Background/Aims

The TRPC1 gene on chromosome 3q22–24 resides within the linkage region for diabetic nephropa-thy (DN) in type 1 (T1D) and type 2 diabetes mellitus (T2D). A recent study has demonstrated that TRPC1 expression is reduced in the kidney of diabetic ZDF- and STZ-treated rats. The present study aimed to evaluate the genetic and functional role of TRPC1 in the development of DN.

Methods

Genetic association study was performed with two independent cohorts, including 1,177 T1D European Americans with or without DN from GoKinD population and 850 African-American subjects with T2D-associated end-stage renal disease (ESRD), or with hypertensive (non-diabetic) ESRD, and nondiabetic controls. Seven tag SNP markers derived from HapMap data (phase II) were genotyped. TRPC1 gene expression was examined using real time RT-PCR.

Results

No significant association of TRPC1 DNA polymorphisms with DN or ERSD was found in GoKinD and African-American populations. TRPC1 gene mRNA expression in kidney was found to be trendily reduced in 12-week and significantly in 26-week-old db/db mice.

Conclusions

TRPC1 genetic polymorphism may not fundamentally contribute to the development of DN, while reduction of the gene expression in kidney may be a late phenomenon of DN as seen in diabetic animal models.

Background

Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.

Methods

Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bearing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes.

Results

AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers.

Conclusion

Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.

Background/Aim:

In in vitro studies, angiotensin (Ang) II has been demonstrated to promote podocyte apoptosis. The present study evaluates the effects of Ang II infusion in rats on podocyte nephrin expression and apoptosis and the molecular mechanisms involved in Ang II-induced proteinuria and mesangial expansion.

Methods:

Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang II (400 ng·kg−1·min−1) by means of a mini-osmotic pump for variable time periods. Systolic blood pressure and urinary protein and albumin excretion rate measurements were carried out on days 7, 14, 21, and 28. The animals were sacrificed on days 14 and 28 and evaluated for serum creatinine, renal pathological changes, podocyte apoptosis, renal nephrin mRNA, and protein expression.

Results:

The Ang II-infused rats developed hypertension and proteinuria. On day 14, the Ang II-infused rats showed narrowing of the slit diaphragm, an increase in podocyte nephrin mRNA and protein expression, and alterations in its distribution along the foot processes. On day 28, the Ang II-infused rats demonstrated the presence of apoptotic podocytes and decreased nephrin mRNA and protein expression. There was a negative correlation between nephrin expression and the numbers of apoptotic podocytes (r = −0.63, p < 0.05).

Conclusion:

These results suggest that changes in nephrin expression may play a role in the pathogenesis of Ang II-induced podocyte apoptosis.

Background/Aims

Several inflammatory biomarkers have been found to be associated with cardiovascular disease or all-cause mortality in dialysis patients, but their usefulness in clinical practice or as surrogate endpoints is not certain. The purpose of the present study was to determine the intrapatient variation of C-reactive protein, IL-6, fetuin-A and albumin in a population of dialysis patients.

Methods

Apparently healthy dialysis patients with either a tunneled dialysis catheter or fistula had monthly assessments of these biomarkers for a total of four determinations, and the intraclass correlation coefficients were calculated as measures of intersubject variance.

Results

Our results showed large within-subject variation relative to the total variation in the measurements (31-46%). Having a tunneled catheter as opposed to a fistula was not significantly associated with mean levels, suggesting that chronic subclinical catheter infection does not explain the variation seen in the biomarkers. In contrast, there was a rapid change in these biomarkers with a clinically apparent acute infection.

Conclusion

These results suggest that these biomarkers have limitations for use as surrogate endpoints in clinical trials due to wide fluctuations, even in apparently clinically healthy individuals.

Background

Clinical practice guidelines recommend alkali therapy in patients with non-dialysis-dependent chronic kidney disease (CKD) and metabolic acidosis to prevent complications from metabolic acidosis. We systematically reviewed the effect of sodium bicarbonate on benefits and harms in patients with CKD.

