamb.

BACKGROUND/PURPOSE: The type of renal dysplasia resulting from obstructive uropathy depends on the completeness of the obstruction and its timing with respect to the stage of glomerulogenesis at the time of the obstruction. The authors created a successful obstructive uropathy model in the female fetal lamb to demonstrate the differing pathogenesis of renal dysplasia. METHODS: Female fetal lambs at 60 and 90 days' gestation had their urethra and urachus ligated transabdominally and were delivered by cesarean section at 145 days (full term). Kidney length and cortical thickness were measured, and samples were examined histologically. In the lambs operated on at 90 days, the urine was collected at delivery and Na and CI were measured and compared with the results obtained from normal full-term lambs. RESULTS: Seven of 10 female lambs had hydronephrosis or dysplastic kidneys. The cortext to kidney length ratio was 10+/-3% in the 90-days hydronephrotic group versus 29+/-6% in the controls (P<.001). Morphologically, the 90-day model had dilatation of the collecting tubules with normal glomerular numbers. The 60-day model had tubular cysts with fibromuscular cuffing and reduced glomerular numbers. The fetal urine Na was 47+/-3.3 mmol/L in controls versus 78+/-24 mmol/L in the hydropnephrotic lambs (P<.05). The urine CI in these lambs was 38+/-8.6 mmol/L in controls versus 55+/-14.5 mmol/L in the hydronephrotic lambs (P<.05). CONCLUSIONS: An obstructive uropathy model was created in female fetal lambs. There were no dysplastic changes in the kidneys in lambs operated on at 90 days' gestation, but there were definite dysplastic changes in those operated on at 60 days. Concentrations of Na and CI in the fetal urine are higher than normal in the 90-day model.