Methods

We searched for randomized controlled trials in MEDLINE (through July 2011), Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, and scientific abstracts. We included trials that compared sodium bicarbonate to standard-of-care therapy or placebo that reported on kidney-related outcomes. We performed random-effects model meta-analyses to compute net changes (for continuous variables) and risk ratios (for binary variables).

Results

Two short-term (≤7 days) crossover trials and 4 long-term (≥2 months) parallel design randomized controlled trials met eligibility (312 patients). All 6 trials prescribed sodium bicarbonate in the alkali-treated group. In the long-term studies, alkali therapy was associated with a net decrease in serum creatinine (−0.07 mg/dL, 95% CI −0.09, −0.05, P<0.001; I2=0), a net improvement in GFR (3.2 mL/min/1.73 m2, 95% CI 1.6, 4.7, P<0.001; I2=0), and a lower incidence of dialysis initiation (risk ratio 0.21, 95% CI 0.08, 0.54; P=0.001; I2=0). No benefit was observed on the serum creatinine or GFR in short-term studies. Alkali therapy was not associated with a higher likelihood of initiating or escalating anti-hypertensive medications.

Conclusions

Alkali therapy is associated with an improvement in kidney function, which may afford a long-term benefit in slowing the progression of CKD. However, differences in study protocols and small sample sizes preclude definitive conclusions.

Background/Aims

Arteriovenous fistulas (AVFs) appear to be clinically superior to catheters as vascular access for maintenance hemodialysis, but higher insertion costs and high disease burden and mortality obscure the issue of whether AVF placement before hemodialysis initiation represents a net cost savings. We aimed to investigate Medicare costs for patients beginning maintenance hemodialysis, as related to timing of arteriovenous fistula (AVF) placement.

Methods

Data were from Medicare claims for incident hemodialysis patients aged ≥ 67 years in 2006. The study period extended from two years before to one year after dialysis initiation. Patients identified as having AVFs were categorized by timing of placement (mature AVF at dialysis initiation, maturing AVF at initiation, post-initiation AVF placement). Because timing may be influenced by factors that also influence overall costs, the model accounted for this non-random treatment assignment. An ordered probit extension of the classic Heckman correction was employed after identifying an appropriate instrumental variable. A cohort with Medicare coverage before and after dialysis initiation was identified, and Medicare claims were used to identify comorbid conditions and treatment costs.

Results

Principal findings are that earlier AVF placement leads to lower costs, with the potential for about $500 million in savings. Additionally, the effect of non-random treatment assignment is real and significant. In our data, the impact of AVF placement timing was understated when treatment selection was ignored.

Conclusions

For appropriate AVF candidates, having a mature AVF in place at the time of dialysis initiation appears to confer a cost savings.

Background/Aims

Our previous studies have shown that supplementation with 17-β estradiol (E2) from the onset of diabetes attenuates diabetic nephropathy. But, E2 is accompanied by feminizing effects as well as adverse side effects on other organs. The current study examined the renoprotective effects of a selective estrogen receptor modulator, raloxifene (RAL), in an experimental model of diabetic nephropathy. RAL activates estrogen receptors and estrogen receptor-mediated cellular events without the side effects of E2.

Methods

The study was performed in Sprague-Dawley non-diabetic (ND), streptozotocin (STZ)-induced diabetic (D) and STZ-induced diabetic+raloxifene (D+RAL) rats (n=6/group).

Results

After 12 weeks of treatment, D was associated with increased albumin excretion (UAE; ND, 4.2±0.4; ND, 41.3±9.0 mg/day), glomerulosclerosis (GSI; ND, 0.26±0.04; D, 1.86±0.80 AU), tubulointerstitial fibrosis (TIFI; ND, 0.37±0.05; D, 2.12±0.50 AU), increased collagen type I (CI; ND, 1.31±0.07; D, 4.65±0.09 ROD), collagen type IV (CIV; ND, 0.64±0.03; D, 1.37±0.11 ROD) and transforming growth factor beta protein expression (TGF-β; ND, 0.65±0.08; D, 1.25±0.10 ROD), increased density of CD68-positive cells (CD68; ND, 1.37±3.02; D, 29.2±1.74 cells/mm2) and increased plasma levels of interleukin-6 (IL-6; ND, 14.8±5.0; D, 51.3±14.0 pg/ml). Treatment with RAL partially or fully attenuated these processes (UAE, 21.0±5.0 mg/day; GSI, 0.40±0.06 AU; TIFI, 0.20±0.04 AU; CI, 2.55±0.49 ROD; CIV, 0.70±0.09 ROD; TGF-β, 0.91±0.08 ROD; CD68, 6.03±2.38 cells/mm2; IL-6, 31.2±5.0 pg/ml).

Conclusions

Our data indicate that treatment with RAL attenuates albuminuria and renal structural changes associated with diabetes.

Background

Alloantigen-independent factors contribute to long-term damage in renal transplant recipients, likely due to ischemia/reperfusion (I/R) injury at transplantation (Tx). I/R injury promotes oxidative stress and inflammation resulting in endothelial injury.

Methods

In this study we investigated the long-term efficacy (22 weeks) of short-term (10 day) endothelial protection therapy (EP) in ‘optimal’ donor kidneys using the male Fisher 344 rat isograft (ISO) model. ISO-EP kidneys were compared to untreated ISO (ISO-UN) kidneys. EP involved dexamethasone to donor, ex vivo treatment of the kidney with deferoxamine and tempol, and administration to the recipient of l-arginine and tempol for 10 days. Rats were sacrificed 22 weeks following Tx and compared to age-matched, normal controls.

Results

Both groups of ISO Tx rats developed similar renal dysfunction and structural damage and renal NADPH-oxidase-dependent O2− production was similarly elevated in ISO-UN and ISO-EP groups vs. controls. In vitro renal cortex NO synthase (NOS) activity was also similar in ISO-UN and ISO-EP rats, despite lower nNOS and eNOS protein abundance in ISO-EP.

Conclusion

I/R injury-induced late graft dysfunction occurs even when optimal donors are used and when short-term EP treatment is given. Increased renal superoxide production is not prevented by short-term EP therapy.

Background

MYH9 is a podocyte-expressed gene encoding nonmuscle myosin IIA that is associated with idiopathic and human immunodeficiency virus-associated focal segmental glomerulosclerosis (FSGS) and hypertensive end-stage renal disease in African Americans.

Methods

Four single nucleotide polymorphisms comprising the major MYH9 E1 risk haplotype were tested for association with estimated glomerular filtration rate (eGFR) and urine albumin:creatinine ratio (ACR) in 2,903 HyperGEN participants (1,458 African Americans (AA) in 895 families and 1,445 European Americans (EA) in 859 families) to determine the role of MYH9 in subclinical nephropathy. Association analyses employed general linear models in unrelated probands and generalized estimating equations in families. Adjustment was performed for age, sex, diabetes, BMI, medications, and mean arterial pressure separately in each race.

Results

Mean (SD) eGFR and ACR were 74.3 (16.0) ml/min/1.73 m2 and 20.3 (119.9) mg/g in EA, and 88.6 (20.9) ml/min/1.73 m2 and 76.8 (394.5) mg/g in AA (both p < 0.0001 across ethnicities). Urine ACR was associated with rs3752462 (p = 0.01) and rs4821481 (p = 0.05) in unrelated AA and with rs4821481 (p = 0.03), rs2032487 (p = 0.04) and the E1 3224 haplotype (p = 0.013) in AA families. Single nucleotide polymorphisms and the haplotype were not associated with ACR in EA or with eGFR in either ethnic group.

Conclusions

MYH9 variants are associated with albuminuria in hypertensive AA. The strength of the association was weaker than that in FSGS and hypertensive end-stage renal disease. MYH9 risk variants appear to be associated with primary FSGS with secondary hypertension, although nephrosclerosis may develop in response to hypertension in subjects homozygous for the MYH9 E1 risk haplotype.

The analysis of change is central to the study of kidney research. In the past 25 years, newer and more sophisticated methods for the analysis of change have been developed, however as of yet these newer methods are underutilized in the field of kidney research. Repeated measures ANOVA is the traditional model that is easy to understand and simpler to interpret, but it may not be valid in complex real-world situations. Problems with the assumption of sphericity, unit of analysis, lack of consideration for different types of change, and missing data, in the repeated measures ANOVA context are often encountered. Multilevel modeling, a newer and more sophisticated method for the analysis of change, overcomes these limitations and provides a better framework for understanding the true nature of change. The present article provides a primer on the use of multilevel modeling to study change. An example from a clinical study is detailed and the method for implementation in SAS is provided.

Background/Aim:

The kd/kd mouse spontaneously develops severe and progressive nephritis leading to renal failure, characterized by cellular infiltration, tubular destruction and glomerular sclerosis. Recent identification of the mutant gene and the observation that podocytes are affected, led to the hypothesis that there are primary renal epithelial cell defects in this strain.

Methods:

Clinical and pathological signs of disease in a large cohort of kd/kd mice were studied by light microscopy, electron microscopy, and biochemical analyses of serum and urine at early stages of disease. Special attention was paid to mice under 140 days of age that had normal blood urea nitrogen (BUN) levels, but had developed albuminuria.

Results:

Although overt glomerular abnormalities are commonly observed either coincident with or after tubulointerstitial nephritis, we now report that albuminuria and visceral epithelial abnormalities, including hyperplasia and podocyte effacement may occur before the onset of either elevated BUN levels or severe interstitial nephritis, and this is accompanied by biochemical perturbations in serum typical of the nephrotic syndrome.

Conclusions:

The results suggest that the defect in kd/kd mice primarily affects both the tubular and glomerular visceral epithelium. The tubular epithelial defect triggers autoimmune interstitial nephritis, whereas a defect in podocytes leads to proteinuria and glomerulosclerosis. Thus, a single mitochondrial abnormality may result in differences in disease expression that vary with the type of epithelial cells. It is likely that the mitochrondrial perturbations in glomerular and tubular epithelia act in concert, through activation of different pathologic pathways, to accelerate disease progression leading to renal failure.

Background:

Primary hyperoxaluria-type 1 (PH-1) is a rare autosomal recessive disorder of glyoxalate metabolism caused by deficiency in the liver-specific peroxisomal enzyme alanine-glyoxalate transaminase 1 (AGT) resulting in the increased oxidation of glyoxalate to oxalate. Accumulation of oxalate in the kidney and other soft tissues results in loss of renal function and significant morbidity. The present treatment options offer some relief in the short term, but they are not completely successful. In the present study, we tested the feasibility of corrective gene therapy for this metabolic disorder.

Methods:

A cDNA library was made from HepG2 cells. PCR primers were designed for the AGT sequence with modifications to preclude mistargeting during gene delivery. Amplified AGT cDNA was cloned as a fusion protein with green fluorescent protein (GFP) using the vector EGFP-C1 (Clontech) for monitoring subcellular distribution. Sequence and expression of the fusion protein was verified. Fusion protein vectors were transfected into hepatocytes by liposomal transfection. AGT expression and subcellular distribution was monitored by GFP fluorescence.

Results:

HepG2 cells express full-length mRNA coding for AGT as confirmed by insert size as well as sequence determination. Selective primers allowed us to generate a modified recombinant GFP-AGT fusion protein. Cellular transfections with Lipofectamine resulted in transfection efficiencies of 60–90%. The recombinant AGT did localize to peroxisomes as monitored by GFP fluorescence.

Conclusions:

The results demonstrate preliminary in vitro feasibility data for AGT transfection into the hepatocytes. To the best of our knowledge, this is the first study to attempt recombinant AGT gene therapy for treatment of primary hyperoxaluria-1.